StAR Gene Research Articles

Transcription of Steroidogenic Acute Regulatory Protein in the Rodent Ovary and Placenta: Alternative Modes of Cyclic Adenosine 3′, 5′-Monophosphate Dependent and Independent Regulation

Steroid hormone synthesis is a vital function of the adrenal cortex, serves a critical role in gonadal function, and maintains pregnancy if normally executed in the placenta. The substrate for the synthesis of all steroid hormones is cholesterol, and its conversion to the first steroid, pregnenolone, by the cholesterol side-chain cleavage cytochrome P450 (CYP11A1) enzyme complex takes place in the inner mitochondrial membranes. Steroidogenic acute regulatory protein (STAR) facilitates the rate-limiting transfer of cholesterol from the outer mitochondrial membrane to CYP11A1 located in the inner organelle membranes. The current study explored the mechanisms controlling transcription of the Star gene in primary cell cultures of mouse placental trophoblast giant cells and rat ovarian granulosa cells examined throughout the course of their functional differentiation. Our findings show that the cis-elements required for Star transcription in the rodent placenta and the ovary are centered in a relatively small proximal region of the promoter. In placental trophoblast giant cells, cAMP is required for activation of the Star promoter, and the cis-elements mediating a maximal response were defined as cAMP response element 2 and GATA. EMSA studies show that placental cAMP-responsive element binding protein (CREB)-1 and activating transcription factor-2 (ATF2) bind to a -81/-78 sequence, whereas GATA-2 binds to a -66/-61 sequence. In comparison, patterns of Star regulation in the ovary suggested tissue-specific and developmental controlled modes of Star transcription. During the follicular phase, FSH/cAMP induced CREB-1 dependent activity, whereas upon luteinization STAR expression becomes cAMP and CREB independent, a functional shift conferred by FOS-related antigen-2 displacement of CREB-1 binding, and the appearance of a new requirement for CCAAT enhancer-binding protein beta and steroidogenic factor 1 that bind to upstream elements (-117/-95). These findings suggest that during evolution, the promoters of the Star gene acquired nonconsensus sequence elements enabling expression of a single gene in different organs, or allowing dynamic temporal changes corresponding to progressing phases of differentiation in a given cell type.

The role of JUN in the regulation of PRKCC-mediated STAR expression and steroidogenesis in mouse Leydig cells

Activator protein 1 (JUN) transcription factors (JUN and FOS) play critical roles in a wide variety of signaling processes including those in the protein kinase C (PRKCC) pathway, a pathway that is instrumental in the expression of the steroidogenic acute regulatory (STAR) protein. In the present study, we determined the functional involvement of one of the key JUN family members, JUN, in the regulation of PRKCC-dependent STAR expression and steroidogenesis. MA-10 mouse Leydig tumor cells treated with an activator of PRKCC, phorbol 12-myristate 13-acetate (PMA), demonstrated increases in the expression of the STAR and CYP11A1 proteins and progesterone synthesis, which coincided with the expression and phosphorylation of JUN (P-JUN). PMA was also capable of enhancing the cAMP analog, (Bu)(2)cAMP, which stimulated JUN, STAR, P-STAR and progesterone levels. The induction of Jun mRNA expression and steroid synthesis by PMA requires de novo protein synthesis. Chromatin immunoprecipitation studies revealed the association of P-JUN with the STAR proximal promoter and that PMA specifically enhanced in vivo P-JUN-DNA interaction. Electrophoretic mobility shift assays and reporter gene analyses demonstrated that JUN binds to the JUN motif (-81/-75 bp) in the STAR promoter, and that JUN-DNA-binding activity was highly correlated with the induction of JUN by PRKCC signaling. Overexpression of JUN increased the PMA-mediated transcription of the Star gene, an event markedly decreased by TAM-67, a dominant negative mutant of JUN. Targeted silencing of endogenous JUN, by small interfering RNA, was correlated with the repression of basal- and PMA-mediated STAR expression and progesterone synthesis. These findings describe the mechanisms by which JUN influences PRKCC signaling and provide additional and novel insight into the regulation of the steroidogenic machinery in mouse Leydig cells.

The Role of A-Kinase Anchoring Protein 121 (AKAP121) in Steroidogenesis and Steroidogenic Acute Regulatory Protein (STAR) Expression.

