[Show abstract][Hide abstract] ABSTRACT: Cytochrome P450 monooxygenase (CYP) superfamily contributes a broad array of biological functions in living organisms. In fungi, CYPs play diverse and pivotal roles in versatile metabolism and fungal adaptation to specific ecological niches. In this report, CYPomes in the 47 genomes of fungi belong to the phyla Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota have been studied. The comparison of fungal CYPomes suggests that generally fungi possess abundant CYPs belonging to a variety of families with the two global families CYP51 and CYP61, indicating individuation of CYPomes during the evolution of fungi. Fungal CYPs show highly conserved characteristic motifs, but very low overall sequence similarities. The characteristic motifs of fungal CYPs are distinguishable from those of CYPs in animals, plants, and especially archaea and bacteria. The four representative motifs contribute to the general function of CYPs. Fungal CYP51s and CYP61s can be used as the models for the substrate recognition sites analysis. The CYP proteins are clustered into 15 clades and the phylogenetic analyses suggest that the wide variety of fungal CYPs have mainly arisen from gene duplication. Two large duplication events might have been associated with the booming of Ascomycota and Basidiomycota. In addition, horizontal gene transfer also contributes to the diversification of fungal CYPs. Finally, a possible evolutionary scenario for fungal CYPs along with fungal divergences is proposed. Our results provide the fundamental information for a better understanding of CYP distribution, structure and function, and new insights into the evolutionary events of fungal CYPs along with the evolution of fungi.
Genome Biology and Evolution 06/2014; · 4.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytochrome P450 1B1 (CYP1B1) is involved in the metabolism of xenobiotic compounds and endogenous metabolites. Disruption of Cyp1b1 in mice results in suppression of high-fat diet (HFD)-induced obesity and an extensive change in hepatic energy regulation despite minimal constitutive expression of CYP1B1 in hepatocytes. Lack of CYP1B1 is correlated with altered lipid metabolism, especially lysophosphatidylcholines, contributing to protection against obesity. Ultra-performance liquid chromatography coupled with electrospray ionization quadrupole mass spectrometry (UPLC-ESI-QTOFMS)-based metabolomics revealed lysophosphatidylcholine 18:0 (LPC 18:0) as a biomarker positively related to HFD-induced obesity. The increased serum LPC 18:0 in wild-type mice is reduced in Cyp1b1-null mice on a HFD, which is reversed in CYP1B1-humanized mice. CYP1B1-humanized mice show higher diet-induced obesity compared to Cyp1b1-null mice, suggesting that human CYP1B1 shows a similar response to HFD as mouse Cyp1b1. In addition, hepatic stearoyl-CoA desaturase 1 (SCD1) expression was decreased in Cyp1b1-null mice, and the attenuated diet-induced obesity and lower serum LPC 18:0 in the Cyp1b1-null mice is elevated after SCD1 overexpression, suggesting that SCD1 is correlated with CYP1B1-induced obesity. These studies establish a biochemical link between cytochromes P450, lipids and metabolic disorders, and suggest that inhibition of CYP1B1 could be target for anti-obesity drugs.
Journal of Proteome Research 03/2014; · 5.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutation in CYP1B1 is reported in patients with congenital glaucoma. However, the underlying mechanisms remain unknown. Here we show increased diurnal intraocular pressure (IOP) in Cyp1b1-deficient (Cyp1b1(-/-)) mice. Cyp1b1(-/-) mice presented ultrastructural irregular collagen distribution in their trabecular meshwork (TM) tissue, along with increased oxidative stress and decreased levels of Periostin (Postn). Increased levels of oxidative stress and decreased levels of Postn were also detected in human glaucomatous TM tissues. Furthermore, Postn-deficient mice exhibited similar TM tissue ultrastructural abnormalities as Cyp1b1(-/-) mice. Administration of antioxidant N-acetylcysteine (NAC) restored structural abnormality of TM tissue in Cyp1b1(-/-) mice. In addition, TM cells prepared from Cyp1b1(-/-) mice exhibited increased oxidative stress, altered adhesion and decreased levels of Postn. These aberrant cellular responses were reversed in the presence of NAC or by restoration of Cyp1b1 expression. Cyp1b1 knockdown or inhibition of CYP1B1 activity in Cyp1b1(+/+) TM cells resulted in a Cyp1b1(-/-) phenotype. Thus, metabolic activity of CYP1B1 contributes to oxidative homeostasis and ultrastructural organization and function of TM tissue through modulation of Postn expression.
