Quantitation of spatial and temporal variability of biomarkers for Barrett's Esophagus.

Chemoprevention and risk-stratification studies in Barrett's esophagus (BE) rely on biomarkers but the variability in their temporal and spatial expression is unknown. If such variability exists, it will impact sampling techniques and sample size calculations. Specimens from three levels of biopsies over two serial endoscopies in nondysplastic BE patients were analyzed for aneuploidy, proliferation markers (Ki67, Mcm2), and cell cycle markers (cyclin A and cyclin D1). A modification of the image cytometry technique, where cytokeratin staining automatically distinguished epithelial and stromal cells, measured aneuploidy on whole tissue sections. Other biomarkers were studied by immunohistochemistry. Coefficient of variability (SD/mean) was calculated; a value <10% indicated low variability. A total of 120 specimens (20 subjects each with three biopsy levels at two time points) from nondysplastic BE patients (71 ± 8.8 years, all Caucasian, 90% males, C5.1M7.5 ± 3.4 cm) were analyzed. The mean interval between endoscopies was 32.8 ± 8.4 months. Aneuploidy had a spatial variability of 6.8% at visit 1 (mean diploid index: 1.1 ± 0.09) and 7.9% at visit 2 (mean diploid index: 1.1 ± 0.06) and a temporal variability of 7.0-8.1% for the three levels. For other biomarkers, the spatial variability ranged from ∼5 to 30% at visit 1 and 11-92% at visit 2 and the temporal variability ranged from 0 to 77%. To conclude, of all the biomarkers, only aneuploidy had both spatial and temporal variability of <10%. Spatial and temporal variability were biomarker dependent and could be as high as 90% even without progression. These data will be useful to design chemoprevention and risk-stratification studies in BE.

Feasibility of mcroRNAs as biomarkers for Barrett's Esophagus progression: a pilot cross-sectional, phase 2 biomarker study.

OBJECTIVES: Risk stratification of Barrett's esophagus (BE) using biomarkers remains an important goal. We evaluated feasibility and clinical accuracy of novel microRNA (miRNA) biomarkers for prediction of BE dysplasia. METHODS: Paired fresh-frozen and hematoxylin/eosin specimens from a prospective tissue repository where only biopsies with the lesion of interest (i.e., intestinal metaplasia (IM) or high-grade dysplasia (HGD)/esophageal adenocarcinoma (EAC)) occupying >50% of biopsy area were included. Tissue miRNA expression was determined by microarrays and validated by quantitative reverse transcription-PCR (qRT-PCR). Three groups were compared-group A, IM tissues from BE patients without dysplasia; group B, IM tissues from group C patients; and group C, dysplastic tissues from BE patients with HGD/EAC. RESULTS: Overall, 22 BE patients, 11 with and without dysplasia (mean age 64 ± 8.2 and 63 ± 11.6 years, respectively, all Caucasian males) were evaluated. Nine miRNAs were identified by high-throughout analysis (miR-15b, -21, -192, -205, 486-5p, -584, -1246, let-7a, and -7d) and qRT-PCR confirmed expression of miR-15b, -21, 486-5p, and let-7a. Two of 4 miRNAs (miR-145 and -203, but not -196a and -375) previously described in BE patients also exhibited differential expression. Sensitivity and specificity of miRNAs for HGD/EAC were miR-15b: 87 and 80%, miR-21: 93 and 70%, miR-203: 87 and 90%, miR-486-5p: 82 and 55%, and miR-let-7a: 88 and 70%. MiRNA-15b, -21, and -203 exhibited field effects (i.e., groups A and B tissues while histologically similar yet exhibited different miRNA expression). CONCLUSIONS: This pilot study demonstrates feasibility of miRNAs to discriminate BE patients with and without dysplasia with reasonable clinical accuracy. However, the specific miRNAs need to be evaluated further in future prospective trials.

MicroRNA control of ovarian function.

