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Pathways Leading to Phosphorylation of P450c17 and to the Posttranslational Regulation of Androgen Biosynthesis
Abstract
Cytochrome P450c17 (P450c17) is the single enzyme that catalyzes steroid 17alpha-hydroxylase and 17,20 lyase activities and hence is the crucial decision-making step that determines the class of steroid made in a steroidogenic cell. Although both activities are catalyzed on a single active site, the ratio of these activities is regulated by posttranslational events. Serine phosphorylation of P450c17 increases 17,20 lyase activity by increasing the enzyme's affinity for its redox partner, P450 oxidoreductase. We searched for the relevant kinase(s) that phosphorylates P450c17 by microarray studies and by testing of kinase inhibitors. Microarrays show that 145 of the 278 known serine/threonine kinases are expressed in human adrenal NCI-H295A cells, only six of which were induced more than 2-fold by treatment with 8-Br-cAMP. Key components of the ERK1/2 and MAPK/ERK kinase (MEK)1/2 pathways, which have been implicated in the insulin resistance of PCOS, were not found in NCI-H295A cells, implying that these pathways do not participate in P450c17 phosphorylation. Treatment with various kinase inhibitors that probe the protein kinase A/phosphatidylinositol 3-kinase/Akt pathway and the calcium/calmodulin/MAPK kinase pathway had no effect on the ratio of 17,20 lyase activity to 17alpha-hydroxylase activity, appearing to eliminate these pathways as candidates leading to the phosphorylation of P450c17. Two inhibitors that target the Rho-associated, coiled-coil containing protein kinase (ROCK)/Rho pathway suppressed 17,20 lyase activity and P450c17 phosphorylation, both in NCI-H295A cells and in COS-1 cells transfected with a P450c17 expression vector. ROCK1 phosphorylated P450c17 in vitro, but that phosphorylation did not affect 17,20 lyase activity. We conclude that members of the ROCK/Rho pathway act upstream from the kinase that phosphorylates P450c17 in a fashion that augments 17,20 lyase activity, possibly acting to catalyze a priming phosphorylation.
... To date no kinase could be identified that is capable of augmenting the 17,20-lyase activity with subsequent phosphorylation of serine and/or threonine residues of CYP17 in vitro. However, the 17,20-lyase activity has been shown to be influenced by two intracellular signalling proteins in a whole-cell milieu, namely protein kinase A (Zhang et al., 1995;Wang et al., 2010;Kempna et al., 2010) and rho-associated coiled-coil containing kinase 1 (ROCK1) (Tee et al., 2008). Differences between in vitro and in vivo results have led to conclusions such as the involvement of the ROCK/Rho signalling pathway upstream of the relevant kinase that phosphorylated CYP17 (Tee et al., 2008). ...
... However, the 17,20-lyase activity has been shown to be influenced by two intracellular signalling proteins in a whole-cell milieu, namely protein kinase A (Zhang et al., 1995;Wang et al., 2010;Kempna et al., 2010) and rho-associated coiled-coil containing kinase 1 (ROCK1) (Tee et al., 2008). Differences between in vitro and in vivo results have led to conclusions such as the involvement of the ROCK/Rho signalling pathway upstream of the relevant kinase that phosphorylated CYP17 (Tee et al., 2008). This conclusion of Tee et al. (2008) was based on the observation that ROCK1 increases 17,20-lyase activity in vivo, but not in vitro, despite its capability to phosphorylate CYP17 in vitro. ...
... Differences between in vitro and in vivo results have led to conclusions such as the involvement of the ROCK/Rho signalling pathway upstream of the relevant kinase that phosphorylated CYP17 (Tee et al., 2008). This conclusion of Tee et al. (2008) was based on the observation that ROCK1 increases 17,20-lyase activity in vivo, but not in vitro, despite its capability to phosphorylate CYP17 in vitro. It is therefore unknown whether ROCK1 truly act upstream of the kinase that phosphorylates CYP17, or whether the observations of Tee et al. (2008) resulted from the experimental setup (in vivo vs. in vitro). ...
This study describes:
• the comparison of the enzymatic activities of the two ovine cytochrome P450 17�-
hydroxylase/17,20-lyase (CYP17) isoforms expressed in non-steroidogenic COS-1 cells.
The Km and Vmax values for the metabolism of pregnenolone and progesterone were
determined, while time-dependent metabolism of pregnenolone, 17-hydroxypregenolone,
progesterone and 17-hydroxyprogesterone was also reported. The cloning and sequencing of
ovine cytochrome b5 is reported and was co-expressed with CYP17. The results showed that
the wild type 1 (WT1) isoform of ovine CYP17 produce more cortisol precursors than the
wild type 2 (WT2) isoform;
• the analysis of the frequency distribution of the CYP17 genotypes within a South African
Merino population, which were divergently selected for (H-line) or against (L-line) the
ability of a ewe to rear multiple offspring per birthing opportunity. It was observed that the
CYP17 frequency distribution was the same within the H- and L-line, with 78.3 %
heterozygous WT1/WT2 and 21.7 % homozygous WT1/WT1. No homozygous WT2/WT2
individuals were identified;
• the development of a UPLC-MS/MS method for the separation and quantification of all
thirteen adrenal steroids that are produced in the adrenal gland;
• the relative contribution of the CYP17 genotypes in the total steroidogenic output in adult
adrenocortical cells from the adrenal glands of H- and L-line sheep, with particular emphasis
on cortisol production. The adrenocortical cells from the H-line sheep showed a marked
higher cortisol production than the L-line, while adrenocortical cells from homozygous
WT1/WT1 sheep also produced more cortisol than heterozygous WT1/WT2 sheep;
• the blood cortisol responses upon the stimulation of the HPA axis by insulin induced
hypoglycaemia of the H- and L-line sheep with known CYP17 genotypes. It was observed
that the CYP17 genotype and selection line are important factors affecting the cortisol
responses of sheep, where L-line heterozygous WT1/WT2 sheep showed the lowest cortisol
response and glucose recovery;
• the association of the CYP17 genotype with behavioural responses of H- and L-line sheep to
flock isolation stress, as well as the association of the CYP17 genotype with ewe
reproduction and lamb output. While reproduction seemed to be unaffected by the CYP17
genotype, the behavioural stress responses of sheep to flock isolation correlated with the
CYP17 genotype, where the heterozygous WT1/WT2 genotype was associated with a wilder
nature.
... Protein phosphatase 2A (PP2A) was shown to dephosphorylate CYP17 and diminish lyase activity and androgen synthesis [66]. Although phosphorylation of CYP17 has been shown to be enhanced by cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) [64,65], and rho-associated, coiled-coil containing protein kinase 1 (ROCK1), this phosphorylation does not increase lyase activity [67]. Only recent studies identified the mitogenactivated protein kinase 14 (MAPK14, p38a) as the responsible kinase enhancing 17,20 lyase activity by phosphorylation [68]. ...
... The exact signaling between phospho-SMADs and CYP17A1 gene (dotted line) remains unknown. Transcriptome profiling of H295A cells revealed that ROCK1 kinase phosphorylates CYP17A1; however, this phosphorylation does not enhance 17,20 lyase activity [67]. Recent p38a (MAPK14) was described to phosphorylate CYP17A1 and confer increased 17,20 lyase activity, which is essential for the production of androgens [68]. ...
... In a recent review we published some preliminary results establishing a possible role for protein kinase C (PKC) and protein kinase D (PKD) signaling in androgen production [86]. Studies for phosphoinositide-3-kinase (PI3K)/protein kinase B (PKB/Akt) signaling revealed mixed results [67,86], so that its role remains unsolved. Additionally, non-GPCR and non-cAMP-dependent signaling pathways were found to influence androgen biosynthesis [85]. ...
... Since progesterone is catalyzed by cytochrome P450c17, it was important to determine the duration of the actions of intrathecal progesterone on the activity of P450c17. Since phosphorylation of P450c17 on serine residues increases the activity of this enzyme (Tee et al., 2008), we examined the CCI-induced changes in the phosphoserine levels of P450c17 using co-immunoprecipitation experiments followed by Western blot analysis. Sciatic nerve injury induced a significant increase in the phospho-serine levels of P450c17 in the lumbar spinal cord dorsal horn at day 1 post-surgery when compared to sham surgery mice ( Figure 4A; **p < 0.01 vs. Sham group; T (6) = 3.939, p = 0.0076). ...
... In the present study, the phospho-serine levels of P450c17 were increased on post-operative day 1, during the early phase of neuropathic pain development following sciatic nerve injury. Since phosphorylation of P450c17 on serine residues increases 17,20-lyase activity (Tee et al., 2008;Tee and Miller, 2013), it is possible that intrathecally administrated progesterone is rapidly metabolized during the induction phase of neuropathic pain through the initial activation of the P450c17 enzyme and that this process may elicit the allodynic action of progesterone treatment on the development of neuropathic pain. ...
Progesterone has been shown to have neuroprotective capabilities against a wide range of nervous system injuries, however there are negative clinical studies that have failed to demonstrate positive effects of progesterone therapy. Specifically, we looked into whether progesterone receptors or its metabolizing enzymes, cytochrome P450c17 and 5α-reductase, are involved in the effects of progesterone on neuropathic pain after chronic constriction injury (CCI) of the sciatic nerve in mice. Intrathecal progesterone administration during the induction phase of chronic pain enhanced mechanical allodynia development and spinal glial fibrillary acidic protein (GFAP) expression, and this enhancement was inhibited by administration of ketoconazole, a P450c17 inhibitor, but not finasteride, a 5α-reductase inhibitor. Furthermore, phospho-serine levels of P450c17 in the spinal cord were elevated on day 1 after CCI operation, but not on day 17. In contrast, intrathecal progesterone administration during the maintenance phase of chronic pain decreased the acquired pain and elevated GFAP expression; this inhibition was restored by finasteride administration, but not by ketoconazole. The modification of mechanical allodynia brought on by progesterone in CCI mice was unaffected by the administration of mifepristone, a progesterone receptor antagonist. Collectively, these findings imply that progesterone suppresses spinal astrocyte activation via 5α-reductase activity during the maintenance phase of chronic pain and has an analgesic impact on the mechanical allodynia associated with the growing neuropathy. Progesterone, however, stimulates spinal astrocytes during the induction stage of peripheral neuropathy and boosts the allodynic impact caused by CCI through early spinal P450c17 activation.
... This activity is depending on the availability of cytochrome P450 oxidoreductase (POR), cytochrome b5 (CYB5). Moreover, the 17,20 lyase activity is promoted via phosphorylation of serine/threonine residues by specific kinases, such as the p38α mitogen-activated protein kinase MAPK [28], increasing the enzyme's affinity for its redox partner [32,45,51]. Finally, the 17β-hydroxysteroid dehydrogenase 3 (Hsd17b3) enzyme catalyzes androstenedione to testosterone conversion, as the final product for DHT synthesis. ...
... Akt is a serine/threonine kinase activated by phosphorylation via hormone binding to LHCGR [5]. However, data from a steroidogenic adrenal cell line excluded that the Akt-responsive kinase p70S6K impacts 17,20 lyase activity [45]. Upregulation of androgen production by the kinase was suggested in Leydig cells treated with a molecule having inhibitory activity on Akt, Milazopam [52]. ...
Androgens are produced by adrenal and gonadal cells thanks to the action of specific enzymes. We investigated the role of protein kinase B (Akt) in the modulation of Δ4 steroidogenic enzymes' activity, in the mouse Leydig tumor cell line mLTC1. Cells were treated for 0-24 h with the 3 × 50% effective concentration of human luteinizing hormone (LH) and choriogonadotropin (hCG), in the presence and in the absence of the specific Akt inhibitor 3CAI. Cell signaling analysis was performed by bioluminescence resonance energy transfer (BRET) and Western blotting, while the expression of key target genes was investigated by real-time PCR. The synthesis of progesterone, 17α-hydroxy (OH)-progesterone and testosterone was measured by immunoassay. Control experiments for cell viability and caspase 3 activation were performed as well. We found that both hormones activated cAMP and downstream effectors, such as extracellularly-regulated kinase 1/2 (Erk1/2) and cAMP response element-binding protein (Creb), as well as Akt, and the transcription of Stard1, Hsd3b1, Cyp17a1 and Hsd17b3 genes, boosting the Δ4 steroidogenic pathway. Interestingly, Akt blockade decreased selectively Cyp17a1 expression levels, inhibiting its 17,20-lyase, but not the 17-hydroxylase activity. This effect is consistent with lower Cyp17a1 affinity to 17α-OH-progesterone than progesterone. As a result, cell treatment with 3CAI resulted in 17α-OH-progesterone accumulation at 16-24 h and decreased testosterone levels after 24 h. In conclusion, in the mouse Leydig cell line mLTC1, we found substantial Akt dependence of the 17,20-lyase activity and testosterone synthesis. Our results indicate that different intracellular pathways modulate selectively the dual activity of Cyp17a1.
... Involvement of the MAPK/ERK signaling (mitogen-activated protein kinase/ extracellular-signal-regulated kinase) in androgen regulation has been described by several investigators [25,26]. In vitro, fasting induced the phosphorylation and thus the consequent activation of the MAPK/ERK pathway, which enhanced P450c17 phosphorylation and its 17,20-lyase activity [14,27]. Alterations in MAPK/ERK signaling have also been described in hyperandrogenic theca cells of PCOS ovaries compared with controls [28]. ...
... Visit 1 was used as baseline, P values are derived from Friedman tests comparing the ranks of the 3 visits. Another suggested common path leading to the hyperandrogenic and metabolic changes observed with PCOS might be serine phosphorylation of the insulin receptor and of the CYP17A1 enzyme [27][28][29]. It has been demonstrated that serine phosphorylation of the CYP17A1 enzyme enhances its 17,20-lyase activity for androgen production, and that serine phosphorylation of the insulin receptor weakens its insulin signaling activity and causes insulin resistance. ...
Background
Fasting is stressful for the human body. It is managed by metabolic adaptations maintaining energy homeostasis and involves steroid hormone biosynthesis, but the exact interplay between energy and steroid metabolism remains elusive. Women with the polycystic ovary syndrome (PCOS) suffer from disturbed metabolism and androgen excess, while in women with anorexia nervosa cortisol and androgen production are decreased. By contrast, starvation of steroidogenic cells shifts adrenal steroid biosynthesis towards enhanced androgen production.
Aim
This study investigated the effect of fasting on steroid production in healthy women.
Methods
Twenty healthy young women fasted for 48 hours; steroid profiles were assessed at baseline, after 24 and 48 hours from plasma and urine samples by liquid and gas chromatography mass spectrometry.
Results
Fasting did not change overall steroidogenesis, although it increased progestagen production and lowered relative mineralocorticoid, glucocorticoid, and androgen production. The largest decrease in urine metabolites was seen for β-cortol, dehydroepiandrosterone and androstenediol; higher levels were found for pregnanediol in urine and progesterone and aldosterone in serum. Activity of CYP17A1 and its 17,20-lyase activity essential for androgen biosynthesis, was decreased after fasting in healthy women as were CYP21A2 and 5α-reductase activities. By contrast, HSD11B1 activity for cortisol inactivation seemed to increase with fasting.
Conclusion
Significant changes in steroid metabolism occurred after 48 hours of fasting in healthy women. In contrast to metabolic changes seen at baseline in PCOS women compared to healthy women, and after starving of steroidogenic cells, no androgen excess was observed after short-term fasting in healthy young women.
... However, a phosphorylation event postulated to be at Ser-258 of P450 17A1 has also been proposed to facilitate the lyase reaction (27)(28)(29)(30), and that charge (negative) is opposite to that of the arginine residues (Arg-347 and Arg-358) proposed to be involved in binding b 5 (3,5,(31)(32)(33)(34)(35)(36). Zebrafish P450 17A1 lyase activity is only slightly enhanced by b 5 , and the related zebrafish P450 17A2 enzyme catalyzes only 17αhydroxylation, not lyase activity, with or without b 5 (37,38). ...
... As mentioned earlier, a phosphorylation event postulated to be at Ser-258 of P450 17A1 has been proposed to facilitate the lyase reaction (27)(28)(29)(30), and that charge (negative) is opposite to that of the arginine residues. What has not been clear from that work is whether the phosphorylation is sufficient in itself to stimulate the lyase reaction or whether the phosphorylation enhances the functional binding of b 5 to produce the enhancement. ...
It has been recognized for >50 years that cytochrome b5 (b5) stimulates some cytochrome P450 (P450)-catalyzed oxidations, but the basis of this function is still not understood well. The strongest stimulation of catalytic activity by b5 is in the P450 17A1 lyase reaction, an essential step in androgen synthesis from 21-carbon (C21) steroids, making this an excellent model system to interrogate b5 function. One of the issues in studying b5-P450 interactions has been the limited solution assay methods. We constructed a fluorescently-labeled variant of human b5 that can be used in titrations. The labeled b5 bound to wild-type P450 17A1 with a Kd of 2.5 nM and rapid kinetics, on the order of 1 s⁻¹. Only weak binding was observed with the clinical P450 17A1 variants E305G, R347H, and R358Q; these mutants are deficient in lyase activity, which has been hypothesized to be due to attenuated b5 binding. Kd values were not affected by the presence of P450 17A1 substrates. A peptide containing the P450 17A1 Arg-347/Arg-358 region attenuated Alexa 488-T70C-b5 fluorescence at higher concentrations. The addition of NADPH-P450 reductase (POR) to an Alexa 488-T70C-b5:P450 17A1 complex resulted in a concentration-dependent, partial restoration of b5 fluorescence, indicative of a ternary P450:b5:POR complex, which was also supported by gel filtration experiments. Overall, these results are interpreted in the context of a dynamic and tight P450 17A1:b5 complex that also binds POR to form a catalytically competent ternary complex, and variants that disrupt this interaction have low catalytic activity.
