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Identification of the molecular switch that regulates access of 5??-DHT to the androgen receptor
Abstract
Pairs of hydroxysteroid dehydrogenases (HSDs) govern ligand access to steroid receptors in target tissues and act as molecular switches. By acting as reductases or oxidases, HSDs convert potent ligands into their cognate inactive metabolites or vice versa. This pre-receptor regulation of steroid hormone action may have profound effects on hormonal response. We have identified the HSDs responsible for regulating ligand access to the androgen receptor (AR) in human prostate. Type 3 3alpha-hydroxysteroid dehydrogenase (aldo-keto reductase 1C2) acts solely as a reductase to convert 5alpha-dihydrotestosterone (DHT), a potent ligand for the AR (K(d)=10(-11)M for the AR), to the inactive androgen 3alpha-androstanediol (K(d)=10(-6)M for the AR); while RoDH like 3alpha-HSD (a short-chain dehydrogenase/reductase (SDR)) acts solely as an oxidase to convert 3alpha-androstanediol back to 5alpha-DHT. Our studies suggest that aldo-keto reductase (AKRs) and SDRs function as reductases and oxidases, respectively, to control ligand access to nuclear receptors.
... The intratumoral formation of potent androgens may render advanced forms of PCa independent of the circulating levels of testosterone and 5α-dihydrotestosterone (5α-DHT). Several members of the short-chain dehydrogenase/reductase (SDR) superfamily were shown to locally activate androgens, including hydroxysteroid dehydrogenase (HSD) 17B3 (SDR12C2) converting androstenedione to testosterone [4,5], and the 3α-HSDs converting 3α-androstanediol (3α-Adiol) to 5α-DHT, i.e., HSD17B6 (SDR9C6), retinol dehydrogenase (RDH) 5 (SDR9C5), RDH16 (SDR9C8), and DHRS9 (SDR9C4) [6][7][8]. Additionally, HSD3B1 (SDR11E1), required for the production of intratumoral androgens from adrenal precursors, has been associated with CRPC [9]. ...
... Experiments using cell-based assays and purified enzymes then suggested that DHRS7 is a multifunctional enzyme and may accept various substrates, including xenobiotics, retinoids, and steroid hormones, such as 5α-DHT [11,14,15]. The latter is of particular interest since other SDR members were reported as playing a role in intracrinology by regulating intratissue and intratumoral androgen concentrations [4,6,7,36]. The finding that DHRS7 is able to inactivate 5α-DHT, the most potent AR ligand in men [3,7], to 3α-Adiol led to the hypothesis that DHRS7 acts as a tumor- ...
... The latter is of particular interest since other SDR members were reported as playing a role in intracrinology by regulating intratissue and intratumoral androgen concentrations [4,6,7,36]. The finding that DHRS7 is able to inactivate 5α-DHT, the most potent AR ligand in men [3,7], to 3α-Adiol led to the hypothesis that DHRS7 acts as a tumor- ...
Prostate cancer (PCa), one of the most common malignancies in men, typically responds to initial treatment, but resistance to therapy often leads to metastases and death. The dehydrogenase/reductase 7 (DHRS7, SDR34C1) is an “orphan” enzyme without known physiological function. DHRS7 was previously found to be decreased in higher-stage PCa, and siRNA-mediated knockdown increased the aggressiveness of LNCaP cells. To further explore the role of DHRS7 in PCa, we analyzed the proteome of LNCaP cells following DHRS7 knockdown to assess potentially altered pathways. Although DHRS7 is able to inactivate 5α-dihydrotestosterone, DHRS7 knockdown did not affect androgen receptor (AR) target gene expression, and its effect on PCa cells seems to be androgen-independent. Importantly, proteome analyses revealed increased expression of epidermal growth factor receptor (EGFR), which was confirmed by RT-qPCR and Western blotting. Comparison of AR-positive LNCaP with AR-negative PC-3 and DU145 PCa cell lines revealed a negative correlation between DHRS7 and EGFR expression. Conversely, EGFR knockdown enhanced DHRS7 expression in these cells. Importantly, analysis of patient samples revealed a negative correlation between DHRS7 and EGFR expression, both at the mRNA and protein levels, and DHRS7 expression correlated positively with patient survival rates. These results suggest a protective role for DHRS7 in PCa.
... As early as sixty years ago, steroid-hormones were recognized to exist in either active or inactive forms which could be enzymatically interconverted in a tissue-specific manner. This concept in steroid-hormone physiology was called pre-receptor control, and implied that inactive metabolites could serve as precursors for metabolic conversion to active ligands, thereby complementing the pool of ligands available for receptor binding in a tissue-specific manner [2,3]. ...
... While AKR1C2 is capable of bidirectional activity (i.e. catalyzing conversion of 3α-diol back to DHT), intracellularly it functions primarily to reduce DHT [3,6]. The reductase activity of AKR1C2, together with the reverse oxidative activity of 3α-HSDs, including HSD17B6, HSD17B10, and RDH5, is a critical molecular switch that regulates tissue androgen levels [3,[6][7][8]. ...
... catalyzing conversion of 3α-diol back to DHT), intracellularly it functions primarily to reduce DHT [3,6]. The reductase activity of AKR1C2, together with the reverse oxidative activity of 3α-HSDs, including HSD17B6, HSD17B10, and RDH5, is a critical molecular switch that regulates tissue androgen levels [3,[6][7][8]. ...
Androgens play an important role in prostate cancer (PCa) development and progression. Accordingly, androgen deprivation therapy remains the front-line treatment for locally recurrent or advanced PCa, but patients eventually relapse with the lethal form of the disease termed castration resistant PCa (CRPC). Importantly, castration does not eliminate androgens from the prostate tumor microenvironment which is characterized by elevated tissue androgens that are well within the range capable of activating the androgen receptor (AR). In this mini-review, we discuss emerging data that suggest a role for the enzymes mediating pre-receptor control of dihydrotestosterone (DHT) metabolism, including AKR1C2, HSD17B6, HSD17B10, and the UGT family members UGT2B15 and UGT2B17, in controlling intratumoral androgen levels, and thereby influencing PCa progression. We review the expression of steroidogenic enzymes involved in this pathway in primary PCa and CRPC, the activity and regulation of these enzymes in PCa experimental models, and the impact of genetic variation in genes mediating pre-receptor DHT metabolism on PCa risk. Finally, we discuss recent data that suggests several of these enzymes may also play an unrecognized role in CRPC progression separate from their role in androgen inactivation.
... AKR1C1 works predominately as a 3-ketosteroid reductase on DHT leading to the formation of 5α-androstane-3β,17β-diol, a pro-apoptotic ligand for estrogen receptor β [52] . AKR1C2 works predominately as a 3-ketosteroid reductase on DHT leading to the formation of the inactive androgen 5α-androstane-3α,17β-diol [53] . Similar reactions are possible with 11-keto-DHT [50] . ...
... All enzymes are listed in italics by their gene names. AKR1C3 , type 5 17β-hydroxysteroid dehydrogenase; HSD11B2, type 2 11β-hydroxysteroid dehydrogenase; and SRD5A1/2 , type 1 and type 2 steroid 5α-reductase (3a-hydroxysteroid oxidase) work as the molecular switch that determines DHT ligand access to the AR in the normal and diseased prostate [53] . HSD17B2 and HSD17B4 by working as 17β-hydroxysteroid oxidases convert T and DHT to their inactive counterparts, e.g., Δ 4 -AD and 5α-androstane-3,17-dione, respectively, and are implicated in the inactivation of these hormones [54,55] . ...
Castration-resistant prostate cancer is the lethal form of prostate cancer and most commonly remains dependent on androgen receptor (AR) signaling. Current therapies use AR signaling inhibitors (ARSI) exemplified by abiraterone acetate, a P450c17 inhibitor, and enzalutamide, a potent AR antagonist. However, drug resistance to these agents occurs within 12-18 months and they only prolong overall survival by 3-4 months. Multiple mechanisms can contribute to ARSI drug resistance. These mechanisms can include but are not limited to germline mutations in the AR, post-transcriptional alterations in AR structure, and adaptive expression of genes involved in the intracrine biosynthesis and metabolism of androgens within the tumor. This review focuses on intracrine androgen biosynthesis, how this can contribute to ARSI drug resistance, and therapeutic strategies that can be used to surmount these resistance mechanisms.
... Prostate tissue also demonstrates epithelial cell expression of phase I (reducing) and phase II (conjugating) DHT catabolizing enzymes that act in concert to regulate access of DHT to the AR. AKR1C1 is the primary enzyme responsible for the irreversible reduction of DHT to the weak metabolite, 5α-androstane-3,17-diol (3α-androstanediol or 3α-diol, a low affinity AR ligand), whereas AKR1C2 catalyzes the reversible conversion of DHT to 5α-androstane-3,17-diol (3β-diol, a pro-apoptotic ligand of estrogen receptor beta, ER) (44). The reductase activity of AKR1C2, coupled with the reverse oxidative activity of specific 3α-HSD enzymes is a critical molecular switch regulating access of DHT to the AR (44)(45)(46)(47). ...
... AKR1C1 is the primary enzyme responsible for the irreversible reduction of DHT to the weak metabolite, 5α-androstane-3,17-diol (3α-androstanediol or 3α-diol, a low affinity AR ligand), whereas AKR1C2 catalyzes the reversible conversion of DHT to 5α-androstane-3,17-diol (3β-diol, a pro-apoptotic ligand of estrogen receptor beta, ER) (44). The reductase activity of AKR1C2, coupled with the reverse oxidative activity of specific 3α-HSD enzymes is a critical molecular switch regulating access of DHT to the AR (44)(45)(46)(47). ...
While androgen deprivation therapy (ADT) remains the primary treatment for metastatic prostate cancer (PCa) since the seminal recognition of the disease as androgen-dependent by Huggins and Hodges in 1941, therapy is uniformly marked by progression to castration-resistant prostate cancer (CRPC) over a period of about 18 months, with an ensuing median survival of 1 to 2 years. Importantly, castration does not eliminate androgens from the prostate tumor microenvironment. Castration resistant tumors are characterized by elevated tumor androgens that are well within the range capable of activating the AR and AR-mediated gene expression, and by steroid enzyme alterations which may potentiate de novo androgen synthesis or utilization of circulating adrenal androgens. The dependence of CRPC on intratumoral androgen metabolism has been modeled in vitro and in vivo, and residual intratumoral androgens are implicated in nearly every mechanism by which AR-mediated signaling promotes castration-resistant disease. These observations suggest that tissue based alterations in steroid metabolism contribute to the development of CRPC and underscore these metabolic pathways as critical targets of therapy. Herein, we review the accumulated body of evidence which strongly supports intracrine (tumoral) androgen synthesis as an important mechanism underlying PCa progression. We first discuss the presence and significance of residual prostate tumor androgens in the progression of CRPC. We review the classical and non-classical pathways of androgen metabolism, and how dysregulated expression of these enzymes is likely to potentiate tumor androgen production in the progression to CRPC. Next we review the in vitro and in vivo data in human tumors, xenografts, and cell line models which demonstrate the capacity of prostate tumors to utilize cholesterol and adrenal androgens in the production of testosterone (T) and dihydrotestosterone (DHT), and briefly review the potential role of exogenous influences on this process. Finally, we discuss the emerging data regarding mechanisms of response and resistance to potent ligand synthesis inhibitors entering clinical practice, and conclude by discussing the implications of these findings for future therapy.
... In the classical pathway of androgen synthesis ( Figure 2, light gray arrows), cholesterol is converted to C-21 precursors (pregnenolone and progesterone), which are converted to the C-19 steroids DHEA (via the sequential hydroxylase and lyase activity of CYP17A1) and AED (through the action of HSD3B1,2 on DHEA), and then acted on by AKR1C3 (or potentially HSD17B3) to generate T, with peripheral con-version of T to 5α-DHT carried out by SRD5A1 or 2 in target tissues [34]. AKR1C2 mediates the reduction of 5α-DHT to 3α-diol (a metabolite with weak/low androgenic activity), whereas AKR1C1 catalyzes the conversion of 5α-DHT to 3β-diol [35]. The glucuronidating enzymes UGT2B15 and UTG2B17 irreversibly terminate the androgen signal by glucuronidation of 3α-diol (as well as T, 5α-DHT and other metabolites) [36][37][38]. ...
... Androsterone generated by the lyase activity of CYP17A1 is then acted upon by HSD17B3 or AKR1C3 to generate 3α-diol followed by, a reverse oxidative step (not required in the classical pathway) to generate 5α-DHT [27]. Candidate enzymes mediating the reverse conversion of 3α-diol to 5α-DHT include RL-HSD (17BHSD6), 17BHSD10, RODH4, RDH5, and DHRS9 [35,40,41]. RL-HSD also acts as an epimerase to convert 3α-diol to 3β-diol, although at much higher substrate concentrations [42], and can directly catalyze conversion of physiologic levels of 5α-DHT to 3β-diol [43]. ...
While androgen deprivation therapy (ADT) remains the primary treatment for metastatic prostate cancer (PCa), castration does not eliminate androgens from the prostate tumor microenvironment, and residual intratumoral androgens are implicated in nearly every mechanism by which androgen receptor (AR)-mediated signaling promotes castration-resistant disease. The uptake and intratumoral (intracrine) conversion of circulating adrenal androgens such as dehydroepiandrosterone sulfate (DHEA-S) to steroids capable of activating the wild type AR is a recognized driver of castration resistant prostate cancer (CRPC). However, less well-characterized adrenal steroids, including 11-deoxcorticosterone (DOC) and 11beta-hydroxyandrostenedione (11OH-AED) may also play a previously unrecognized role in promoting AR activation. In particular, recent data demonstrate that the 5α-reduced metabolites of DOC and 11OH-AED are activators of the wild type AR. Given the well-recognized presence of SRD5A activity in CRPC tissue, these observations suggest that in the low androgen environment of CRPC, alternative sources of 5α-reduced ligands may supplement AR activation normally mediated by the canonical 5α-reduced agonist, 5α-DHT. Herein we review the emerging data that suggests a role for these alternative steroids of adrenal origin in activating the AR, and discuss the enzymatic pathways and novel downstream metabolites mediating these effects. We conclude by discussing the potential implications of these findings for CRPC progression, particularly in context of new agents such as abiraterone and enzalutamide which target the AR-axis for prostate cancer therapy.