Post-transcriptional gene regulation, a regulatory mechanism classically involved in female and male germ cell function has recently been implicated in control of somatic cells of the ovary and testis. Recent advancements in this field may be attributed primarily to the discovery and study of microRNAs (miRNA), small RNA transcripts that can influence mRNA expression via post-transcriptional gene regulatory mechanisms. In the ovary, targeted deletion of Dicer 1, a key enzyme in miRNA biogenesis, provided the first empirical evidence that miRNA/siRNA were critically involved in multiple aspects of ovarian function (folliculogenesis, oocyte maturation, ovulation, and luteal function). Functional studies of miRNA in the ovary have mostly focused on granulosa cells during the critical period of the ovarian cycle surrounding the ovulatory surge of luteinizing hormone (LH). Specific miRNA have been implicated in ovarian responses, due to their transcriptional induction by the LH surge (i.e., miR-21, -132 and -212) or through bioinformatic approaches (miR-224, -17-5p and let-7b). Numerous other miRNA are highly abundant in ovarian somatic tissues, suggesting that we have much to discover with respect to the role of miRNA and regulation of ovarian function. This review will recap the key observations of these early studies and provide insight into future experiments that might further our understanding of ovarian function.

Differentiation-induced post-transcriptional control of B7-H1 in human trophoblast cells.

Trophoblast expression of immunomodulatory proteins in the human placenta is among the mechanisms that are critical for ensuring lymphocyte tolerance to the semi-allogeneic fetus. High levels of B7-H1 on trophoblast cells together with the known role of this protein in establishment of peripheral tolerance suggest that B7-H1 mediates immunological protection of the placenta during gestation. In this study, we investigated the molecular mechanisms of regulation of B7-H1 in trophoblast cells by epidermal growth factor (EGF), a key regulator of trophoblast cell differentiation. EGF increased B7-H1 protein levels within 24 h and mRNA levels within 4h of the initiation of treatment; by 24 h B7-H1 mRNA levels were similar between control and EGF-treated cells. Analysis of two different potential promoter regions revealed strong promoter activity in response to IFN-gamma. In contrast, no promoter activity could be induced by EGF, suggesting that this cytokine regulates B7-H1 expression post-transcriptionally in trophoblast cells. EGF-induced B7-H1 protein expression was completely blocked in the presence of inhibitors of the PI3Kinase/Akt/mTOR pathway, a pathway known to regulate gene expression at the translational level. Finally, analysis of monosomal and polysomal mRNA fractions of untreated and EGF-treated term trophoblast cells revealed that EGF induces a shift towards the translatable fractions and away from the untranslated fractions. These results highlight a novel mechanism for regulation of B7 family proteins in the placenta.

MicroRNA in the ovary and female reproductive tract.

Posttranscriptional gene regulation plays a vital role in male and female germ cell function, but our understanding of this regulatory process in somatic cells and its effect on reproductive tissue development and function is not understood. In mammalian cells, microRNA (miRNA) are key posttranscriptional regulators and function by modulating translation or degradation of their target mRNA. Mature miRNA are synthesized through a multi-step process that concludes with the cleavage of stem-loop pre-miRNA by the RNase III enzyme, Dicer1. To determine the extent of miRNA regulation and establish a baseline, miRNA profiling has indicated the presence of large numbers of miRNA within reproductive tissues and cells. Moreover, several studies have indicated that miRNA expression in reproductive tissues varies in response to pituitary and gonadal hormones. To understand the role that miRNA-mediated posttranscriptional gene regulation plays in female reproduction, a global Dicer1 hypomorph mouse and several tissue-specific Dicer1 knockout mice have been studied. Interestingly, when Dicer1 expression is decreased in reproductive tissues or cells, the females are infertile. This review discusses all the work regarding miRNA regulation within the mammalian female reproductive system published to date.

Aberrant cholesterol transport and impaired steroidogenesis in Leydig cells lacking estrogen sulfotransferase.

Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes the sulfoconjugation and inactivation of estrogens. It is expressed abundantly in the mammalian testes in which it may modulate the activity of locally produced estrogen. We demonstrate here that testicular Leydig cells from mice rendered deficient in EST expression by targeted gene deletion acquire a phenotype of increased cholesterol ester accumulation and impaired steroidogenesis with natural aging or in response to estrogen challenge. Abnormal accumulation of cholesterol ester in the mutant Leydig cells correlated with induced expression of the scavenger receptor type B class I, and cultured EST-deficient but not wild-type Leydig cells avidly uptook high-density lipoprotein cholesterol ester ex vivo. EST-deficient Leydig cells in culture produced 50-70% less testosterone than wild-type cells. This deficiency was reversed by androstenedione but not progesterone supplementation, indicating that reduced activities of 17-alpha-hydroxylase-17, 20-lyase were responsible. This conclusion was corroborated by decreased expression levels of 17-alpha-hydroxylase-17, 20-lyase but not of other key steroidogenic enzymes in the mutant cells. These results suggest that EST plays a physiologic role in protecting Leydig cells from estrogen-induced biochemical lesions and provide an example of critical regulation of tissue estrogen sensitivity by a ligand-transformation enzyme rather than through estrogen receptors.