... However, the 17,20-lyase reaction has been proposed to use a ferric peroxide (Fe(II)O 2-) rather than the compound I (Cpd I) that normally is used in P450 reactions [155][156][157]. It has been reported that the cyt b5 and phosphorylation of P450 17A1 can only stimulate the lyase reaction and not the 17a-hydroxylation reaction [99,[158][159][160][161]. Recent NMR studies have revealed that the human P450 17A1 has the same binding site for both CPR as for cyt b5 [109], however, unlike CPR the cyt b5 stimulates the lyase reaction without transferring electrons [162]. ...
... The hydroxylase activity of P450 17A1 is reported to be preserved during phosphorylation and dephosphorylation process, however, the serine phosphorylation of P450 17A1 is reported to increase 17,20 lyase activity by enhanced affinity for CPR and this activity is diminished after dephosphorylation [223]. It has been suggested that the phosphorylation leads to an increase in negatively charged residues of P450 17A1 that could enhance the duration of the electrostatic association with CPR [160]. Pandey et al. in 2005, demonstrated that serine phosphorylation of P450 17A1 modifies the allosteric action of cyt b5 that in turn augments the 17,20-lyase reaction [223]. ...
Cytochrome b5 (cyt b5) is a small hemoprotein that plays a significant role in the modulation of activities of an important steroidogenic enzyme, cytochrome P450 17α-hydroxylase/17,20-lyase (P450 17A1, CYP17A1). Located in the zona fasciculata and zona reticularis of the adrenal cortex and in the gonads, P450 17A1 catalyzes two different reactions in the steroidogenic pathway; the 17α-hydroxylation and 17,20-lyase, in the endoplasmic reticulum of these respective tissues. The activities of P450 17A1 are regulated by cyt b5 that enhances the 17,20-lyase reaction by promoting the coupling of P450 17A1 and cytochrome P450 reductase (CPR) allosterically. Cyt b5 can also act as an electron donor to enhance the 16-ene-synthase activity of human P450 17A1. In this review, we discuss the many roles of cyt b5 and focus on the modulation of CYP17A1 activities by cyt b5 and the mechanisms involved.
... In addition, PP2A was found negatively regulated by phosphoprotein SET. Initial studies suggested rho-associated, coiledcoil containing protein kinase 1 (ROCK1) as the kinase phosphorylating CYP17; however this kinase is only able to phosphorylate CYP17 without enhancing its lyase activity (Tee et al., 2008). Recently, the 'real' kinase phosphorylating CYP17 has been identified (Tee and Miller, 2013). ...
... Implication of PKB signaling has been reported in human theca cell steroidogenesis (Munir et al., 2004). However, when inhibiting PI3K in H295R or H295A cells, we (unpublished data) and others (Tee et al., 2008) found no effect on CYP17-lyase activity. However, assessment of PKB signaling in starved H295R cells revealed significantly decreased phosphorylation of PKB Ser473 and Thr308 suggesting inhibited signaling through this pathway under starvation conditions (Fig. 3B). ...
Regulation of androgen production is poorly understood. Adrenarche is the physiologic event in mid-childhood when the adrenal zona reticularis starts to produce androgens through specific expression of genes for enzymes and cofactors necessary for androgen synthesis. Similarly, expression and activities of same genes and products are deregulated in hyperandrogenic disorders such as the polycystic ovary syndrome (PCOS). Numerous studies revealed involvement of several signaling pathways stimulated through G-protein coupled receptors or growth factors transmitting their effects through cAMP- or non-cAMP-dependent signaling. Overall a complex network regulates androgen synthesis targeting involved genes and proteins at the transcriptional and post-translational levels. Newest players in the field are the DENND1A gene identified in PCOS patients and the MAPK14 which is the kinase phosphorylating CYP17 for enhanced lyase activity. Next generation sequencing studies of PCOS patients and transcriptome analysis of androgen producing tissues or cell models provide newer tools to identify modulators of androgen synthesis.
... High levels of plasma free fatty acids cause increased synthesis of androgens in the ovary as well as in the zona reticularis of the adrenal gland. Insulin stimulates androgenesis by stimulating P450c17 activity in zona reticularis of the adrenal gland to produce DHEA and androstenedione [151]. Hyperinsulinemia causes decreased expression of SHBG by hepatocytes (see above), thus further increasing free testosterone levels. ...
Increase in body weight due to excess accumulation of fat can lead to obesity, a chronic, progressive, relapsing, multifactorial, neurobehavioral disease caused by adipose tissue dysfunction. Obesity often results in adverse biomechanical, metabolic, psychosocial, and economic consequences. In humans, effects of obesity are diverse and interrelated and can be classified on the basis of organ/organ system affected. Physical problems associated with weight gain are musculoskeletal problems, respiratory problems, lower limb venous diseases, skin-related problems, and stress incontinence in females. Metabolic conditions caused by obesity include gout, insulin resistance and metabolic syndrome, type 2 diabetes mellitus, certain cancers, CVD, fatty liver, gall bladder disease, etc. Obesity is known to affect the reproductive health. Hypogonadism and pseudo-gynecomastia are more common in males with obesity. Decreased fertility is reported in both the sexes. Polycystic ovarian syndrome (PCOS), anovulation, endometrial hyperplasia, and increased risk of complications in pregnancy have been reported in females. Persons with obesity have increased healthcare expense, pay more insurance premium, take more illness-related leaves, thus suffering economic loss due to their condition. Persons with obesity are often considered legitimate targets for teasing and bullying, which may cause social isolation, depression, eating disorders, etc. Obesity affects the morbidity and mortality. This chapter deals with the different consequences of obesity.
... P=0.012) because hyperinsulinemia directly increases ovary and adrenal androgen production in PCOS women by inhancing cytochtome P450c 17α enzyme activity in both glands . Secretion of insulin may also stimulate adrenal androgen secretion by disregulation of 17α hydroxylase and 17, 20 hydroxylase activity (16). In this study, serum insulin level is higher in PCOS patients than in controls the difference is statistically significant. ...
FIBCOG Summary: Background: Polycystic ovarian syndrome (PCOS) is one of the most common cause of anovulation during reproductive life.Resistin can increase ovarian androgen production by directly stimulating ovarian theca cell or indirectly by augmenting pancreatic-B cell production of insulin. Patients and Methods: Sixty patients with PCOS who were non diabetic and not taking any medicine for the last three months were involved in the study .Thirty normal fertile female serves as control group. Fasting blood samples were aspirated from all individuals from 3 rd-6 th day of the menstrual cycle to measure resistin, insulin, glucose, LH, FSH, TT3, TT4, Prolactin , Total Testosterone and lipid profile, by ELISA and routine methods. Results: mean serum resistin concentration was increased in women with PCOS compared with the control group (Mean ±SD) (19.83 ± 6.101 vs 9.36 ± 2.17) ng/ml. Serum resistin concentration correlated positively with BMI, which is divided into two subgroups. The first with BMI < 25 kg/m 2 and the second with BMI ≥25kg/m 2 in both control and patient groups. In BMI < 25kg/m 2 serum resistin concentration for the control group was (8.90 ± 1.76) and (14.66 ± 2.09) for patients group ,while BMI ≥ 25 kg/m 2 serum resistin concentration for the control group was (10.62 ± 1.76) and (21.55 ± 5.40) ng/ml for patients group. Resistin also correlated positively with Insulin, LH, LH/FSH ratio and total Testosteron in women with PCOS but not in control. Fasting insulin level was higher in PCOS group compared with the control group (Mean ±SD) (27.45 ± 4.47 vs 13.27 ± 3.80) mIU/ml.The Fasting serum glucose was also higher in PCOS group compared with the control group (Mean ±SD) (125.27 ± 28.63 vs 92.63 ± 13.99) mg/dl. Total Testosterone level was elevated in the PCOS group compared with the control group (1.04 ± 0.37 vs 0.52 ± 0.25) ng/ml.Total Testosterone correlated positively with BMI, Resistin, Insulin, LH, and LH/FSH ratio. Conclusion: PCOS women with BMI >25 kg/m² were found to have a marked increase level of Resistin ,Insulin , Glucose ,LH ,and Total Testosterone .and a decrease level in their insulin sensitivity i.e increased insulin resistance.These data indicate that abnormal resistin secretion in obese PCOS women may play a role in causing ovarian hyperandrogenism and hyperinsulinemia. Therefore fasting serum resistin level could be helpful in diagnosing PCOS patient.
... (Gilling et al., 1994). Clinical features of hypothyroidism, BMI, USG abdomen and pelvis, serum TSH, free T3, Free T4 levels, serum prolactin levels, serum estrogen the collected blood specimen were carefully labeled and then they transported to the pathology laboratory carefully (Tee et al., 2008). 88 specimens from 300 patients were included in the study, keeping in view that all the randomly selected specimens must be positive for showing suggestive signs of PCOS. ...
Polycystic ovary syndrome (PCOS) is known as a widespread endocrine disorder that is affecting up to 12 percent of all women. There is a noteworthy relationship of symptoms in between thyroid diseases and PCOS. Thyroid gland is very important endocrine gland and has special effects equally on androgen and estrogen metabolism. Obesity is also related to polycystic ovary syndrome and hypothyroidism which is resulting in insulin resistance. The main objectives of this study were to reveals about the prevalence of the hypo-thyroidism and hyper-prolactinemia in between women with and without polycystic ovary syndrome. The level of thyroid hormone was measured in the obese and polycystic ovary patients. This was a case of control study. The Blood samples for the measurement of hormonal level in age of (16 to 40) females, which is collected from the department of Gynecology and obstetrics in DHQ Hospital Bahawal Nagar. Questionnaire was also filled by the patients. The blood samples were stored at-20•c until the thyroid stimulating hormone (TSH) were performed, free tri-idothyrionine (FT3), free tetre-idothydrionine (FT4), Estrogen, Prolactin and an increased level of TSH Estrogen but decreased FT4 and FT3 level in Polycystic Ovary Syndrome group as compared to Obese PCOS group. Remarkable upraise of TSH level in PCOS Obese females as interconnected to the PCOS females, obese females and the normal females was supposed. Prolactin level was found higher in PCOS and Obese-PCOS female group but lower in obese and normal female group. it was found that many of the PCOS obese having females had higher level of estrogen but low level in PCOS female as compared to PCOS obese in which level are low too. The thyroid antibodies were higher in PCOS obese group as well in PCOS group. Thus thyroid profile analysis can help in the treatment and providing the better insight into symptomatology. [Amjab M, Shafeeq A, Quarshi JA, Ali Q, Malik A. Evaluation of clinical and hormonal parameters in obese polycystic ovarian syndrome from Bahawalnagar city, Pakistan. Life Sci J 2020;17(5):85-91].
... These results indicate that POR is one of the gonadotropin-regulatable genes in ovarian granulosa cells, and this regulation should cause the augmentation of estrogen production by gonadotropins. It was also reported in adrenocortical cell lines that ACTH or cAMP increases the expression of POR [118,119]. In addition, Hall and colleagues showed in vitro that the activity of purified CYP17A1 was increased by the addition of POR proteins in a dose-dependent manner, resulting in increased androgen production [120][121][122]. ...
Ovaries represent one of the primary steroidogenic organs, producing estrogen and progesterone under the regulation of gonadotropins during the estrous cycle. Gonadotropins fluctuate the expression of various steroidogenesis-related genes, such as those encoding steroidogenic enzymes, cholesterol deliverer, and electronic transporter. Steroidogenic factor-1 (SF-1)/adrenal 4-binding protein (Ad4BP)/NR5A1 and liver receptor homolog-1 (LRH-1) play important roles in these phenomena via transcriptional regulation. With the aid of cAMP, SF-1/Ad4BP and LRH-1 can induce the differentiation of stem cells into steroidogenic cells. This model is a useful tool for studying the molecular mechanisms of steroidogenesis. In this article, we will provide insight into the transcriptional regulation of steroidogenesis-related genes in ovaries that are revealed from stem cell-derived steroidogenic cells. Using the cells derived from the model, novel SF-1/Ad4BP- and LRH-1-regulated genes were identified by combined DNA microarray and promoter tiling array analyses. The interaction of SF-1/Ad4BP and LRH-1 with transcriptional regulators in the regulation of ovarian steroidogenesis was also revealed.
... In PCOS patients, dysregulation between 17α-hydroxylase and 17,20-lyase activities has been frequently reported [23,51]. Higher 17,20-lyase activity has been attributed to the phosphorylation of Ser/Thr residues, cytochrome b5 activity, or yet to the insulin or estradiol levels [47,[52][53][54][55]. In addition, 17,20-lyase activity seems to be lower in ovaries than in adrenals [49]. ...
The phenotypic complex of patients with definitive diagnosis of polycystic ovary syndrome may include patients with normal and high serum androgen levels. Patients with hyperandrogenemia seem to present higher risk of changes to the glucose and lipid metabolism and, eventually, of earlier development of cardiovascular diseases than normoandrogenemic patients or healthy women. From a laboratory and clinical point of view, it is important to check androgen levels in patients with polycystic ovary syndrome. The identification of partial insufficiency of a given corticosteroidogenic enzyme is also relevant to understand the physiopathology of androgen increase in polycystic ovary syndrome. Therefore, the present review analyzes the functions of the different enzymes involved in the ovary and adrenal steroidogenesis in normal cycling women and in patients with polycystic ovary syndrome. In addition, it emphasizes appropriate reason for investigating eventual enzyme deficiency to provide rationale for prescription and follow-up of women with polycystic ovary syndrome.
... Phosphorylation increases enzyme activity, affecting exclusively the lyase reaction, while having no effect on allylic hydroxylation [103]. As for AR, dephosphorylation has an opposite regulatory effect [104]. Mutagenesis studies pinpointed Ser427 and Thr341 as possible phosphorylation sites [105]. ...
Cytochromes P450 (CYP450s) promote the biosynthesis of steroid hormones with major impact on the onset of diseases such as breast and prostate cancers. By merging distinct functions into the same catalytic scaffold, steroidogenic CYP450s enhance complex chemical transformations with extreme efficiency and selectivity. Mammalian CYP450s and their redox partners are membrane-anchored proteins, dynamically associating to form functional machineries. Mounting evidence signifies that environmental factors are strictly intertwined with CYP450s catalysis. Atomic-level simulations have the potential to provide insights into the catalytic mechanism of steroidogenic CYP450s and on its regulation by environmental factors, furnishing information often inaccessible to experimental means. In this review, after an introduction of computational methods commonly employed to tackle these systems, we report the current knowledge on three steroidogenic CYP450s—CYP11A1, CYP17A1, and CYP19A1—endowed with multiple catalytic functions and critically involved in cancer onset. In particular, besides discussing their catalytic mechanisms, we highlight how the membrane environment contributes to (i) regulate ligand channeling through these enzymes, (ii) modulate their interactions with specific protein partners, (iii) mediate post-transcriptional regulation induced by phosphorylation. The results presented set the basis for developing novel therapeutic strategies aimed at fighting diseases originating from steroid metabolism dysfunction.
... The pathogenetic link between prenatal androgen exposition and AH in adult life might be also related to hyperinsulinism. On the one hand, p38α and other MAPKs are final components of a three-kinase cascade [128] and hyperinsulinism is a potential trigger of that MAP3K pathway, likely involving the insulin-stimulated Rho associated coiled-coil containing protein kinase 1 (ROCK-1) [129,130]. On the other, gestational androgen excess of female rhesus monkeys induced by T administration increases insulin secretion in their offspring by favoring hyperglycemia in dams [131] or by ulterior metabolic derangements such as abdominal visceral fat deposition or obesity [132]. In conceptual agreement, treatment of prenatally androgenized rhesus monkeys with the insulin sensitizer drug pioglitazone normalizes DHEAS responses to cosyntropin stimulation [133]. ...
Methods:
The aim of this review is to update the pathogenesis and consequences of AH in PCOS, from molecular genetics to the clinical setting.
Results:
Mounting evidence derived from animal models suggests that genetically or enviromentally determined prenatal androgen excess, by influencing the hormonal and metabolic phenotype of susceptible female fetuses later in life, may be the capital event for the development of AH in PCOS. Because human placental aromatase activity is likely to prevent any deleterious effect of maternal hyperandrogenemia on the fetus, inheritance of the maternal steroidogenic defect is the more likely culprit, even though other factors such as changes in placental steroidogenesis itself or its nutritional efflux may also be involved in the building a deregulated enzymatic pathway from utero to adult life. Anyhow, the most important issue is whether or not AH influences the cardiometabolic risk of women with PCOS. On the one hand, AH has shown a controversial relationship with carbohydrate metabolism and adiposity, and is also associated with abnormalities in blood pressure regulation in these patients. On the other hand, DHEAS may exert a beneficial effect on the lipid profile of both lean and obese patients. Lastly, available studies in women with PCOS cast doubt upon a protective role of DHEAS levels on subclinical atherosclerosis, despite opposite data from the general population.