... In this regard, it has been shown that, besides acting directly by binding to androgen receptors, DHT may also act indirectly through its metabolite 5-androstane- 3,17-diol (3-diol)91011. The conversion of DHT to 3-diol in the normal prostate is favored by the higher (13-fold) expression of 3-hydroxysteroid dehydrogenase (3-HSD), compared to the stereo-specific 3-HSD, which produces 3- diol from DHT111213. The 3-diol is greatly oxidized back to DHT, thus working mostly as a source of this potent androgen [11,131415 . By contrast, the 3-diol formation is virtually irreversible, because most of this metabolite is rapidly hydroxylated by cytochrome P450-7B1 (CYP7B1), forming the water-soluble 6-or 7-triol, which terminates the 3-diol action [11,161718. ...
... The conversion of DHT to 3-diol in the normal prostate is favored by the higher (13-fold) expression of 3-hydroxysteroid dehydrogenase (3-HSD), compared to the stereo-specific 3-HSD, which produces 3- diol from DHT111213. The 3-diol is greatly oxidized back to DHT, thus working mostly as a source of this potent androgen [11,131415 . By contrast, the 3-diol formation is virtually irreversible, because most of this metabolite is rapidly hydroxylated by cytochrome P450-7B1 (CYP7B1), forming the water-soluble 6-or 7-triol, which terminates the 3-diol action [11,161718. ...
Prostate is one of the major targets for dihydrotestosterone (DHT), however this gland is also recognized as a nonclassical target for estrogen as it expresses both types of estrogen receptors (ER), especially ERbeta. Nevertheless, the concentrations of aromatase and estradiol in the prostate are low, indicating that estradiol may not be the only estrogenic molecule to play a role in the prostate. It is known that DHT can be metabolized to 5alpha-androstane-3beta,17beta-diol (3beta-diol), a hormone that binds to ERbeta but not to AR. The concentration of 3beta-diol in prostate is much higher than that of estradiol. Based on the high concentration of 3beta-diol and since this metabolite is a physiological ERbeta ligand, we hypothesized that 3beta-diol would be involved in the regulation of ERbeta expression. To test this hypothesis, adult male rats were submitted to castration followed by estradiol, DHT or 3beta-diol replacement. ERbeta and AR protein levels in the prostate were investigated by immunohistochemistry and Western blotting assays. The results showed that after castration, the structure of the prostate was dramatically changed and ERbeta and AR protein levels were decreased. Estradiol had just minor effects on the parameters analyzed. DHT-induced partial recovery of ERbeta while it was the most effective inductor of AR expression. Replacement with 3beta-diol-induced the highest levels of ERbeta, but was comparatively less effective in recovering the AR expression and the gland structure. These results offer evidence that one functional role of 3beta-diol in the prostate may be autoregulation of its natural receptor, ERbeta.
... Our findings raise an intriguing possibility that a functionally homologous system exists in mammals-we found that in mouse, several genes with putative hormone-degrading or -altering functions are most highly expressed in brain endothelium relative to other endothelial cells (Fig. S10K) (160)). Ark1c14 and H2-Ke6 (aka 17β-HSD), which both degrade testosterone (161)(162)(163), displayed higher expression in brain vascular endothelium than in endothelial cells from four other tissues, and compared to whole brain (Fig. S10K, left). These data suggest that BBB-based hormonal degradation may be a broader mechanism for regulating brain hormone levels. ...
Here we reveal an unanticipated role of the blood-brain-barrier (BBB) in regulating complex social behavior in ants. Using scRNA-seq we find localization in the BBB of a key hormone-degrading enzyme called Juvenile hormone esterase (Jhe), and we show that this localization governs the level of Juvenile Hormone (JH3) entering the brain. Manipulation of the Jhe level reprograms the brain transcriptome between ant castes. While ant Jhe is retained and functions intracellularly within the BBB, we show that Drosophila Jhe is naturally extracellular. Heterologous expression of ant Jhe into the Drosophila BBB alters behavior in fly to mimic what is seen in ant. Most strikingly, manipulation of Jhe levels in ant reprograms complex behavior between worker castes. Our study thus uncovers a novel, potentially conserved role of the BBB serving as a molecular gatekeeper for a neurohormonal pathway that regulates social behavior.
... The confined space effect enables cost-effective screens of low affinity binders, such as those anticipated in fragment libraries, that escape detection by activity-based assays or other biophysical techniques (164). The greater flexibility in detection and elaboration of "hits" discovery through the application of FBDD using RM-NMR will provide ways to differentiate AKR1C3 from its close homolog AKR1C2, which inactivates DHT (165). Such agents offer the promise to eliminate AR ligand synthesis in CRPC and also block the transactivation of the AR-FL enabled by the coactivator function of AKR1C3 without affecting AKR1C2. ...
Castration resistant prostate cancer (CRPC) continues to be androgen receptor (AR) driven. Inhibition of AR signaling in CRPC could be advanced using state-of-the-art biophysical and biochemical techniques. Structural characterization of AR and its complexes by cryo-electron microscopy would advance the development of N-terminal domain (NTD) and ligand binding domain (LBD) antagonists. The structural basis of AR function is unlikely to be determined by any single structure due to the intrinsic disorder of its NTD which not only interacts with coregulators but likely accounts for the constitutive activity of AR-splice variants (SV) which lack the LBD and emerge in CRPC. Using different AR constructs lacking the LBD their effects on protein folding, DNA binding, and transcriptional activity could reveal how interdomain coupling explains the activity of AR-SVs. The AR also interacts with co-regulators that promote chromatin looping. Elucidating the mechanisms involved can identify vulnerabilities to treat CRPC which do not involve targeting the AR. Phosphorylation of the AR coactivator MED-1 by CDK7 is one mechanism that can be blocked by the use of CDK7 inhibitors. CRPC gains resistance to AR signaling inhibitors (ARSI). Drug resistance may involve AR-SVs but their role requires their reliable quantification by SILAC-mass spectrometry during disease progression. ARSI drug resistance also occurs by intratumoral androgen biosynthesis catalyzed by AKR1C3 (type 5 17β-hydroxysteroid dehydrogenase), which is unique in that its acts as a coactivator of AR. Novel bifunctional inhibitors that competitively inhibit AKR1C3 and block its coactivator function could be developed using reverse-micelle-NMR and fragment-based drug discovery.
... 106 The AR is expressed at various levels by a variety of leukocytes such as neutrophils and macrophages. 107 Most recently, the AR was identified on ILC2s and signaling through this receptor reduced the susceptibility to IL-33-driven and alternaria extract-driven lung inflammation in part by reducing the expansion and reactivity of ILC2s. 95,108 Signaling through the AR pathway provides support for the protective role of androgens in allergic asthma and the dimorphic switch that occurs after puberty. ...
Group 2 innate lymphoid cells (ILC2s) are a recently described subset of innate lymphocytes with important immune and homeostatic functions at multiple tissue sites, especially the lung. These cells expand locally after birth and during postnatal lung maturation and are present in the lung and other peripheral organs. They are modified by a variety of processes and mediate inflammatory responses to respiratory pathogens, inhaled allergens and noxious particles. Here, we review the emerging roles of ILC2s in pulmonary homeostasis and discuss recent and surprising advances in our understanding of how hormones, age, neurotransmitters, environmental challenges, and infection influence ILC2s. We also review how these responses may underpin the development, progression and severity of pulmonary inflammation and chronic lung diseases and highlight some of the remaining challenges for ILC2 biology.
... They include testosterone, the majority of which (98%) becomes irreversibly converted to the active metabolite 5alpha-dihydrotestosterone (DHT). DHT binds with higher affinity than testosterone to the androgen receptor (AR), which is expressed at different levels in leukocytes, regulating transcription of target genes (100). When evaluating testosterone effects, it should be keep in mind that some could be mediated via the ER pathway, as testosterone can be converted into E1 or E2 by the enzyme aromatase. ...
Multiple sclerosis (MS) is a chronic inflammatory disease of the Central Nervous System (CNS) affecting young people and leading to demyelination and neurodegeneration. The disease is clearly more common in women in whom incidence has been rising. Gender differences include: earlier disease onset and more frequent relapses in women; and faster progression and worse outcomes in men. Hormone‐related physiological conditions in women such as puberty, pregnancy, puerperium and menopause also exert significant influence both on disease prevalence as well as on outcomes.
Hormonal and/or genetic factors are therefore believed to be involved in regulating the course of disease. In this review, we discuss clinical evidence of sex hormone (estrogens, progesterone, prolactin and testosterone) impact on MS, and attempt to elucidate hormonal and immunological mechanisms potentially underlying these changes. We also review current knowledge on the relationship between sex hormones and resident CNS cells, and provide new insights in the context of MS. Understanding these molecular mechanisms may contribute to the development of new and safer treatments for both men and women.
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... Most testosterone (98%) is irreversibly converted to an active metabolite, dihydrotestosterone (86), which binds with higher affinity than testosterone to ARs, which are expressed at various levels by leukocytes (87). In innate immune cells, such as neutrophils, AR signaling maintains cellular differentiation via induction of G-CSF signaling through activation of ERK1/2 and STAT3 (88). ...
New attention to sexual dimorphism in normal mam-malian physiology and disease has uncovered a previ-ously unappreciated breadth of mechanisms by which females and males differentially exhibit quantitative phenotypes. Thus, in addition to the established mod-ifying effects of hormones, which prenatally and postpubertally pattern cells and tissues in a sexually dimorphic fashion, sex differences are caused by extra-gonadal and dosage effects of genes encoded on sex chromosomes. Sex differences in immune responses, especially during autoimmunity, have been studied pre-dominantly within the context of sex hormone effects. More recently, immune response genes have been local-ized to sex chromosomes themselves or found to be reg-ulated by sex chromosome genes. Thus, understanding how sex impacts immunity requires the elucidation of complex interactions among sex hormones, sex chromo-somes, and immune response genes. In this Brief Re-view, we discuss current knowledge and new insights into these intricate relationships in the context of viral infections.
... Probably the most extensively studied SDRs are hydroxysteroid dehydrogenases (HSDs) with key roles in adrenal and gonadal steroidogenesis, including 3b-HSDs and 17b-HSDs, as well as enzymes with 3a-HSD, 11b-HSD and 17b-HSD activities catalyzing the metabolism of steroids in peripheral tissues and thereby controlling local steroid hormone action [4]. Generally, 3a-HSDs are assigned to the family of aldo-keto reductases (AKR); however, several SDRs are reported to have 3a-HSD activity such as 17b-HSD6, 17b-HSD10 or members of the retinol dehydrogenase (RODH) subfamily [5,6]. ...
Several members of the short-chain dehydrogenase/reductase (SDR) enzyme family play fundamental roles in adrenal and gonadal steroidogenesis as well as in the metabolism of steroids, oxysterols, bile acids, and retinoids in peripheral tissues, thereby controlling the local activation of their cognate receptors. Some of these SDRs are considered as promising therapeutic targets, for example to treat estrogen-/androgen-dependent and corticosteroid-related diseases, whereas others are considered as anti-targets as their inhibition may lead to disturbances of endocrine functions, thereby contributing to the development and progression of diseases. Nevertheless, the physiological functions of about half of all SDR members are still unknown. In this respect, i n silico tools are highly valuable in drug discovery for lead molecule identification, in toxicology screenings to facilitate the identification of hazardous chemicals, and in fundamental research for substrate identification and enzyme characterization. Regarding SDRs, computational methods have been employed for a variety of applications including drug discovery, enzyme characterization and substrate identification, as well as identification of potential endocrine disrupting chemicals (EDC). This review provides an overview of the efforts undertaken in the field of virtual screening supported identification of bioactive molecules in SDR research. In addition, it presents an outlook and addresses the opportunities and limitations of computational modeling and in vitro validation methods.
... 36,37 These agents do not inhibit COX isozymes, nor do they inhibit AKR1C1 and AKR1C2, which are required for the inactivation of 5α-DHT within the prostate. [38][39][40] Naproxen, (S)-2-(6-methoxynaphthalen-2-yl)propanoic acid is a NSAID that is used clinically to block cyclooxygenase (COX) mediated inflammation. It is also a potent AKR1C3 inhibitor that inhibits the AKR1C3 catalyzed reduction of the bioreductive drug PR-104 in multiple human cancer cells lines and a lung cancer xenograft model. ...
Type 5 17β-hydroxysteroid dehydrogenase, aldo-keto reductase 1C3 (AKR1C3) converts Δ4-androstene-3,17-dione and 5α-androstane-3,17-dione to testosterone (T) and 5α-dihydrotestosterone, respectively in castration resistant prostate cancer (CRPC). In CRPC, AKR1C3 is implicated in drug resistance, and enzalutamide drug resistance can be surmounted by indomethacin a potent inhibitor of AKR1C3. We examined a series of naproxen analogs and find that (R)-2-(6-methoxynaphthalen-2-yl)butanoic acid (in which the methyl group of R-naproxen was replaced by an ethyl group) acts a potent AKR1C3 inhibitor that displays selectivity for AKR1C3 over other AKR1C enzymes. This compound was devoid of inhibitory activity on COX isozymes and blocked AKR1C3 mediated production of T and induction of PSA in LNCaP-AKR1C3 cells as a model of a CRPC cell line. R-Profens are substrate selective COX-2 inhibitors and block the oxygenation of endocannabinoids, and in the context of advanced prostate cancer R-profens could inhibit intratumoral androgen synthesis and act as analgesics for metastatic disease.
... We also assayed 11KT and 11KDHT in LNCaP cells after 48 h to determine free and conjugated steroid levels in order to compare the conjugation of the C 19 and C11-oxy C 19 steroids (Fig. 8). As discussed in the aforementioned text, AST and 3aADIOL, the inactive derivatives of 5aDIONE and DHT, may either be conjugated or may undergo reactivation by RL-HSDs (17bHSD6) or 17bHSD10 [15,20,47]. Although AST is conjugated at C3 only and 3aADIOL at both C3 and C17, the former was detected in the conjugated form (0.97 mM) only, indicating that the steroid had not been converted to 5aDIONE (Supplemental Fig. 4c). ...
... AKR1C2 is the primary peripheral enzyme involved in the inactivation of 5a-DHT (K d = 10 À11 M for the AR) to 3a-diol (K d = 10 À6 M for the AR) and deprives the AR of its ligand [45]. This is supported by transient transfection studies in COS-1 and PC-3 cells [24], expression profiling in prostate stromal and epithelial cells [44]; and expression analysis in prostate biopsy material. ...
... Surgery or radiation are therefore 'standard of care' for organ-confined PCa, and the use of androgen deprivation therapies is generally applied only in advanced cases of PCa in an attempt to control androgen-sensitive metastatic cancer. Importantly, the development of CRPC is, in part, the result of increased intraprostatic androgen synthesis within the tumor tissue itself, including increased conversion of circulating DHEA(S) to androgens as well as de novo androgen synthesis Fung et al., 2006;Penning et al., 2007;Bauman et al., 2006;Chang et al., 2013). Studies of responses to castration, in both human tissues and mice bearing human tumor xenografts, demonstrate a distinct upregulation of steroid synthesis machinery with increased androgenic steroid profiles in prostate tumor tissues . ...