Steroidogenic acute regulatory protein: an update on its regulation and mechanism of action.

Steroidogenic acute regulatory (StAR) protein controls the rate-limiting step in steroidogenesis: the transport of cholesterol from the outer to the inner mitochondrial membrane. Early studies indicated that rate of transcription of the StAR gene is a primary determinant of steroidogenesis. The transcription factors that govern basal and cAMP-dependent StAR expression are reviewed, as are new findings regarding chromatin modifications associated with activation of the StAR promoter. Molecular genetic studies of congenital lipoid adrenal hyperplasia, a rare disease caused by mutations in the StAR gene, and structure-function studies defined two major domains within the StAR protein, the N-terminal mitochondrial targeting sequence and the C-terminal StAR-related lipid transfer (START) domain, which promotes the translocation of cholesterol between the two mitochondrial membranes. Several models of StAR's mechanism of action have been proposed based on a combination of structure/function studies or on the crystal structure of a related START domain. The models-intermembrane shuttle hypothesis, and cholesterol desorption hypothesis-are discussed with respect to the known biochemical and biophysical events associated with steroidogenesis and the structure of StAR.

Expression of steroidogenic acute regulatory protein in the human corpus luteum throughout the luteal phase.

The expression of the steroidogenic acute regulatory protein (StAR) in the human corpus luteum (CL) was examined throughout the luteal phase. The primary 1.6-kb StAR transcript was in greater abundance in early (3.1-fold) and mid (2.2-fold) luteal phase CL compared with late luteal phase CL. The larger StAR transcript (4.4 kb) was found in early and midluteal phase CL, but was not detected in late luteal phase specimens. Mature StAR protein (30 kDa) was present in lower amounts within late CL compared with early and midluteal phase CL. The StAR preprotein (37 kDa) was also detected in greater abundance in early and midluteal CL. Immunohistochemistry revealed that StAR staining was most prominent in thecal-lutein cells throughout the luteal phase. The intensity of the signal for StAR exhibited significant changes throughout the luteal phase, being most intense during the midluteal phase and least during the late luteal phase. Plasma progesterone concentrations were highly correlated (r = 0.73 and r = 0.79) with luteal expression of the preprotein and mature StAR isoforms, respectively, throughout the luteal phase. To examine the LH dependency of StAR expression, the GnRH antagonist, Cetrorelix, was administered during the midluteal phase. Cetrorelix caused a decline in serum LH levels within 2 h, which, in turn, caused a pronounced decline in plasma progesterone within 6 h. The StAR 4.4-kb transcript was not detectable, and the 1.6-kb transcript was reduced by approximately 50% within 24 h of Cetrorelix treatment. The mature 30-kDa StAR protein level declined approximately 30% after Cetrorelix treatment. We conclude that 1) StAR mRNA and protein are highly expressed in early and midluteal phase CL; 2) StAR protein is present in both thecal-lutein and granulosa-lutein cells throughout the luteal phase; 3) StAR protein levels in the CL are highly correlated with plasma progesterone levels; 4) declining StAR mRNA and protein levels are characteristic of late luteal phase CL; and 5) suppression of LH levels during the midluteal phase results in a marked decline in plasma progesterone and a diminished abundance of StAR transcripts in the CL without a corresponding significant decline in StAR protein. Collectively, these data are consistent with the idea that StAR gene expression is a key determinant of luteal progesterone during the normal menstrual cycle. However, the pharmacologically induced withdrawal in the midluteal phase of LH support diminishes luteal progesterone output by mechanisms others than reduced StAR protein levels.

Quantitative analysis of the hormone-induced hyperacetylation of histone H3 associated with the steroidogenic acute regulatory protein gene promoter.