Conclusions:
AH is frequent in patients with PCOS yet unraveling its consequences for the management of this disorder requires future longitudinal studies.
... This rapid change implies that etifoxine does not activate steroidogenic enzyme gene transcription but rather acts at a post-translational level, likely through serine (Ser) and/or threonine (Thr) phosphorylation of the enzymes. In particular, it is clearly established that phosphorylation of of Ser 106 and Thr 112 residues in human P450 C17 stimulates the activity of the enzyme [69][70][71][72][73][74]. Interestingly, a rapid response in the activity of 3α-HSD has been observed in the rat brain after administration of fluoxetine [75][76][77][78], which like etifoxine exerts anxiolytic properties [79,80]. ...
Neurosteroids can modulate the activity of the GABAA receptors, and thus affect anxiety-like behaviors. The non-benzodiazepine anxiolytic compound etifoxine has been shown to increase neurosteroid concentrations in brain tissue but the mode of action of etifoxine on neurosteroid formation has not yet been elucidated. In the present study, we have thus investigated the effect and the mechanism of action of etifoxine on neurosteroid biosynthesis using the frog hypothalamus as an experimental model. Exposure of frog hypothalamic explants to graded concentrations of etifoxine produced a dose-dependent increase in the biosynthesis of 17-hydroxypregnenolone, dehydroepiandrosterone, progesterone and tetrahydroprogesterone, associated with a decrease in the production of dihydroprogesterone. Time-course experiments revealed that a 15-min incubation of hypothalamic explants with etifoxine was sufficient to induce a robust increase in neurosteroid synthesis, suggesting that etifoxine activates steroidogenic enzymes at a post-translational level. Etifoxine-evoked neurosteroid biosynthesis was not affected by the central-type benzodiazepine (CBR) receptor antagonist flumazenil, the translocator protein (TSPO) antagonist PK11195 or the GABAA receptor antagonist bicuculline. In addition, the stimulatory effects of etifoxine and the triakontatetraneuropeptide TTN, a TSPO agonist, were additive, indicating that these two compounds act through distinct mechanisms. Etifoxine also induced a rapid stimulation of neurosteroid biosynthesis from frog hypothalamus homogenates, a preparation in which membrane receptor signalling is disrupted. In conclusion, the present study demonstrates that etifoxine stimulates neurosteroid production through a membrane receptor-independent mechanism.
... Kyoto Encyclopedia of Genes and Genomes (KEGG) network pathway analysis [75][76][77] from RNA-Seq expression profiles of the GWAS candidates in normal and PCOS theca cells revealed that DENND1A, ZNF217, YAP1, and HMGA2 are associated in an inter-related pathway. A review of the literature has also revealed that the GWAS candidates, LHCGR, INSR, DENND1A, RAB5B, mediate overlapping intracellular signaling pathways, including the PI3K, PKB, and/or MAPK signaling cascades [14,[78][79][80][81][82]. Comparison of the activation states of the AKT/PKB and MAPK/MEK/ERK signaling pathways in normal and PCOS theca cells has provided evidence that alteration of these signaling cascades may underlie increased androgen production and CYP17A1 gene expression in PCOS [14,78]. ...
Polycystic ovary syndrome (PCOS) is a common endocrinopathy characterized by increased ovarian androgen biosynthesis, anovulation, and infertility. PCOS has a strong heritable component based on familial clustering and twin studies. Genome-wide association studies (GWAS) identified several PCOS candidate loci including LHCGR, FSHR, ZNF217, YAP1, INSR, RAB5B, and C9orf3. We review the functional roles of strong PCOS candidate loci focusing on FSHR, LHCGR, INSR, and DENND1A. We propose that these candidates comprise a hierarchical signaling network by which DENND1A, LHCGR, INSR, RAB5B, adapter proteins, and associated downstream signaling cascades converge to regulate theca cell androgen biosynthesis. Future elucidation of the functional gene networks predicted by the PCOS GWAS will result in new diagnostic and therapeutic approaches for women with PCOS.
Copyright © 2014 Elsevier Ltd. All rights reserved.
... For x-ray crystallographic experiments, zebrafish P450 17A1 and 17A2 were expressed and purified as described previously for E. coli recombinant bovine P450 21A2 (36), including Ni 2 - nitrilotriacetate, DEAE, and SP-Sepharose chromatography steps. The proteins were each eluted from an SP-Sepharose Fast Flow FPLC column (GE Healthcare) with 50 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol (v/v), 0.1 mM dithiothre- itol, 0.1 mM EDTA, 0.004% (w/v) 3,6,9,12,15,18,21,24,27-nonaox- anonatriacontan-1-ol (C12E9) detergent (Anatrace, Maumee, OH), and either 500 mM NaCl (for P450 17A1) or 250 mM NaCl (for P450 17A2). Purified zCYP17s were obtained in yields between 300 and 600 nmol/liter culture. ...
Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals a single enzyme, P450 17A1, catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s: zebrafish P450 17A1 catalyzes both 17α-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17α-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the 2-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17α-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1, but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and, for P450 17A2, with Prog. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116-119) showed only a few differences near the active site, despite only ~50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternate ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity.
Copyright © 2014, The American Society for Biochemistry and Molecular Biology.
Semaphorins are a family of evolutionarily conserved morphogenetic molecules that were initially found to be associated with axonal guidance. Semaphorin 4C (Sema4C), a member of the fourth subfamily of semaphorins, has been demonstrated to play multifaceted and important roles in organ development, immune regulation, tumour growth and metastasis. However, it is completely unknown whether Sema4C is involved in the regulation of ovarian function. We found that Sema4C was widely expressed in the stroma, follicles and corpus luteum of mouse ovaries, and its expression was decreased at distinct foci in ovaries of mice of mid-to-advanced reproductive age. Inhibition of Sema4C by the ovarian intrabursal administration of recombinant adeno-associated virus (AAV)-shRNA significantly reduced oestradiol, progesterone and testosterone levels in vivo. Transcriptome sequencing analysis showed changes in pathways related to ovarian steroidogenesis and the actin cytoskeleton. Similarly, knockdown of Sema4C by siRNA interference in mouse primary ovarian granulosa cells (GCs) or thecal interstitial cells (TICs) significantly suppressed ovarian steroidogenesis and led to actin cytoskeleton disorganization. Importantly, the cytoskeleton-related pathway RHOA/ROCK1 was simultaneously inhibited after downregulation of Sema4C. Furthermore, treatment with a ROCK1 agonist after siRNA interference stabilized the actin cytoskeleton and reversed the inhibitory effect on steroid hormones described above. In conclusion, Sema4C may play an important role in ovarian steroidogenesis through regulation of the actin cytoskeleton via the RHOA/ROCK1 signalling pathway. These findings shed new light on the identification of dominant factors involved in the endocrine physiology of female reproduction.
Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age. Despite its incidence, the syndrome is poorly understood and remains underdiagnosed, and female patients are diagnosed with a delay. The heterogenous nature of this complex disorder results from the combined occurrence of genetic, environmental, endocrine, and behavioral factors. Primary clinical manifestations of PCOS are derived from the excess of androgens (anovulation, polycystic ovary morphology, lack of or scanty, irregular menstrual periods, acne and hirsutism), whereas the secondary manifestations include multiple metabolic, cardiovascular, and psychological disorders. Dietary and lifestyle factors play important roles in the development and course of PCOS, which suggests strong epigenetic and environmental influences. Many studies have shown a strong association between PCOS and chronic, low-grade inflammation both in the ovarian tissue and throughout the body. In the vast majority of PCOS patients, elevated values of inflammatory markers or their gene markers have been reported. Development of the vicious cycle of the chronic inflammatory state in PCOS is additionally stimulated by hyperinsulinemia and obesity. Changes in DNA methylation, histone acetylation and noncoding RNA levels are presented in this review in the context of oxidative stress, reactive oxygen species, and inflammatory signaling in PCOS. Epigenetic modulation of androgenic activity in response to inflammatory signaling is also discussed.
Polycystic ovary syndrome is a common endocrinopathy, with hyperandrogenism being the cardinal feature, but which is also highly associated with insulin resistance and metabolic disorders. Despite present evidence, the exact role of insulin resistance and associated compensatory hyperinsulinemia in PCOS hyperandrogenism is still debated. The existence of non-insulin-resistant PCOS phenotype in a subset of women with typical hyperandrogenism suggests that insulin resistance is not a requisite for the development of this syndrome. Nevertheless, several lines of emerging evidences suggest that impairment in insulin signaling may be implicated in androgen overproduction at the level of androgen-producing organs. After an historical review, this chapter will address in vivo studies assessing insulin action in women with PCOS. It will focus on the main results describing the effects of insulin resistance on hyperandrogenism in PCOS and then discuss the different insulin signaling anomalies occurring in both metabolic and mitogenic insulin signaling pathways. In addition, how these signaling dysfunctions may hypothetically influence androgen synthesis in these women will be addressed. Finally, the lipotoxicity theory will be discussed. This chapter will describe how increased exposure of lean organ to circulating fatty acids could lead to the development of insulin resistance in PCOS, as it does in other conditions such as type 2 diabetes. More importantly, literature suggesting potential direct effects of lipotoxicity on androgen production and potential underlying mechanisms will be presented. Through this chapter, whether insulin resistance may be the main cause of hyperandrogenism in PCOS or whether it could be the consequence of an upstream dysfunction, namely, lipotoxicity will be assessed.
PCOS, also known as Stein-Leventhal syndrome, is a common endocrine disease characterized by two of the following three characteristics: Once the associated endocrinological and gynecological diseases have been ruled out, oligo-ovulation or anovulation, ii) clinical and/or biochemical indications of hyperandrogenism, or iii) polycystic ovaries should be considered. Cardiovascular disease (CVD) risk factors are common in women with polycystic ovarian syndrome (PCOS). The Androgen Excess and Polycystic Ovary Syndrome (AE-PCOS) Society established a panel to offer evidence-based evaluations of research examining the PCOS-CVD risk connection and to produce CVD prevention recommendations. The main pathophysiological abnormality in polycystic ovarian syndrome is a source of much debate (PCOS). Chronic anovulation in conjunction with androgen excess, hyperinsulinemia, and changes in gonadotropin production are now widely accepted as symptoms of this disease in women. Polycystic ovarian syndrome (PCOS) is linked to obesity and low-grade inflammation, and it may raise the risk of cardiovascular disease (CVD). This study examines the assessment of cardiovascular disease risk in women with PCOS.
Androgen and estrogen synthesis is necessary for human growth and sexual maturation. Hormone-dependent cancers, such as breast and prostate cancer, however, use these sex steroids to drive cellular proliferation. Cytochrome P450 17A1 (CYP17A1), an essential enzyme for sex steroid synthesis, represents a clinically established drug target. Inhibiting CYP17A1 decreases androgen and estrogen biosynthesis and thereby blocks the growth of hormone-dependent cancers. The first part of this dissertation characterizes the previously unreported interaction between the FDA approved CYP17A1 inhibitor, abiraterone, and the estrogen receptor (ER). We show for the first time that abiraterone is a weak ER agonist in preclinical models of ER-positive breast cancer cells. Abiraterone induces cellular growth and expression of the ER response gene, GREB1, by binding to ER, and these effects are inhibited with the ER antagonist fulvestrant (ICI 182,780). To further investigate the impact of CYP17A1 expression in breast cancer, we engineered ER-positive MCF-7 cells to express CYP17A1 (MCF-7/CYP17A1). Progesterone treatment induces cell growth and GREB1 expression in these cells but not in the parental MCF-7 cells, which do not express CYP17A1. Tandem mass spectrometry (LC-MS/MS) analysis confirmed that following progesterone treatment, MCF-7/CYP17A1 cells synthesize downstream steroid products that require CYP17A1 activity including 17OH-progesterone, androstenedione, and testosterone. Treatment of these cells with either abiraterone or a novel CYP17A1 inhibitor decreases progesterone metabolite-induced GREB1 expression in a dose-dependent manner. In addition to studies of CYP17A1 in breast cancer, we further hypothesized that characterization of CYP17A1 genetic variants may lead to insights on enzyme structure and function. We therefore utilized a HEK-293T cell-based expression system to characterize the enzymatic properties of two CYP17A1 gene variants, D216H (rs200063521) and G162R (rs141821705). Our results show that the D216H variant selectively alters 16OH-progesterone production, while no effect on 17OH-progesterone synthesis was observed. In contrast, the G162R substitution exhibits decreased CYP17A1 protein stability compared to wild-type. Proteasome inhibition with MG132 indicated that this variant is preferentially ubiquitinated and degraded prematurely. Overall, these studies have broadened our understanding of CYP17A1 enzymatic activity in breast cancer, as well as led to new insights into how CYP17A1 structure relates to enzyme function and stability.
In humans, reticularis cells of the adrenal cortex fuel the production of androgen steroids, constituting the driver of numerous morphological changes during childhood. These steps are considered a precocious stage of sexual maturation and are grouped under the term “adrenarche”. This review describes the molecular and enzymatic characteristics of the zona reticularis, along with the possible signals and mechanisms that control its emergence and the associated clinical features. We investigate the differences between species and discuss new studies such as genetic lineage tracing and transcriptomic analysis, highlighting the rodent inner cortex’s cellular and molecular heterogeneity. The recent development and characterization of mouse models deficient for Prkar1a presenting with adrenocortical reticularis-like features prompt us to review our vision of the mouse adrenal gland maturation. We expect these new insights will help increase our understanding of the adrenarche process and the pathologies associated with its deregulation.
Unexplained hyperandrogenic oligoanovulation is a main feature of polycystic ovary syndrome (PCOS). P450c17 phosphorylation selectively increases 17,20-lyase activity and androgen biosynthesis but minimally affects 17α-hydroxylase. Studies have recently identified mitogen-activated protein kinase 14 (MAPK14, p38α) as the kinase responsible for enhancing 17,20-lyase activity through P450c17 phosphorylation. We investigated whether oxidant-induced oxidative stress increases 17,20-lyase activity through oxidant-sensitive p38α signaling pathways. NCI-H295R adrenal cells were treated with three oxidants, palmitate, H2O2 and 4-hydroxy-2-nonenal (HNE), to simulate the excessive oxidative stress of PCOS. Oxidant exposure significantly induced dehydroepiandrosterone production and increased p38α phosphorylation and activation, but the effect on 17α-hydroxyprogesterone production was far less clear. None of the treatments altered the expression of P450c17 or its necessary factors POR and b5. LC-MS/MS revealed increased DHEA production in NCI-H295R cells. Both p38α inhibition and siRNA-mediated silencing attenuated H2O2- or 0.45–0.75 mM PA-mediated augmentation of DHEA production with relatively stable 17OHP levels, indicating that activated p38α mediates oxidative stress-induced 17,20-lyase activation and androgen synthesis stimulation, which may underlie hyperandrogenism in PCOS.
Introduction Early detection of early rather than advanced stage ovarian cancer is emphasized for its correlation to significantly improved patient outcome. Despite improvements in median survival through surgical advances and new chemotherapeutic regimens, the overall survival for women with stage III/IV EOC has remained poor. However, women diagnosed with disease confined to the ovary (stage I) require less morbid surgical intervention, may not require adjuvant chemotherapy, have a significantly improved quality of life, and most importantly, have an overall 5-year survival rate approximating 90%. Enhanced understanding of ovarian cancer molecular biology, etiology, and associated risk factors and pathologies are important in identifying clinically validated tools to save women’s lives. Symptoms: new advancements and discoveries Within the past decade, the concept of ovarian cancer as a "silent killer" has been challenged by numerous investigators. An important study by Goff et al. identified symptoms presenting before the diagnosis of ovarian cancer, in an effort to minimize delay in detection [1]. Of the 1,725 women with ovarian cancer surveyed, 95% recalled experiencing symptoms before their diagnosis. Of those women, 30% had symptoms within 2 months of diagnosis, 35% had symptoms between 3 and 6 months prior, and 35% reported having symptoms for more than 6 months before discovering the cancer. Although patients with late stage disease were more likely to be symptomatic versus those with early stage disease, women with the latter were only asymptomatic 11% of the time. The specific symptoms were mostly abdominal (77%), gastrointestinal (70%), constitutional (50%), urinary (34%), and pelvic (26%) [1].
Introduction Endometriosis has been traditionally defined as the presence of endometrial glands and stroma in ectopic locations. This disease affects approximately 6 to 10% of reproductive-aged women, resulting in dysmenorrhea, dyspareunia, chronic pelvic pain, and/or infertility [1]. Endometriosis is a debilitating condition, posing quality-of-life issues for the individual patient. The prevalence of endometriosis in women experiencing pain, infertility, or both can be as high as 50%. The disorder represents a major cause of gynecologic hospitalization in the United States resulting in over $3 billion in inpatient health care costs in 2004 alone [2,3]. The significant individual and public health concerns associated with endometriosis are paramount to understanding its pathogenesis. The first recorded description of pathology consistent with endometriosis was provided by Shroen in 1690 [4,5]. Despite decades of extensive clinical and scientific investigation, the exact pathogenesis of this enigmatic disorder still remains largely unknown. Theories regarding pathogenesis of endometriosis Numerous theories detailing the development of endometriosis have been described. These theories can generally be classified into two groups: (1) Nonendometrial, those that propose that implants arise from tissues other than the endometrium, and (2) Endometrial, those that propose that implants arise from uterine endometrium (Table 14.1).