... In contrast to early reports [14,15], but in line with Hofland and co-workers [16], we found low expression levels of CYP11A1, CYP17A1 and HSD3B2 in both untreated and CRPC metastases. High CYP11A1, CYP17A1 and HSD3B2 mRNA levels were instead found in non-malignant prostate tissue, probably due to high synthesis of those enzymes in normal prostate stromal cells, and the same was true for SRD5A2 and HSD17B6 [27,28,29,30]. From this we would like to conclude that adrenal-derived androgens probably contribute to growth of CRPC bone metastases by their intrametastatic conversion into more potent androgens, while de novo synthesis of androgens from cholesterol within metastases is less likely, except maybe in individual cases with correlated expression of CYP11A1, CYP17A1, and HSD3B2. ...
Intra-tumoral steroidogenesis and constitutive androgen receptor (AR) activity have been associated with castration-resistant prostate cancer (CRPC). This study aimed to examine if CRPC bone metastases expressed higher levels of steroid-converting enzymes than untreated bone metastases. Steroidogenic enzyme levels were also analyzed in relation to expression of constitutively active AR variants (AR-Vs) and to clinical and pathological variables.
Untreated, hormone-naïve (HN, n = 9) and CRPC bone metastases samples (n = 45) were obtained from 54 patients at metastasis surgery. Non-malignant and malignant prostate samples were acquired from 13 prostatectomy specimens. Transcript and protein levels were analyzed by real-time RT-PCR, immunohistochemistry and immunoblotting. No differences in steroidogenic enzyme levels were detected between CRPC and HN bone metastases. Significantly higher levels of SRD5A1, AKR1C2, AKR1C3, and HSD17B10 mRNA were however found in bone metastases than in non-malignant and/or malignant prostate tissue, while the CYP11A1, CYP17A1, HSD3B2, SRD5A2, and HSD17B6 mRNA levels in metastases were significantly lower. A sub-group of metastases expressed very high levels of AKR1C3, which was not due to gene amplification as examined by copy number variation assay. No association was found between AKR1C3 expression and nuclear AR staining, tumor cell proliferation or patient outcome after metastases surgery. With only one exception, high AR-V protein levels were found in bone metastases with low AKR1C3 levels, while metastases with high AKR1C3 levels primarily contained low AR-V levels, indicating distinct mechanisms behind castration-resistance in individual bone metastases.
Induced capacity of converting adrenal-gland derived steroids into more potent androgens was indicated in a sub-group of PC bone metastases. This was not associated with CRPC but merely with the advanced stage of metastasis. Sub-groups of bone metastases could be identified according to their expression levels of AKR1C3 and AR-Vs, which might be of relevance for patient response to 2(nd) line androgen-deprivation therapy.
... Both steroids have little activity at the AR, however 3β-Diol has a moderate ability to bind and activate ERbeta (Kuiper et al., 1997; Weihua et al., 2001). 3β-Diol is synthesized from DHT by the actions of multiple enzymes including 3αHSD, 17αHSD, and 3βHSD, whereas 3β Diol is metabolized to 6α-and 7α-triol by the actions of CYP7b1 (Figure 1B; Jin and Penning, 2001; Gangloff et al., 2003; Steckelbroeck et al., 2004; Penning et al., 2007). In contrast, 3α-Diol is synthesized from DHT through the actions of 3αHSD. ...
Activation of the hypothalamo-pituitary-adrenal (HPA) axis is a basic reaction of animals to environmental perturbations that threaten homeostasis. These responses are ultimately regulated by neurons residing within the paraventricular nucleus of the hypothalamus (PVN). Within the PVN, corticotropin-releasing hormone (CRH), vasopressin (AVP) and oxytocin (OT) expressing neurons are critical as they can regulate both neuroendocrine and autonomic responses. Estradiol (E2) and testosterone (T) are well known reproductive hormones, however, they have also been shown to modulate stress reactivity. In rodent models, evidence shows that under some conditions E2 enhances stress activated ACTH and corticosterone secretion. In contrast, T decreases the gain of the HPA axis. The modulatory role of testosterone was originally thought to be via 5 alpha reduction to the potent androgen, dihydrotestosterone, whereas E2 effects were thought to be mediated by both estrogen receptors alpha (ERα) and beta (ERβ). However, DHT has been shown to be metabolized to the ERβ agonist, 5alpha- androstane 3beta,17beta diol (3b-Diol). The actions of 3β-Diol on the HPA axis are mediated by ERbeta which inhibits the PVN response to stressors. In gonadectomized rats, ERbeta agonists reduce CORT and ACTH responses to restraint stress, an effect that is also present in wild-type but not ERbeta knockout mice. The neurobiological mechanisms underlying the actions of ERbeta to alter HPA reactivity are not currently known. CRH, AVP and OT have all been shown to be regulated by estradiol and recent studies indicate an important role of ERbeta in these regulatory processes. Moreover, activation of the CRH and AVP promoters have been shown by 3β-Diol binding to ERbeta and this is thought to be through alternate pathways of gene regulation. Based on available data, a novel and important role for 3beta Diol in the regulation of the HPA axis is suggested.
... In a study comparing CRPC with primary tumors, changes in the relative expression of numerous transcripts involved in androgen biosynthesis were observed, including CYP17A1 (16.9-fold increase), HSD3B2 (7.5-fold increase), AKR1C3 (8.0-fold increase), SRD5A1 (2.6-fold increase), and SRD5A2 (9.4-fold decrease) (Montgomery, Mostaghel et al. 2008). Other studies of CRPC tumors have demonstrated similar findings suggestive of intracrine steroidogenesis, including the expression of HSD17B3 and AKR1C3 (Stanbrough, Bubley et al. 2006;Hofland, van Weerden et al. 2010), as well the expression of cholesterol biosynthetic enzymes including squalene epoxidase (SQLE), the rate limiting enzyme in cholesterol biosynthesis (Holzbeierlein, Lal et al. 2004), and the loss of enzymes mediating DHT catabolism such as AKR1C1 and AKR1C2 (Ji et al. 2007;Penning et al. 2007). These studies suggest that upregulated intratumoral steroidogenesis plays a role in facilitating CRPC survival in a castrate environment. ...
The transcriptional programs regulated through the activity of the androgen receptor (AR) modulate normal prostate development and the maintenance of prostatic functions at maturity. AR signaling also controls key survival and growth functions operative in prostate cancer. Inhibiting the AR program remains the key target in the treatment of advanced prostate cancer, and suppressing AR also holds great potential for preventing the development or progression of early stage prostate cancer. In this review, we detail molecular mechanisms of AR activity, cellular components contributing to the maintenance of AR signaling despite AR-ligand suppression, and discuss treatment strategies designed to target components of resistance to AR-directed therapeutics.
... The classical pathway for DHT synthesis is conversion in the testis of the major adrenal androgen androstenedione to T, followed by irreversible 5a-reduction of T to DHT by 5a-reductase type 2 in prostate and other, but not all, androgen target tissues ( Fig. 1; ref. 29). Studies in the beagle dog (30) and tammar wallaby (31,32) indicate an alternative backdoor pathway of DHT synthesis that uses androstanediol as precursor instead of T. Androstanediol is the major degradation product of DHT from the reductive 3a-hydroxysteroid dehydrogenase (HSD) activity of 3a-HSD aldo-keto reductases 1C (AKR1C; Fig. 1), enzymes with both 3-and 17-ketosteroid reductase activity (33)(34)(35)(36)(37)(38). AR binds androstanediol with moderate affinity, but androstanediol must be converted to DHT to induce transactivation by wild-type AR. ...
High-affinity binding of dihydrotestosterone (DHT) to the androgen receptor (AR) initiates androgen-dependent gene activation, required for normal male sex development in utero, and contributes to prostate cancer development and progression in men. Under normal physiologic conditions, DHT is synthesized predominantly by 5α-reduction of testosterone, the major circulating androgen produced by the testis. During androgen deprivation therapy, intratumoral androgen production is sufficient for AR activation and prostate cancer growth, even though circulating testicular androgen levels are low. Recent studies indicate that the metabolism of 5α-androstane-3α, 17β-diol by 17β-hydroxysteroid dehydrogenase 6 in benign prostate and prostate cancer cells is a major biosynthetic pathway for intratumoral synthesis of DHT, which binds AR and initiates transactivation to promote prostate cancer growth during androgen deprivation therapy. Drugs that target the so-called backdoor pathway of DHT synthesis provide an opportunity to enhance clinical response to luteinizing-hormone-releasing hormone (LHRH) agonists or antagonists, AR antagonists, and inhibitors of 5α-reductase enzymes (finasteride or dutasteride), and other steroid metabolism enzyme inhibitors (ketoconazole or the recently available abiraterone acetate).
... Alternatively, 5α-DHT can be formed by the "backdoor pathway" in which 3α-androstanediol is oxidized to 5α-DHT via RoDH-like 3α-HSD. In this pathway, Δ 4 -androstene-3,17-dione and testosterone are not precursors of 5α-DHT [84,85]. This pathway starts with the conversion of pregnenolone to progesterone catalyzed by 3β-HSD2 (HSD3B2), formation of 17α-hydroxyprogesterone catalyzed by 17αhydroxylase (CYP17A1), 5α-reduction to yield 5α-pregnane-17α-ol-3,20-dione (catalyzed by 5α-reductase isoforms), 3-ketone reduction to yield 5α-pregnane-3α,17α-diol-20-one (catalyzed by AKR1C2) followed by CYP-17,20-lyase (CYP17A1) to yield androsterone. ...
Prostate cancer remains a leading cause of cancer death, as there are no durable means to treat advanced disease. Treatment of non-organ-confined prostate cancer hinges on its androgen dependence. First-line therapeutic strategies suppress androgen receptor (AR) activity, via androgen ablation and direct AR antagonists, whereas initially effective, incurable, 'castration-resistant' tumors arise as a result of resurgent AR activity. Alterations of AR and/or associated regulatory networks are known to restore receptor activity and support resultant therapy-resistant tumor progression. However, recent evidence also reveals an unexpected contribution of the AR ligand, indicating that alterations in pathways controlling androgen synthesis support castration-resistant AR activity. In this report, the mechanisms underlying the lethal pairing of AR deregulation and aberrant androgen synthesis in prostate cancer progression will be discussed.
... The regulation of ligand occupancy of nuclear receptors is often governed by pairs of HSDs. For example regulation of the ER is governed by type 1 17β-HSD (which reduces E1 to E2) and by type 2/4 17β-HSD (which oxidizes E2 to E1) [44][45][46][47], while regulation of the AR is governed by AKR1C2 (which reduces 5α-DHT to 3α-diol) and by HSD17B6 (which oxidizes 3α-diol to DHT) [48,49]. Thus a component of measuring intra-tumoral levels of steroid hormones is to distinguish between the intracrine formation of ketosteroids from hydroxysteroids. ...
Advances in liquid chromatography-mass spectrometry (LC-MS) can be used to measure steroid hormone metabolites in vitro and in vivo. We find that LC-electrospray ionization (ESI)-MS using a LCQ ion trap mass spectrometer in the negative ion mode can be used to monitor the product profile that results from 5alpha-dihydrotestosterone (DHT)-17beta-glucuronide, DHT-17beta-sulfate, and tibolone-17beta-sulfate reduction catalyzed by human members of the aldo-keto reductase (AKR) 1C subfamily and assign kinetic constants to these reactions. We also developed a stable isotope dilution LC-electron capture atmospheric pressure chemical ionization (ECAPCI)-MS method for the quantitative analysis of estrone (E1) and its metabolites as pentafluorobenzyl (PFB) derivatives in human plasma in the attomole range. The limit of detection for E1-PFB was 740attomole on column. Separations can be performed using normal-phase LC because ionization takes place in the gas phase rather than in solution. This permits efficient separation of the regioisomeric 2- and 4-methoxy-E1. The method was validated for the simultaneous analysis of plasma E2 and its metabolites: 2-methoxy-E2, 4-methoxy-E2, 16alpha-hydroxy-E2, estrone (E1), 2-methoxy-E1, 4-methoxy-EI, and 16alpha-hydroxy-E1 from 5pg/mL to 2000pg/mL. Our LC-MS methods have sufficient sensitivity to detect steroid hormone levels in prostate and breast tumors and should aid their molecular diagnosis and treatment.
... Accurate measurement of these androgens in serum remains important to confirm the diagnosis of androgen deficiency states [2] and the therapeutic efficacy of DHT [3] or 5α reductase inhibitors [4]. Furthermore, there is growing interest in the primary DHT metabolites , 5α-androstane-3α,17ß-diol (3αDiol) and 5α-androstane- 3β,17β-diol (3βDiol), which have androgenic and estrogenic properties by back-conversion to DHT or binding to the estrogen receptor β, respectively567. Similarly, estradiol (E 2 ) and estrone (E 1 ) are the major estrogens involved in the development and maintenance of the female phenotype and pregnancy, as well as having important indirect roles in men via aromatization of T within tissues. ...
Immunoassays are widely used to quantify steroid hormones in biological samples. However, they lack specificity, especially at low levels. This study aimed to develop a sensitive LC-MS/MS method to measure serum androgens and estrogens without derivatization within a single run.
A stable-isotope dilution LC-MS/MS method was established using atmospheric pressure photoionization to quantify testosterone (T), dihydrotestosterone (DHT), estradiol (E2) and estrone (E1) from serum. Sample preparation involved liquid-liquid extraction (LLE) with hexane:ethyl acetate (3:2) containing deuterated internal standards. Accuracy was assessed by spiked recovery of serum pools, and imprecision by quality controls.
Using 200 microL serum, limits of quantification were 0.3 pg (1.5 pg/mL) E(1), 0.5 pg (2.5 pg/mL) E(2), 2 pg (10 pg/mL) T and 10 pg (50 pg/mL) DHT. Accuracy (93-110%) and precision (median 4%, all <15%) were determined to be well within acceptable limits for bioanalytical method validation. An analysis time of less than 10 min allowed up to 150 samples (600 analytes) to be processed per day.
The method is sufficiently sensitive and precise to accurately quantify serum T levels in females and E(2) in males, and is readily adapted to tissue and non-human samples.
... Dies zeigt, dass die Androgenaktivität beim alternden Mann, aufgrund von Änderungen im Androgen-1 Abb. 1. Androgenmetabolismus in der BPH. Sexualsteroidhormone werden in der Prostata durch Hydroxysteroid Dehydrogenasen (HSD), welche vorwiegend als Ketosteroid Reduktasen oder als Hydroxysteroid Oxidasen wirken, metabolisiert [116]. DHT ist das wichtigste pro-proliferativ wirkende und höchst-aktive Androgen der Prostata. ...