Transcriptional regulation of steroidogenic acute regulatory protein (StAR) determines adrenal and gonadal cell steroidogenesis. Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with the StAR promoter. MA-10 cells treated with 8-bromo-cAMP had increased acetylated histone H3 associated with the proximal (but not distal) StAR promoter, nascent StAR transcripts, and progesterone production within 15 min, whereas StAR mRNA increased at 30 min. At 360 min, steroidogenesis remained elevated, but mRNA, nascent RNA, and StAR promoter-associated H3 acetylation all declined. StAR promoter-associated H4 acetylation was unchanged by 8-bromo-cAMP treatment of MA-10 cells. In vivo analysis of macaque and human granulosa cells showed that luteinization was associated with increased StAR promoter-associated H3 acetylation. We conclude that acetylation of H3 (but not H4) associated with the proximal promoter is associated with StAR gene transcription, that chromatin modification occurs in discrete regions of the promoter, that the initial steroidogenic response to 8-bromo-cAMP occurs prior to increased StAR mRNA accumulation, and that MA-10 cell StAR gene transcription and promoter-associated H3 acetylation are biphasic during a 6-h treatment period. The union of the chromatin immunoprecipitation assay with quantitative real-time polymerase chain reaction described and validated here should enhance the analysis of gene expression.

Conditional response of the human steroidogenic acute regulatory protein gene promoter to sterol regulatory element binding protein-1a.

The steroidogenic acute regulatory protein (StAR) gene controls the rate-limiting step in the biogenesis of steroid hormones, delivery of cholesterol to the cholesterol side-chain cleavage enzyme on the inner mitochondrial membrane. We determined whether the human StAR promoter is responsive to sterol regulatory element-binding proteins (SREBPs). Expression of SREBP-1a stimulated StAR promoter activity in the context of COS-1 cells and human granulosa-lutein cells. In contrast, expression of SREBP-2 produced only a modest stimulation of StAR promoter activity. One of the SREBP-1a response elements in the StAR promoter was mapped in deletion constructs and by site-directed mutagenesis between nucleotides -81 to -70 from the transcription start site. This motif bound recombinant SREBPs in electrophoretic mobility shift assays, but with lesser affinity than a low density lipoprotein receptor SREBP-binding site. An additional binding site for the transcriptional modulator, yin yang 1 (YY1), was observed within the SREBP-binding site (nucleotides -73 to -70). Mutation of the YY1-binding site increased the responsiveness of the StAR promoter to exogenous SREBP-1a, but did not alter the affinity for SREBP-1a binding in electrophoretic mobility gel shift assays. Manipulations that altered endogenous mature SREBP-1a levels (e.g. culture in lipoprotein-deficient medium and addition of 27-hydroxycholesterol) did not affect StAR promoter function, but influenced low density lipoprotein receptor promoter activity. We conclude that 1) the human StAR promoter is conditionally responsive to SREBP-1a such that promoter activity is up-regulated in the presence of high levels of SREBP-1a, but is unaffected when mature SREBP levels are suppressed; and 2) the human StAR promoter is selectively responsive to SREBP-1a.

Steroidogenic acute regulatory protein (StAR) and the intramitochondrial translocation of cholesterol.

The steroidogenic acute regulatory (StAR) protein regulates the rate limiting step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane. Insight into the structure and function of StAR was attained through molecular genetic studies of congenital lipoid adrenal hyperplasia, a rare disease caused by mutations in the StAR gene. Subsequent functional analysis defined two major domains within the StAR protein, the N-terminal mitochondrial targeting sequence and the C-terminus, which promotes the translocation of cholesterol between the two mitochondrial membranes. Two models of StAR's mechanism of action, (1) stimulation of cholesterol desorption from the outer mitochondrial membrane and (2) an intermembrane shuttle hypothesis, are discussed with respect to the known biochemical and biophysical events associated with the process of steroidogenesis and the structure of StAR. StAR gene expression is regulated primarily at the transcriptional level, and the roles of transcription factors that govern basal and cAMP-dependent StAR expression including SF-1, C/EBP beta, Sp1 and GATA-4 are reviewed.

Changes in expression of vascular endothelial growth factor and angiopoietin-1 and -2 in the macaque corpus luteum during the menstrual cycle.