Introduction Surgical assessment and histologic evaluation are the only means by which a neoplasm can be classified as benign or malignant, primary, or metastatic. When an early primary ovarian cancer is diagnosed, the next goal is determining the extent of disease or stage. Surgical staging is required to define those patients in whom surgery alone may be curative and those who will require adjuvant therapy, and to determine the modality, intensity, and duration of such treatment. Accurate surgical staging also permits assignment of prognosis, allows comparison of cure rates, and defines subsequent surveillance. In the 70 to 75% of patients who present with advanced ovarian cancer, the goal of laparotomy is also to remove as much tumor as possible through a process of surgical "cytoreduction" to maximize response to chemotherapy, and improve survival. We offer epithelial ovarian cancer as a model; the principles of treatment also apply to ovarian germ-cell tumors, stromal tumors, and other primary ovarian cancers.
Introduction Germ cell tumors (GCT) are a fascinating group of tumors exhibiting a large variety of pathological patterns and occurring in clinical settings that often include developmental, genetic, and hormonal disorders. These tumors are unique because they involve gonads of both sexes, ovaries and testes, and are seen mostly in young patients. Actually, they represent about 60% of all ovarian tumors diagnosed in patients under the age of 30. With the exception of dermoid cysts (benign, mature cystic teratomas), they are uncommon neoplasms; fortunately, the most common are benign and conversely, the malignant tumors are the least common. Their clinical outlook has changed dramatically for the better, over the past decades, due to their responsiveness to chemotherapy. The pathologic characteristics of ovarian germ cell tumors are also unique: they are composed of multiple tissues, occurring in various degrees of maturation from their cells of origin (the germ cells) as well as in various histologic tissue combinations. Some germ cell tumors are associated with sex-cord tissue derivatives. Germ cell tumors represent one-third of all malignant tumors seen in young females and about 20% of all ovarian tumors in Europe and North America. In Asia and Africa, they represent a much larger proportion of ovarian tumors because the prevalence of epithelial-stromal-derived tumors is much lower.
Introduction The American Cancer Society estimates that 21,990 women will be diagnosed with ovarian cancer and 15,460 will die of the disease in 2011 [1]. Of these 21,990 patients, 20% will have stage I disease, for which 5-year survival rates approach 90%. However, numerous studies have shown that a significant percentage of patients with apparent early stage (stage I) ovarian cancer actually harbor microscopic metastatic disease. Consequently, the benefits of surgical staging for epithelial ovarian carcinoma have been well established. Traditionally, it has been recommended that a comprehensive surgical staging procedure for epithelial ovarian and fallopian tube cancers include a total abdominal hysterectomy, bilateral salpingo-oophorectomy, peritoneal cytologic washings, biopsies of adhesions and peritoneal surfaces, omentectomy, and retroperitoneal lymph node sampling from the pelvic and para-aortic regions through a generous vertical midline laparotomy incision [2]. With the advent of minimally invasive surgical techniques, surgeons are now able to perform all of the necessary procedures for comprehensive surgical staging, including pelvic and para-aortic lymphadenectomies and omentectomies, using conventional videolaparoscopy or robotic-assisted videolaparoscopy in selected patients.
Introduction Menopause is the "landmark" in a woman’s life when she experiences permanent cessation of menstruation. The collective follicle capacity of the ovaries to secrete adequate estradiol diminishes to a level at which proliferation of an endometrium adequate to produce menstruation is no longer achievable. But menopause is justifiably more than simply loss of menses or even reproductive competence. Although some ovarian steroid secretion continues transiently beyond this critical point and limited extra-gonadal production of estrogen may persist, at menopause the ability of the ovaries to function as endocrine organs capable of providing sufficient hormone to sustain the estrogen-dependent biologic aspects of a wide variety of tissues has also ceased. Reviewed in Chapter 2, estrogen modulates a broad array of non-reproductive functions including bone and mineral metabolism, cardiovascular function, fuel and metabolic homeostasis, neuropsychiatric balance, and the risk of progression of age-related neurodegenerative diseases. The consequences of age combined with a prolonged hypoestrogenic state and the various reactive strategies available to meet these challenges will be reviewed in this chapter. It will deal with the definitions of the various phases surrounding menopause and the sometimes paradoxical endocrinology of each stage. In addition, the increased metabolic, endocrine, cardiovascular, and cancer risks accompanying aging and how these might be affected by a prolonged state of hypoestrogenism will be analyzed. The benefits-risks of hormonal replenishment and an individual-appropriate clinical management algorithm is provided. The effort will be inclusive but not exhaustive; it should encourage the reader to pursue more encyclopedic resources [1-6], which are emphasized in the text.
Introduction Ovarian cancer presents as familial clusters and as sporadic cases without strong family history. As summarized elsewhere in this book, rare mutations conferring substantially increased ovarian cancer risk (up to 50%) occur in several genes including BRCA1 and BRCA2 [9,10], mismatch repair genes such as MLH1 and MSH2 [11], and other DNA repair genes, such as RAD51C [12]. Yet, these high-risk variants are very rare (less than 1% in most populations), and they are estimated to account for less than 40% of the excess familial risk of ovarian cancer [13]. Here, we describe the genetic etiology of sporadic ovarian cancer (i.e., ovarian cancers that do not arise from within high-risk familial clusters). We review evidence that ovarian cancer is genetic, data on particular genes which have been studied as candidates, results from searches throughout the genome for ovarian cancer risk factors, strategies for follow-up of genomic regions harboring risk loci, and approaches for the identification and characterization of additional genetic factors. Evidence for low-penetrance loci The role of inherited factors in ovarian cancer susceptibility is suggested by numerous epidemiologic studies reporting increased risks from 2- to 10-fold for women with family history of ovarian cancer [14]. In fact, compared with confirmed demographic, reproductive, and lifestyle factors, positive family history is the strongest risk factor for ovarian cancer. Increased familial risk can be explained, in part, by shared environmental factors; however, studies of twins and migrants provide additional support for genetic heritability of the disease. For example, identical twins are more likely to have the same ovarian cancer disease state than fraternal twins [15]. In addition, genetic epidemiologic models suggest that a large number of additional rare high-risk variants, such as BRCA1 or BRCA2 is unlikely to exist [16].
Introduction Aside from primary ovarian malignancies, ovaries are a frequent site of metastases from other primary tumors, mostly from the gastrointestinal tract, breast, and other gynecological organs. Metastatic tumors to the ovaries are an important group of ovarian neoplasms, and their correct pathological interpretation is paramount for the right treatment of the patient. It can be very difficult for a pathologist to diagnose metastatic disease of the ovary because it often mimics a primary ovarian malignancy. Evaluating the metastatic nature of an ovarian tumor depends on the clinician’s and pathologist’s knowledge about the frequency of metastases of different primary tumors, a complete clinical history, a careful evaluation and re-evaluation of the gross pathology of the specimen and also, use of special stains and immuno-histochemistry. The diagnosis of metastatic ovarian tumor should be considered when the anatomical distribution of the disease is atypical for primary ovarian cancer, when the patient has another tumor outside the ovaries and when both ovaries are involved by the tumor, although unilaterality of the tumor is not a definite argument against one’s metastatic nature (different studies show bilaterality of ovarian metastatic tumors in approximately 70% of cases) [1,2]. Other tumoral findings suggestive of metastasis are: size less than 10 cm, tumor grossly visible on the surface of the ovary, presence of multiple tumor nodules often growing in a desmoplastic stroma, and lymphatic and/or blood vessels invasion (more pronounced in the ovarian hilum).
Disorders of the ovary can lead to a wide range of endocrinologic and malignant conditions, many of which are linked with fertility. This comprehensive, yet succinct book presents a multidisciplinary approach to address the major issues in diagnosing and managing ovarian disorders. Beginning with the complex functioning of the normal ovary, the editors address many of the major issues in women's health. New chapters on ovarian cysts, menopause, the aging ovary, early detection and risk assessment of ovarian cancer, screening, stage I ovarian cancer and many other topics have been added to this third edition. Assisted reproductive techniques, diagnostic imaging modalities, minimally invasive surgery, and chemotherapy have advanced dramatically and the chapters have been updated accordingly. This well-documented volume has been fully updated with contemporary references and chapters written by current leaders in their field. A must-read for gynecologists, oncologists, obstetricians, pathologists and researchers in human reproductive sciences.
Disorders of the ovary can lead to a wide range of endocrinologic and malignant conditions, many of which are linked with fertility. This comprehensive, yet succinct book presents a multidisciplinary approach to address the major issues in diagnosing and managing ovarian disorders. Beginning with the complex functioning of the normal ovary, the editors address many of the major issues in women's health. New chapters on ovarian cysts, menopause, the aging ovary, early detection and risk assessment of ovarian cancer, screening, stage I ovarian cancer and many other topics have been added to this third edition. Assisted reproductive techniques, diagnostic imaging modalities, minimally invasive surgery, and chemotherapy have advanced dramatically and the chapters have been updated accordingly. This well-documented volume has been fully updated with contemporary references and chapters written by current leaders in their field. A must-read for gynecologists, oncologists, obstetricians, pathologists and researchers in human reproductive sciences.
Introduction In recent studies of the origin(s) of pelvic serous carcinoma, two concepts have emerged: the fallopian tube as a major source for these tumors and a carcinogenic sequence in the distal fallopian tube. The first has immediate implications for both the early detection and classification of this disease. The second impacts on the pathogenesis of tubal malignancies and has important implications for the histologic diagnosis. In particular, the discovery of benign-appearing secretory cell outgrowths (SCOUTs) in the fallopian tube that contain functional gene perturbations shared with cancer requires a reassessment of the concept of intra-epithelial neoplasia. This exercise requires the separation of innocuous clonal expansions or outgrowths of no clinical significance from those with the potential, albeit low, to metastasize. In this chapter, we introduce the term "intra-epithelial neoplasia" to denote the latter, preferring to relegate epithelial processes of lesser degree to a descriptive category that although linked to cancer, do not belong in the diagnostic lexicon. In this spectrum may lie the genetic changes that mark the acquisition of the metastatic phenotype. We emphasize that, while this progression is most closely linked to serous carcinomas, it can occur with other phenotypes, including endometrioid and, rarely, mucinous neoplasia. Finally it is important to superimpose this new information on the existing concept of ovarian and peritoneal carcinoma. In both instances, it is useful to look at both low- and high-grade serous neoplasms and the potential contribution of the fallopian tube in their pathogenesis.
Introduction Chapter 1 of this volume chronicled the integration of the hypothalamic-anterior pituitary-ovarian follicle events involving intrinsic and endocrine factors driving gonadogenesis, primordial follicle formation, follicle arrest, reactivation, recruitment, selection, dominance, ovulation, and corpus luteum function [1]. The crucial importance of the timely appearance, concentrations, and sequence of gonadotropins FSH and LH was emphasized. However, simply adequate gonadotropin availability does not insure ovulatory or reproductive success. The follicle must also be at the appropriate stage of functional maturity to respond to any of the selection steps leading to pre-ovulatory dominance let alone respond to the ovulation stimulus. In the normal cycle, gonadotropin release and final maturation of the follicle coincide because the timing of the gonadotropin surge is controlled by the level of gonadal feedback signals which reflect the degree of dominant follicle growth and functional maturity. The requisite feedback relationships in both the inter-cycle early follicle selection phase and at ovulation are tightly controlled, generally permitting only one follicle to reach the point of ovulation (Fig. 11.1) [2]. This is the salient feature of the evolutionary pressure to survive as a species - the human uterus cannot sustain a litter of embryos successfully, nor has the human mother the mammary capacity to feed more than two suckling dependent neonates. Superimposed on these structural constraints is the capacity to transiently postpone reproduction when negative internal or environmental factors challenge the system - illness, starvation, chronic danger, and stress - by inducing acute or chronic anovulation.
Introduction The subject of ovarian cysts and tumors in the young patient has often been overlooked. Our house staff-resident teaching programs in gynecology teach the traditional gynecologic problems of the mature woman. Training of pediatricians also has overlooked gynecologic problems. The young patient in all aspects is completely different from the mature woman. The young patient has frequent functional physiologic benign cysts (simple follicle cysts; complex corpus luteum cysts), most of which are asymptomatic and self-limited. They are often discovered by coincidence when a pelvic or abdominal ultrasound examination is done on a child for other purpose, usually pain. It takes clinical judgment and experience to decide whether the pain is due to the cyst or perhaps due to another cause, such as a gastrointestinal problem. Ovarian cysts are often a normal developmental occurrence in neonates, children, and adolescents. They are common, frequently regress, and are seldom associated with malignancy [1]. Benign mature teratoma (dermoid cyst) is the most common cystic ovarian neoplasm in the adolescent. They are solid masses which are often palpable and with symptoms of pressure, increased abdominal girth, and with or without pain. About one-third of solid neoplasms may be malignant. The goal of surgical treatment of the young patient aside from preserving her health is to preserve fertility if at all possible. The latter is often possible because the malignant ovarian tumors including germ cell cancer (MOGC) usually do not involve the opposite ovary.
Introduction In the United States, bilateral salpingo-oophorectomy (BSO) at the time of hysterectomy for benign disease is commonly done to prevent the subsequent development of ovarian cancer. Almost all BSOs (87%) are done at the time of hysterectomy [1]. Oophorectomy rates appear to have peaked recently, with 55% of hysterectomies accompanied by the procedure in 1999 compared with 39-45% in more recent years [2-4]. Recent data show age remains the strongest predictor of elective BSO, with 40% of women 40-44 years old, 78% of women 50-54 years old, and 68% of women 55 years or older having had BSO at hysterectomy [5]. Despite the common practice of "risk-reducing" or prophylactic oophorectomy at the time of hysterectomy for benign disease, an increasing body of evidence suggests removal of normal ovaries has minimal overall benefit for those women who are not at an increased risk of breast or ovarian cancer. Several large cohort studies suggest that, while removing normal ovaries reduces the risk of ovarian cancer to almost zero, prophylactic oophorectomy does not appear to increase overall survival. This chapter will address the history of risk-reducing oophorectomy, assess the evidence for the practice, and consider its appropriateness in light of recent studies.
Introduction The ovaries are paired almond-shaped organs located along each pelvic sidewall. Normal ovarian volumes range up to 20 mL and 8-10 mL in pre-menopausal and post-menopausal women, respectively [1]. Each ovary is covered by surface epithelium and encloses numerous follicles, containing germ cells (eggs), within the ovarian stromal tissue [2]. With each menstrual cycle, a follicle matures and releases its ovum into the fallopian tube and then becomes a corpus luteum, which in the absence of pregnancy involutes to form a corpus albicans. Ovarian cancer is the ninth most common cancer among women worldwide. It is the fifth most common cause of female cancer-related death. Ovarian cancer causes more deaths than any other cancer of the female reproductive tract. In 2013, in the United States there will be an estimated 22,240 new cases of ovarian cancer diagnosed and 14,030 deaths from the disease [3]. Risk factors for the development of ovarian cancer include advanced age, nulliparity, early menarche, late menopause, and long-term hormone replacement therapy [4]. Furthermore, approximately 10% of ovarian cancers are related to genetic mutations with the breast and ovarian cancer genes, BRCA1 and BRCA2, and Lynch II syndrome accounting for the majority of these cases [3-5].
Introduction The surface ovarian epithelium is considered to be the most common origin of ovarian neoplasms, both benign and malignant. Composed of a single layer of cuboidal cells, as an extension of the peritoneal mesothelium, the ovarian surface epithelium is closely related to the adjacent ovarian cortical stroma and some tumors arising in the area include both epithelial and stromal elements. Recent studies suggest that the fallopian tube epithelium, benign or malignant, that implants on the ovary is the source of low-grade and high-grade serous carcinoma [1]. Benign ovarian tumors of epithelial origin are fortunately far more common than malignant ovarian tumors. Borderline ovarian malignancy will be discussed in a separate chapter (Chapter 5) due to their complex and often controversial clinical-pathologic correlations. The histogenesis of ovarian epithelial tumors, their classification, grading, and inter-relationship has recently been the object of extensive research and of fundamental changes, based on new molecular and genetic discoveries. This process is still unfolding, although several established facts have contributed to a revision of previously accepted histopathologic categories.
Introduction In early 2006, a third "positive" phase 3 randomized trial examining intra-peritoneal cisplatin-based chemotherapy as primary treatment of small-volume residual advanced ovarian cancer was reported [1]. This outcome led the United States National Cancer Institute to issue a "Clinical Announcement" describing the impact of this management strategy on outcome in this malignancy [2]. Since that time, several favorable and unfavorable critiques regarding this strategy have been published [3-7], revealing the controversy surrounding this novel management strategy. This chapter will review the basic biological foundation supporting intra-peritoneal chemotherapy in the management of ovarian cancer, clinical trial data supporting its routine use, and possible strategies to improve both the efficacy and toxicity associated with regional drug delivery in this setting. Rationale for intra-peritoneal chemotherapy and early phase clinical trials The theoretical rationale for delivering regional chemotherapy as treatment of ovarian cancer has previously been described in detail [8-12]. In brief, the arguments include the following considerations: (a) anatomic localization and natural history of the malignancy [9,10]; (b) pharmacokinetic advantage associated with the intra-peritoneal delivery of anti-neoplastic agents with known activity in ovarian cancer [11], and pre-clinical evidence for the impact of a clinically relevant "dose-response" effect for specific cytotoxic drugs against ovarian cancer at concentrations that are potentially achievable with regional delivery, but not after systemic administration [12].