Molecular and cellular biology 08/2013; · 6.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Four dioxin-inducible enzymes--NAD(P)H:quinone oxidoreductase-1 (NQO1) and three cytochromes P450 (CYP1A1, CYP1A2 & CYP1B1)--are implicated in both detoxication and metabolic activation of various endobiotics and xenobiotics. NQO1 is generally regarded as a cytosolic enzyme; whereas CYP1 proteins are located primarily in endoplasmic reticulum (ER), CYP1A1 and CYP1A2 proteins are also targeted to mitochondria. This lab has generated Cyp1a1(mc/mc) and Cyp1a1(mtt/mtt) knock-in mouse lines in which CYP1A1 protein is targeted exclusively to ER (microsomes) and mitochondria, respectively. Comparing dioxin-treated Cyp1(+/+) wild-type, Cyp1a1(mc/mc), Cyp1a1(mtt/mtt), and Cyp1a1(-/-), Cyp1b1(-/-) and Nqo1(-/-) knockout mice, in the present study we show that [a] NQO1 protein locates to cytosol, ER and mitochondria, [b] CYP1B1 protein (similar to CYP1A1 and CYP1A2 proteins) traffics to mitochondria as well as ER, and [c] NQO1 and CYP1B1 targeting to mitochondrial or ER membranes is independent of CYP1A1 presence in that membrane.
Biochemical and Biophysical Research Communications 05/2013; · 2.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Perivascular supporting cells, including pericytes and smooth muscle cells (PC/SMC), have an integral role during angiogenesis and control vascular remodeling, maturation, and stabilization of neoteric vessels. We recently showed that a Cyp1B1 deficiency in mice results in the attenuation of angiogenesis in vivo and the pro-angiogenic activity of endothelial cells in vitro. However, the contribution of PC/SMC, and more specifically the cell autonomous effects of Cyp1B1 in these processes, needs further investigation. Here we demonstrate that PC constitutively expressed Cyp1B1, and that a deficiency in Cyp1B1 was associated with enhanced proliferation, and decreased apoptosis. Mechanistically, the lack of Cyp1B1 was associated with increased oxidative stress and sustained NF-κB activation, which was reversed by the antioxidant N-acetylcysteine. These changes were also concomitant with alterations in PC migration, adhesion, and expression of various extracellular matrix proteins, including thrombospondin-2. Cyp1B1-deficient PC also expressed decreased levels of vascular endothelial growth factor. Together, our results suggest an important role for Cyp1B1 expression in the regulation of PC proliferation, migration, and survival through modulation of the intracellular oxidative state and NF-κB expression and/or activity. Thus, a lack of Cyp1B1 in PC may have a significant role in vascular dysfunction and integrity, contributing to the attenuation of angiogenesis.Laboratory Investigation advance online publication, 8 April 2013; doi:10.1038/labinvest.2013.55.
[Show abstract][Hide abstract] ABSTRACT: Cytochrome P4501B1 (Cyp1b1) is expressed specifically in certain neural crest (NC) cells during embryogenesis. Mesenchymal progenitor cells that develop from NC cells are modeled here by mouse C3H10T1/2 and 3T3-L1 cells. Dexamethasone in combination with methylisobutylxanthine (DM) induces Cyp1b1 and a 6.7 kb mouse Cyp1b1 promoter-luciferase reporter in each cell type prior to adipogenesis. An 18 base sequence (at -6.11 kb) (PaxE) which was essential for this reporter stimulation in 3T3-L1 cells bound the transcription factor Pax6. This is shown by gel mobility shifts and sequence mutations. Heterologous vector expression of Pax6 in 3T3-L1 cells enhanced DM stimulated Cyp1b1 promoter activity through cooperation with two Sp1 sites in the proximal promoter region. Chromatin immunoprecipitation showed that DM stimulated binding of Pax6 adjacent to Sp1 in the proximal promoter more than in the PaxE region. The Cyp1b1 induction by DM in C3H10T1/2 cells was more rapid but independent of Pax6. The far upstream enhancer region (FUER) found in rat Cyp1b1 responded to DM but was inactive in the mouse promoter due to key sequence changes. The expression patterns of Pax6 and Cyp1b1 frequently overlap during mouse embryogenesis. The relationship between Pax6 and Cyp1b1 expression warrants further investigation, particularly in the NC.