Benign prostatic hyperplasia (BPH) and benign prostatic enlargement (BPE) are among the most frequent medical disorders of elderly men and cause a number of annoying symptoms of the lower urinary tract (LUTS), leading to reduced quality of life and severe complications, including acute urinary retention. Nodular overgrowth of the epithelium and in particular the fibromuscular tissue is observed in the transition zone and periurethral areas. In particular, functional and phenotypic transdifferentiation of fibroblasts into myofibroblasts is a hallmark of the tissue remodeling in the benign hyperplastic prostate. BPH/BPE have a complex pathophysiology with a multitude of endocrine and local factors involved. Two risk factors, namely aging and circulating androgens, contribute significantly to risk of BPH/BPE. One of the primary initiating mechanisms appears to be a consequence of age-related changes in systemic sex steroid hormone levels accompanied by alterations in local androgen metabolism. This results in the disruption of the delicate balance of interacting growth factor signaling pathways and stromal/epithelial interactions generating a growth promoting and tissue remodeling microenvironment that leads to an increase in prostate volume. Secondarily, altered cytokine and chemoattractant production by the remodeled stroma promotes local inflammation that may further contribute to disease progression via lymphocyte-derived inflammatory cytokines and reactive oxygen/nitrogen species. Local hypoxia as a result of increased oxygen demands of proliferating cells may induce low levels of reactive oxygen species promoting neovascularization and fibroblast-to-myofibroblast transdifferentiation. Medical therapies for LUTS due to BPH/BPE have changed little over the past 15 years with mainstay treatments being alpha-adrenoreceptor blockade and 5alpha-reductase inhibitors. We provide an in depth view of the mechanisms underlying BPH/BPE and relate new research findings to the clinical picture with the prospect of novel therapeutic targets, including selective hormone antagonists/agonists, anti-stromal therapy, vitamin-D analogues and approaches to redress the redox imbalance.
Here, we reveal an unanticipated role of the blood-brain barrier (BBB) in regulating complex social behavior in ants. Using scRNA-seq, we find localization in the BBB of a key hormone-degrading enzyme called juvenile hormone esterase (Jhe), and we show that this localization governs the level of juvenile hormone (JH3) entering the brain. Manipulation of the Jhe level reprograms the brain transcriptome between ant castes. Although ant Jhe is retained and functions intracellularly within the BBB, we show that Drosophila Jhe is naturally extracellular. Heterologous expression of ant Jhe into the Drosophila BBB alters behavior in fly to mimic what is seen in ants. Most strikingly, manipulation of Jhe levels in ants reprograms complex behavior between worker castes. Our study thus uncovers a remarkable, potentially conserved role of the BBB serving as a molecular gatekeeper for a neurohormonal pathway that regulates social behavior.
Aldo-keto reductase 1C3 (AKR1C3) is overexpressed in castration-resistant prostate cancer where it acts to drive proliferation and aggressiveness by producing androgens. The reductive action of the enzyme leads to chemoresistance development against various clinical antineoplastics across a range of cancers. Herein, we report the continued optimization of selective AKR1C3 inhibitors and the identification of 5r, a potent AKR1C3 inhibitor (IC50 = 51 nM) with >1216-fold selectivity for AKR1C3 over closely related isoforms. Due to the cognizance of the poor pharmacokinetics associated with free carboxylic acids, a methyl ester prodrug strategy was pursued. The prodrug 4r was converted to free acid 5r in vitro in mouse plasma and in vivo. The in vivo pharmacokinetic evaluation revealed an increase in systemic exposure and increased the maximum 5r concentration compared to direct administration of the free acid. The prodrug 4r demonstrated a dose-dependent effect to reduce the tumor volume of 22Rv1 prostate cancer xenografts without observed toxicity.
The amide functional group plays a key role in the composition of biomolecules, including many clinically approved drugs. Bioisosterism is widely employed in the rational modification of lead compounds; being used to increase potency, enhance selectivity, improve pharmacokinetic properties, eliminate toxicity and to acquire novel chemical space to secure intellectual property. The introduction of a bioisostere leads to structural changes in molecular size, shape, electronic distribution, polarity, pKa, dipole or polarizability, which can be either favorable or detrimental to biological activity. This approach has opened up new avenues in drug design and development resulting in more efficient drug candidates introduced onto the market as well as in the clinical pipeline. Herein, we review the strategic decisions in selecting an amide bioisostere (the why), synthetic routes to each (the how) and success stories of each bioisostere (the implementation) to provide a comprehensive overview of this important toolbox for medicinal chemists.
Androgens are critical drivers of prostate cancer. In this chapter we first discuss the canonical pathways of androgen metabolism and their alterations in prostate cancer progression, including the classical, backdoor and 5α-dione pathways, the role of pre-receptor DHT metabolism, and recent findings on oncogenic splicing of steroidogenic enzymes. Next, we discuss the activity and metabolism of non-canonical 11-oxygenated androgens that can activate wild-type AR and are less susceptible to glucuronidation and inactivation than the canonical androgens, thereby serving as an under-recognized reservoir of active ligands. We then discuss an emerging literature on the potential non-canonical role of androgen metabolizing enzymes in driving prostate cancer. We conclude by discussing the potential implications of these findings for prostate cancer progression, particularly in context of new agents such as abiraterone and enzalutamide, which target the AR-axis for prostate cancer therapy, including mechanisms of response and resistance and implications of these findings for future therapy.
Aldo–keto reductase 1C3 (AKR1C3) catalyzes the synthesis of 9α,11β-prostaglandin (PG) F2α and PGF2α prostanoids that sustain the growth of myeloid precursors in the bone marrow. The enzyme is overexpressed in acute myeloid leukemia (AML) and T-cell acute lymphoblastic leukemia (T-ALL). Moreover, AKR1C3 confers chemotherapeutic resistance to the anthracyclines; first-line agents for the treatment of leukemias. The highly homologous isoforms AKR1C1 and AKR1C2 inactive 5-dihydroprogesterone and their inhibition would be undesirable. We report herein, the identification of AKR1C3 inhibitors that demonstrate exquisite isoform selectivity for AKR1C3 over the other closely related isoforms to the order of >2800-fold. Biological evaluation of our isoform selective inhibitors revealed a high degree of synergistic drug action in combination with the clinical leukemia therapeutics daunorubicin and cytarabine in in vitro cellular models of AML and primary patient-derived T-ALL cells. Our developed compounds exhibited >100-fold dose reduction index that results in complete resensitization of a daunorubicin-resistant AML cell line to the chemotherapeutic and >100-fold dose reduction of cytarabine in both AML cell lines and primary T-ALL cells.
Aldo-keto reductases (AKRs) are monomeric NAD(P)(H) dependent oxidoreductases that play pivotal roles in the biosynthesis and metabolism of steroids in humans. AKR1C enzymes acting as 3-keto-, 17-keto- and 20-keto-steroid reductases are involved in the pre-receptor regulation of ligands for the androgen, estrogen and progesterone receptor and are considered drug targets to treat steroid hormone dependent malignancies and endocrine disorders. By contrast AKR1D1 is the only known steroid 5β-reductase and is essential for bile-acid biosynthesis, the generation of ligands for the farnesoid-X-receptor, and the 5β-dihydrosteroids that have their own biological activity. This article will review the crystal structures of these AKRs, their kinetic and catalytic mechanisms, AKR genomics (gene expression, splice variants, polymorphic variants, and inherited genetic deficiencies), distribution in steroid target tissues, roles in steroid hormone action and disease, and inhibitor design.
Introduction: AKR1C3 is a drug target in hormonal and hormonal independent malignancies and acts as a major peripheral 17β -hydroxysteroid dehydrogenase to yield the potent androgens testosterone and dihydrotestosterone, and as a prostaglandin (PG) F synthase to produce proliferative ligands for the PG FP receptor. AKR1C3 inhibitors may have distinct advantages over existing therapeutics for the treatment of castration resistant prostate cancer, breast cancer and acute myeloid leukemia.
Area Covered: This article reviews the patent literature on AKR1C3 inhibitors using SciFinder which identified inhibitors in the following chemical classes: N-phenylsulfonyl-indoles, N-(benzimidazoylylcarbonyl)- N-(indoylylcarbonyl)- and N-(pyridinepyrrolyl)- piperidines, N-benzimidazoles and N-benzindoles, repurposed nonsteroidal antiinflammatory drugs (indole acetic acids, N-phenylanthranilates and aryl propionic acids), isoquinolines, and nitrogen and sulfur substituted estrenes. The article evaluates inhibitor AKR potency, specificity, efficacy in cell-based and xenograft models and clinical utility. The advantage of bifunctional compounds that competitively inhibit AKR1C3 and block its androgen receptor (AR) coactivator function or act as AKR1C3 inhibitors and direct acting AR antagonists are discussed.
Expert Opinion: A large number of potent and selective inhibitors of AKR1C3 have been described however, preclinical optimization, is required before their benefit in human disease can be assessed.
The maintenance of steroid homeostasis in the prostate is critical, with perturbation of steroidogenesis contributing to the modulation of active ligands in the androgen pool. In this scenario, enzymes catalysing the biosynthesis, inactivation and conjugation of steroids are the key players, regulating active ligand levels and in so doing, the activation of the androgen receptor (AR). The glucuronidation of potent ligands renders them unable to bind the AR, allowing the secretion of conjugated steroids. Uridine diphosphate glucuronosyltransferase 2B type 28 (UGT2B28), one of the UGT enzymes catalyzing the glucuronidation of androgens, has recently been given a prominent role in the regulation of prostate steroidogenesis—one which stands in contrast to the accepted dogma that lower androgen levels resulting from increased conjugation are associated with decreased prostate cancer (PCa) risk and disease progression. Increased DHT and its precursors, T and androstenediol, were reported to be associated with increased UGT2B28 tumor expression levels, linked to lower PSA levels but higher Gleason scores and increased PCa risk. In addition, the complete deletion of UGT2B28, was associated with decreased T, DHT and glucuronide derivatives when compared to patients carrying both alleles. UGT2B28 is encoded by a single gene giving rise to UGT2B28 type I which catalyses androgen glucuronidation and, due to alternative splicing, also produces two distinct transcripts, UGT2B28 type II and III. Type II with its premature stop codon, is devoid of the cofactor binding domain while type III is devoid of the substrate binding domain, both catalytically inactive, truncated proteins. Increased UGT2B28 mRNA expression was reported in primary tumours, and while variable nuclear and strong cytoplasmic staining were distinctive of tumour cells, the expression levels and compartmentalization of the specific protein isoforms remain unknown. While increased expression of type I would contribute towards lowering androgen levels, increased expression of types II and III would not. The abundance of type III transcripts in multiple tissues may provide insight into a regulatory role with truncated isoforms possibly affecting androgen levels by regulating substrate and/or co-factor availability, dimerization or the formation of protein complexes with other UGTs, while protein-protein interaction may also impact cascade signaling pathways in PCa development and disease progression.
The accumulation of high concentrations of signalling androgens within prostate tumours that progress despite use of androgen-deprivation therapy is a clinically important mechanism of the development of castration-resistant prostate cancer. In the past 5 years, data from a number of studies have increased our understanding of the enzymes and substrates involved in intratumoural androgen biosynthesis, and have implicated three competing pathways, which are likely to account for these observations. These pathways ('canonical', 'backdoor' and '5α-dione'), which can all ultimately generate the potent signalling androgen, dihydrotestosterone, involve many of the same enzymes, but differ in terms of substrate preference, reaction sequence and the organs and tissues in which they occur. For this reason, the relative importance of each pathway to the development and progression of prostate cancer remains controversial. In this Review, we describe the current understanding of androgen synthesis and the evidence for its role in castration resistance, and examine the evidence supporting and or rebutting the relevance of each pathway to patients with prostate cancer.
Human 3-alpha hydroxysteroid dehydrogenase type 3 (3α-HSD3) plays an essential role in the inactivation of the most potent androgen 5α-DHT. Our study attempts to obtain the important structure of 3α-HSD3 in complex with 5α-DHT and to investigate the role of 3α-HSD3 in breast cancer cells. We report the crystal structure of human 3α-HSD3•NADP+•A-dione/epi-ADT complex, which was obtained by co-crystallization with 5α-DHT in the presence of NADP+. Although 5α-DHT was introduced during the crystallization, oxidoreduction of 5α-DHT occurred. The locations of A-dione and epi-ADT were identified in the steroid binding sites of two 3α-HSD3 molecules per crystal asymmetric unit. An overlay showed that A-dione and epi-ADT were oriented upside down and flipped relative to each other, providing structural clues for 5α-DHT reverse binding in the enzyme with the generation of different products. Moreover, we report the crystal structure of the 3α-HSD3•NADP+•4-dione complex. When a specific siRNA (100 nM) was used to suppress 3α-HSD3 expression without interfering with 3α-HSD4, which shares a highly homologous active site, 5α-DHT concentration increased, whereas MCF7 cell growth was suppressed. Our study provides structural clues for 5α-DHT reverse binding within 3α-HSD3, and first demonstrates that downregulation of 3α-HSD3 decreases MCF7 breast cancer cell growth.
Androgen deprivation is still the standard systemic therapy for locally advanced or metastatic prostate cancer (PCa), but
patients invariably relapse with a more aggressive form of PCa that has been termed castration-recurrent PCa (CRPCa). The
androgen receptor (AR) is expressed at high levels in most cases of CRPCa, and these tumors resume their expression of multiple
AR regulated genes, which indicates that AR transcriptional activity becomes reactivated at this stage of the disease. Mechanisms
that may contribute to AR reactivation in CRPCa include increased AR protein expression, AR mutations, increased expression
of transcriptional coactivator proteins, and activation of signal transduction pathways that can enhance AR responses to low
levels of androgens. Recent data indicate that a further mechanism for AR reactivation in CRPCa cells may be through increased
intracellular synthesis of testosterone and 5α-dihydrotestosterone (DHT). The enzymes that mediate androgen synthesis and
metabolism in normal prostate and in PCa, and evidence indicating that their increased expression contributes to the development
of CRPCa, are outlined in this chapter. The early use of therapies that more aggressively block androgen production may enhance
responses to androgen deprivation therapy, and prevent or delay the adaptations that eventually lead to CRPCa.