A-kinase anchoring proteins (AKAPs) serve to coordinate and enhance the effects of the second messenger cAMP by recruiting the cAMP-dependent protein kinase (PKA) to discrete subcellular compartments and positioning the kinase near potential substrates. The characterization of the mitochondria-specific PKA-anchoring protein AKAP121 has led us to predict that cAMP signaling to the mitochondria is a key transduction element in regulating the activity of STAR. STAR regulates the rate-limiting step in steroidogenesis by permitting the transfer of cholesterol form the outer- to the inner-mitochondrial membrane, and we have previously demonstrated that Star gene expression and activity are tightly coupled to PKA activity, although very minimal concentrations of cAMP can be sufficient in permitting maximal steroidogenesis. We predict that AKAP121, which can recruit both the PKA holoenzyme as well as mRNAs bearing unique stem-loop motifs to the outer-mitochondrial membrane, serves to enhance the post-transcriptional regulation of STAR. Using MA-10 mouse Leydig tumor cells as a model, we have tested this hypothesis to reveal that cAMP-induced STAR expression and steroidogenesis closely follow AKAP121 expression. Overexpression of AKAP121 in these cells results in a potent increase in STAR protein abundance and steroidogenesis, while silencing the endogenous AKAP121 strongly attenuates STAR-mediated steroidogenesis. We have also extended these experiments using KK1 cells, an immortalized mouse luteinized granulosa cell line, in which STAR expression showed a similar dependency upon the presence of AKAP121. An important observation made in these experiments was that AKAP121-dependent changes in STAR expression were effected post-transcriptionally. To define the mechanism through which AKAP121 is enhancing STAR expression, we sought to resolve what complexes AKAP121 might establish at the mitochondria. Affinity purification experiments using MA-10 cells have revealed that AKAP121 preferentially associates with type II PKA. Moreover, similar affinity purification schemes in transfected COS cells were used to detect Star mRNA bound to AKAP121. Notably, the silencing of AKAP121 in MA-10 cells also corresponded with a reduction in the localization of type II PKA regulatory subunit alpha at the mitochondria. Following the observation that AKAP121 is directing type II PKA to the mitochondria, we used analogs that preferentially activate either type I or type II PKA to demonstrate that the phosphorylation of STAR is highly sensitive to type II PKA activity interestingly though, the Star promoter shows little response to type II PKA, and instead is acutely induced by type I PKA. Our currently data suggests that both type I and type II PKA serve in different capacities related to the expression of STAR. Importantly, AKAP121 appears to coordinate the effects of type II PKA at the outer-mitochondrial membrane either as STAR is being translated at or translocated into the mitochondria. We predict that this serves to potentiate the post-transcriptional synthesis and/or the phosphorylation of STAR, and suggest that this may serve as a broader mechanism by which steroidogenic tissues differently integrate and interpret cAMP signaling. (supported by NIH grant HD17481 and grant # B1-0028 from the Robert A. Welch Foundation)

Gene array and real time PCR analysis of the adrenal sensitivity to adrenocorticotropic hormone in pig

BackgroundVariability in hypothalamic-pituitary-adrenal (HPA) axis activity has been shown to be influenced by genetic factors and related to great metabolic differences such as obesity. The aim of this study was to investigate molecular bases of genetic variability of the adrenal sensitivity to ACTH, a major source of variability, in Meishan (MS) and Large White (LW) pigs, MS being reported to exhibit higher basal cortisol levels, response to ACTH and fatness than LW. A pig cDNA microarray was used to identify changes in gene expression in basal conditions and in response to ACTH stimulation.ResultsGenotype and/or ACTH affected the expression of 211 genes related to transcription, cell growth/maintenance, signal transduction, cell structure/adhesion/extra cellular matrix and protein kinase/phosphatase activity. No change in the expression of known key regulator proteins of the ACTH signaling pathway or of steroidogenic enzymes was found. However, Mdh2, Sdha, Suclg2, genes involved in the tricarboxylic acid (TCA) pathway, were over-expressed in MS pigs. Higher TCA cycle activity in MS than in LW may thus result in higher steroidogenic activity and thus explain the typically higher cortisol levels in MS compared to LW. Moreover, up-regulation of Star and Ldlr genes in MS and/or in response to ACTH suggest that differences in the adrenal function between MS and LW may also involve mechanisms requisite for cholesterol supply to steroidogenesis.ConclusionThe present study provides new potential candidate genes to explain genetic variations in the adrenal sensitivity to ACTH and better understand relationship between HPA axis activity and obesity.