Archives of Biochemistry and Biophysics 01/2013; · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Female sex steroid hormones, estradiol-17β (E2-17β) and progesterone (P4) regulate reproductive function and gene expression in a broad range of tissues. Given the central role of the liver in regulating homeostasis including steroid hormone metabolism, we sought to understand how E2-17β and P4 interact to affect global gene expression in liver. Ovariectomized cows (n = 8) were randomly assigned to 4 treatment groups applied in a replicated Latin Square design: 1) No hormone supplementation, 2) E2-17β treatment (ear implant), 3) P4 treatment (intravaginal inserts), and 4) E2-17β combined with P4. After 14 d of treatment, liver biopsies were collected, allowing 28 d intervals between periods. Changes in gene expression in the liver biopsies were monitored using bovine-specific arrays. Treatment with E2-17β altered expression of 479 genes, P4 472 genes, and combined treatment significantly altered expression of 468 genes. In total, 578 genes exhibited altered expression including a remarkable number (346 genes) that responded similarly to E2-17β, P4, or combined treatment. Additional evidence for similar gene expression actions of E2-17ß and/or P4 were: principal component analysis placed almost every treatment array at a substantial distance from controls; Venn diagrams indicated overall treatment effects for most regulated genes; clustering analysis indicated the two major clusters had all treatments up-regulating (172 genes) or down-regulating (173 genes) expression. Thus, unexpectedly, common biological pathways were regulated by E2-17β and/or P4 in liver. This indicates that the mechanism of action of these steroid hormones in the liver might be either indirect or might occur through non-genomic pathways. This unusual pattern of gene expression in response to steroid hormones is consistent with the idea that there are classical and non-classical tissue-specific responses to steroid hormone actions. Future studies are needed to elucidate putative mechanism(s) responsible for overlapping actions of E2-17β and P4 on the liver transcriptome.
PLoS ONE 01/2013; 8(9):e73552. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bone marrow (BM) hematopoietic cells are selectively sensitive to polycyclic aromatic hydrocarbons (PAH) in vivo. 7,12-Dimethylbenz(a)anthracene (DMBA), but not benzo(a)pyrene (BP), depletes BM hematopoietic cells in C57BL/6 mice. This difference is due to a BP-selective aryl hydrocarbon receptor (AhR)-mediated recovery. Colony-forming unit assays show suppression of lymphoid progenitors by each PAH within 6 h but a subsequent recovery, exclusively after BP treatment. Suppression of myeloid progenitors (6 h) occurs only for DMBA. Each progenitor responded equally to DMBA and BP in congenic mice expressing the PAH-resistant AhR (AhR(d)). AhR, therefore, mediates this BP recovery in each progenitor type. These PAH suppressions depend on Cyp1b1-mediated metabolism. Paradoxically, few genes responded to DMBA, whereas 12 times more responded to BP. Progenitor suppression by DMBA, therefore, occurs with minimal effects on the general BM population. Standard AhR-mediated stimulations (Cyp1a1, Cyp1b1, Ahrr) were similar for each PAH and for the specific agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin but were absent in AhR(d) mice. A group of 12 such AhR responses was sustained from 6 to 24 h. A second, larger set of BP responses (chemokines, cytokines, cyclooxygenase 2) differed in two respects; DMBA responses were low and BP responses declined extensively from 6 to 24 h. A third cluster exhibited BP-induced increases in protective genes (Nqo1, GST-mu) that appeared only after 12 h. Conversion of BP to quinones contributes oxidative signaling not seen with DMBA. We propose that genes in this second cluster, which share oxidative signaling and AhR activation, provide the AhR-dependent protection of hematopoietic progenitors seen for BP.