Introduction The major functions of the human ovary are the development, nurture, and release of a mature oocyte ready for fertilization and successful propagation of the species. In support of these processes, the ovary secretes steroid hormones which stimulate growth and development of organs of reproduction; are critically involved in the elaborate endocrine interchange which directs orderly, repetitive cyclic ovulations; and finally supports successful uterine implantation, placentation, and the corpus luteum-dependent phase of pregnancy. A description of how the ovary and its secretions achieve these reproductive functions is the focus of the first two chapters of this volume. In addition, it is now clear from observations in prolonged physiologic (menopause) and non-physiologic (gonadal failure) hypogonadal states that ovarian steroid secretions have important influences on a variety of non-reproductive organ systems which determine the quality of life and the life expectancy of women. A more complete treatment of these issues is dealt with in Chapter 2, How Steroid Hormones Work, and Chapter 20, Menopause. General principles The physiologic responsibilities of the ovary are the periodic release of gametes (oocytes) and the timely sequential secretion of the hormones estradiol (E2), progesterone (P), and the inhibins A and B. These endocrine signals integrate the hypothalamus, anterior pituitary, and ovaries in a continuous repetitive process of follicle recruitment, rescue, maturation, selection, ovulation, corpus luteum formation, function, and regression. But the ovary cannot be viewed as a static, purely endocrine organ whose size and function waxes and wanes, depending on the stimulatory input of tropic hormones. Rather, the female gonad is a heterogeneous, constantly changing organ with cyclicity measured in days and weeks, with each phase governed by a specific anatomic subunit.
Introduction Endometriosis is a common gynecological disease that affects 6% to 44% of all women of reproductive age [1]. It is defined as the presence of endometrial tissue, consisting of both glandular epithelium and stroma outside the uterus. The most frequent sites of implantation are the pelvic viscera and the peritoneum. Three different clinical entities of endometriosis can be distinguished: peritoneal endometriosis, ovarian endometriosis, and deep invasive (or infiltrating) endometriosis. They vary in appearance from a few minimal lesions on otherwise intact pelvic organs to large ovarian endometriotic cysts that affect tubo-ovarian anatomy and extensive adhesions often involving the bowel, bladder, and ureters. It is associated with pelvic pain and subfertility, and has a detrimental impact on quality of life [2,3]. Etiology Although signs and symptoms of endometriosis have been described since 1896 by von Recklinghausen, its widespread occurrence was acknowledged during the 20th century. Even 120 years after its first description, the pathogenesis of this condition remains poorly understood. Endometriosis is an estrogen-dependent disease. Three main theories have been proposed to explain the histogenesis. Ectopic transplantation of endometrial tissue Coelomic metaplasia Induction.
Disorders of the ovary can lead to a wide range of endocrinologic and malignant conditions, many of which are linked with fertility. This comprehensive, yet succinct book presents a multidisciplinary approach to address the major issues in diagnosing and managing ovarian disorders. Beginning with the complex functioning of the normal ovary, the editors address many of the major issues in women's health. New chapters on ovarian cysts, menopause, the aging ovary, early detection and risk assessment of ovarian cancer, screening, stage I ovarian cancer and many other topics have been added to this third edition. Assisted reproductive techniques, diagnostic imaging modalities, minimally invasive surgery, and chemotherapy have advanced dramatically and the chapters have been updated accordingly. This well-documented volume has been fully updated with contemporary references and chapters written by current leaders in their field. A must-read for gynecologists, oncologists, obstetricians, pathologists and researchers in human reproductive sciences.
Turner syndrome (TS) affects approximately one in 2500 live-born females [1]. This disorder presents the clinician with a challenging array of genetic, developmental, endocrine, cardiovascular, psychosocial, and reproductive issues. For the purpose of this chapter, Turner syndrome will be used to describe the patient with an abnormality of the chromosomal karyotype involving loss of part or all of the X chromosome associated with phenotypic abnormalities that include short stature and the potential for or the presence of ovarian failure. Definition The diagnosis of TS requires the presence of characteristic physical features in phenotypic females coupled with complete or partial absence of the second sex chromosome, with or without cell line mosaicism [2-4]. Individuals with a 45, X cell population but without clinical features are not considered to have TS. Phenotypic males are also excluded from the diagnosis of TS, regardless of karyotype. Whether to diagnose individuals with sex chromosome structural abnormalities as having TS requires clinical judgment. Abnormalities such as ring X and Xq isochromosomes are common in patients with classic TS features, and many of these patients have phenotypes indistinguishable from that of patients with apparently non-mosaic monosomy X (45, X) [4]. Patients with small distal short arm deletions (Xp-) including the SHOX gene frequently have short stature and other TS-associated skeletal anomalies, but most are at low risk of ovarian failure and should generally not be diagnosed with TS if band Xp22.3 is not deleted [5]. Individuals with deletions of the long arm distal to Xq24 frequently have primary or secondary amenorrhea without short stature or other TS features [6]; the diagnosis of premature ovarian failure is more appropriate for them. Table 3.1 summarizes the cytogenetic findings in TS patients.
Polycystic ovary syndrome is a common endocrinopathy of unclear etiology. The cardinal feature of the syndrome is hyperandrogenism. However, it is also highly associated with metabolic disorders that have in common the development of insulin resistance. Despite the evidences in literature, the exact roles of insulin resistance and associated compensatory hyperinsulinemia for hyperandrogenism of PCOS are still debated. The existence of non-insulin-resistant PCOS phenotype in a subset of women with typical hyperandrogenism suggests that insulin resistance is not a requisite for the development of this syndrome. Nevertheless, several evidences suggest that impairment in insulin signaling may be implicated in androgen overproduction at the level of androgen producing organs. After a short historical reminder, this chapter will first address in vivo studies assessing insulin action in PCOS women. We will focus on the main results describing the effects of insulin resistance on hyperandrogenism in PCOS. We will then discuss the different insulin signaling anomalies occurring in both metabolic and mitogenic insulin signaling pathways. In addition, we will describe how these signaling dysfunctions may hypothetically influence androgen synthesis in these women. Finally, we will discuss the lipotoxicity theory. We will describe how tissue fatty acid overflow could lead to the development of insulin resistance in PCOS, as it does in other conditions such as type 2 diabetes. More importantly, we will present recent literature suggesting potential direct effects of lipotoxicity on androgen production and address potential underlying mechanisms. Through this chapter, we will discuss whether insulin resistance may be the main cause of hyperandrogenism in PCOS or whether it could be the consequence of an upstream dysfunction, namely lipotoxicity. © 2014 Springer Science+Business Media New York. All rights are reserved.
Polycystic ovary syndrome (PCOS) is a complex genetic disorder caused by interplay between several 'susceptibility' genes and environment factors. In the past few years, numerous studies of genomics and transcriptomics attempted to discover genes affecting PCOS. Pre-genome wide association study (GWAS) plays a stepping stone effect on the progress of PCOS, even though most of the strongest associations are for loci rather than functional variants. A trend towards large-scale GWAS has succeeded in identifying many additional novel PCOS loci. Most of the PCOS-associated regions are shared with other diseases or symptoms, as well as with metabolism, inflammation or insulin signaling-related traits, or cancer. Moreover, susceptibility genes for early diagnosis of PCOS are expected to offer the prevention of long-term risk of obesity, cardiovascular disease, and type 2 diabetes (T2DM) as well. Furthermore, considerable advanced new technical approaches such as GWAS and next-generation sequencing will provide new opportunities in the molecular analysis of PCOS, which can, in the long term, lead to new therapeutic treatments for the disorder. The present review discusses heterogeneous clinical manifestations of PCOS, controversies surrounding the diagnosis of PCOS, and the recent findings of pre-GWAS and GWAS studies on PCOS, highlighting the relevant candidate gene families and their potential functional pathways relevant for PCOS.
Chinese hamster ovary cells overexpressing the human insulin receptor were transfected with cDNAs encoding protein kinase C isoenzymes alpha, betaI, gamma, and epsilon as well as an inactive alpha. Overexpression of these protein kinase Cs did not affect expression of the insulin receptor or insulin-stimulated tyrosine phosphorylation of the receptor. However, in response to phorbol esters, cells overexpressing isoenzymes alpha, betaI, and gamma, but not epsilon or inactive alpha, exhibited 3-4-fold higher levels of insulin receptor phosphorylation. This increased phosphorylation occurred exclusively on serines and threonine. Tryptic peptide maps indicated that this phosphorylation was primarily on serines 1305/1306 and threonine 1348 as well as several other unidentified sites. This phorbol ester-stimulated phosphorylation did not inhibit activation of the insulin receptor kinase when the receptor was activated in situ but assayed in vitro. However, in cells overexpressing protein kinase Calpha, it did inhibit an in vivo monitor of the activation of the insulin receptor kinase, the insulin-stimulated increase in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase activity. These results indicate that increased protein kinase Calpha activity can inhibit insulin-stimulated responses and support the hypothesis that excessive protein kinase Calpha is involved in the insulin resistance observed in non-insulin-dependent diabetics.
In the biosynthesis of steroid hormones, P450c17 is the single enzyme that catalyzes both the 17α-hydroxylation of 21-carbon
steroids and the 17,20-lyase activity that cleaves the C17-C20 bond to produce C19sex steroids. Cytochrome b
5 augments the 17,20-lyase activity of cytochrome P450c17 in vitro, but this has not been demonstrated in membranes, and the mechanism of this action is unknown. We expressed human P450c17,
human P450-oxidoreductase (OR), and/or human cytochromeb
5 in Saccharomyces cerevisiae and analyzed the 17α-hydroxylase and 17,20-lyase activities of the resulting yeast microsomes. Yeast expressing only P450c17
have 17α-hydroxylase and trace 17,20-lyase activities toward both Δ4 and Δ5 steroids. Coexpression of human OR with P450c17 increases the V
max of both the 17α-hydroxylase and 17,20-lyase reactions 5-fold; coexpression of human b
5 with P450c17 also increases theV
max of the 17,20-lyase reactions but not of the 17α-hydroxylase reactions. Simultaneous expression of humanb
5 with P450c17 and OR, or addition of purified human b
5 to microsomes from yeast coexpressing human P450c17 and OR, further increases theV
max of the 17,20-lyase reaction without altering 17α-hydroxylase activity. Genetically engineered yeast and mixing experiments
demonstrate that OR is both necessary and sufficient for microsomal 17,20-lyase activity. Addition of purified human holo-b
5, apo-b
5, or cytochrome c to microsomes containing both human P450c17 and OR demonstrate that the stimulatory action ofb
5 does not require electron transfer fromb
5 to P450c17. These data suggest that humanb
5 acts principally as an allosteric effector that interacts primarily with the P450c17·OR complex to stimulate 17,20-lyase
activity.
Steroid 17 alpha-hydroxylase deficiency is caused by defects in cytochrome P450c17, the single enzyme that has 17-alpha hydroxylase and 17,20-lyase activities. We describe a rapid and efficient polymerase chain reaction tactic for identifying these genetic lesions and identify Ser106----Pro as the cause of 17 alpha-hydroxylase deficiency in two unrelated homozygous patients from Guam. We used site-directed mutagenesis of the normal P450c17 cDNA to construct the Pro106 mutant, and expressed both the normal and mutant sequences in monkey COS-1 cells and in yeast. Expression of the normal sequence permitted the cells to convert pregnenolone to 17-OH pregnenolone, progesterone to 17-OH progesterone, and 17-OH pregnenolone to dehydroepiandrosterone, showing the normal sequence conferred both 17 alpha-hydroxylase and 17,20-lyase activities. Expression of the mutant sequence generated P450c17 mRNA, but conferred none of these activities, proving that the Ser106----Pro mutation abolished the 17 alpha-hydroxylase and 17,20-lyase activities. An HhaI restriction site created by the mutation should permit screening of large populations.
We established a continuous cell line, NCI-H295, from an invasive primary adrenocortical carcinoma. The cell line was established in a fully defined medium (HITES) and later could be adapted for growth in a simple medium supplemented only with selenium, insulin, and transferrin and devoid of serum, steroids, fibroblast growth factor, and a source of exogenous cholesterol. NCI-H295 cells had a relatively long population doubling time and were tumorigenic when inoculated s.c. into athymic nude mice. The cultured cells had ultrastructural features of steroid-secreting cells and contained complex cytogenetic abnormalities including the presence of multiple marker chromosomes. Steroid analyses (radioimmunoassays and mass spectrometry), performed 7 to 9 years after culture initiation, demonstrated secretion of more than 30 steroids characteristic of adrenocortical cells. Total unconjugated steroid secretion in serum-supplemented medium was 2.83 micrograms/10(6) cells/24 h and about 4-fold less in serum-free medium. The major pathway of pregnenolone metabolism in NCI-H295 cells is androgen synthesis, with formation of dehydroepiandrosterone, androstenedione, testotesterone, and at least three sulfated androgens, as well as estrogens. In addition, formation of cortisol, corticosterone, aldosterone, and 11 beta-hydroxyandrostenidione indicated the presence of 11 beta-hydroxylase. Thus, multiple pathways of steroidogenesis are expressed by NCI-H295 cells, including formation of corticosteroids, mineralocorticoids, androgens, and estrogens. Our findings indicate the presence in NCI-H295 cells of all of the major adrenocortical enzyme systems, including 11 beta-hydroxylase, desmolase, 21 alpha-hydroxylase, 17 alpha-hydroxylase, 18-hydroxylase, lyase, sulfokinase, and aromatase. The NCI-H295 cell line should prove of value in studying the regulation, metabolic pathways, and enzymes involved in steroid formation and secretion. In addition, it may provide insights into the biology and treatment of adrenocortical carcinoma.
The effect of 8-bromo-cAMP and forskolin on the phosphorylation state and protein kinase activity of the insulin receptor was evaluated in cultured IM-9 lymphoblasts. 8-Bromo-cAMP (1 mM) or forskolin (10 microM) enhanced the phosphorylation of the insulin receptor purified from 32P-labeled cells by affinity chromatography on wheat germ agglutinin-agarose and immunoprecipitation with monoclonal antibody. In the absence of insulin, phosphorylation of the beta subunit of the receptor was increased approximately 2-fold by raising intracellular cAMP. Phosphoamino acid analysis of the beta subunit following treatment of cells with forskolin revealed an increase in phosphoserine and phosphothreonine residues. In contrast, the insulin-stimulated phosphorylation of the receptor occurred on serine, threonine, and tyrosine residues and was diminished by prior exposure of cells to forskolin. Pulse-chase experiments indicated that forskolin did not enhance the turnover of phosphate on the receptor of cells previously exposed to insulin. Furthermore, extracts from forskolin-treated cells did not differ from control extracts in their capacity to dephosphorylate 32P-labeled receptor isolated from cells treated with insulin. The insulin-dependent tyrosine protein kinase activity of the receptor isolated from forskolin-treated cells was approximately 50% as active as the receptor isolated from either control or insulin-treated cells. This was assessed using both histone and a peptide synthesized in accordance with the deduced amino acid sequence of a potential autophosphorylation site of the human receptor (Thr-Arg-Asp-Ile-Tyr-Glu-Thr-Asp-Tyr-Tyr-Arg-Lys) as substrates for the protein kinase reaction. These results suggest that agents that raise intracellular cAMP increase phosphorylation of the insulin receptor on serine and threonine residues, reduce insulin-mediated receptor phosphorylation on tyrosine, serine, and threonine residues, and inhibit the insulin-dependent tyrosine protein kinase activity of the receptor. Thus cAMP may attenuate insulin action by altering the state of phosphorylation of the insulin receptor.
P450c17 is the single enzyme mediating both 17 alpha-hydroxylase (steroid 17 alpha-monooxygenase, EC 1.14.99.9) and 17,20 lyase activities in the synthesis of steroid hormones. It has been suggested that different P450c17 isozymes mediate these activities in the adrenal gland and testis. We sequenced 423 of the 509 amino acids (83%) of the porcine adrenal enzyme; based on this partial sequence, a 128-fold degenerate 17-mer was synthesized and used to screen a porcine adrenal cDNA library. This yielded a 380-base cloned cDNA, which in turn was used to isolate several human adrenal cDNAs. The longest of these, lambda hac17-2, is 1754 base pairs long and includes the full-length coding region, the complete 3'-untranslated region, and 41 bases of the 5'-untranslated region. This cDNA encodes a protein of 508 amino acids having a predicted molecular weight of 57,379.82. High-stringency screening of a human testicular cDNA library yielded a partial clone containing 1303 identical bases. RNA gel blots and nuclease S1-protection experiments confirm that the adrenal and testicular P450c17 mRNAs are indistinguishable. These data indicate that the testis possesses a P450c17 identical to that in the adrenal. The human amino acid sequence is 66.7% homologous to the corresponding regions of the porcine sequence, and the human cDNA and amino acid sequences are 80.1 and 70.3% homologous, respectively, to bovine adrenal P450c17 cDNA. Both comparisons indicate that a central region comprising amino acid residues 160-268 is hypervariable among these species of P450c17. Comparison of the amino acid sequence of P450c17 with two other human steroidogenic cytochromes P450 show much greater homology with P450c21 (28.9%), another microsomal enzyme, than with P450scc (12.3%), a mitochondrial enzyme.