Dynamic expression of the RNA-binding protein Sam68 during mouse pre-implantation development

The STAR protein Sam68 (KHDRBS1) is involved in several aspects of post-transcriptional mRNA metabolism. Herein, we have investigated the expression and subcellular localization of Sam68 during early mouse embryogenesis. We found that mouse oocytes express high levels of Sam68 mRNA, low levels of the transcript for Khdrbs2 (current symbol for Slm-1) and no Khdrbs3 (current symbol for Slm-2), two highly homologous STAR genes. Sam68 protein is expressed throughout oocyte meiotic maturation and early embryogenesis. It is released in the cytoplasm upon meiotic resumption and it slowly accumulates in the nucleus after fertilization. Unlike what was observed for other RNA-binding proteins, nuclear accumulation of Sam68 was independent of de novo mRNA transcription. However, we found that inhibition of mRNA translation by either cycloheximide or puromycin in one-cell embryos caused the accumulation of Sam68 in cytoplasmic granules. Analysis of these granules by deconvolution microscopy demonstrated that they are sites of accumulation for proteins involved in the initiation of mRNA translation, such as eIF4A1, eIF4E and eIF4G. These granules contained RNA and were dissolved by treatment with RNase A. Other proteins expressed by the zygote, like the splicing factor SC35 or the cytoplasmic kinase ERK2, did not accumulate in such structures after treatment with inhibitors of mRNA translation, indicating that the localization of Sam68 and of the translation initiation factors in these granules is a specific event. These results indicate that Sam68 is involved in translational regulation of maternal mRNAs in the zygote and in the early signaling events triggered by fertilization.

Inhibitory Effects of Cholesterol Sulfate on Progesterone Production in Human Granulosa-like Tumor Cell Line, KGN

Cholesterol sulfate (CS) is a component of cell membranes that plays a role in stabilizing the cell membrane. We previously reported that CS increased in the endometrium during implantation, suggesting that CS plays an important role in reproduction. It has been reported that CS regulates progesterone and pregnenolone production in the placenta, adrenal glands and ovary. The regulatory mechanisms of steroid hormone production by CS, however, are still unknown. In the present study, we investigated the effect of CS on the expression of progesterone production-related genes in KGN cells, derived from human granulosa-like tumor. KGN cells were cultured with CS (10 muM) or cholesterol (10 muM) in the presence of 8-bromo-cAMP (1 mM). Progesterone levels in the culture media were measured by enzyme linked fluorescent assay at 24 h after treatment of CS and cAMP. Total RNAs were extracted for quantitative real time RT-PCR with specific primer of StAR protein, P450scc, HSD3B2, ferredoxin and ferredoxin reductase. Whole cell lysates were extracted for western blot analysis with antibody for StAR protein. Progesterone concentration in the culture medium increased to 38-fold by treatment of cAMP. CS significantly reduced progesterone concentration by 30% compared with those of cAMP treatment (p<0.05), while cholesterol did not change the progesterone concentration. CS treatment down-regulated the expression of StAR mRNA and P450scc mRNA was to 54% and 60%, respectively (p<0.05). Western blot analysis revealed that the amount of StAR protein was also reduced by CS treatment. The expression of HSD3B2 mRNA was up-regulated to 3.4-fold by treatment of cAMP. The expression of ferredoxin and ferredoxin reductase mRNA was not affected by CS treatment. These data implied that CS has an inhibitory effect on progesterone production by regulating the expression of StAR and P450scc gene expression.