[Show abstract][Hide abstract] ABSTRACT: The steroidogenic acute regulatory (StAR) protein is generated in rodents from 1.6 kb and 3.5 kb mRNA formed by alternative polyadenylation. The zinc finger protein, TIS11B (also Znf36L1), is elevated by cAMP in adrenal cells in parallel with StAR mRNA. TIS11b selectively destabilizes the 3.5 kb mRNA through AU-rich sequences at the end of the 3'UTR. siRNA suppression shows that TIS11b surprisingly increases StAR protein and cholesterol metabolism. StAR transcription is directly activated by PKA phosphorylation. cAMP responsive element binding (CREB) protein 1 phosphorylation is a key step leading to recruitment of the co-activator, CREB binding protein (CBP). A second protein, CREB regulated transcription coactivator (TORC/CRTC), enhances this recruitment, but is inhibited by salt inducible kinase (SIK). Basal StAR transcription is constrained through this phosphorylation of TORC. PKA provides an alternative stimulation by phosphorylating SIK, which prevents TORC inactivation. PKA stimulation of StAR nuclear transcripts substantially precedes TORC recruitment to the StAR promoter, which may, therefore, mediate a later step in mRNA production. Inhibition of SIK by staurosporine elevates StAR transcription and TORC recruitment to maximum levels, but without CREB phosphorylation. TORC suppression by SIK evidently limits basal StAR transcription. Staurosporine and cAMP stimulate synergistically. SIK targets the phosphatase, PP2a (activation), and Type 2 histone de-acetylases (inhibition), which may each contribute to suppression. Staurosporine stimulation through SIK inhibition is repeated in cAMP stimulation of many steroidogenic genes regulated by steroidogenic factor 1 (SF-1) and CREB. TIS11b and SIK may combine to attenuate StAR expression when hormonal stimuli decline.
Molecular and Cellular Endocrinology 12/2010; 336(1-2):53-62. · 4.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have recently shown that deletion of constitutively expressed CYP1B1 is associated with attenuation of retinal endothelial cell (EC) capillary morphogenesis (CM) in vitro and angiogenesis in vivo. This was largely caused by increased intracellular oxidative stress and increased production of thrombospondin-2, an endogenous inhibitor of angiogenesis. Here, we demonstrate that endothelium nitric oxide synthase (eNOS) expression is dramatically decreased in the ECs prepared from retina, lung, heart, and aorta of CYP1B1-deficient (CYP1B1(-/-)) mice compared with wild-type (CYP1B1(+/+)) mice. The eNOS expression was also decreased in retinal vasculature of CYP1B1(-/-) mice. Inhibition of eNOS activity in cultured CYP1B1(+/+) retinal ECs blocked CM and was concomitant with increased oxidative stress, like in CYP1B1(-/-) retinal ECs. In addition, expression of eNOS in CYP1B1(-/-) retinal ECs or their incubation with a nitric oxide (NO) donor enhanced NO levels, lowered oxidative stress, and improved cell migration and CM. Inhibition of CYP1B1 activity in the CYP1B1(+/+) retinal ECs resulted in reduced NO levels and attenuation of CM. In contrast, expression of CYP1B1 increased NO levels and enhanced CM of CYP1B1(-/-) retinal ECs. Furthermore, attenuation of CYP1B1 expression with small interfering RNA proportionally lowered eNOS expression and NO levels in wild-type cells. Together, our results link CYP1B1 metabolism in retinal ECs with sustained eNOS activity and NO synthesis and/or bioavailability and low oxidative stress and thrombospondin-2 expression. Thus CYP1B1 and eNOS cooperate in different ways to lower oxidative stress and thereby to promote CM in vitro and angiogenesis in vivo.
[Show abstract][Hide abstract] ABSTRACT: In this study we used colony forming unit (CFU) assays to demonstrate rapid suppression (within 6h) of lymphoid (CFU-preB) and myeloid (CFU-GM) progenitor cells in DMBA-treated mice. The duration of these changes were consistent with the blood levels of DMBA and its metabolites that were achieved by either IP or oral DMBA administration. CFU-GM and CFU-preB activities returned to control levels by 2 and 7 days after oral DMBA exposure, respectively, but remained suppressed through 7 days after IP DMBA administration. The continued presence of low levels of DMBA in the bloodstream following IP administration was associated with sustained suppression of CFU-preB, total bone marrow lymphoid cells and peripheral blood lymphocytes. The changes noted above were not observed in Cyp1b1 null mice, demonstrating the need for local DMBA metabolism in the bone marrow by Cyp1b1 to impair bone marrow CFU-preB and CFU-GM. Furthermore, these data provide evidence that myeloid-lineage cells are restored more quickly than lymphoid-lineage cells after DMBA exposure.