Die Benigne Prostata Hyperplasie/Vergrößerung (BPH/BPE) ist der häufigste gutartige Tumor des alternden Mannes. Dieser gilt
als eine Ursache einer Reihe von Symptomen des unteren Urogenitaltraktes (LUTS, Lower Urinary Tract Symptoms). Die BPH/BPE
führt zu Einschränkungen der Lebensqualität sowie zu medizinischen Komplikationen bis hin zur akuten Harnverhaltung. Noduläres
Wachstum, vorwiegend des fibromuskulären Gewebes der Transitionszone und der periurethralen Anteile, sowie funktionelle Änderungen
der Zellen sind charakteristisch für die beobachtete Gewebsreorganisation in der BPH/BPE. Die Pathophysiologie der BPH/BPE
ist komplex und es ist hierbei eine Anzahl endokriner und lokaler Faktoren involviert. Zwei Risikofaktoren sind essentiell
für die Entwicklung der BPH/BPE, Altern und Androgene. Einen bedeutenden Einfluss auf die Entstehung scheinen die ab dem 35.
Lebensjahr einsetzenden Veränderungen der Sexualsteroidhormon-Serumspiegel und -ratios zu haben. Diese laufen parallel zu
Veränderungen im lokalen Sexualsteroidhormon-Stoffwechsel und zu Störungen in Wachstumsfaktor Signaltransduktionswegen in
Stroma und Epithel. Das veränderte Stroma fördert über eine veränderte Zytokinproduktion lokale inflammatorische Prozesse,
die das Fortschreiten der Erkrankung über inflammatorische Zytokine lymphozytären Ursprungs begünstigen. Lokale Hypoxie, ausgelöst
durch zelluläre Proliferation, führt zur Produktion von Sauerstoffradikalen, welche eine Neovaskularisierung und die charaktreristische
Transdifferenzierung von Fibroblasten zu glatten Muskelzellen bzw. Myofibroblasten auslösen. Die medikamentösen Standardtherapien
von LUTS auf Grund von BPH/BPE haben sich seit 15 Jahren wenig geändert. Es sind dies die Blockade α-adrenerger Rezeptoren
und die Hemmung des DHT-konvertierenden Enzyms 5α-Reduktase. In unserer Arbeit fassen wir die vermutlichen molekularen Mechanismen
zur Entstehung der BPH/BPE zusammen und zeigen neue Wege zur therapeutischen Intervention auf. Zukünftige Ansätze beinhalten
möglicherweise selektive Hormon Antagonisten/Agonisten, anti-stromal Therapie, Vitamin-D Analoga und Substanzen zur Wiederherstellung
des Redox Gleichgewichts.
Benign prostatic hyperplasia (BPH) and benign prostatic enlargement (BPE) are among the most frequent medical disorders of
elderly men and cause a number of annoying symptoms of the lower urinary tract (LUTS), leading to reduced quality of life
and severe complications, including acute urinary retention. Nodular overgrowth of the epithelium and in particular the fibromuscular
tissue is observed in the transition zone and periurethral areas. In particular, functional and phenotypic transdifferentiation
of fibroblasts into myofibroblasts is a hallmark of the tissue remodeling in the benign hyperplastic prostate. BPH/BPE have
a complex pathophysiology with a multitude of endocrine and local factors involved. Two risk factors, namely aging and circulating
androgens, contribute significantly to risk of BPH/BPE. One of the primary initiating mechanisms appears to be a consequence
of age-related changes in systemic sex steroid hormone levels accompanied by alterations in local androgen metabolism. This
results in the disruption of the delicate balance of interacting growth factor signaling pathways and stromal/epithelial interactions
generating a growth promoting and tissue remodeling microenvironment that leads to an increase in prostate volume. Secondarily,
altered cytokine and chemoattractant production by the remodeled stroma promotes local inflammation that may further contribute
to disease progression via lymphocyte-derived inflammatory cytokines and reactive oxygen/nitrogen species. Local hypoxia as
a result of increased oxygen demands of proliferating cells may induce low levels of reactive oxygen species promoting neovascularization
and fibroblast-to-myofibroblast transdifferentiation. Medical therapies for LUTS due to BPH/BPE have changed little over the
past 15 years with mainstay treatments being α-adrenoreceptor blockade and 5α-reductase inhibitors. We provide an in depth
view of the mechanisms underlying BPH/BPE and relate new research findings to the clinical picture with the prospect of novel
therapeutic targets, including selective hormone antagonists/agonists, anti-stromal therapy, vitamin-D analogues and approaches
to redress the redox imbalance.
Oestradiol exerts a profound influence upon multiple brain circuits. For the most part, these effects are mediated by oestrogen receptor (ER)α. We review here the roles of ERβ, the other ER isoform, in mediating rodent oestradiol-regulated anxiety, aggressive and sexual behaviours, the control of gonadotrophin secretion, and adult neurogenesis. Evidence exists for: (i) ERβ located in the paraventricular nucleus underpinning the suppressive influence of oestradiol on the stress axis and anxiety-like behaviour; (ii) ERβ expressed in gonadotrophin-releasing hormone neurones contributing to oestrogen negative-feedback control of gonadotrophin secretion; (iii) ERβ controlling the offset of lordosis behaviour; (iv) ERβ suppressing aggressive behaviour in males; (v) ERβ modulating responses to social stimuli; and (vi) ERβ in controlling adult neurogenesis. This review highlights two major themes; first, ERβ and ERα are usually tightly inter-related in the oestradiol-dependent control of a particular brain function. For example, even though oestradiol feedback to control reproduction occurs principally through ERα-dependent mechanisms, modulatory roles for ERβ also exist. Second, the roles of ERα and ERβ within a particular neural network may be synergistic or antagonistic. Examples of the latter include the role of ERα to enhance, and ERβ to suppress, anxiety-like and aggressive behaviours. Splice variants such as ERβ2, acting as dominant negative receptors, are of further particular interest because their expression levels may reflect preceeding oestradiol exposure of relevance to oestradiol replacement therapy. Together, this review highlights the predominant modulatory, but nonetheless important, roles of ERβ in mediating the many effects of oestradiol upon adult brain function.
The androgen receptor (AR) mediates the growth of benign and malignant prostate in response to dihydrotestosterone (DHT). In patients undergoing androgen deprivation therapy for prostate cancer, AR drives prostate cancer growth despite low circulating levels of testicular androgen and normal levels of adrenal androgen. In this report, we demonstrate the extent of AR transactivation in the presence of 5α-androstane-3α,17β-diol (androstanediol) in prostate-derived cell lines parallels the bioconversion of androstanediol to DHT. AR transactivation in the presence of androstanediol in prostate cancer cell lines correlated mainly with mRNA and protein levels of 17β-hydroxysteroid dehydrogenase 6 (17β-HSD6), one of several enzymes required for the interconversion of androstanediol to DHT and the inactive metabolite androsterone. Levels of retinol dehydrogenase 5, and dehydrogenase/reductase short-chain dehydrogenase/reductase family member 9, which also convert androstanediol to DHT, were lower than 17β-HSD6 in prostate-derived cell lines and higher in the castration-recurrent human prostate cancer xenograft. Measurements of tissue androstanediol using mass spectrometry demonstrated androstanediol metabolism to DHT and androsterone. Administration of androstanediol dipropionate to castration-recurrent CWR22R tumor-bearing athymic castrated male mice produced a 28-fold increase in intratumoral DHT levels. AR transactivation in prostate cancer cells in the presence of androstanediol resulted from the cell-specific conversion of androstanediol to DHT, and androstanediol increased LAPC-4 cell growth. The ability to convert androstanediol to DHT provides a mechanism for optimal utilization of androgen precursors and catabolites for DHT synthesis.
Prolonged exposure to estrogens is a significant risk factor for the development of breast cancer. Estrogens exert carcinogenic effects by stimulating cell proliferation or through oxidative metabolism that forms DNA-damaging species. In the present study, we aimed to provide a better understanding of estrogen metabolism and actions in breast cancer, and to characterize model breast cancer cell lines. We determined the expression profiles of the genes for the estrogen and progesterone receptors, and for 18 estrogen-metabolizing enzymes in eight cell lines: MCF-7, MCF-10A, T47D, SKBR3, MDA-MB-231, MDA-MB-361, Hs-578T and Hs-578Bst cells. Similar gene expression profiles of these receptors and enzymes for the formation of estradiol via the aromatase and sulfatase pathways were observed in the MCF-7 and T47D metastatic cell lines. The MDA-MB-361 cells expressed ESR1, ESR2 and PGR as well, but differed in expression of the estrogen-metabolizing enzymes. In the MDA-MB-231 and SKBR3 cells, all of these estrogen-forming enzymes were expressed, although the lack of ESR1 and the low levels of ESR2 expression suggested that the estrogens can only act via non-ER mediated pathways. In the non-tumorigenic MCF-10A cell line, the key enzymes of the aromatase pathway were not expressed, and the sulfatase pathway also had a marginal role. The comparison between gene expression profiles of the non-tumorigenic Hs-578Bst cells and the cancerous Hs-578T cells revealed that they can both form estrogens via the sulfatase pathway, while the aromatase pathway is less important in the Hs-578Bst cells. The Hs-578T cells showed low levels of ESR1, ESR2 and PGR expression, while only ESR1 and ESR2 expression was detected in the Hs-578Bst cells. Our data show that the cell lines examined provide the full range of model systems and should further be compared with the expression profiles of breast cancer specimens.
Sex hormones are increasingly recognized as regulators of lung development. Respiratory distress syndrome (RDS) is the leading cause of morbidity in preterm neonates and occurs with a higher incidence in males. The mechanisms underlying the effects of androgens on lung development and the occurrence of RDS are only partially deciphered, and positive roles of estrogens on surfactant production and alveologenesis are relevant to our understanding of pulmonary diseases. This manuscript reviews current knowledge on androgen and estrogen metabolism and on relevant hormone targets in the fetal lung. Further investigations are needed to elucidate mechanisms orchestrating sex hormone effects on lung development. These studies aim to decrease mortality and morbidity associated with RDS and other pathologies related to lung immaturity at birth.
To summarize recent advances in androgen biosynthesis and metabolism in peripheral tissues (e.g., liver and prostate) and how these can be exploited therapeutically.
Human liver catalyzes the reduction of circulating testosterone to yield four stereoisomeric tetrahydrosteroids. Recent advances have assigned the enzymes responsible for these reactions and elucidated their structural biology. Data also suggest that for 5alpha-dihydrotestosterone (5alpha-DHT), conjugation reactions (phase II) may precede ketosteroid reduction (phase I) reactions. Human prostate is the site of benign prostatic hyperplasia and prostate cancer, which occur in the aging male. Although the importance of local androgen biosynthesis in these diseases is accepted, recent advances have identified enzymes that regulate ligand access to the androgen-receptor; a 'backdoor pathway' to 5alpha-DHT that does not require testosterone acting as an intermediate; and the finding that castrate-resistant prostate cancer (CRPC) has undergone an adaptive response to androgen deprivation, which involves intratumoral testosterone and 5alpha-DHT biosynthesis that can be targeted using inhibitors of (CYP17-hydroxylase/17,20-lyase), aldo-keto reductase 1C3, and 5alpha-reductase type 1 and type 2.
Enzyme isoforms responsible for the biosynthesis and metabolism of androgens in liver and prostate have been identified and those responsible for the biosynthesis of androgens in CRPC can be therapeutically targeted.
Aldo-keto reductase (AKR) 1C2 is a human, cytosolic enzyme that has an important role in the deactivation of the potent androgen dihydrotestosterone (DHT). AKR1C2 can regulate the extent and duration of activation of the androgen receptor by catalyzing the reduction of DHT to the less potent receptor ligand 3alpha-diol. In this study, we functionally characterize in vitro the effect of 11 naturally occurring nonsynonymous single nucleotide polymorphisms on the ability of AKR1C2 to reduce DHT to 3alpha-diol. The wild-type and variant enzymes were expressed using a transfected insect cell system, and their kinetic activities were measured using both a specific fluorogenic probe and DHT as substrates. This functional characterization demonstrates that several variant AKR1C2 proteins have significantly reduced or altered reductase activities as shown by their measured kinetic parameters. Data from our two separate in vitro studies revealed significant reductions in V(max) for two variants (F46Y and L172Q) and significantly lower apparent K(m) values for three variants (L172Q, K185E, and R258C) compared with the wild type. These results provide evidence that several naturally occurring nonsynonymous single nucleotide polymorphisms in AKR1C2 result in reduced enzyme activities. These variant AKR1C2 alleles may represent one factor involved in the variable degradation of DHT in vivo.
Hydroxysteroid dehydrogenases (HSDs) are essential for the biosynthesis and mechanism of action of all steroid hormones. We report the complete kinetic mechanism of a mammalian HSD using rat 3alpha-HSD of the aldo-keto reductase superfamily (AKR1C9) with the substrate pairs androstane-3,17-dione and NADPH (reduction) and androsterone and NADP(+) (oxidation). Steady-state, transient state kinetics, and kinetic isotope effects reconciled the ordered bi-bi mechanism, which contained 9 enzyme forms and permitted the estimation of 16 kinetic constants. In both reactions, loose association of the NADP(H) was followed by two conformational changes, which increased cofactor affinity by >86-fold. For androstane-3,17-dione reduction, the release of NADP(+) controlled k(cat), whereas the chemical event also contributed to this term. k(cat) was insensitive to [(2)H]NADPH, whereas (D)k(cat)/K(m) and the (D)k(lim) (ratio of the maximum rates of single turnover) were 1.06 and 2.06, respectively. Under multiple turnover conditions partial burst kinetics were observed. For androsterone oxidation, the rate of NADPH release dominated k(cat), whereas the rates of the chemical event and the release of androstane-3,17-dione were 50-fold greater. Under multiple turnover conditions full burst kinetics were observed. Although the internal equilibrium constant favored oxidation, the overall K(eq) favored reduction. The kinetic Haldane and free energy diagram confirmed that K(eq) was governed by ligand binding terms that favored the reduction reactants. Thus, HSDs in the aldo-keto reductase superfamily thermodynamically favor ketosteroid reduction.
Androgen and androgen receptor (AR) are involved in growth of normal prostate and development of prostatic diseases including prostate cancer. Androgen deprivation therapy is used for treating advanced prostate cancer. This therapeutic approach focuses on suppressing the accumulation of potent androgens, testosterone and 5alpha-dihydrotestosterone (5alpha-DHT), or inactivating the AR. Unfortunately, the majority of patients with prostate cancer eventually advance to androgen-independent states and no longer respond to the therapy. In addition to the potent androgens, 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), reduced from 5alpha-DHT through 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs), activated signaling may represent a novel pathway responsible for the progression to androgen-independent prostate cancer. Androgen sensitive human prostate cancer LNCaP cells were used to compare 5alpha-DHT and 3alpha-diol activated androgenic effects. In contrast to 5alpha-DHT, 3alpha-diol regulated unique patterns of beta-catenin and Akt expression as well as Akt phosphorylation in parental and in AR-silenced LNCaP cells. More significantly, 3alpha-diol, but not 5alpha-DHT, supported AR-silenced LNCaP cells and AR negative prostate cancer PC-3 cell proliferation. 3alpha-diol-activated androgenic effects in prostate cells cannot be attributed to the accumulation of 5alpha-DHT, since 5alpha-DHT formation was not detected following 3alpha-diol administration. Potential accumulation of 3alpha-diol, as a result of elevated 3alpha-HSD expression in cancerous prostate, may continue to support prostate cancer growth in the presence of androgen deprivation. Future therapeutic strategies for treating advanced prostate cancer might need to target reductive 3alpha-HSD to block intraprostatic 3alpha-diol accumulation.