Regulation of Kruppel-like factor 4, 9, and 13 genes and the steroidogenic genes LDLR, StAR, and CYP11A in ovarian granulosa cells

Kruppel-like factors (KLFs) are important Sp1-like eukaryotic transcriptional proteins. The LDLR, StAR, and CYP11A genes exhibit GC-rich Sp1-like sites, which have the potential to bind KLFs in multiprotein complexes. We now report that KLF4, KLF9, and KLF13 transcripts are expressed in and regulate ovarian cells. KLF4 and 13, but not KLF9, mRNA expression was induced and then repressed over time (P < 0.001). Combined LH and IGF-I stimulation increased KLF4 mRNA at 2 h (P < 0.01), whereas LH decreased KLF13 mRNA at 6 h (P < 0.05), and IGF-I reduced KLF13 at 24 h (P < 0.01) compared with untreated control. KLF9 was not regulated by either hormone. Transient transfection of KLF4, KLF9, and KLF13 suppressed LDLR/luc, StAR/luc, and CYP11A/luc by 80-90% (P < 0.001). Histone-deacetylase (HDAC) inhibitors stimulated LDLR/luc five- to sixfold and StAR/luc and CYP11A/luc activity twofold (P < 0.001) and partially reversed suppression by all three KLFs (P < 0.001). Deletion of the zinc finger domain of KLF13 abrogated repression of LDLR/luc. Lentiviral overexpression of the KLF13 gene suppressed LDLR mRNA (P < 0.001) and CYP11A mRNA (P = 0.003) but increased StAR mRNA (P = 0.007). Collectively, these data suggest that KLFs may recruit inhibitory complexes containing HDAC corepressors, thereby repressing LDLR and CYP11A transcription. Conversely, KLF13 may recruit unknown coactivators or stabilize StAR mRNA, thereby explaining enhancement of in situ StAR gene expression. These data introduce new potent gonadal transregulators of genes encoding proteins that mediate sterol uptake and steroid biosynthesis.

Occurrence and biological effect of exogenous steroids in the Elkhorn River, Nebraska, USA

Recent studies of surface waters in North America, Japan and Europe have reported the presence of steroidogenic agents as contaminants. The current study has three objectives: 1) to determine if steroidogenic compounds are present in the Elkhorn River, 2) to determine if sediments collected from the Elkhorn River can act as a source of steroidogenic compounds to aquatic organisms, and 3) to determine if site-specific biological effects are apparent in the hepatic gene expression of fathead minnows. Evidence was obtained using three approaches: 1) deployment of polar organic chemical integrative samplers (POCIS), 2) deployment of caged fathead minnows, and 3) a laboratory experiment in which POCIS and fish were exposed to sediments from the deployment sites. Deployment sites included: the Elkhorn River immediately downstream from a Nebraska wastewater treatment plant, two waterways (Fisher Creek and Sand Creek) likely to be impacted by runoff from cattle feeding operations, and a reference site unlikely to be impacted by waste water inputs. The POCIS extracts were analyzed for a number of natural steroids and metabolites, as well as four different synthetic steroids: ethinylestradiol, zearalonol, 17β-trenbolone and melengestrol acetate. Estrogenic and androgenic metabolites, as well as progesterone and trace levels of melengestrol acetate were detected in POCIS deployed at each site. POCIS deployed in tanks containing field sediments from the four sites did not accumulate the synthetic steroids except for ethinylestradiol, which was detected in the aquarium containing sediments collected near the wastewater treatment plant. Fish deployed in Sand Creek and at the wastewater treatment plant experienced significantly elevated levels of gene expression for two genes (StAR and P450scc) relative to those deployed in Fisher Creek. Fish exposed to the sediments collected from Sand Creek had significantly higher levels of hepatic StAR and P450scc gene expression than did fish exposed to sediments from the two other field sites, as well as the no-sediment control tank. In conclusion: 1) detectable levels of steroidogenic compounds were detected in passive samplers deployed in the Elkhorn River, 2) sediments do not appear to be a significant source for steroidogenic compounds, and 3) site-specific differences were found in mRNA expression among the different treatment groups of fish; however, a functional explanation for these differences is not readily forthcoming.

Lack of vasoactive intestinal peptide reduces testosterone levels and reproductive aging in mouse testis