[Show abstract][Hide abstract] ABSTRACT: Star is expressed in steroidogenic cells as 3.5- and 1.6-kb transcripts that differ only in their 3'-untranslated regions (3'-UTR). In mouse MA10 testis and Y-1 adrenal lines, Br-cAMP preferentially stimulates 3.5-kb mRNA. ACTH is similarly selective in primary bovine adrenocortical cells. The 3.5-kb form harbors AU-rich elements (AURE) in the extended 3'-UTR, which enhance turnover. After peak stimulation of 3.5-kb mRNA, degradation is seen. Star mRNA turnover is enhanced by the zinc finger protein ZFP36L1/TIS11b, which binds to UAUUUAUU repeats in the extended 3'-UTR. TIS11b is rapidly stimulated in each cell type in parallel with Star mRNA. Cotransfection of TIS11b selectively decreases cytomegalovirus-promoted Star mRNA and luciferase-Star 3'-UTR reporters harboring the extended 3'-UTR. Direct complex formation was demonstrated between TIS11b and the extended 3'-UTR of the 3.5-kb Star. AURE mutations revealed that TIS11b-mediated destabilization required the first two UAUUUAUU motifs. HuR, which also binds AURE, did not affect Star expression. Targeted small interfering RNA knockdown of TIS11b specifically enhanced stimulation of 3.5-kb Star mRNA in bovine adrenocortical cells, MA-10, and Y-1 cells but did not affect the reversals seen after peak stimulation. Direct transfection of Star mRNA demonstrated that Br-cAMP stimulated a selective turnover of 3.5-kb mRNA independent of AURE, which may correspond to these reversal processes. Steroidogenic acute regulatory (STAR) protein induction was halved by TIS11b knockdown, concomitant with decreased cholesterol metabolism. TIS11b suppression of 3.5-kb mRNA is therefore surprisingly coupled to enhanced Star translation leading to increased cholesterol metabolism.
[Show abstract][Hide abstract] ABSTRACT: Reactive species derived from cell oxygenation processes play an important role in vascular homeostasis and the pathogenesis of many diseases including retinopathy of prematurity. We show that CYP1B1-deficient (CYP1B1(-/-)) mice fail to elicit a neovascular response during oxygen-induced ischemic retinopathy. In addition, the retinal endothelial cells (ECs) prepared from CYP1B1(-/-) mice are less adherent, less migratory, and fail to undergo capillary morphogenesis. These aberrant cellular responses were completely reversed when oxygen levels were lowered or an antioxidant added. CYP1B1(-/-) ECs exhibited increased oxidative stress and expressed increased amounts of the antiangiogenic factor thrombospondin-2 (TSP2). Increased lipid peroxidation and TSP2 were both observed in retinas from CYP1B1(-/-) mice and were reversed by administration of an antioxidant. Reexpression of CYP1B1 in CYP1B1(-/-) ECs resulted in down-regulation of TSP2 expression and restoration of capillary morphogenesis. A TSP2 knockdown in CYP1B1(-/-) ECs also restored capillary morphogenesis. Thus, CYP1B1 metabolizes cell products that modulate intracellular oxidative stress, which enhances production of TSP2, an inhibitor of EC migration and capillary morphogenesis. Evidence is presented that similar changes occur in retinal endothelium in vivo to limit neovascularization.
[Show abstract][Hide abstract] ABSTRACT: Cytochrome P450 1B1 (Cyp1b1) metabolism contributes to physiologic functions during embryogenesis but also to carcinogenic activation of polycyclic aromatic hydrocarbons (PAH). We generated Cyp1b1-deficient mice carrying the Min allele of the adenomatous polyposis coli gene. These Cyp1b1-deficient Min mice developed twice as many tumors as Min controls, which, however, remained similar in size and histology. Tumors from older (130 days) Cyp1b1-deficient Min mice selectively exhibited focal areas of nuclear atypia associated with less organized epithelia. The metabolism of endogenous substrates by Cyp1b1, therefore, suppresses tumor initiation but also affects progression. Treatment of Min mice with 7,12-dimethylbenzanthracene (DMBA) doubled both tumor multiplicity and size within 20 days but not when mice lacked Cyp1b1. This was paralleled by an abnormal staining of crypts with beta-catenin, phospho-IkappaB kinase, and RelA, which may represent an early stage of tumorigenesis similar to aberrant crypt formation. Cyp1b1 deletion did not affect circulating DMBA and metabolites. Cyp1b1 expression was higher in the tumors compared with normal small intestines. Increased tumorigenesis may, therefore, arise from generation of DMBA metabolites by Cyp1b1 in the developing tumors. Benzo(a)pyrene (BP), which is similarly activated by Cyp1b1 in vitro, did not affect tumorigenesis in Min mice. By contrast, BP and DMBA each suppressed tumor multiplicity in the absence of Cyp1b1. Cyp1b1 metabolism of DMBA and endogenous oxygenation products may each affect a tumor-promoting nuclear factor-kappaB activation, whereas Ah receptor activation by PAH affects suppression. Tumorigenesis may, therefore, depend on activation of PAH by Cyp1b1 and on offsetting suppression by Cyp1b1 of endogenous tumor-enhancing substrates.