Residual tissue androgens are consistently detected within the prostate tumors of castrate individuals and are thought to play a critical role in facilitating the androgen receptor-mediated signaling pathways leading to disease progression. The source of residual tumor androgens is attributed in part to the uptake and conversion of circulating adrenal androgens. Whether the de novo biosynthesis of androgens from cholesterol or earlier precursors occurs within prostatic tumors is not known, but it has significant implications for treatment strategies targeting sources of androgens exogenous to the prostate versus 'intracrine' sources within the prostatic tumor. Moreover, increased expression of androgen-metabolizing genes within castration-resistant metastases suggests that up-regulated activity of endogenous steroidogenic pathways may contribute to the outgrowth of 'castration-adapted' tumors. These observations suggest that a multi-targeted treatment approach designed to simultaneously ablate testicular, adrenal and intracrine contributions to the tumor androgen signaling axis will be required to achieve optimal therapeutic efficacy.
The kinetic parameters, steroid substrate specificity and identities of reaction products were determined for four homogeneous recombinant human 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) isoforms of the aldo-keto reductase (AKR) superfamily. The enzymes correspond to type 1 3 alpha-HSD (AKRIC4), type 2 3 alpha(17 beta)-HSD (AKR1C3), type 3 3 alpha-HSD (AKR1C2) and 20 alpha(3 alpha)-HSD (AKR1C1), and share at least 84% amino acid sequence identity. All enzymes acted as NAD(P)(H)-dependent 3-, 17- and 20-ketosteroid reductases and as 3 alpha-, 17 beta- and 20 alpha-hydroxysteroid oxidases. The functional plasticity of these isoforms highlights their ability to modulate the levels of active androgens, oestrogens and progestins. Salient features were that AKR1C4 was the most catalytically efficient, with k(cat)/K-m values for substrates that exceeded those obtained with other isoforms by 10-30-fold. In the reduction direction, all isoforms inactivated 5 alpha-dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one; 5 alpha-DHT) to yield 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-androstanediol). However, only AKR1C3 reduced Delta(4)-androstene-3,17-dione to produce significant amounts of testosterone. All isoforms reduced oestrone to 17 beta-oestradiol, and progesterone to 20 alpha-hydroxy-pregn-4-ene-3,20-dione (20 alpha-hydroxyprogesterone). In the oxidation direction, only AKR1C2 converted 3a-androstanediol to the active hormone 5 alpha-DHT. AKR1C3 and AKR1C4 oxidized testosterone to Delta(4)-androstene-3,17-dione. All isoforms oxid ized 17 beta-oestradiol to oestrone, and 20 alpha-hydroxyprogesterone to progesterone. Discrete tissue distribution of these AKR1C enzymes was observed using isoform-specific reverse transcriptase-PCR. AKR1C4 was virtually liver-specific and its high k(cat)/K-m allows this enzyme to form 5 alpha/5 beta-tetrahydrosteroids robustly. AKR1C3 was most prominent in the prostate and mammary glands. The ability of AKR1C3 to interconvert testosterone with Delta(4)-androstene-3,17-dione, but to inactivate 5 alpha-DHT, is consistent with this enzyme eliminating active androgens from the prostate. In the mammary gland, AKR1C3 will convert Delta(4)-androstene-3,17-dione to testosterone (a substrate aromatizable to 17 beta-oestradiol), oestrone to 17 beta-oestradiol, and progesterone to 20 alpha-hydroxyprogesterone, and this concerted reductive activity may yield a. pro-oesterogenic state. AKR1C3 is also the dominant form in the uterus and is responsible for the synthesis of 3 alpha-androstanediol which has been implicated as a parturition hormone. The major isoforms in the brain, capable of synthesizing anxiolytic steroids, are AKR1C1 sand AKR1C2. These studies are in stark contrast with those in rat where only a single AKR with positional- and stereospecificity for 3 alpha-hydroxysteroids exists.
Spontaneous prostatic hyperplasia in the beagle appears to progress with age from a glandular to a cystic histological appearance. Prostatic hyperplasia can be induced in young beagles with intact testes by treatment for 4 mo with either dihydrotestosterone or 5 alpha-androstane-3 alpha, 17 beta-diol, alone, or with either of these steroids in combination with 17 beta-estradiol. In contrast, the induction of prostatic hyperplasia in young castrated beagles, in which the gland had been allowed to involute for 1 mo, requires the administration of both 17 beta-estradiol and either 5 alpha-androstane-3 alpha, 17 beta-diol or dihydrotestosterone. Testosterone and 17 beta-estradiol, either singly or in combination, did not produce the hyperplastic condition in intact or castrated beagles. The experimentally induced prostatic hyperplasia is identical in pathology to the glandular hyperplasia that occurs naturally in the aging dog with intact testes. However, cystic hyperplasia was not produced by any of the treatments tested in young animals.
Rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) (EC 1.1.1.50) is an NAD(P)(+)-dependent oxidoreductase that is potently inhibited at its active site by non-steroidal anti-inflammatory drugs (NSAIDs). Initial-velocity and product-inhibition studies performed in either direction at pH 7.0 are consistent with a sequential ordered Bi Bi mechanism in which pyridine nucleotide binds first and leaves last. This mechanism is supported by fluorescence titrations of the E-NADH complex, and by the failure to detect the binding of either [3H]androsterone or [3H]androstanedione to free enzyme by equilibrium dialysis. Dead-end inhibition studies with NSAIDs also support this mechanism. Initial-velocity studies with indomethacin show that this drug is an uncompetitive inhibitor against NAD+, but a potent competitive inhibitor against androsterone, indicating the ordered formation of an E.NAD+.indomethacin complex. Calculation of the individual rate constants reveals that the binding and release of pyridine nucleotide is rate-limiting and that isomerization of the central complex is favoured in the forward direction. Equilibrium dialysis experiments with [14C]indomethacin reveal the presence of two abortive NSAID complexes, a high-affinity ternary complex corresponding to E.NAD+.indomethacin (Kd = 1-2 microM for indomethacin) and a low-affinity binary complex corresponding to E.indomethacin (Kd = 22 microM for indomethacin). Since indomethacin has a low affinity for free enzyme, the formation of this abortive binary complex does not complicate kinetic measurements which are made in the presence of NAD+, but may contribute to the inhibition of the enzyme by NSAIDs. Using either pro-R-[4-3H]NADH or pro-S-[4-3H]NADH as cofactor, radiolabelled androsterone was formed only when the pro-R-[4-3H]NADH was used, confirming that purified 3 alpha-HSD is a Class A dehydrogenase.
Intracellular levels of active steroid hormones are determined by their relative rates of synthesis and breakdown. In the case of the potent androgen dihydrotestosterone, synthesis from the precursor testosterone is mediated by steroid 5alpha-reductase, whereas breakdown to the inactive androgens 5alpha-androstane-3alpha, 17beta-diol (3alpha-adiol), and androsterone is mediated by reductive 3alpha-hydroxysteroid dehydrogenases (3alpha-HSD) and oxidative 17beta-hydroxysteroid dehydrogenases (17beta-HSD), respectively. We report the isolation by expression cloning of a cDNA encoding a 17beta-HSD6 isozyme that oxidizes 3alpha-adiol to androsterone. 17beta-HSD6 is a member of the short chain dehydrogenase/reductase family and shares 65% sequence identity with retinol dehydrogenase 1 (RoDH1), which catalyzes the oxidation of retinol to retinal. Expression of rat and human RoDH cDNAs in mammalian cells is associated with the oxidative conversion of 3alpha-adiol to dihydrotestosterone. Thus, 17beta-HSD6 and RoDH play opposing roles in androgen action; 17beta-HSD6 inactivates 3alpha-adiol by conversion to androsterone and RoDH activates 3alpha-adiol by conversion to dihydrotestosterone. The synthesis of an active steroid hormone by back conversion of an inactive metabolite represents a potentially important mechanism by which the steady state level of a transcriptional effector can be regulated.
We report the cDNA sequence and catalytic properties of a new member of the short chain dehydrogenase/reductase superfamily. The 1134-base pair cDNA isolated from the human liver cDNA library encodes a 317-amino acid protein, retinol dehydrogenase 4 (RoDH-4), which exhibits the strongest similarity with rat all-trans-retinol dehydrogenases RoDH-1, RoDH-2, and RoDH-3, and mouse cis-retinol/androgen dehydrogenase (</=73% identity). The mRNA for RoDH-4 is abundant in adult liver, where it is translated into RoDH-4 protein, which is associated with microsomal membranes, as evidenced by Western blot analysis. Significant amounts of RoDH-4 message are detected in fetal liver and lung. Recombinant RoDH-4, expressed in microsomes of Sf9 insect cells using BacoluGold Baculovirus system, oxidizes all-trans-retinol and 13-cis-retinol to corresponding aldehydes and oxidizes the 3alpha-hydroxysteroids androstane-diol and androsterone to dihydrotestosterone and androstanedione, respectively. NAD+ and NADH are the preferred cofactors, with apparent Km values 250-1500 times lower than those for NADP+ and NADPH. All-trans-retinol and 13-cis-retinol inhibit RoDH-4 catalyzed oxidation of androsterone with apparent Ki values of 5.8 and 3.5 microM, respectively. All-trans-retinol bound to cellular retinol-binding protein (type I) exhibits a similar Ki value of 3.6 microM. Unliganded cellular retinol-binding protein has no effect on RoDH activity. Citral and acyclic isoprenoids also act as inhibitors of RoDH-4 activity. Ethanol is not inhibitory. Thus, we have identified and characterized a sterol/retinol-oxidizing short chain dehydrogenase/reductase that prefers NAD+ and recognizes all-trans-retinol as substrate. RoDH-4 can potentially contribute to the biosynthesis of two powerful modulators of gene expression: retinoic acid from retinol and dihydrotestosterone from 3alpha-androstane-diol.
Development of the male urogenital tract in mammals is mediated by testicular androgens. It has been tacitly assumed that testosterone acts through its intracellular metabolite dihydrotestosterone (DHT) to mediate this process, but levels of these androgens are not sexually dimorphic in plasma at the time of prostate development. Here we show that the 3 alpha-reduced derivative of DHT, 5 alpha-androstane-3 alpha,17 beta-diol (5 alpha-adiol), is formed in testes of tammar wallaby pouch young and is higher in male than in female plasma in this species during early sexual differentiation. Administration of 5 alpha-adiol caused formation of prostatic buds in female wallaby pouch young, and in tissue minces of urogenital sinus and urogenital tubercle radioactive 5 alpha-adiol was converted to DHT, suggesting that circulating 5 alpha-adiol acts through DHT in target tissues. We conclude that circulating 5 alpha-adiol is a key hormone in male development.
We report characterization of a novel member of the short chain dehydrogenase/reductase superfamily. The 1513-base pair cDNA
encodes a 319-amino acid protein. The corresponding gene spans over 26 kilobase pairs on chromosome 2 and contains five exons.
The recombinant protein produced using the baculovirus system is localized in the microsomal fraction of Sf9 cells and is
an integral membrane protein with cytosolic orientation of its catalytic domain. The enzyme exhibits an oxidoreductase activity
toward hydroxysteroids with NAD+ and NADH as the preferred cofactors. The enzyme is most efficient as a 3α-hydroxysteroid dehydrogenase, converting 3α-tetrahydroprogesterone
(allopregnanolone) to dihydroprogesterone and 3α-androstanediol to dihydrotestosterone with similar catalytic efficiency (V
max values of 13–14 nmol/min/mg microsomal protein and K
m values of 5–7 μm). Despite ∼44–47% sequence identity with retinol/3α-hydroxysterol dehydrogenases, the enzyme is not active toward retinols.
The corresponding message is abundant in human trachea and is present at lower levels in the spinal cord, bone marrow, brain,
heart, colon, testis, placenta, lung, and lymph node. Thus, the new short chain dehydrogenase represents a novel type of microsomal
NAD+-dependent 3α-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution.
Steroid target tissues regulate the local level of steroid hormone that can bind and trans-activate nuclear receptors (a process known as intracrine modulation). This pre-receptor regulation can be achieved by hydroxysteroid dehydrogenases (HSDs). For each sex hormone there is a pair of HSD isoforms which act either as reductases or oxidases to convert potent steroid hormones into their cognate inactive metabolites, or vice-versa. In this manner, HSDs can function as molecular switches to regulate steroid hormone action. Because these HSDs show tissue-specific expression, inhibitors of these enzymes are predicted to cause tissue-specific responses to steroid hormones. These inhibitors would represent a new class of therapeutics called 'selective intracrine modulators' (SIMs). SIMs are expected to have the same tissue-specific effects as selective steroid receptor modulators but a different mode of action as their effects are enzyme- and not receptor-mediated. HSDs responsible for these interconversions belong to two protein superfamilies: the short-chain dehydrogenases/reductases; and the aldo-keto reductases. Crystal structures exist for HSDs in both families, making rational design of SIMs a reality. Broad-based criteria have been established which must be fulfilled to validate each HSD isoform as a potential SIM target.
The source of NADPH-dependent cytosolic 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity is unknown to date. This important reaction leads e.g. to the reduction of the potent androgen 5alpha-dihydrotestosterone (DHT) into inactive 3beta-androstanediol (3beta-Diol). Four human cytosolic aldo-keto reductases (AKR1C1-AKR1C4) are known to act as non-positional-specific 3alpha-/17beta-/20alpha-HSDs. We now demonstrate that AKR1Cs catalyze the reduction of DHT into both 3alpha- and 3beta-Diol (established by (1)H NMR spectroscopy). The rates of 3alpha- versus 3beta-Diol formation varied significantly among the isoforms, but with each enzyme both activities were equally inhibited by the nonsteroidal anti-inflammatory drug flufenamic acid. In vitro, AKR1Cs also expressed substantial 3alpha[17beta]-hydroxysteroid oxidase activity with 3alpha-Diol as the substrate. However, in contrast to the 3-ketosteroid reductase activity of the enzymes, their hydroxysteroid oxidase activity was potently inhibited by low micromolar concentrations of the opposing cofactor (NADPH). This indicates that in vivo all AKR1Cs will preferentially work as reductases. Human hepatoma (HepG2) cells (which lack 3beta-HSD/Delta(5-4) ketosteroid isomerase mRNA expression, but express AKR1C1-AKR1C3) were able to convert DHT into 3alpha- and 3beta-Diol. This conversion was inhibited by flufenamic acid establishing the in vivo significance of the 3alpha/3beta-HSD activities of the AKR1C enzymes. Molecular docking simulations using available crystal structures of AKR1C1 and AKR1C2 demonstrated how 3alpha/3beta-HSD activities are achieved. The observation that AKR1Cs are a source of 3beta-tetrahydrosteroids is of physiological significance because: (i) the formation of 3beta-Diol (in contrast to 3alpha-Diol) is virtually irreversible, (ii) 3beta-Diol is a pro-apoptotic ligand for estrogen receptor beta, and (iii) 3beta-tetrahydrosteroids act as gamma-aminobutyric acid type A receptor antagonists.