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide have long been considered as putative regulators of testicular functions. In vitro evidence suggests that VIP could play an important role in testosterone biosynthesis. However, the endogenous role of VIP on testicular functions remained to be demonstrated. In C57BL/6 mice exhibiting a complete disruption of the VIP gene, we first observed here that serum testosterone levels were lower than those of WT littermates. At the age of 4 months, this phenotype was accompanied with a reduction of expression of StAR and 3-beta-hydroxysteroid dehydrogenase (3beta-HSD) in the testis. In addition, serum levels of FSH but not LH were reduced in young knock-out (KO) males. Testicular anatomy also revealed a higher percentage of degenerated seminiferous tubules in the 4-month-old VIP KO animals when compared with WT. In 15-month-old animals, control males showed typical testicular aging, including a severe degeneration of seminiferous tubules, a dramatic decrease in serum levels of testosterone, and a reduction in StAR and 3beta-HSD gene expression. In age-matching VIP KO males, the levels of serum testosterone and steroidogenic enzymes were still very low. Interestingly, in contrast to that observed in young mice, testicular degeneration at 15 months was significantly less severe in VIP KO than WT mice. All together, these results suggest that 1) VIP is an important factor for regulating testosterone biosynthesis and FSH secretion and 2) VIP may influence testicular aging.

Endothelial cell-mediated regulation of aldosterone release from human adrenocortical cells

Endothelial cells play an important role in the development and functioning of endocrine tissue and endothelial cell-derived factors have been shown to regulate mineralocorticoid release in bovine adrenal cells. In the present study, we analysed the role of human endothelial cells in the synthesis and release of aldosterone from adrenocortical cells (NCI-H295R). Endothelial cell-induced aldosterone release was rapid and lasted as a long-term effect over a period of 48h. This stimulant effect was influenced by the duration of endothelial cell conditioning and decreased linearly with increasing dilutions of the conditioned medium. At the molecular level, an increase in the mRNA transcripts of aldosterone synthase and StAR could be observed. Cellular interaction with endothelial cell-factors enhanced the activation of CRE, and the promoter activity of both StAR and SF-1 reporter genes. In conclusion, human endothelial cells are important intra-adrenal regulators of human aldosterone synthesis and release.

Dephosphorylation of TORC initiates expression of the StAR gene

Cyclic AMP responsive element (CRE) binding protein (CREB) is known to activate transcription when its Ser133 is phosphorylated. However, transducer of regulated CREB activity (TORC), a CREB specific co-activator, upregulates CREB activity in a phospho-Ser133-independent manner. Interestingly, TORC is also regulated by phosphorylation; the phospho-form is inactive, and the dephospho-form active. When PKA phosphorylates CREB, it inhibits TORC kinases simultaneously and accelerates dephosphorylation of TORC. We show in this report that staurosporine, a kinase inhibitor, induces the expression of the StAR gene in Y1 adrenocortical cells, possibly a result of an increase in the population of dephospho-TORC. The expression of the StAR gene is known to be regulated by SF-1 and CREB, and the co-activators CBP/p300 may mediate the actions of both factors. Our experiments using KG501, a disruptor of the interaction between phospho-CREB and CBP/p300, also support the importance of TORC in the regulation of StAR gene expression.

Protein-Protein Interactions Mediate Mitochondrial Cholesterol Transport and Steroid Biosynthesis

Transport of cholesterol into the mitochondria is the rate-determining, hormone-sensitive step in steroid biosynthesis. Here we report that the mechanism underlying mitochondrial cholesterol transport involves the formation of a macromolecular signaling complex composed of the outer mitochondrial membrane translocator protein (TSPO), previously known as peripheral-type benzodiazepine receptor; the TSPO-associated protein PAP7, which binds and brings to mitochondria the regulatory subunit RIalpha of the cAMP-dependent protein kinase (PKARIalpha); and the hormone-induced PKA substrate, steroidogenic acute regulatory protein (StAR). Hormone treatment of MA-10 Leydig cells induced the co-localization of TSPO, PAP7, PKARIalpha, and StAR in mitochondria, visualized by confocal microscopy, and the formation in living cells of a high molecular weight multimeric complex identified using photoactivable amino acids. The hormone-induced recruitment of exogenous TSPO in this complex was found to parallel the increased presence of 7-azi-5alpha-cholestan-3beta-ol in the samples. Co-expression of Tspo, Pap7, PkarIalpha, and Star genes resulted in the stimulation of steroid formation in both steroidogenic MA-10 and non-steroidogenic COS-F2-130 cells that were engineered to metabolize cholesterol. Disruption of these protein-protein interactions and specifically the PKARIalpha-PAP7 and PAP7-TSPO interactions, using PAP7 mutants where the N0 area homologous to dual A-kinase-anchoring protein-1 or the acyl-CoA signature motif were deleted or using the peptide Ht31 known to disrupt the anchoring of PKA, inhibited both basal and hormone-induced steroidogenesis. These results suggest that the initiation of cAMP-induced protein-protein interactions results in the formation of a multivalent scaffold in the outer mitochondrial membrane that mediates the effect of hormones on mitochondrial cholesterol transport and steroidogenesis.