Cancer Research 10/2008; 68(18):7394-402. · 8.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mouse embryo fibroblast cell lines 3T3-L1 and C3H10T1/2 differentiate to adipocytes that exhibit similar insulin regulation of lipogenesis. These cell lines, however, differ appreciably in the processes that produce the major regulator PPAR gamma. Each line is stimulated by a mixture of insulin, dexamethasone, and methylisobutylxanthine (IDM). In the first 24h, IDM activates each cell type to produce similar regulatory changes and cell contraction. However, the increase in PPARy is delayed by 24h in typical 3T3-L1 cells compared with C3H10T1/2 cells. This delay is caused by the need for one or two rounds of cell division (clonal expansion) for PPAR gamma synthesis in 3T3-L1 cells. This expansion also occurs in C3H10T1/2 cells, but is not needed for PPAR gamma synthesis and differentiation. Other 3T3-L1 sublines have been described that follow the C3H10T1/2 pattern of differentiation. Culture conditions and inhibitors are described here that remove clonal expansion in C3H10T1/2 cells. With these constraints the cells retain full commitment to differentiation. This distinction is significant because many agents suppress differentiation in 3T3-L1 cells through inhibition of clonal expansion. Other effects on differentiation may be seen in C3H10T1/2 cells that are obscured in 3T3-L1 cells due to this inhibition of proliferation. Human preadipocytes do not need clonal expansion for adipogenesis, thus paralleling C3H10T1/2 cells.
[Show abstract][Hide abstract] ABSTRACT: Obesity enhances prostate cancer (PC) metastasis to bone. Factors secreted from adipocytes enhance PC growth. We hypothesized that feeding a high fat diet to C57BL/6 mice would enhance bone marrow (BM) fibroblastic progenitor differentiation to adipocytes. Mice were fed isocaloric low (LFD) and high (HFD) fat diets for up to 11 weeks postweaning. BM cells were isolated and cultured in serum-free RPMI 1640 media (PC media), generating BM conditioned media (BM-CM). The HFD increased the adipocyte population. BM-CM-LFD inhibited LNCaP cell growth. The HFD partially reversed this suppression. Cytochrome P4501B1 (CYP1B1), which is expressed in BM and in prostate tumor stroma and epithelia, is suspected to influence PC. CYP1B1-/- BM-CM increased the proportion of DU145 cells in S-phase relative to C57BL/6 media. We conclude that BM cells secrete PC inhibitory paracrine factors, but also generate a stimulatory component in response to the HFD. CYP1B1 metabolism positively affects PC growth by metabolizing growth inhibitory factors.
[Show abstract][Hide abstract] ABSTRACT: cAMP stimulation of rodent steroidogenic cells produces two StAR transcripts, a major 3.5 kb and a minor 1.6 kb mRNA, differing only in their 3' untranslated regions (3' UTR). They exhibit very different responses to stimulation and removal of 8-Br-cAMP, with the 3.5 kb form increasing and declining much more rapidly than the 1.6 kb form. The 3' end of the 3.5 kb StAR mRNA contains three conserved AU-rich element (AURE) motifs that mediate fast mRNA turnover in over 900 genes in the human genome. In this paper, we explore post-transcriptional regulation in steroidogenic and non-steroidogenic cells using expression vectors containing StAR or luciferase with different StAR 3' UTRs. We show that the basal steady-state levels of StAR or luciferase protein and mRNA are five to eight times lower with the 3' UTR of 3.5 kb StAR compared with that of the 1.6 kb 3' UTR. Examination of transcript stability by direct mRNA transfection showed only a 1.5-fold increase in the rate of cytoplasmic decay of the 3.5 kb mRNA relative to the 1.6 kb mRNA. However, the long 3' UTR caused a fivefold decrease in the rate of appearance of mature cytoplasmic mRNA despite transcription from the same promoter. This is attributed to less efficient nuclear processing of immature transcripts prior to export to cytoplasm. Selective 3' UTR sequence substitutions, deletions, and mutations showed that this loss of expression is produced additively by specific sequences in a 700-base basal instability region and by non-specific length effects. These mechanisms are selectively enhanced in steroidogenic cells. The AURE contribute a smaller basal destabilization effect selective for steroidogenic cells that is removed by their mutations. Inclusion of introns in the 3.5 kb StAR vector enhances StAR expression, suggesting the effects of introns complexes on nuclear processing. Br-cAMP provides an additional means to rapidly modulate StAR expression independent of transcription by attenuating the nuclear and cytoplasmic instability mechanisms within the extended 3' UTR.