The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function of the insulin receptor.
The C21 side-chain cleavage enzymes from porcine adrenal and testicular microsomes have been purified and shown to resemble each other very closely (Nakajin, S., Shinoda, M., Hanui, M., Shively, J.E., and Hall, P.F. (1984) J. Biol. Chem. 259, 3971-3976). We have investigated the reason for the low levels of lyase activity shown by adrenal microsomes as compared to testicular microsomes. Competition for substrate with 21-hydroxylase in adrenal microsomes was excluded by studies showing that antibodies to 21-hydroxylase do not increase lyase activity in spite of almost complete inhibition of 21-hydroxylation. Reconstitution of the purified testicular enzyme in lipids extracted from adrenal and testicular microsomes excluded a specific effect of lipids on lyase activity. On the other hand, addition of porcine hepatic P-450 reductase to microsomes from adrenal and testis increased the activity of lyase relative to hydroxylase. The same effect is seen when reductase is added to the pure enzymes. As the concentration of reductase increases, lyase activity increases relative to hydroxylase until the rates of the activities become almost equal. Vmax is the same for both activities (hydroxylase and lyase) of the two enzymes (6.3-6.5 nmol/min/nmol of P-450). Km for reductase is approximately the same for the hydroxylase activities (0.4-0.6 microM) and for the lyase activities (1.7-2.0 microM) of the two enzymes. Antibodies to reductase, when added to testicular microsomes, inhibit both activities, but inhibition of lyase is greater than that of hydroxylase. The enzyme activity of reductase in testicular microsomes is 3-4 times higher than that of adrenal microsomes (0.29 and 0.08 nmol/min/mg of protein, respectively). These findings may account for the greater activity of lyase in testicular as opposed to adrenal microsomes.
The beta subunit of purified insulin receptor is phosphorylated on a serine residue by purified preparations of protein kinase C (ATP: protein phosphotransferase, EC 2.7.1.37). This phosphorylation is inhibited by antibodies to protein kinase C and stimulated by phospholipids, diacylglycerol, and Ca2+. The phosphorylation of the receptor by protein kinase C does not affect its insulin-binding activity but does inhibit by 65% the receptor's intrinsic tyrosine-specific protein kinase activity (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112). These results indicate that activators of protein kinase C, such as phorbol esters, desensitize cells to insulin by direct protein kinase C action on the insulin receptor.
The properties and the purity of a cytochrome P-450 (17 alpha-hydroxylase) from porcine adrenal microsomes have been examined following a report that the corresponding enzyme from bovine adrenocortical microsomes is inactive as a 17 alpha-hydroxylase and fails to show a high spin spectrum on addition of substrate, once the enzyme has been purified (Bumpus, J. A., and Dus, K. M. (1982) J. Biol. Chem. 257, 12696-12704). The purity of the porcine enzyme was demonstrated by electrophoresis on polyacrylamide with sodium dodecyl sulfate, immunoelectrophoresis, and NH2-terminal amino acid sequence (16 residues). The pure enzyme shows Mr = 54,000, heme content of greater than 0.8 nmol/nmol of protein, and absorption spectra typical of cytochrome P-450. The enzyme is active with both delta 4 (progesterone) and delta 5 (pregnenolone) substrates as a 17 alpha-hydroxylase and with the corresponding 17 alpha-hydroxysteroids as a C17,20-lyase. All four substrates produce typical type I spectra with the enzyme (so-called high spin form). We conclude that: 1) porcine adrenal microsomes contain a 17 alpha-hydroxylase/C17,20-lyase which is a single protein molecule readily purified to an enzymatically active form; 2) the C17,20-lyase activity is largely suppressed in the microsomes; and 3) the enzyme closely resembles that found in testicular microsomes. We propose that this enzyme be referred to as the adrenal C21 steroid side chain cleavage enzyme.
Microsomal cytochrome P450c17 catalyzes both steroid 17 alpha-hydroxylase activity and scission of the C17-C20 steroid bond (17,20-lyase) on the same active site. Adrenal 17 alpha-hydroxylase activity is needed to produce cortisol throughout life, but 17,20-lyase activity appears to be controlled independently in a complex, age-dependent pattern. We show that human P450c17 is phosphorylated on serine and threonine residues by a cAMP-dependent protein kinase. Phosphorylation of P450c17 increases 17,20-lyase activity, while dephosphorylation virtually eliminates this activity. Hormonally regulated serine phosphorylation of human P450c17 suggests a possible mechanism for human adrenarche and may be a unifying etiologic link between the hyperandrogenism and insulin resistance that characterize the polycystic ovary syndrome.
We investigated the cellular mechanisms of the unique disorder of insulin action found in the polycystic ovary syndrome (PCOS). Approximately 50% of PCOS women (PCOS-Ser) had a significant increase in insulin-independent beta-subunit [32P]phosphate incorporation (3.7-fold, P < 0.05 vs other groups) in skin fibroblast insulin receptors that was present in serine residues while insulin-induced tyrosine phosphorylation was decreased (both P < 0.05 vs other groups). PCOS skeletal muscle insulin receptors had the same abnormal phosphorylation pattern. The remaining PCOS women (PCOS-n1) had basal and insulin-stimulated receptor autophosphorylation similar to control. Phosphorylation of the artificial substrate poly GLU4:TYR1 by the PCOS-Ser insulin receptors was significantly decreased (P < 0.05) compared to control and PCOS-n1 receptors. The factor responsible for excessive serine phosphorylation appeared to be extrinsic to the receptor since no insulin receptor gene mutations were identified, immunoprecipitation before autophosphorylation corrected the phosphorylation defect and control insulin receptors mixed with lectin eluates from affected PCOS fibroblasts displayed increased serine phosphorylation. Our findings suggest that increased insulin receptor serine phosphorylation decreases its protein tyrosine kinase activity and is one mechanism for the post-binding defect in insulin action characteristic of PCOS.
Chinese hamster ovary cells overexpressing the human insulin receptor were transfected with cDNAs encoding protein kinase C isoenzymes alpha, beta I, gamma, and epsilon as well as an inactive alpha. Overexpression of these protein kinase Cs did not affect expression of the insulin receptor or insulin-stimulated tyrosine phosphorylation of the receptor. However, in response to phorbol esters, cells overexpressing isoenzymes alpha, beta I, and gamma, but not epsilon or inactive alpha, exhibited 3-4-fold higher levels of insulin receptor phosphorylation. This increased phosphorylation occurred exclusively on serines and threonine. Tryptic peptide maps indicated that this phosphorylation was primarily on serines 1305/1306 and threonine 1348 as well as several other unidentified sites. This phorbol ester-stimulated phosphorylation did not inhibit activation of the insulin receptor kinase when the receptor was activated in situ but assayed in vitro. However, in cells overexpressing protein kinase C alpha, it did inhibit an in vivo monitor of the activation of the insulin receptor kinase, the insulin-stimulated increase in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase activity. These results indicate that increased protein kinase C alpha activity can inhibit insulin-stimulated responses and support the hypothesis that excessive protein kinase C is involved in the insulin resistance observed in non-insulin-dependent diabetics.
Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, phosphorylates myosin-binding subunit (MBS) of myosin phosphatase and thereby inactivates the phosphatase activity in vitro. Rho-kinase is thought to regulate the phosphorylation state of the substrates including myosin light chain (MLC), ERM (ezrin/radixin/moesin) family proteins and adducin by their direct phosphorylation and by the inactivation of myosin phosphatase. Here we identified the sites of phosphorylation of MBS by Rho-kinase as Thr-697, Ser-854 and several residues, and prepared antibody that specifically recognized MBS phosphorylated at Ser-854. We found by use of this antibody that the stimulation of MDCK epithelial cells with tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF) induced the phosphorylation of MBS at Ser-854 under the conditions in which membrane ruffling and cell migration were induced. Pretreatment of the cells with Botulinum C3 ADP-ribosyltransferase (C3), which is thought to interfere with Rho functions, or Rho-kinase inhibitors inhibited the TPA- or HGF-induced MBS phosphorylation. The TPA stimulation enhanced the immunoreactivity of phosphorylated MBS in the cytoplasm and membrane ruffling area of MDCK cells. In migrating MDCK cells, phosphorylated MBS as well as phosphorylated MLC at Ser-19 were localized in the leading edge and posterior region. Phosphorylated MBS was localized on actin stress fibers in REF52 fibroblasts. The microinjection of C3 or dominant negative Rho-kinase disrupted stress fibers and weakened the accumulation of phosphorylated MBS in REF52 cells. During cytokinesis, phosphorylated MBS, MLC and ERM family proteins accumulated at the cleavage furrow, and the phosphorylation level of MBS at Ser-854 was increased. Taken together, these results indicate that MBS is phosphorylated by Rho-kinase downstream of Rho in vivo, and suggest that myosin phosphatase and Rho-kinase spatiotemporally regulate the phosphorylation state of Rho-kinase substrates including MLC and ERM family proteins in vivo in a cooperative manner.
NCI-H295 is a recently described human adrenocortical carcinoma cell line that makes a variety of steroid hormones. We sought to determine if steroidogenesis in these cells employs the same enzymes as those used in normal adrenal steroidogenesis, and if the genes encoding those enzymes exhibit characteristic responsiveness to activators of the protein kinase-A and -C pathways of intracellular second messengers. Northern blots show that NCI-H295 cells contain abundant mRNAs for three key steroidogenic enzymes, cytochrome P450scc, cytochrome P450c17, and cytochrome P450c21. These mRNAs accumulated in a time- and dose-dependent fashion in response to 8-bromo-cAMP (8Br-cAMP), forskolin, cholera toxin, and 3-isobutyl-1-methylxanthine, all activators of the protein kinase-A pathway. Nuclear run-on assays and actinomycin-D transcriptional inhibition experiments show that cAMP regulates the expression of all three genes primarily at the transcriptional level. Inhibition of protein synthesis with cycloheximide did...
RNA interference (RNAi) has recently emerged as a specific and efficient method to silence gene expression in mammalian cells either by transfection of short interfering RNAs (siRNAs; ref. 1) or, more recently, by transcription of short hairpin RNAs (shRNAs) from expression vectors and retroviruses. But the resistance of important cell types to transduction by these approaches, both in vitro and in vivo, has limited the use of RNAi. Here we describe a lentiviral system for delivery of shRNAs into cycling and non-cycling mammalian cells, stem cells, zygotes and their differentiated progeny. We show that lentivirus-delivered shRNAs are capable of specific, highly stable and functional silencing of gene expression in a variety of cell types and also in transgenic mice. Our lentiviral vectors should permit rapid and efficient analysis of gene function in primary human and animal cells and tissues and generation of animals that show reduced expression of specific genes. They may also provide new approaches for gene therapy.
Adrenarche is considered to occur as a result of intra-adrenal changes in steroidogenic enzymes involved in C19 steroid production. The present study was conducted because developmental changes in steroidogenic enzymes have not been examined well in human postnatal adrenal.
Twenty-four specimens of nonpathological human adrenals from 7 months to 62 years retrieved from autopsy files. Immunohistochemistry for P450 side-chain cleavage (P450scc), 17α hydroxylase (P450c17), dehydroepiandrosterone sulfotransferase (DHEA-ST), P450 oxidoreductase, cytochrome b5, and 3β-hydroxysteroid dehydrogenase (3βHSD) was per-formed in these specimens, and the immuno-intensity was evaluated using CAS 200 computed image analysis system.
Immunoreactivity of P450scc was marked in the zona glomerulosa, fasciculata and reticularis in the adrenal glands of all the cases examined. P450c17 and DHEA-ST immunoreactivity was weak in the zona fasciculata and reticularis in the adrenals of age 7 months to 5 years, but thereafter became prominent in the zona reticularis. Immunoreactivity of P450 oxidoreductase and cytochrome b5, components of the electron transfer system hypothesized to regulate the 17–20 lyase activity of P450c17, was weak in all three zones of adrenal cortex from 7 months to 5 years, and became more marked in the zona reticularis after age 5 years. 3βHSD immunoreactivity was marked in all three zones of the adrenal cortex from 7 months to 8 years but thereafter decreased in the zona reticularis.
These data suggest that the human adrenal zona reticularis markedly begins to develop morphologically and functionally at around 5 years of age. The increased level of P450c17, DHEA-ST, P450 oxidoreductase, and cytochrome b5, and the decreased level of 3βHSD in the reticularis is likely to contribute to increased C19 steroid production during adrenarche.
The polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism and insulin resistance, but the connection between these two features has been unclear. Androgen synthesis is regulated in part by the ratio of the 17α-hydroxylase and 17,20 lyase activities of P450c17. Three separate lines of evidence show that the ratio of lyase to hydroxylase activity is regulated by electron flow from P450 oxidoreductase. Lyase activity and androgen synthesis are particularly dependent on the serine phosphorylation of P450c17. Serine phosphorylation of the insulin receptor β chain causes insulin resistance, and some PCOS women have hyperphosphorylated receptors. We hypothesize that an overactive serine/threonine kinase hyperphosphorylates both the insulin receptor and P450c17 in PCOS, accounting for the characteristic insulin resistance and hyperandrogenism of this disease.
We have investigated the involvement of the MAPK signaling pathway in increased androgen biosynthesis and CYP17 gene expression in women with polycystic ovary syndrome (PCOS). A comparison of MAPK kinase (MEK1/2) and ERK1/2 phosphorylation in propagated normal and PCOS theca cells, revealed that MEK1/2 phosphorylation was decreased more than 70%, and ERK1/2 phosphorylation was reduced 50% in PCOS cells as compared with normal cells. Infection with dominant-negative MEK1 increased CYP17 mRNA and dehydroepiandrosterone (DHEA) abundance, whereas constitutively active MEK1 reduced DHEA production and CYP17 mRNA abundance. Similarly, the MEK inhibitor, PD98059, increased CYP17 mRNA accumulation and CYP17 promoter activity to levels observed in PCOS cells. Remarkably, in theca cells maintained in the complete absence of insulin, ERK1/2 phosphorylation was decreased in PCOS theca cells as compared with normal theca cells, and CYP17 mRNA and DHEA synthesis were increased in PCOS theca cells. These studies demonstrate that in PCOS cells reduced levels of activated MEK1/2 and ERK1/2 are correlated with increased androgen production, irrespective of the insulin concentration. These findings implicate alterations in the MAPK pathway in the pathogenesis of excessive ovarian androgen production in PCOS.
. Serum ACTH, Cortisol and dehydroepiandrosterone (DHEA) were determined in 200 girls and 80 boys. In girls, serum DHEA showed significant increases between all bone age groups from the youngest one, 7.5 years, to 12.5 years. A plateau was then seen up to 15.5 years of age, followed by a continuous increase to the oldest group (18.5 years). In boys, a progressive increase in DHEA was also seen from the youngest age group (8.5 years), but a period of a more rapid increase did not commence until after 12.5 years of age and it then continued to the oldest group. The level of DHEA in boys was significantly lower than in girls until the oldest group, in concert with the earlier pubertal development in girls. The importance of DHEA in initiating the early physical signs of normal puberty seems also to be different in the two sexes, since serum DHEA in girls was almost double that seen in boys, when compared according to the stage of pubic hair growth. Serum Cortisol showed a small progressive increase in girls, the concentrations postmenarche being significantly higher than premenarche. In boys, a decrease was seen up to 12.5 years of age and an increase occurred from 16.5 years onwards. In both sexes, ACTH and Cortisol levels showed an inverse but nonsignificant relationship to each other. Serum ACTH levels in the different age groups showed no significant changes.
Distinguishing between ovarian and adrenal causes of androgen excess may be difficult. We have found that women with the polycystic ovary syndrome have supranormal plasma 17-hydroxyprogesterone responses to the gonadotropin-releasing hormone agonist nafarelin. We determined the usefulness of testing with nafarelin to distinguish ovarian causes of hyperandrogenism in women.
We studied 40 consecutive women with hyperandrogenism who had oligomenorrhea, hirsutism, or acne. All 40 underwent testing with nafarelin, dexamethasone, and corticotropin with measurement of circulating concentrations of gonadotropins and steroid hormones, and 19 underwent ovarian ultrasonography.
The plasma 17-hydroxyprogesterone response to nafarelin was supranormal in 23 of the 40 women (58 percent), and the plasma androgen response to corticotropin was elevated in 23; 13 women had both abnormalities. Only one woman had conclusive evidence of a steroidogenic block; she had nonclassic adrenal 21-hydroxylase deficiency. Of the 23 women with abnormal responses to nafarelin, only 11 (48 percent) had elevated base-line serum luteinizing hormone concentrations. Of the 13 women with abnormal responses to nafarelin who underwent ultrasonography, 7 (54 percent) had polycystic ovaries. Peak plasma 17-hydroxyprogesterone concentrations after nafarelin administration correlated closely with plasma free testosterone concentrations after dexamethasone administration (r = 0.75, P less than 0.001).
Approximately half of women with oligomenorrhea, hirsutism, or acne have an abnormal response to the gonadotropin-releasing hormone agonist nafarelin, suggesting an ovarian cause of their androgen excess.