Androgen-dependent prostate diseases initially require 5alpha-dihydrotestosterone (DHT) for growth. The DHT product 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), is inactive at the androgen receptor (AR), but induces prostate growth, suggesting that an oxidative 3alpha-hydroxysteroid dehydrogenase (HSD) exists. Candidate enzymes that posses 3alpha-HSD activity are type 3 3alpha-HSD (AKR1C2), 11-cis retinol dehydrogenase (RODH 5), L-3-hydroxyacyl coenzyme A dehydrogenase , RODH like 3alpha-HSD (RL-HSD), novel type of human microsomal 3alpha-HSD, and retinol dehydrogenase 4 (RODH 4). In mammalian transfection studies all enzymes except AKR1C2 oxidized 3alpha-diol back to DHT where RODH 5, RODH 4, and RL-HSD were the most efficient. AKR1C2 catalyzed the reduction of DHT to 3alpha-diol, suggesting that its role is to eliminate DHT. Steady-state kinetic parameters indicated that RODH 4 and RL-HSD were high-affinity, low-capacity enzymes whereas RODH 5 was a low-affinity, high-capacity enzyme. AR-dependent reporter gene assays showed that RL-HSD, RODH 5, and RODH 4 shifted the dose-response curve for 3alpha-diol a 100-fold, yielding EC(50) values of 2.5 x 10(-9) M, 1.5 x 10(-9) M, and 1.0 x 10(-9) M, respectively, when compared with the empty vector (EC(50) = 1.9 x 10(-7) M). Real-time RT-PCR indicated that L-3-hydroxyacyl coenzyme A dehydrogenase and RL-HSD were expressed more than 15-fold higher compared with the other candidate oxidative enzymes in human prostate and that RL-HSD and AR were colocalized in primary prostate stromal cells. The data show that the major oxidative 3alpha-HSD in normal human prostate is RL-HSD and may be a new therapeutic target for treating prostate diseases.
11β-hydroxysteroid dehydrogenases (11β-HSDs) catalyze the interconversion of active glucocorticoids (cortisol, corticosterone) and inert 11-keto forms (cortisone, 11-dehydrocorticosterone). 11β-HSD type 2 has a well recognized function as a potent dehydrogenase that rapidly inactivates glucocorticoids, thus allowing aldosterone selective access to otherwise nonselective mineralocorticoid receptors in the distal nephron. In contrast, the function of 11β-HSD type 1 has, until recently, been little understood. 11β-HSD1 is an ostensibly reversible oxidoreductase in vitro, which is expressed in liver, adipose tissue, brain, lung, and other glucocorticoid target tissues. However, increasing data suggest that 11β-HSD1 acts as a predominant 11β-reductase in many intact cells, whole organs, and in vivo. This reaction direction locally regenerates active glucocorticoids within expressing cells, exploiting the substantial circulating levels of inert 11-keto steroids. While the biochemical determinants of the reactio...
Tritiated 5α-androstane-3α,17β-diol (3α-diol) and 5α-androstane-3β,17β-diol (3β-diol) respectively were administered to patients with benign prostatic hypertrophy (bph) undergoing prostatectomy. In prostate and skeletal muscle homogenates and in plasma the total radioactivity content as well as the formation of metabolites were measured. Histological examination of each ectomized prostate was performed to evaluate the cellular composition of the tissue.
After 3α-diol injection, a higher uptake of radioactivity in the prostate was obtained than after 3β-diol. Within 30 min the 3α-isomer was very efficiently converted to 5α-DHT, while most of the 3β-isomer remained unchanged. There was, however, also after administration of the 3β-diol a substantial biconversion to 5α-DHT as has been confirmed by recrystallization to constant specific radioactivity. Only after 3β-diol epiandrosterone was detected in small but significant amounts. 3α-diol administration resulted in distinct concentrations of 3β-diol, whereas the conversion of 3β-diol to the 3α-isomer was insignificant. When comparing the histological composition of the prostatic tissue with the accumulation of radioactivity and the formation of metabolites only a weak correlation between glandular structure and radioactivity uptake after 3α-diol administration could be revealed.
Intracellular levels of active steroid hormones are determined by their relative rates of synthesis and breakdown. In the
case of the potent androgen dihydrotestosterone, synthesis from the precursor testosterone is mediated by steroid 5α-reductase,
whereas breakdown to the inactive androgens 5α-androstane-3α,17β-diol (3α-adiol), and androsterone is mediated by reductive
3α-hydroxysteroid dehydrogenases (3α-HSD) and oxidative 17β-hydroxysteroid dehydrogenases (17β-HSD), respectively. We report
the isolation by expression cloning of a cDNA encoding a 17β-HSD6 isozyme that oxidizes 3α-adiol to androsterone. 17β-HSD6
is a member of the short chain dehydrogenase/reductase family and shares 65% sequence identity with retinol dehydrogenase
1 (RoDH1), which catalyzes the oxidation of retinol to retinal. Expression of rat and human RoDH cDNAs in mammalian cells
is associated with the oxidative conversion of 3α-adiol to dihydrotestosterone. Thus, 17β-HSD6 and RoDH play opposing roles
in androgen action; 17β-HSD6 inactivates 3α-adiol by conversion to androsterone and RoDH activates 3α-adiol by conversion
to dihydrotestosterone. The synthesis of an active steroid hormone by back conversion of an inactive metabolite represents
a potentially important mechanism by which the steady state level of a transcriptional effector can be regulated.
Tritiated 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) and 5alpha-androstane-3beta,17beta-diol (3beta-diol) respectively were administered to patients with benign prostatic hypertrophy (bph) undergoing prostatectomy. In prostate and skeletal muscle homogenates and in plasma the total radioactivity content as well as the formation of metabolites were measured. Histological examination of each ectomized prostate was performed to evaluate the cellular composition of the tissue. After 3alpha-diol injection, a higher uptake of radioactivity in the prostate was obtained than after 3beta-diol. Within 30 min the 3alpha-isomer was very efficiently converted to 5alpha-DHT, while most of the 3beta-isomer remained unchanged. There was, however, also after administration of the 3beta-diol a substantial biconversion to 5alpha-DHT as has been confirmed by recrystallization to constant specific radioactivity. Only after 3beta-diol epiandrosterone was detected in small but significant amounts. 3alpha-diol administration resulted in distinct concentrations of 3beta-diol, whereas the conversion of 3beta-diol to the 3alpha-isomer was insignificant. When comparing the histological composition of the prostatic tissue with the accumulation of radioactivity and the formation of metabolites only a weak correlation between glandular structure and radioactivity uptake after 3alpha-diol administration could be revealed.
The conditions under which dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-androstanediol) induce prostatic growth in the castrate dog have been investigated in animals in which the initial size of the prostate was either normal or large. When androgen replacement was commenced immediately after castration of dogs with preexisting prostatic hypertrophy, dihydrotestosterone was as effective as 3 alpha-androstanediol in maintaining prostate weight. Under all other conditions examined (replacement commenced immediately after castration in dogs with small prostates or 2 weeks after castration in dogs with large prostates), 3 alpha-androstanediol but not dihydrotestosterone induced significant prostatic growth. The maximal effect of 3 alpha-androstanediol in inducing prostatic growth was observed in 12 weeks. The administration of 17 beta-estradiol enhanced the growth-promoting action of 3 alpha-androstanediol, but did not affect the action of dihydrotestosterone or influence 3 alpha-hydroxysteroid dehydrogenase activity in prostate. Thus, the synergistic effect of 17 beta-estradiol and 3 alpha-androstanediol seems to be independent of any effect on the interconversion of the two androgens. The activity of the 3 alpha-hydroxysteroid dehydrogenase in dog prostate was high under all conditions of accelerated prostatic growth and appeared to be under androgenic control.
Mineralocorticoid receptors, both when in tissue extracts and when recombinant-derived, have equal affinity for the physiological mineralocorticoid aldosterone and for the glucocorticoids cortisol and corticosterone, which circulate at much higher concentrations than aldosterone. Such receptors are found in physiological mineralocorticoid target tissues (kidney, parotid, and colon) and in nontarget tissues such as hippocampus and heart. In mineralocorticoid target tissues the receptors are selective for aldosterone in vivo because of the presence of the enzyme 11 beta-hydroxy-steroid dehydrogenase, which converts cortisol and corticosterone, but not aldosterone, to their 11-keto analogs. These analogs cannot bind to mineralocorticoid receptors.
To study androgen-mediated differentiation in the rat ventral prostate, we separated the two principal cell types (epithelial and stromal) derived from prostates of immature and mature rats on two continuous Percoll gradients. Cells were immediately placed in culture medium. Testosterone metabolism by the two prostatic cell types was evaluated using [3H]testosterone and quantifying the formation of 5 alpha-[3H]dihydrotestosterone (5 alpha-DHT) and 5 alpha-[3H]androstane-(3 alpha or 3 beta), 17 beta-diols. In epithelial cells from both immature and mature rat prostates the major testosterone metabolites were 5 alpha-DHT and 5 alpha-androstane-3 alpha, 17 beta-diol. Stromal cells metabolized less testosterone than did the epithelial cells. Differences in the relative levels of the various metabolites were observed for the two age groups. To examine in more detail the changes in testosterone metabolism observed in vitro both types of cells and unfractionated cells from immature and mature rat prostates were assayed for testosterone 5 alpha-reductase (using testosterone as substrate) and 3 alpha-hydroxysteroid dehydrogenase (using 5 alpha-DHT as substrate) activities (expressed as pmol substrate reduced/min per 10(6) cells). In immature rats both 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase activities were localized in the epithelial cell fraction (17 and 52 respectively); stromal cells showed lower 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase activity (4 and 4). Relative to epithelial cells from immature rats epithelial cells from mature rats showed a decrease in 5 alpha-reductase (7) and an increase in 3 alpha-hydroxysteroid dehydrogenase (160) activity while stromal 5 alpha-reductase showed little change (3) and 3 alpha-hydroxysteroid dehydrogenase increased to 22.(ABSTRACT TRUNCATED AT 250 WORDS)
In normal physiology 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) protects the mineralocorticoid receptor (MR) from glucocorticoid excess. In the rat, however, 11 beta-OHSD mRNA and activity is widespread, suggesting that it may also play a role in regulating ligand access to the glucocorticoid receptor (GR). We have studied the role of the 11 beta-OHSD in modulating corticosteroid hormone action in rat pituitary GH3 cells (glucocorticoids inhibit prolactin gene transcription) and renal epithelial NRK-52E cells (mineralocorticoids increase Na-K ATPase subunit gene expression) in culture. Both cell lines express high levels of 11 beta-OHSD activity, and Northern/Western blot analyses using a rat cDNA probe and antisera raised against rat liver 11 beta-OHSD reveal a single 1.4 Kb mRNA encoding an enzyme of molecular size 34 kDa. In GH3 cells, prolactin gene transcription was unaffected by corticosterone (B) in doses of 10(-8) to 10(-6) M. When 11 beta-OHSD activity was inhibited with the licorice derivative, glycyrrhetinic acid (GE); however, 10(-6) M B inhibited prolactin (PRL) mRNA levels to the same degree as an equimolar concentration of the GR agonist RU 28362. This effect was blocked by co-incubation with the GR antagonist RU 38486. In NRK-52E cells, co-incubation with B and GE resulted in a marked increase in alpha 1/beta 1 Na-K ATPase subunit mRNA levels when compared with GE and/or B alone and this effect could be blocked by administration of the MR antagonist RU 26752.(ABSTRACT TRUNCATED AT 250 WORDS)
Human brain short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) has been demonstrated to be a unique 3alpha-hydroxysteroid dehydrogenase (HSD) that can convert 5alpha-androstane-3alpha, 17beta-diol (3alpha-adiol) to dihydrotestosterone (DHT), whose affinity to the androgen receptor is 10(5)-fold higher than that of 3alpha-adiol. The catalytic efficiency of human SCHAD for this oxidative 3alpha-HSD reaction was estimated to be 164 min(-1) mM(-1), about 10-fold higher than that measured for the backward reaction. Thus, human brain SCHAD may function in androgen metabolism as a new kind of 3alpha-HSD by counteracting all other known 3alpha-HSDs, which would unidirectionally catalyze the reduction of DHT to the almost inactive 3alpha-adiol. Human SCHAD is identical to an amyloid-beta binding protein (ERAB) involved in Alzheimer's disease, which was previously reported to be associated with the endoplasmic reticulum. This protein is, in fact, localized in mitochondria, not endoplasmic reticulum, as evidenced by immunocytochemical studies and its noncleavable mitochondrial targeting sequence and lack of endoplasmic reticulum targeting signals or transmembrane segments. These results prompt the suggestion that the mitochondrion plays not only an essential role in the initial step of steroidogenesis, but also important roles in the intracellular homeostasis of sex steroid hormones. Northern blot analysis revealed that the human SCHAD gene is expressed in both gonadal and peripheral tissues including the prostate whose growth notably requires DHT, the most potent androgen. This study represents the first report of a 3alpha-HSD that could act to generate DHT from 3alpha-adiol and thereby maintain intracellular DHT levels. We propose that inhibitors of the 3alpha-HSD activity of human brain SCHAD could be useful for the treatment of benign prostatic hyperplasia and other disorders involving DHT metabolism, in combination with known inhibitors of steroid 5alpha-reductases.