Lipoid adrenal hyperplasia caused by earlier unknown 21STAR gene mutations

Lipoid adrenal hyperplasia (LAH) is a most severe type of congenital adrenal cortical dysfunction (CACD). In this type of CACD, there is defect in the conversion of cholesterol to pregnenolone, as a consequence the production of all steroid hormones was impaired in both the adrenals and gonads. Defects of the STAR gene encoding for a steroidogenic acute regulatory (StAR) protein underlie the disease in most cases. Until the present time, there have been no reports on cases of LAH in the Russian literature. The diagnosis of LAH was established by the authors in three genetic girls aged 2.3 years, 6 and 7 months who had a normal structure of the external genitalia and in whom the disease was marked by manifestations of primary hypoadrenocorticism at the age of 21, 2, and 10 days, respectively. A hormonal study failed to show elevated levels of Cortisol and 17-hydroxyprogesterone in response to adrenocorticotropic hormone stimulation. A molecular genetic study revealed the following STAR gene defects: P129AC/W250X, IVS5-1G, and W147X, respectively. Р129ЛС, IVSS, and W147X mutations have not been earlier described. The data of the observation emphasizes the need of including LAH into the algorithm of differential diagnosis of CACD in children with the normal female structure of the external genitalia. The molecular genetic analysis should be prominent in diagnosing LAH, by taking into account the lack of hormonal markers of the disease.

Previtellogenic Oocyte Growth in Salmon: Relationships among Body Growth, Plasma Insulin-Like Growth Factor-1, Estradiol-17beta, Follicle-Stimulating Hormone and Expression of Ovarian Genes for Insulin-Like Growth Factors, Steroidogenic-Acute Regulatory Protein and Receptors for Gonadotropins, Growth Hormone, and Somatolactin1

Body growth during critical periods is known to be an important factor in determining the age of maturity and fecundity in fish. However, the endocrine mechanisms controlling oogenesis in fish and the effects of growth on this process are poorly understood. In this study interactions between the growth and reproductive systems were examined by monitoring changes in various components of the FSH-ovary axis, plasma insulin-like growth factor 1 (Igf1), and ovarian gene expression in relation to body and previtellogenic oocyte growth in coho salmon. Samples were collected from females during two hypothesized critical periods when growth influences maturation in this species. Body growth during the fall-spring months was strongly related to the degree of oocyte development, with larger fish possessing more advanced oocytes than smaller, slower growing fish. The accumulation of cortical alveoli in the oocytes was associated with increases in plasma and pituitary FSH, plasma estradiol-17beta, and ovarian steroidogenic acute regulatory protein (star) gene expression, whereas ovarian transcripts for growth hormone receptor and somatolactin receptor decreased. As oocytes accumulated lipid droplets, a general increase occurred in plasma Igf1 and components of the FSH-ovary axis, including plasma FSH, estradiol-17beta, and ovarian mRNAs for gonadotropin receptors, star, igf1, and igf2. A consistent positive relationship between plasma Igf1, estradiol-17beta, and pituitary FSH during growth in the spring suggests that these factors are important links in the mechanism by which body growth influences the rate of oocyte development.

Adrenocorticotropin-Dependent Changes in SF-1/DAX-1 Ratio Influence Steroidogenic Genes Expression in a Novel Model of Glucocorticoid-Producing Adrenocortical Cell Lines Derived from Targeted Tumorigenesis