Ten hirsute women with polycystic ovarian syndrome (PCO) and nine with idiopathic hirsutism (IH) underwent selective ovarian suppression with leuprolide for 5-6 months and then were randomized to receive, in addition, dexamethasone or placebo for 4 more months. Serum hormone levels and hair growth rates were determined before and after each treatment period. During the initial treatment period with leuprolide alone, testosterone decreased by 54 +/- 6% (mean +/- SEM) in PCO and by 36 +/- 3% in IH (P = 0.02). Androstenedione decreased by 53 +/- 6% in PCO and by 31 +/- 7% in IH (P = 0.02). Androstanediol glucuronide (Adiol-G) decreased by 14 +/- 6% in PCO and by 7 +/- 3% in IH. There was no change in dehydroepiandrosterone sulfate (DHEAS). While initial serum androgen levels were higher in PCO than in IH, they were similar after ovarian suppression in the two groups. After ovarian suppression, Adiol-G was more consistently correlated with testosterone and androstenedione than was DHEAS, suggesting that Adiol-G may be a better marker than DHEAS of adrenal androgen secretion. Hair growth rates decreased by 37 +/- 6% in PCO and by 14 +/- 10% in IH (P = 0.07). The change in hair growth correlated with the change in androstenedione (r = 0.66; P = 0.002), but not significantly with the change in testosterone (r = 0.29; P = 0.2). After the addition of dexamethasone therapy (0.5 mg daily), testosterone, androstenedione, and DHEAS levels fell to near or below assay detection limits, while Adiol-G decreased by 80 +/- 3%. Hair growth rates decreased slightly more in women during dexamethasone (46 +/- 6%) than during placebo (26 +/- 9%; P = 0.18). In summary, the ovary was the major source of circulating testosterone and androstenedione in PCO. The adrenal contributed a substantial minority of these hormones in PCO and was the major source of androgen secretion in IH. Adrenal hyperandrogenism was common in both IH and PCO. Hair growth rates correlated best with changes in serum androstenedione levels. Adiol-G, which was derived primarily from adrenal precursors, was a better marker of adrenal androgen secretion than was DHEAS in these subjects.
To investigate the basis of polycystic ovary syndrome, we examined the responses of patients to nafarelin, a specific gonadotropin-releasing-hormone agonist, given to stimulate pituitary and gonadal secretion. We compared 16 normal women in the follicular phase, 5 normal men, 8 women with polycystic ovary syndrome, and 1 woman with polycystic ovary syndrome caused by a 3 beta-hydroxysteroid dehydrogenase deficiency. After 100 micrograms of nafarelin was given subcutaneously, serum follicle-stimulating hormone and luteinizing hormone increased rapidly to peak levels within four hours. The women with polycystic ovary syndrome had a pattern similar to that of the men, with greater early luteinizing-hormone responses (30 minutes to 1 hour) and lower peak follicle-stimulating-hormone responses than normal women (P less than 0.05). Patients with polycystic ovary syndrome responded to gonadotropin stimulation with normal to increased production of plasma estrogens and increased levels of androstenedione at 16 to 24 hours (P less than 0.05). Elevated production of 17 alpha-hydroxyprogesterone was found in all the women with polycystic ovary syndrome and in the men. These abnormal responses were unchanged by pretreatment with dexamethasone to suppress adrenal function. In the patient with the 3 beta-hydroxysteroid dehydrogenase deficiency, both basal and stimulated plasma levels of delta 5-3 beta-hydroxysteroids before the enzymatic block were elevated, whereas plasma levels of 17 alpha-hydroxyprogesterone and androstenedione--the steroids immediately beyond the block--were low. We conclude that women with polycystic ovary syndrome have masculinized pituitary and ovarian responses to stimulation by nafarelin. Our findings suggest that the regulation of the ovarian 17-hydroxylase and C-17,20-lyase activities is abnormal in such women.
Conversion of cholesterol to pregnenolone in man is mediated by a single enzyme, P450scc. To study possible regulation of the single P450scc gene in ovarian steroid synthesis, we incubated human granulosa cells with potential hormonal stimulators, measured P450scc mRNA accumulation by hybridization to³²P-labeled human P450scc cDNA, and compared the results to secretion of progesterone into the culture medium. Primary cultures of human granulosa cells were optimally responsive after 8-14 days of culture. Incubation with hCG (1.0–100 ng/ml), FSH (1.0-50 ng/ml), and (Bu)2cAMP (0.02–2.0 mM) increased P450scc mRNA accumulation and progesterone secretion in dose-dependent fashions. Maximal stimulation increased P450scc mRNA accumulation and progesterone secretion to 490% and 240% of control values, respectively, with hCG, to 166% and 168% with FSH, and to 495% and 380% with (Bu)2cAMP. PRL (to 100 ng/ml), ACTH (10⁻⁶M), and butyric acid (2 mM) had no significant effect on progesterone secretion or P450scc mRNA accumulation. These data indicate gonadotropin- specific stimulation of cAMP-mediated regulation of P450scc mRNA accumulation in human granulosa cells, presumably mediated by increased P450scc gene transcription.
Ovarian estrogen synthesis may require both thecal and granulosa cells, although this two-cell theory of estrogen synthesis is unproven in man. To examine this theory, we probed the same blots used in the experiments described above with³²P-labeled human P450cl7 cDNA (P450cl7 is the single enzyme mediating both 17α-hydroxylase and 17,20-lyase activities). Only miniscule amounts of P450cl7 mRNA were found in the human granulosa cells, and the amounts did not increase in response to any of the above stimuli. These data strongly support the two-cell theory of human ovarian estrogen synthesis.
Introduction STEROID hormones are familiar clinically and physiologically as regulators of physiological processes. Five groups of steroid hormones are generally recognized according to their physiological behavior: mineralocorticoids, which instruct the renal tubules to retain sodium; glucocorticoids, which are named for their carbohydratemobilizing properties but have many other effects as well; estrogens, which induce female secondary sexual characteristics; progestins, which are essential for reproduction; and androgens, which induce male secondary sexual characteristics. These classes of steroid hormones are structurally similar and arise from a common series of pathways. They are distinguished by their actions on one or more specific steroid hormone receptors. The hormone/receptor complexes function as tissue-specific transcriptional regulators of distinct domains of genes and, consequently, exert their broad array of physiological effects. (For reviews, see Refs. 1 and 2.) The pathways by which the...
Cortisol production requires the activity of only 17 alpha-hydroxylase, whereas the formation of sex steroids requires both 17 alpha-hydroxylase and 17,20-lyase activities. Studies in reconstituted enzyme systems have suggested that a single steroid hydroxylase, 17 alpha-hydroxylase cytochrome P-450 (P-450(17) alpha), catalyzes both activities. By expression of bovine adrenocortical P-450(17 alpha) in COS 1 (transformed monkey kidney) cells, which normally contain no detectable P-450(17) alpha, it has now been established in situ that a single polypeptide chain does catalyze both the 17 alpha-hydroxylase and the 17,20-lyase reactions. This heterologous system supports 17 alpha-hydroxylation of pregnenolone and progesterone with equal efficiency, but catalyzes about five times as much 17,20-lyase activity when 17 alpha-hydroxypregnenolone is the substrate than when 17 alpha-hydroxyprogesterone is the substrate. For these activities to be observed in COS 1 cells, newly synthesized apocytochrome P-450(17) alpha must bind heme and insert into the endoplasmic reticulum such that endogenous cytochrome P-450 reductase can support hydroxylation. Thus, COS 1 cells are a useful system for expression and study of various forms of cytochrome P-450.
One hundred nineteen euprolactinemic anovulatory infertility patients who were being evaluated for induction of ovulation with clomiphene citrate were studied to determine the prevalence of increased adrenal androgen (AA) secretion in this group. Fifty percent of these patients exhibited increased AA secretion, as evidenced by an elevated serum dehydroepiandrosterone sulfate (DHEA-S) level. Seventy-seven percent of these women with elevated levels of DHEA-S were nonhirsute . Twenty-six patients with elevated serum DHEA-S levels underwent adrenocorticotropic hormone (ACTH) stimulation tests in order to determine a possible mechanism(s) for the increase in DHEA-S. Plasma ACTH, as well as total, low-density lipoprotein, and high-density lipoprotein cholesterol were also measured. These levels were normal and did not correlate with the elevated levels of DHEA-S. Seven of 16 patients (34%) had exaggerated responses of serum DHEA-S and of 17-OH pregnenolone to ACTH stimulation. In six of these seven patients, our data suggested the occurrence of a mild deficiency of 3 beta-ol dehydrogenase-isomerase. All of these six patients were considered to have polycystic ovary syndrome. While these data do not explain the increased AA secretion in the majority of patients with elevated levels of DHEA-S, we suggest that serum DHEA-S is frequently elevated in anovulatory infertile patients.
The long term effects of nightly dexamethasone administration on basal levels and diurnal fluctuations of circulating gonadotropins, androgens, and cortisol were studied by frequent sampling in four women with polycystic ovarian disease and a similar number of normal women. Basal LH, testosterone, and androstenedione levels were elevated in the patients with polycystic ovarian disease. There were significant diurnal variations of all steroids measured in both groups, with the exception of androstenedione and androstenediol in the polycystic ovarian disease and control subjects, respectively. Nightly dexamethasone administration for 1 month resulted in marked suppression of dehydroepiandrosterone, androstenediol, and cortisol. For testosterone the mean percent decreases of the 24-h transverse means were 15% and 46% for the polycystic ovarian disease and normal subjects, respectively. For androstenedione the mean percent decreases were only 7% and 20%, respectively. The diurnal variation of all steroids disappeared with dexamethasone. These results support the concept that in patients with polycystic ovarian disease the majority of delta 5-androgens is adrenal while the preponderance of elevated testosterone and androstenedione is ovarian in origin. These results do not support the use of long term dexamethasone as an effective agent in suppressing the elevated levels of testosterone and androstenedione in patients with this disease.
I. Introduction IN THE fasted state, glucose homeostasis depends upon the balance between hepatic glucose production and glucose utilization by the major insulin-dependent tissues, such as liver, adipose, and muscle, and by insulin-independent tissues, such as brain and kidney. This balance is tightly regulated by pancreatic hormones. Thus, in normal individuals, the response to increased plasma glucose levels is an increase in secretion of insulin from β-cells of the pancreatic islets. This increase in circulating insulin levels stimulates glucose transport into peripheral tissues and inhibits hepatic gluconeogenesis. In type II diabetes there are at least two fundamental defects: one is a decrease in the ability of peripheral tissues to respond to insulin (insulin resistance), and the second is impaired β-cell function, which results from long-term hyperglycemia. It appears that both genetic and environmental factors are responsible for the progression from normal glucose tolerance to type II diabetes (...
NCI-H295 is a recently described human adrenocortical carcinoma cell line that makes a variety of steroid hormones. We sought to determine if steroidogenesis in these cells employs the same enzymes as those used in normal adrenal steroidogenesis, and if the genes encoding those enzymes exhibit characteristic responsiveness to activators of the protein kinase-A and -C pathways of intracellular second messengers. Northern blots show that NCI-H295 cells contain abundant mRNAs for three key steroidogenic enzymes, cytochrome P450scc, cytochrome P450c17, and cytochrome P450c21. These mRNAs accumulated in a time- and dose-dependent fashion in response to 8-bromo-cAMP (8Br-cAMP), forskolin, cholera toxin, and 3-isobutyl-1-methylxanthine, all activators of the protein kinase-A pathway. Nuclear run-on assays and actinomycin-D transcriptional inhibition experiments show that cAMP regulates the expression of all three genes primarily at the transcriptional level. Inhibition of protein synthesis with cycloheximide did not prevent the cAMP-induced accumulation of P450scc or P450c17 mRNAs, but did inhibit accumulation of P450c21 mRNA, suggesting that cAMP is acting through a mechanism dependent on protein synthesis to promote accumulation of P450c21 mRNA. Stimulation of the protein kinase-C pathway with phorbol ester decreased P450scc and P450c17 mRNAs, but stimulated the accumulation of P450c21 mRNA. RNase protection experiments, Northern blot hybridizations, and reverse transcription-polymerase chain reaction show that NCI-H295 cells express both the 11 beta-hydroxylase (P450c11 beta) encoded by the P450c11B1 gene and the aldosterone synthetase (P450c11AS) encoded by the P450c11B2 gene. 8Br-cAMP increased the abundance of both of these mRNAs with similar kinetics, with maximal accumulation of both after about 24 h. NCI-H295 cells also contain the mRNAs for aromatase and insulin-like growth factor-II. 8Br-cAMP increased the abundance of aromatase mRNA and decreased the abundance of IGF-II mRNA. These studies show that NCI-H295 cells express most of the enzymes needed for human adrenal steroidogenesis, and that the genes encoding these enzymes respond to stimulation of second messenger pathways in a manner similar to that of human adrenals. NCI-H295 cells appear to be a good model for studying the molecular regulation of human adrenal steroidogenesis.
Cytochrome P450c17 (EC 1.14.99.9) catalyzes both 17 alpha-hydroxylase and 17,20-lyase activities in mammalian steroidogenesis and also has some 16 alpha-hydroxylase activity. The ratio of 17 alpha-hydroxylase to 17,20-lyase activity differs in the adrenal and testis and is developmentally regulated at adrenarche, but the nature of the enzyme's active site and the differential regulation of its two principal activities are unknown. The spontaneous human P450c17 mutation Ser106-->Pro eliminates all enzymatic activity. We used site-directed mutagenesis to construct expression vectors for the conservative P450c17 mutations Ser106-->Thr and Ser106-->Ala. When expressed in transfected COS-1 cells, these mutants retain only 20-30% of the 17 alpha-hydroxylase and 17,20-lyase activities, but retain 60% of the 16 alpha-hydroxylase activity of the Ser106 wild type. Thus, the amino acid occupying position 106 greatly affects enzymatic activity. Ser is found at position 106 in P450c17 in all mammals and birds studied, but the corresponding residue (position 112) in fish (trout) is Thr. Both the trout Thr112 wild type and a Thr112-->Ser trout mutant had equivalent 16 alpha-hydroxylase, 17 alpha-hydroxylase, and 17,20-lyase activities, although these were only 5%, 5%, and 10%, respectively, of human Ser106. To catalyze its activities, P450c17 must receive electrons from NADPH via a flavoprotein termed P450 reductase. We examined the influence of the ratio of P450c17 to P450 reductase on enzymatic activity by cotransfecting COS-1 cells with varying amounts of vectors expressing each protein. The endogenous P450 reductase of COS-1 cells was sufficient to confer maximal 17 alpha-hydroxylase activity. P450 reductase produced from the transfected expression vector did not increase the conversion of [14C]progesterone to 17 alpha- or 16 alpha-hydroxyprogesterone, indicating that the endogenous immunodetectable P450 reductase of COS-1 cells was sufficient to confer maximal 17 alpha-hydroxylase activity. By contrast, the additional P450 reductase produced by the expression vector increased 17,20-lyase activity about 3-fold. Thus, the availability of reducing equivalents is a crucial factor in regulating 17,20-lyase activity. P450 reductase also increased the 17,20-lyase activity of the Thr106 and Ala106 mutants. These data suggest that the essential role of Ser106 is in the active site, rather than in interacting with P450 reductase, and that electron transfer may play an important role in regulating the 17,20-lyase activity of P450c17.
Based on indirect evidence, it has often been assumed that the zona reticularis of the adult human adrenal cortex is the source of the adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS), but direct tests of this concept have been few. Using the techniques of cell culture, Northern blotting, and RIA, we compared the properties of separated adult zonal cells to those of fetal zone cells, a cell type well known to secrete large amounts of DHEA(S) due to its low expression of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD). In nine glands from donors of a wide age range, the zona fasciculata and zona reticularis were separated and dissociated, and the cells were placed in culture. After 5 days, serum was removed by a 24-h period in serum-free defined medium followed by a 24-h exposure to cAMP analogs, with the optional addition of insulin, also in serum-free medium. The separated fasciculata and reticularis cells showed large differences in the DHEA(S)/cortisol (F) production ratios from added pregnenolone precursor, consistent with the synthesis of only F and essentially no DHEA(S) by fasciculata cells and with the synthesis of mostly DHEA(S) with little or no F by both reticularis cells and fetal zone cells. The different patterns of steroidogenesis were accompanied by a much lower level of expression of type II 3 beta HSD in reticularis cells, similar to that in fetal zone cells. In contrast, other genes were similarly regulated in the two adult zones and in the fetal zone by both cAMP and insulin. The levels of messenger ribonucleic acids for 17 alpha-hydroxylase, cholesterol side-chain cleavage enzyme, 21-hydroxylase, and 11 beta-hydroxylase responded to cAMP and insulin in both reticularis cells and fetal zone cells in the same pattern as that previously established in fasciculata cells. The central role of the limited expression of 3 beta HSD in the DHEA(S)-synthesizing property of reticularis cells was established by inhibition of 3 beta HSD in fasciculata cells with trilostane, which caused them to increase their DHEA/F production ratio to a level exceeding even that in fetal zone cells. There did not appear to any age-related changes in gene expression that could account for the large age-related decline in DHEA(S) biosynthesis in humans in either reticularis or fasciculata cells. Thus, the most likely cause of the age-related decline in adrenal androgen biosynthesis is an age-related decline in the number of functional reticularis cells, without a major change in the differentiated properties of the zonal cells as a function of age.