To determine the temporal expression of vascular growth factors during the lifespan of the primate corpus luteum, experiments were designed to detect mRNA for vascular endothelial growth factor (VEGF), angiopoietin (Ang)-1 and Ang-2 and to localize protein expression for VEGF in macaque luteal tissue during the menstrual cycle. Corpora lutea (n = 3-5/stage) were collected during the early (3-5 days post-luteinizing hormone surge), mid- (6-8 days), mid-late (10-12 days), and late (14-16 days) luteal phase and at menstruation (17-18 days). Reverse transcription-polymerase chain reaction products equated to cDNA for VEGF, Ang-1 and Ang-2 in all corpora lutea. VEGF mRNA levels increased (P: < 0.05) from early to mid-luteal phase and declined in the late luteal phase and at menstruation. Immunostaining for VEGF was detected in the cytoplasm of steroidogenic luteal cells, with the most intense staining in the early luteal phase. Ang-1 and Ang-2 mRNA expression was low in the early to mid-luteal phase but increased (P: < 0.05) at late luteal phase before declining at menstruation. These data suggest transcriptional control of VEGF, Ang-1 and Ang-2, as well as post-transcriptional control of VEGF, in macaque corpus luteum. Dynamic expression of angiogenic/angiostatic factors appears critical for development, maintenance and regression of the luteal microvasculature during the menstrual cycle.

Providing progesterone for pregnancy: control of cholesterol flux to the side-chain cleavage system.

Progesterone, which is required to support human gestation, is derived initially from the corpus luteum and subsequently from the placenta. The rate-limiting step in progesterone synthesis is the delivery of cholesterol to the mitochondrial cholesterol side-chain cleavage system. The steroidogenic acute regulatory protein (StAR) mediates this process in the corpus luteum, whereas in the placenta, which does not express StAR, a StAR homologue, MLN64, may accomplish this function. StAR expression is regulated in the ovary at the transcriptional level by a cAMP-activated signal transduction system and StAR activity is also increased acutely by protein kinase A-mediated phosphorylation. These long-term (transcriptional) and short-term (post-translational, that is, phosphorylation) mechanisms govern luteal steroidogenic activity. The StAR protein has two key functional domains. The StAR C-terminal domain increases cholesterol movement to cytochrome P450scc by promoting sterol desorption from the sterol-rich outer mitochondrial membrane, driving it to the relatively sterol-poor inner membrane. The N-terminal domain mitochondrial targeting sequence directs the StAR protein to the mitochondria.

Endocrine and paracrine-autocrine regulation of the human corpus luteum during the mid-luteal phase.

Human corpora lutea undergo an extremely rapid period of growth, development and regression during the course of non-fertile cycles. The tissue consists of steroidogenic (parenchymal) and non-steroidogenic (stromal) cells. In women and other primates, steroid hormone production by corpora lutea depends on the presence of pituitary-derived LH. Nevertheless, there is also intra-luteal regulation of steroid synthesis. Steroidogenic luteal cells and non-steroidogenic cells interact via endocrine and paracrine pathways, and by contact-dependent pathways (gap junctions). Thus, hormones and locally produced factors including steroids, growth factors, cytokines, reactive oxygen species and nitric oxide may modulate luteal lifespan. The factors regulating regression and rescue of the corpus luteum are not understood completely. This review describes the expression of two representative intragonadal peptides that may influence luteal regression (interleukin 1 beta) and luteal rescue (steroidogenic acute regulatory protein).

Allelic variants of the follistatin gene in polycystic ovary syndrome.

In an earlier study of 37 candidate genes for polycystic ovary syndrome (PCOS), the strongest evidence for genetic linkage was found with the region of the follistatin gene. We have now carried out studies to detect variation in the follistatin gene and assess its relevance to PCOS. By sequencing the gene in 85 members of 19 families of PCOS patients, we found sequence variants at 17 sites. Of these, 16 sites have variants that are too rare to make a major contribution to susceptibility; the only common variant is a single base pair change in the last exon at a site that is not translated. In our sample of 249 families, the evidence for linkage between PCOS and this variant is weak. We also examined the expression of the follistatin gene; messenger RNA levels in cultured fibroblasts from PCOS and control women did not differ appreciably. We conclude that contributions to the etiology of PCOS from the follistatin gene, if any, are likely to be small.

Insulin and insulin-like growth factor-I and -II modulate human granulosa-lutein cell steroidogenesis: enhancement of steroidogenic acute regulatory protein (StAR) expression.