We established the first adrenocortical tumor cell lines with complete zona fasciculata (ZF) cell phenotype from tumors induced in transgenic mice by large T-antigen of simian virus 40 under the control of the aldose reductase-like akr1b7 gene promoter. Adrenocortical tumor cell lines produced high amounts of corticosterone and were responsive to ACTH. All genes that are supportive for glucocorticoid synthesis including cyp21a1 and cyp11b1 were expressed, and most of them were transiently up-regulated by ACTH at transcriptional level: stimulation culminated after 3-6 h and returned to basal levels after 24 h. Taking advantage of these cells, we have examined the effect of ACTH on DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on X-chromosome, gene 1) and SF-1 (steroidogenic factor 1), two transcription factors known to respectively repress and activate adrenocortical steroidogenesis by acting on common target genes. According to their antagonistic activities, DAX-1 mRNA and protein levels were transiently down-regulated by ACTH, whereas those of SF-1 were stimulated, with kinetics paralleling those of steroidogenic genes expression, notably of two known SF-1 target genes, star and akr1b7. This suggests an essential role of SF-1/DAX-1 proteins ratio to achieve proper ACTH control of steroidogenic gene expression in cells derived from ZF. This was confirmed in mice adrenals, where repression of dax-1 gene and concomitant up-regulation of sf-1, star, and akr1b7 genes were observed in response to ACTH stimulation. In conclusion, using both unique differentiated cell lines and in vivo approaches, we provide the first evidence that hormonally induced changes in SF-1/DAX-1 ratio are part of the molecular arsenal of ZF cells to fine tune ACTH responsiveness.

Replacement of Alanine with Asparagic Acid at Position 203 in Human Steroidogenic Acute Regulatory Protein Impairs the Ability to Enhance Steroidogenesis in vitro

Steroidogenic acute regulatory protein (StAR) is a 30-kDa phosphorylated protein that rapidly appears in mitochondria of steroidogenic cells following tropic stimulation, and is required in the acute regulation of steroidogenesis. It was reported that mutations in the STAR gene encoding StAR cause congenital lipoid adrenal hyperplasia (CLAH), an autosomal recessive disorder characterized by impaired synthesis of all adrenal and gonadal steroid hormones. We previously reported a D203A polymorphism in the STAR gene in Japanese patients with CLAH as well as in normal Japanese subjects. In the present study, we analyzed the ability of the A203 StAR and D203 StAR to stimulate steroidogenesis using the in vitro functional expression system. The A203 StAR caused a twelve-fold increase in pregnenolone secretion over COS-1 cells transfected with an NH2-cholesterol side-chain cleavage enzyme (P450scc)-adrenodoxin reductase-adrenodoxin-COOH fusion protein expressing plasmid (F2) and an empty vector, whereas the D203 StAR increased pregnenolone production no more than threefold. Western blot analysis detected mainly two species of StAR consisting of the 37-kDa precursor and the 30-kDa mature form. Together, these results indicate that the alanine at position 203 in human StAR is functionally important and that the D203 StAR is extremely unlikely to be a polymorphism.

Involvement of Protein Kinase C and Cyclic Adenosine 3′,5′-Monophosphate-Dependent Kinase in Steroidogenic Acute Regulatory Protein Expression and Steroid Biosynthesis in Leydig Cells1

This study investigated the roles of the protein kinase C (PKC) and protein kinase A (PKA) pathways in regulating constitutive steroidogenesis and steroidogenic acute regulatory (STAR; herein designated by its common name, StAR) protein in R2C Leydig tumor cells. Inhibition of PKC and phospholipase C resulted in significant decreases in steroid production, phosphorylation of cAMP-responsive element binding (CREB) protein, and Star gene transcription under basal conditions in R2C cells. These observations were corroborated in MA-10 and mLTC-1 Leydig tumor cell lines, in which activation of PKC by phorbol-12-myristate-13-acetate (PMA, 10 nM) increased CREB phosphorylation and total StAR (tot-StAR) protein expression. However, induction of StAR protein by PMA did not result in the expected concomitant increase in steroids because PKC failed to phosphorylate StAR, the biologically active form of the protein. However, in conjunction with PMA, minor increases in PKA activity using submaximal doses of (Bu)2cAMP (0.05-0.1 mM; a concentration range insufficient for induction of StAR), were able to stimulate dramatic increases in both phospho-StAR (P-StAR) and steroid production. Human chorionic gonadotropin stimulation also resulted in a further enhancement in P-StAR and progesterone production when added to PMA-treated MA-10 cells. Similar results for tot-StAR and P-StAR expression were observed in primary cultures of immature rat Leydig cells treated with PMA and submaximal doses of (Bu)2cAMP. In summary, the present study demonstrates that basal activities of both PKC and PKA play important roles in the constitutive steroidogenic characteristics of R2C cells. This study also demonstrates for the first time a role for PMA-induced PKC in StAR protein regulation and the requirement for submaximal doses of cAMP to produce steroids in Leydig cells.