Human NCI-H295 cells, which express all of the genes for the steroidogenic enzymes in a hormonally regulated fashion, should be an ideal system in which to study the transcriptional regulation of these genes. Using deletional promoter/reporter constructions for the human P450scc and P450c17 genes, we identified the regions conferring basal and cAMP-induced transcription of these two genes in NCI-H295 human adrenal cells. In the P450scc gene, both basal and cAMP-induced transcriptional activation elements lie within the first 79 bp upstream (-79) from the transcriptional start site. In the P450c17 promoter, both basal and cAMP-responsive elements lie within the first upstream 63 bp, and a second basal element lies between -184 and -206 bp. The locations of these elements are substantially different from the locations of elements that appear to be functionally equivalent when these human gene promoters are transfected into mouse adrenal Y1, mouse testicular MA-10, or human choriocarcinoma JEG-3 cells. These data indicate that the transcriptional regulation of these genes in their native species and cell type differs substantially from their regulation in cells from other species and tissues, and suggests that the results from transfection experiments examining genes for steroidogenic enzymes in heterologous cells may not reflect events in vivo.
P450c17 is a single microsomal enzyme that catalyzes two distinct steroid biosynthetic activities: 17 alpha-hydroxylase and 17,20 lyase. Human beings have only one gene that encodes only one form of P450c17. Three clinical observations indicated that these were independently regulated activities. First, several cases of isolated 17,20 lyase deficiency were reported, in which 17 alpha-hydroxylase activity was spared. Second, most adrenal steroidogenesis in children stops after 17 alpha-hydroxylation, thus permitting the synthesis of cortisol, whereas most gonadal steroidogenesis proceeds to C19 sex steroids as a result of both activities. Third, the 17,20 lyase activity of the human adrenal is developmentally activated during adrenarche. To catalyze these two activities, P450c17 must receive reducing equivalents from electron donors (redox partners). Previous observations showed that the molar ratio of P450 oxidoreductase to P450c17 was 3-fold higher in the testis than in the adrenal, and that increasing the molar ratio of the redox partner to P450c17 would increase the ratio of 17,20 lyase activity to 17 alpha-hydroxylase. We have recently shown that P450c17 must be phosphorylated on serine and threonine residues by a cAMP-dependent protein kinase to acquire 17,20 lyase activity. We have also recently found two cases of isolated 17,20 lyase deficiency that have mutations of residues in the proposed redox partner binding site. Together, these studies suggest a unified view of the regulation of 17,20 lyase activity. The ratio of 17,20 lyase to 17 alpha-hydroxylase activity of P450c17 is regulated by the availability of reducing equivalents flowing to the enzyme. This can be increased by increasing the molar concentration of electron-donating redox partners, such as P450 oxidoreductase or possibly cytochrome b5, as appears to be the case in the gonads. Alternatively, the affinity of P450c17 for redox partners may be selectively increased by Ser/Thr phosphorylation, or selectively decreased by certain mutations in the redox partner binding site, in either case altering an electrostatic interaction between P450c17 and the redox partner. This model is consistent with all present observations about the biochemistry, genetics, enzymology, and clinical phenomenology of P450c17.
p160ROCK is a serine/threonine protein kinase that binds selectively to GTP-Rho and is activated by this binding. To identify its function, we transfected HeLa cells with wild type and mutants of p160ROCK and examined morphology of the transfected cells. Transfection with wild type and mutants containing the kinase domain and the coiled-coil forming region induced focal adhesions and stress fibers, while no induction was observed with a kinase-defective mutant or a mutant containing only the kinase domain. Furthermore, Rho-induced formation of focal adhesions and stress fibers was inhibited by co-expression of a mutant defective in both kinase and Rho-binding activities. Rho, however, still induced an increase in F-actin content in these cells. These results suggest that p160ROCK works downstream of Rho to induce formation of focal adhesions and that Rho-induced actin polymerization is mediated by other effector(s).
The integration of multiple transmembrane signals is especially important during development and maintenance of the nervous system, communication between cells of the immune system, evolution of transformed cells, and metabolic control (Hunter 1997). Tyrosine phosphorylation plays a key role in many of these processes by directly controlling the activity of receptors or enzymes at early steps in signaling cascades, or by the assembly of multicomponent signaling complexes around activated receptors or their cellular substrates (Pawson 1995). In most if not all cases, initialization of the signaling cascade controlled by growth factor and cytokine receptors originates with multisite tyrosine phosphorylation catalyzed directly by kinases activated during ligand-induced dimerization of specific membrane receptors (Schlessinger 1988; Helding 1995). In many cases, tyrosine autophosphorylation sites in activated receptors directly bind signaling proteins containing Src homology-2 domains (SH2 proteins). In other cases, tyrosine autophosphorylation increases the activity of the receptor kinase, which mediates tyrosine phosphorylation of cytosolic substrates or docking proteins that recruit SH2 proteins into multipotential signaling complexes (Myers and White 1995). The network is further elaborated through other modules which mediate protein-protein or protein-lipid interactions, including PTB, PDZ, SH3, WW, and PH domains.
In the biosynthesis of steroid hormones, P450c17 is the single enzyme that catalyzes both the 17alpha-hydroxylation of 21-carbon steroids and the 17,20-lyase activity that cleaves the C17-C20 bond to produce C19 sex steroids. Cytochrome b5 augments the 17,20-lyase activity of cytochrome P450c17 in vitro, but this has not been demonstrated in membranes, and the mechanism of this action is unknown. We expressed human P450c17, human P450-oxidoreductase (OR), and/or human cytochrome b5 in Saccharomyces cerevisiae and analyzed the 17alpha-hydroxylase and 17,20-lyase activities of the resulting yeast microsomes. Yeast expressing only P450c17 have 17alpha-hydroxylase and trace 17,20-lyase activities toward both Delta4 and Delta5 steroids. Coexpression of human OR with P450c17 increases the Vmax of both the 17alpha-hydroxylase and 17,20-lyase reactions 5-fold; coexpression of human b5 with P450c17 also increases the Vmax of the 17,20-lyase reactions but not of the 17alpha-hydroxylase reactions. Simultaneous expression of human b5 with P450c17 and OR, or addition of purified human b5 to microsomes from yeast coexpressing human P450c17 and OR, further increases the Vmax of the 17,20-lyase reaction without altering 17alpha-hydroxylase activity. Genetically engineered yeast and mixing experiments demonstrate that OR is both necessary and sufficient for microsomal 17,20-lyase activity. Addition of purified human holo-b5, apo-b5, or cytochrome c to microsomes containing both human P450c17 and OR demonstrate that the stimulatory action of b5 does not require electron transfer from b5 to P450c17. These data suggest that human b5 acts principally as an allosteric effector that interacts primarily with the P450c17.OR complex to stimulate 17, 20-lyase activity.
Adrenarche is the increased adrenal production of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) that occurs during the prepubertal period. To date, the exact mechanism initiating adrenarche is unknown, although many factors have been postulated. In the present study, we examined the hypothesis that alterations in intra-adrenal expression of 3beta-hydroxysteroid dehydrogenase (3betaHSD) or 21-hydroxylase (CYP21) within the inner reticularis zone leads to the increased production of 19-carbon (C19) steroids. After conversion of cholesterol to pregnenolone, 17alpha-hydroxylase/17,20-lyase (CYP17) can metabolize pregnenolone through to DHEA. The enzyme 3betaHSD competes for substrate with CYP17 and effectively removes steroid precursor from the pathway leading to DHEA. On the other hand, deficiency in CYP21 expression is known to cause excessive production of adrenal C19 steroids, suggesting that CYP21 could play a role in adrenarche. Thus, a decrease in 3betaHSD or CYP21 expression would allow substrate to flow toward the synthesis of DHEA. To determine whether adrenarche results from a decreased expression of 3betaHSD or CYP21 in the reticularis, immunohistochemical localization of 3betaHSD and CYP21 was performed, and staining intensities compared using adrenal glands from children ages 4 months to 4 yr (n = 12), ages 5-7 yr (n = 9), ages 8-13 yr (n = 9), and adults ages 25-56 yr (n = 8). There were no differences in the zonal expression of CYP21. No difference in 3betaHSD staining was observed between the glomerulosa and fasciculata from any age group. However, children age 8 yr and older show a significant decrease in 3betaHSD expression in reticularis as compared with the fasciculata. No significant difference was noted for 3betaHSD levels between the fasciculata and reticularis for children age 7 yr or younger. The level of 3betaHSD expression in the reticularis continued to decrease in the adult adrenals examined. These findings suggest that as children mature there is a decreased level of 3betaHSD in the adrenal reticularis that may contribute to the increased production of DHEA and DHEAS seen during adrenarche.
Cytochrome P450c17 catalyzes steroid 17alpha-hydroxylase and 17,20-lyase activities and hence is a key enzyme in the production of human glucocorticoids and sex steroids. These two activities are catalyzed in a single substrate-binding site but are regulated independently in human physiology. We have recently shown that cytochrome b5 facilitates 17,20-lyase activity by allosterically promoting the interaction of P450c17 with P450 oxidoreductase (OR) and that the human P450c17 mutations, R347H and R358Q, selectively destroy 17,20-lyase activity while sparing 17alpha-hydroxylase activity. We transfected COS-1 cells with vectors for these P450c17 mutants and found that an excess of OR and b5 restored a small amount of 17,20-lyase activity, suggesting the mutations interfere with electron donation. To determine whether these mutations selectively interfere with the interaction of P450c17 and its electron-donating system, we expressed each P450cl7 mutant in yeast with or without OR, b5, or both, and measured enzyme kinetics in yeast microsomes using pregnenolone and 17alpha-hydroxypregnenolone as substrates. The apparent Michaelis-Menten (Km) values for the R347H mutant with and without coexpressed OR were 0.2 and 0.6 microM, respectively, and for the R358Q mutant with and without OR they were 0.3 and 0.4 microM, respectively; these values did not differ significantly from the wild-type values of 0.4 and 0.8 microM with and without OR, respectively. Furthermore, coincubation with 17alpha-hydroxypregnenolone showed a competitive mechanism for interference of catalysis. The similar kinetics and the competitive inhibition prove that the mutations did not affect the active site. Coexpression of the mutants with OR yielded insignificant 17,20-lyase activity, but addition of a 30:1 molar excess cytochrome b5 to these microsomes restored partial 17,20-lyase activity, with the R358Q mutant achieving twice the activity of the R347H mutant. These data indicate that both mutations selectively interfere with 17,20-lyase activity by altering the interaction of P450c17 with OR, thus proving that the lyase activity was disrupted by interfering with electron transfer. Furthermore, the data offer the first evidence that R347 is a crucial component of the site at which b5 interacts with the P450c17 x OR complex to promote electron transfer.
Microsomal 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17) catalyzes both the 17αhydroxylase reaction required to produce cortisol, the major glucocorticoid in many animals, and the 17,20-lyase activity required for the production of androgens in all animals. In rodents such as rat, which utilize corticosterone as the major glucocorticoid, P450c17 is expressed predominantly in the gonads, and is absent in the adrenal. In other species including humans, P450c17 is expressed in both adrenal and gonads and participates in both glucocorticoid and androgen production. Rat and human forms of P450c17 are 69% identical at the amino acid level. Based on the differences in physiological roles between P450c17 in these two species, it could be predicted that major differences would be observed in their hydroxylase activities. Contrary to this hypothesis, using partially purified, recombinant human and rat P450c17, we found that the most significant differences lie in their lyase activities. Lyase activities demonstrate that the rat enzyme favors Δ4 (progesterone) substrates while the human enzyme favors Δ5 (pregnenolone) substrates. This substrate preference is also observed in the ability of steroids to decrease uncoupled H2O2 production and to increase stability during turnover. Cytochrome b5, a microsomal electron-transfer protein, enhances lyase activities of rat and human P450c17. However, the most dramatic stimulatory effect is on the human HO-PROG lyase activity. This enhancement of activities is not associated with electron transfer. These differences in biochemical properties between the two forms of P450c17 indicate that human P450c17 has evolved as an enzyme system that limits androgen production to the gonads where a favorable bs:P450c17 ratio exists. Even though orthologous forms of P450c17 are capable of catalyzing the same enzymatic activities, specific physiological requirements of each species ensure biochemical differences between these enzymes.
P450c17 (17alpha-hydroxylase/17,20-lyase) catalyzes steroid 17alpha-hydroxylase and 17,20-lyase activities in the biosynthesis of androgens and estrogens. These two activities are differentially regulated in a tissue-specific and developmentally programmed manner. To visualize the active site topology of human P450c17 and to study the structural basis of its substrate specificity and catalytic selectivity, we constructed a second-generation computer-graphic model of human P450c17. The energetics of the model are comparable to those of the principal template of the model, P450BMP, as determined from its crystallographic coordinates. The protein structure analysis programs PROCHECK, WHATIF, and SurVol indicate that the predicted P450c17 structure is reasonable. The hydrophobic active site accommodates both delta4 and delta5 steroid substrates in a catalytically favorable orientation. The predicted contributions of positively charged residues to the redox-partner binding site were confirmed by site-directed mutagenesis. Molecular dynamic simulations with pregnenolone, 17-OH-pregnenolone, progesterone, and 17-OH-progesterone docked into the substrate-binding pocket demonstrated that regioselectivity of the hydroxylation reactions is determined both by proximity of hydrogens to the iron-oxo complex and by the stability of the carbon radicals generated after hydrogen abstraction. The model explains the activities of all known naturally occurring and synthetic human P450c17 mutants. The model predicted that mutation of lysine 89 would disrupt 17,20-lyase activity to a greater extent than 17alpha-hydroxylase activity; expression of a test mutant, K89N, in yeast confirmed this prediction. Hydrogen peroxide did not support catalysis of the 17,20-lyase reaction, as would be predicted by mechanisms involving a ferryl peroxide. Our present model and biochemical data suggest that both the hydroxylase and lyase activities proceed from a common steroid-binding geometry by an iron oxene mechanism. This model will facilitate studies of sex steroid synthesis and its disorders and the design of specific inhibitors useful in chemotherapy of sex steroid-dependent cancers.
CYP17 is a microsomal enzyme embodying two distinct activities, 17alpha-hydroxylase and 17,20-lyase, essential for the synthesis of cortisol and sex hormone precursors, respectively. The two activities are differentially regulated in a tissue and developmental stage-dependent fashion. Leptin might play a role in such differential control. Low dose leptin caused a significant increase in 17,20-lyase activity in adrenal NCI-H295R cells expressing leptin (OB) receptor (OB-R), without significant sustained influence on the 17alpha-hydroxylase activity. To analyze the time dependence of this leptin effect, the impact of long and short-term leptin treatment was studied. To assess the relationship with the OB-R signal transduction pathway, the same experiments were performed in intact cells and in a reconstituted system. The long- and short-term studies in intact cells and in microsomes suggest that the 17alpha-hydroxylase activity of CYP17 can be promptly stimulated by leptin, but that the effect is transient. In contrast, physiological doses of leptin steadily enhance 17,20-lyase activity. This influence is direct, OB-R specific and dependent on the integrity of the signal transduction pathway. The 17,20-lyase activity stimulation relies on phosphate incorporation, as demonstrated by the loss of leptin-dependent 17,20-lyase stimulation after phosphate removal, and by the fact that the DHEA production appears to be related exclusively to the presence of phosphorylated CYP17, independently from novel protein synthesis. The mechanism underlying the observed events seems to involve CYP17 phosphorylation, a feature of the OBR signal transduction pathway, and a process already shown to be crucial for 17,20-lyase activity.
Adrenarche is considered to occur as a result of intra-adrenal changes in steroidogenic enzymes involved in C19 steroid production. The present study was conducted because developmental changes in steroidogenic enzymes have not been examined well in human postnatal adrenal. Twenty-four specimens of nonpathological human adrenals from 7 months to 62 years retrieved from autopsy files. Immunohistochemistry for P450 side-chain cleavage (P450scc), 17alpha hydroxylase (P450c17), dehydroepiandrosterone sulfotransferase (DHEA-ST), P450 oxidoreductase, cytochrome b5, and 3beta-hydroxysteroid dehydrogenase (3betaHSD) was per-formed in these specimens, and the immuno-intensity was evaluated using CAS 200 computed image analysis system. Immunoreactivity of P450scc was marked in the zona glomerulosa, fasciculata and reticularis in the adrenal glands of all the cases examined. P450c17 and DHEA-ST immunoreactivity was weak in the zona fasciculata and reticularis in the adrenals of age 7 months to 5 years, but thereafter became prominent in the zona reticularis. Immunoreactivity of P450 oxidoreductase and cytochrome b5, components of the electron transfer system hypothesized to regulate the 17-20 lyase activity of P450c17, was weak in all three zones of adrenal cortex from 7 months to 5 years, and became more marked in the zona reticularis after age 5 years. 3betaHSD immunoreactivity was marked in all three zones of the adrenal cortex from 7 months to 8 years but thereafter decreased in the zona reticularis. These data suggest that the human adrenal zona reticularis markedly begins to develop morphologically and functionally at around 5 years of age. The increased level of P450c17, DHEA-ST, P450 oxidoreductase, and cytochrome b5, and the decreased level of 3betaHSD in the reticularis is likely to contribute to increased C19 steroid production during adrenarche.