11beta-hydroxysteroid dehydrogenases (11beta-HSDs) catalyze the interconversion of active glucocorticoids (cortisol, corticosterone) and inert 11-keto forms (cortisone, 11-dehydrocorticosterone). 11beta-HSD type 2 has a well recognized function as a potent dehydrogenase that rapidly inactivates glucocorticoids, thus allowing aldosterone selective access to otherwise nonselective mineralocorticoid receptors in the distal nephron. In contrast, the function of 11beta-HSD type 1 has, until recently, been little understood. 11beta-HSD1 is an ostensibly reversible oxidoreductase in vitro, which is expressed in liver, adipose tissue, brain, lung, and other glucocorticoid target tissues. However, increasing data suggest that 11beta-HSD1 acts as a predominant 11beta-reductase in many intact cells, whole organs, and in vivo. This reaction direction locally regenerates active glucocorticoids within expressing cells, exploiting the substantial circulating levels of inert 11-keto steroids. While the biochemical determinants of the reaction direction are not fully understood, insights to its biological importance have been afforded by use of inhibitors in vivo, including in humans, and the generation of knockout mice. Such studies suggest 11beta-HSD1 effectively amplifies glucocorticoid action at least in the liver, adipose tissue, and the brain. Inhibition of 11beta-HSD1 represents a potential target for therapy of disorders that might be ameliorated by local reduction of glucocorticoid action, including type 2 diabetes, obesity, and age-related cognitive dysfunction.
11-cis-Retinol dehydrogenase catalyzes the oxidation of cis-retinols, a rate-limiting step in the biosynthesis of 9-cis-retinoic acid. It is also active toward 3alpha-hydroxysteroids, and thus might be involved in steroid metabolism. To better understand the role of this enzyme, we produced stable transfectants expressing 11-cis-retinol dehydrogenase in human embryonic kidney 293 cells. In vitro enzymatic assays have demonstrated that, with an appropriate exogenous cofactor, the enzyme catalyzes the interconversion of 5alpha-androstane-3alpha,17beta-diol and dihydrotestosterone and that of androsterone and androstanedione. However, using intact transfected cells, we found that the enzyme catalyzes reactions only in the oxidative direction. Thus, it is possible that 5alpha-androstane-3alpha,17beta-diol (an inactive androgen) can be converted into dihydrotestosterone, the most potent androgen, by the action of 11-cis-retinol dehydrogenase. This reaction could constitute a non-classical pathway of production of active androgens in the peripheral tissues. We also showed that all-trans-, 9-cis- and 13-cis-retinol inhibit the oxidative 3alpha-hydroxysteroid steroid activity of 11-cis-retinol dehydrogenase with similar K(i) values. Since all-trans-retinol is a precursor of cis-retinols, its inhibitory effect on the activity suggests that it could play an important role in modulating the formation of 9-cis-retinoic acid. In addition, we examined the effect of several known enzyme modulators, namely carbenoxolone, phenylarsine oxide and phosphatidylcholine, on 11-cis-retinol dehydrogenase activity. Taken together, our results suggest that, in humans, this enzyme might play a role in the biosynthesis of both 9-cis-retinoic acid and dihydrotestosterone.
Four human aldo-keto reductases (AKRs) that belong to the AKR1C subfamily function in vitro as 3-keto-, 17-keto- and 20-ketosteroid reductases or as 3alpha-, 17beta- and 20alpha- hydroxysteroid oxidases to varying degrees. By acting as ketosteroid reductases or hydroxysteroid oxidases these AKRs can either convert potent sex hormones (androgens, estrogens and progestins) into their inactive metabolites or they can form potent hormones by catalyzing the reverse reaction. In this manner they may regulate occupancy and trans-activation of steroid hormone receptors. Tissue distribution studies previously indicated that AKR1C2 (type 3 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD)) and AKR1C3 (type 2 3alpha-HSD) are highly expressed in human prostate. An assessment of the directionality of these AKR1C isozymes in a cellular environment would help identify which isozymes are responsible for 5alpha-dihydrotestosterone (5alpha-DHT) formation or its elimination in the prostate. An imbalance in 5alpha-DHT levels has been implicated in development of prostate carcinoma and benign prostatic hyperplasia. We focused our attention on AKR1C2 since this is the isoform that will oxidize 3alpha-androstanediol (3alpha-diol) to 5alpha-DHT in vitro, suggesting it could elevate 5alpha-DHT levels. To determine whether AKR1C2 preferentially functions as a reductase or an oxidase in a cellular context, we transiently transfected AKR1C2 (pcDNA3-AKR1C2) into COS-1 cells and stably transfected pcDNA3-AKR1C2 and pLNCX-AKR1C2 constructs into PC-3 and LNCaP cells, respectively. COS-1 is a monkey kidney cell line, while PC-3 and LNCaP cells are androgen receptor (-) and (+) prostate adenocarcinoma cell lines, respectively. In transient COS-1-AKR1C2 and in stable PC3-AKR1C2 transfectants, AKR1C2 functioned as a 3-ketosteroid reductase inactivating 5alpha-DHT. In androgen dependent human prostate cancer cells LNCaP, it was not possible to ascertain the preferred direction of AKR1C2 by stable transfection due to the high rate of 5alpha-DHT and 3alpha-diol glucuronidation. Based on these findings AKR1C2 may diminish 5alpha-DHT and prevent this ligand from activating the androgen receptor in situ.
Human aldo-keto reductases (AKRs) of the AKR1C subfamily function in vitro as 3-keto-, 17-keto-, and 20-ketosteroid reductases or as 3alpha-, 17beta-, and 20alpha-hydroxysteroid oxidases. These AKRs can convert potent sex hormones (androgens, estrogens, and progestins) into their cognate inactive metabolites or vice versa. By controlling local ligand concentration AKRs may regulate steroid hormone action at the prereceptor level. AKR1C2 is expressed in prostate, and in vitro it will catalyze the nicotinamide adenine dinucleotide (NAD(+))-dependent oxidation of 3alpha-androstanediol (3alpha-diol) to 5alpha-dihydrotestosterone (5alpha-DHT). This reaction is potently inhibited by reduced NAD phosphate (NADPH), indicating that the NAD(+): NADPH ratio in cells will determine whether AKR1C2 makes 5alpha-DHT. In transient COS-1-AKR1C2 and in stable PC-3-AKR1C2 transfectants, 5alpha-DHT was reduced by AKR1C2. However, the transfected AKR1C2 oxidase activity was insufficient to surmount the endogenous 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity, which eliminated 3alpha-diol as androsterone. PC-3 cells expressed retinol dehydrogenase/3alpha-HSD and 11-cis-retinol dehydrogenase, but these endogenous enzymes did not oxidize 3alpha-diol to 5alpha-DHT. In stable LNCaP-AKR1C2 transfectants, AKR1C2 did not alter androgen metabolism due to a high rate of glucuronidation. In primary cultures of epithelial cells, high levels of AKR1C2 transcripts were detected in prostate cancer, but not in cells from normal prostate. Thus, in prostate cells AKR1C2 acts as a 3-ketosteroid reductase to eliminate 5alpha-DHT and prevents activation of the androgen receptor. AKR1C2 does not act as an oxidase due to either potent product inhibition by NADPH or because it cannot surmount the oxidative 17beta-HSD present. Neither AKR1C2, retinol dehydrogenase/3alpha-HSD nor 11-cis-retinol dehydrogenase is a source of 5alpha-DHT in PC-3 cells.
Human 17beta-hydroxysteroid dehydrogenases (17betaHSDs) catalyze the interconversion of weak and potent androgen and estrogen pairs. Although the reactions using purified enzymes can be driven in either direction, these enzymes appear to function unidirectionally in intact cells: only reductive reactions for 17betaHSD1 and 17beta HSD3 and only oxidative reactions for 17betaHSD2. We show that, after exhaustive incubations with either 17beta-hydroxy- or 17-ketosteroid, the medium for HEK-293 cells expressing 17betaHSD1 or 17betaHSD3 contains a 92:8 ratio of reduced:oxidized steroid. Similarly, 17betaHSD2 yields a >95:5 ratio of oxidized:reduced steroids for both androgens and estrogens. Dual-isotope kinetic measurements show that the rates of the forward and reverse reactions are identical at these functional equilibrium states in intact cells for all three 17betaHSD isoforms, and these rates are much faster than those estimated from single-isotope flux studies. Mutation L36D converts 17betaHSD1 to an oxidative enzyme in intact cells, reversing the equilibrium distribution of estradiol:estrone to 5:95; however, the rates of the forward and reverse reactions at equilibrium are equal and comparable to those of the wild-type enzymes. The co-expression of 17betaHSD2 paradoxically increases the potency of estrone in transactivation assays, demonstrating the physiological relevance of "backwards" metabolism to estradiol. We conclude that 17betaHSD types 1, 2, and 3 catalyze both oxidative and reductive reactions in HEK-293 cells at intrinsic rates that are much faster than those estimated from single-isotope studies. These 17betaHSD isoforms do not drive steroid flux in one direction but rather may achieve functional equilibria in intact cells, reflecting thermodynamically driven steroid distributions.
Hydroxysteroid dehydrogenases (HSDs) interconvert potent and relatively inactive forms of individual steroid hormones using nicotinamide cofactors NADPH/NADP(+) and NADH/NAD(+) [nicotinamide adenine dinucleotide (phosphate), reduced/oxidized forms]. Although reactions with purified enzymes in vitro may be driven in either direction depending on the assay conditions, HSD enzymes appear to function in one direction or the other in intact cells. At least for some of these enzymes, however, the apparent unidirectional metabolism actually reflects bidirectional catalysis that reaches a pseudoequilibrium state with a strong directional preference. This directional preference, in turn, derives from intracellular concentration gradients for the nicotinamide cofactors and the relative affinities of each HSD for these cofactors. Because the concentrations of free cofactor exceed those of steroids by many orders of magnitude, the activities of these enzymes are predominantly driven by cofactor abundance, which is linked to intermediary metabolism. Consequently, the amount of active steroids in cells containing HSDs may be modulated by cofactor abundance and, hence, intracellular redox state. We will review the evidence linking cofactor handling and HSD activity, speculate on additional ways that intracellular metabolism can alter HSD activity and, thus, hormone potency, and discuss fruitful avenues of further investigation.
Human prostate adenocarcinoma (CaP) and benign prostatic hyperplasia (BPH) have epithelial and stromal cell origins, respectively. To determine whether the androgen signal is processed differently in these cell types the expression of transcripts for enzymes that control ligand access to the androgen receptor (AR) were measured. Transcripts for type 2 5alpha-reductase, ketosteroid reductases [aldo-keto reductase (AKR)1C1-AKR1C4], the major oxidative 3alpha-hydroxysteroid dehydrogenase (HSD) retinol dehydrogenase (RODH)-like 3alpha-HSD (RL-HSD) and nuclear receptors [AR, estrogen receptor (ER)alpha, and ERbeta] were determined in whole human prostate and in cultures of primary epithelial cells (PEC) and primary stromal cells (PSC) from normal prostate, CaP and BPH by real-time RT-PCR. Normal PEC (n=14) had higher levels of AKR1C1 (10-fold, P<0.001), AKR1C2 (115-fold, P<0.001) and AKR1C3 (6-fold, P<0.001) than normal PSC (n=15), suggesting that reductive androgen metabolism occurs. By contrast, normal PSC had higher levels of AR (8-fold, P<0.001) and RL-HSD (21-fold, P<0.001) than normal PEC, suggesting that 3alpha-androstanediol is converted to 5alpha-dihydrotestosterone to activate AR. In CaP PEC (n=14), no significant changes in transcript levels vs. normal PEC were observed. In BPH PSC (n=21) transcripts for AR (2-fold, P<0.001), AKR1C1 (4-fold, P<0.001), AKR1C2 (10-fold P<0.001), AKR1C3 (4-fold, P<0.001) and RL-HSD (3-fold, P<0.003) were elevated to increase androgen response. Differences in the AR:ERbeta transcript ratios (eight in normal PEC vs. 280 in normal PSC) were maintained in PEC and PSC in diseased prostate. These data suggest that CaP may be more responsive to an ERbeta agonist and BPH may be more responsive to androgen ablation.
Human type 3 3alpha-hydroxysteroid dehydrogenase, or aldo-keto reductase (AKR) 1C2, eliminates the androgen signal in human prostate by reducing 5alpha-dihydrotestosterone (DHT, potent androgen) to form 3alpha-androstanediol (inactive androgen), thereby depriving the androgen receptor of its ligand. The k(cat) for the NADPH-dependent reduction of DHT catalyzed by AKR1C2 is 0.033 s(-1). We employed transient kinetics and kinetic isotope effects to dissect the contribution of discrete steps to this low k(cat) value. Stopped-flow experiments to measure the formation of the AKR1C2.NADP(H) binary complex indicated that two slow isomerization events occur to yield a tight complex. A small primary deuterium isotope effect on k(cat) (1.5) and a slightly larger effect on k(cat)/K(m) (2.1) were observed in the steady state. In the transient state, the maximum rate constant for the single turnover of DHT (k(trans)) was determined to be 0.11 s(-1) for the NADPH-dependent reaction, which was approximately 4-fold greater than the corresponding k(cat) x k(trans) was significantly reduced when NADPD was substituted for NADPH, resulting in an apparent (D)k(trans) of 3.5. Thus, the effects of isotopic substitution on the hydride transfer step were masked by slow events that follow or precede the chemical transformation. Transient multiple-turnover reactions generated curvilinear reaction traces, consistent with the product formation and release occurring at comparable rates. Global fitting analysis of the transient kinetic data enabled the estimate of the rate constants for the three-step cofactor binding/release model and for the minimal ordered bi-bi turnover mechanism. Results were consistent with a kinetic mechanism in which a series of slow events, including the chemical step (0.12 s(-1)), the release of the steroid product (0.081 s(-1)), and the release of the cofactor product (0.21 s(-1)), combine to yield the overall observed low turnover number.
Transcript profiling of the androgen signal in normal prostate, benign prostatic hyperplasia, and prostate cancer
- D P Buaman
- S Steckelbroeck
- D M Peehl
- T M Penning
Buaman DP, Steckelbroeck S, Peehl DM, Penning TM. Transcript profiling of the androgen signal in
normal prostate, benign prostatic hyperplasia, and prostate cancer. Endocrinology. 2006bEpub Ahead
of Print September 7, 2006
Cells were incubated with 0.1 μM [ 3 H]-3α-diol. Percent conversion was normalized to β-galacosidase activity, and conversion to 5α-DHT and 5α-androstane-3,17-dione were combined due to the endogenous 17β-HSD present
- Bauman
ERAB (▽) and pcDNA3 (▲). Cells were incubated with 0.1
μM [ 3 H]-3α-diol. Percent conversion was normalized to β-galacosidase activity, and
conversion to 5α-DHT and 5α-androstane-3,17-dione were combined due to the endogenous
17β-HSD present. Adapted from Bauman, et al. 2006a.
Human 3α-hydroxysteroid dehydrogenase isoforms (AKR1C1–AKR1C4) of the aldo keto reductase superfamily: functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones
- Penning