Genetic Interactions of Drosophila melanogaster arrest Reveal Roles for Translational Repressor Bruno in Accumulation of Gurken and Activity of Delta

arrest mutants have pleiotropic phenotypes, ranging from an early arrest of oogenesis to irregular embryonic segmentation defects. One function of arrest is in translational repression of oskar mRNA; this biochemical activity is presumed to be involved in other functions of arrest. To identify genes that could provide insight into how arrest contributes to translational repression or that may be targets for arrest-dependent translational control, we screened deficiency mutants for dominant modification of the arrest phenotype. Only four of the many deficiencies tested, which cover approximately 30% of the genome, modified the starting phenotype. One enhancer, identified fortuitously, is the Star gene. Star interaction with arrest results in excess Gurken protein, supporting the model that gurken is a target of repression. Two modifiers were mapped to individual genes. One is Lk6, which encodes a protein kinase predicted to regulate the rate-limiting initiation factor eIF4E. The second is Delta. The interaction between arrest and Delta mimics the phenotype of homozygous Delta mutants, suggesting that arrest could positively control Delta activity. Indeed, arrest mutants have significantly reduced levels of Delta protein at the interface of germline and follicle cells.

Characterization of gene expression in mucinous cystic neoplasms of the pancreas using oligonucleotide microarrays.

Mucinous cystic neoplasms (MCNs) of the pancreas are uncommon neoplasms usually located in the body or tail of the pancreas and usually in females (>90% of cases). Clinically, they are often misdiagnosed as non-neoplastic pseudocysts leading to failed opportunities for curative resection. To better understand the biology of MCNs and to identify markers of the disease, we performed global gene expression profiling of MCNs using oligonucleotide microarrays. Using laser capture microdissection applied to frozen sections, RNA was extracted from the neoplastic epithelium of MCNs, from the adjacent 'ovarian-type' stroma of MCNs, from histologically normal pancreatic ductal epithelium, from pancreatic acinar tissue and from fibrous stroma in pancreata affected by chronic pancreatitis. Each RNA sample was subjected to two rounds of linear amplification followed by hybridization with U133A gene chips (Affymetrix). The expression patterns of selected genes were confirmed by quantitative RT-PCR and by immunohistochemistry using tissue microarrays containing 19 resected MCNs. A total of 114 known genes were overexpressed in the neoplastic epithelium compared to normal pancreatic ductal epithelium (>3-fold) including S100P, PSCA, c-myc, STK6/STK15, cathepsin E and pepsinogen C. Activation of the Notch pathway in the epithelial component of MCNs was evident by the demonstration of overexpression of Jagged1 and the downstream Notch pathway member Hes1. In the 'ovarian-type' stroma, several genes involved in estrogen metabolism were overexpressed including STAR and ESR1 genes. Some of the genes identified as overexpressed in these neoplasms may be useful as markers that can distinguish MCNs from non-neoplastic pancreatic cystic lesions.

A Family of Two Patients with Congenital Lipoid Adrenal Hyperplasia Due to StAR Mutation

We are reporting the case of two sisters born to nonrelated French Canadian parents. Patient A is of female phenotype with 46,xy, and patient B with 46,xx. The children had severe manifestations of mineralocorticoid deficiency at the age of 11 and 4.5 months, respectively. Residual cortisol secretion seemed present until the age of 3 years for patient A and until 15 months in the case of her sister. Both patients responded to glucocorticoid and Florinef treatment. Patient A did not show any androgen secretion and gonadectomy was performed at the age of 13.4 years; estrogen therapy was started at the age of 14 years resulting in a good breast development and an increase of growth velocity. In patient B, a progressive development of secondary sex characters occurred at 11.6 years of age followed at 14 years by menarche associated with a normal secretion of LH, FSH and estradiol; regular menstruations continued up to her last visit at the age of 25 years. We identified a homozygous L275P mutation on the StAR gene of both patients and a heterozygous L275P mutation on that of their mother and father. In transfection analysis in COS‐1 cells, the mutant L275P was well‐expressed, but its StAR activity was 87% impaired. The remaining activity of the L275P StAR mutant is consistent with the moderate severity of clinical onset of manifestations.