Journal of Molecular Endocrinology 03/2007; 38(1-2):159-79. · 3.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Stimulation of PPARgamma1 and adipogenesis in multipotential C3H10T1/2 cells by the combination of dexamethasone and 3-isobutyl-1-methylxanthine (DM) is suppressed by 2,3,7,8 tetrachlorodibenzodioxin (TCDD) (10 nM). This suppression requires sustained activation of extracellular signal-regulated kinase (Erk)1/2. We show that it arises from an effect of TCDD on epidermal growth factor (EGF) signaling. DM initiates an early loss of cell adhesion that is reversed by this TCDD/EGF synergy. Src kinase activity was completely essential for adhesion restoration, sustained Erk activation, and suppression of peroxisome proliferator-activated receptor (PPAR)gamma1. MEK/Erk activity did not contribute, however, to TCDD-induced adhesion. Stimulation of adhesion may therefore precede elevation of Erk. Adhesion is produced by interaction of alphabeta integrins with extracellular matrix proteins and subsequent Src-mediated phosphorylation of focal adhesion kinase (FAK, Tyr576/577) and paxillin (Tyr118). TCDD enhanced the steady state Src-mediated phosphorylation of FAK but not of paxillin. Protein tyrosine phosphatase (PTPase) inhibition by orthovanadate (OVA) showed that this Src activity is highly restricted by PTPases. Partial inhibition of PTPases by OVA mimicked TCDD in producing EGF- and Src-dependent effects on cell adhesion and PPARgamma1 suppression. TCDD may therefore induce a protein that enhances Src effectiveness at adhesion sites. Rho kinase (ROCK) inhibition blocked TCDD/EGF stimulation of clustered focal adhesion complexes without affecting either sustained Erk activation or suppression of PPARgamma1. Thus, this ROCK-mediated clustering of integrin complexes is not needed for the effects of TCDD on Erk and PPARgamma1. A minimal cholesterol depletion with beta-methylcyclodextrin attenuated TCDD effects on PPARgamma1 and Erk activation. TCDD intervention is therefore linked to extracellular proteins. It indicates that TCDD-enhanced stimulation of EGF signaling to Erk may derive from the initial alphabeta integrin complexes.
[Show abstract][Hide abstract] ABSTRACT: Steroidogenic acute regulatory (StAR) protein is an important regulatory protein in steroidogenesis and rapidly undergoes proteolysis after import into the mitochondria. In this study, we determined the proteolytic cleavage sites and investigated the effects on the stimulation of steroidogenic activity of the blockage of these sites by mutation. The cleaved StAR proteins, which were purified using an anti-StAR immobilized column, reacted with antiserum against the StAR C-terminal oligopeptide. The molecular weights of the purified proteins were determined by MALDI-TOF mass spectrometry, and were found to be identical to those of the 40-285 and 55-285 amino-acid-regions of the StAR protein. To confirm the identification of the cleavage sites, we constructed site-directed mutants of bovine StAR cDNA, which contained the amino acids R37A/R38A/L40A and/or R53A/R54A/R55A. These mutant StAR proteins expressed in COS-1 cells were not cleaved at positions 39-40 and 54-55, and were processed at sites different from those in the wild-type StAR protein. These mutant proteins stimulated pregnenolone formation at almost the same rate as the wild-type StAR protein in COS-1 cells, which suggests that the cholesterol transfer activity was not affected by the mutation.
Biochimica et Biophysica Acta 11/2006; 1764(10):1561-7. · 4.66 Impact Factor