Insulin and insulin-like growth factors (IGF)-I and -II stimulate granulosa cell steroidogenesis. Since steroidogenic acute regulatory protein (StAR) regulates the rate-limiting step in steroid hormone biosynthesis, the ability of insulin and IGF to modulate StAR protein and mRNA expression was examined in two human granulosa cell culture systems: (i) proliferating granulosa-lutein cells and (ii) luteinized-granulosa cells derived during in-vitro fertilization (IVF). In proliferating granulosa-lutein cells, IGF-I and IGF-II increased StAR protein approximately 4-5-fold, while insulin and 8-bromoadenosine 3',5'-cAMP (8-Br-cAMP) increased amounts of StAR protein 2.5- and 6-fold respectively. A combination of IGFs/insulin and 8-Br-cAMP increased StAR 7-9-fold. Luteinized granulosa cells also had increased StAR expression after treatment with IGF-I (2. 8-fold), IGF-II (3-fold), insulin (2.5-fold) and 8-Br-cAMP (4. 5-fold). Progesterone production generally followed a pattern similar to StAR protein in both cell systems. In proliferating granulosa-lutein cells, both IGF-I and insulin increased StAR mRNA (3-fold) and 8-Br-cAMP increased StAR mRNA 4-fold, whereas a combination of IGF-I and 8-Br-cAMP had an additive effect on StAR mRNA expression (7-fold). Transient transfection of proliferating granulosa-lutein cells with StAR promoter-luciferase reporter constructs demonstrated that IGF-I, IGF-II, and insulin had no effect on the StAR promoter activity, while 8-Br-cAMP stimulated StAR promoter function. The results indicate that: (i) IGFs and insulin stimulate StAR mRNA and protein expression in human granulosa-lutein cells; (ii) IGF-I and 8-Br-cAMP have an additive effect on StAR gene expression; and (iii) IGF-I increases StAR mRNA and protein by a mechanism that does not involve activation of the proximal StAR gene promoter.

The steroidogenic acute regulatory protein (StAR): a window into the complexities of intracellular cholesterol trafficking.

Stimulation of steroid-producing cells of the gonads and adrenals with tropic hormone results in a marked increase in steroid hormone synthesis within minutes. The rate-limiting step in this acute steroidogenic response is the transport of cholesterol from the outer to the inner mitochondrial membrane, where the first committed step in steroid synthesis is performed by the side-chain cleavage enzyme system. This process of cholesterol translocation is blocked by inhibitors of protein synthesis, suggesting that the effect of trophic hormones, acting through the intermediacy of cAMP, most likely involves the de novo synthesis of a protein that is rapidly inactivated. The recently identified steroidogenic acute regulatory (StAR) protein appears to be the most likely candidate for the "labile" protein, based on the following observations: 1) Expression of StAR in COS-1 cells engineered to contain the cholesterol side-chain cleavage system substantially augments pregnenolone formation; 2) StAR protein is expressed almost exclusively in steroid-producing cells, except the trophoblast of the human placenta, and its presence is correlated with steroid hormone production; 3) StAR mRNA increases in response to cAMP; 4) StAR is a target for serine phosphorylation mediated by protein kinase A, a process that is essential for maximizing StAR activity; and 5) lack of functional StAR causes the autosomal recessive disease, congenital lipoid adrenal hyperplasia, characterized by markedly impaired gonadal and adrenal steroid hormone synthesis. Studies on the mechanism of action of StAR revealed that import into mitochondria is not essential to its steroidogenesis-enhancing activity and more likely represents a means of rapidly inactivating StAR. Truncation mutations and site-directed mutations established that the C-terminus of the StAR protein contains the functionally important domains. The demonstration of steroidogenic activity of recombinant StAR protein on isolated mitochondria from bovine corpus luteum using protein that lacks the mitochondrial targeting sequence confirmed that StAR import is not essential for its steroidogenic activity and suggested that StAR acts directly on the outer mitochondrial membrane in the absence of intermediary cytosolic factors. Evidence that StAR functions as a cholesterol transfer protein raises the possibility that StAR acts directly on lipids of the outer mitochondrial membrane, probably stimulating cholesterol desorption from the sterol-rich outer membrane and its movement to the relatively sterol-poor inner membrane.

CCAAT/enhancer-binding proteins regulate expression of the human steroidogenic acute regulatory protein (StAR) gene.

Two putative CCAAT/enhancer-binding protein (C/EBP) response elements were identified in the proximal promoter of the human steroidogenic acute regulatory protein (StAR) gene, which encodes a key protein-regulating steroid hormone synthesis. Expression of C/EBPalpha and -beta increased StAR promoter activity in COS-1 and HepG2 cells. Cotransfection of C/EBPalpha or -beta and steroidogenic factor 1, a transcription factor required for cAMP regulation of StAR expression, into COS-1 augmented 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP)-stimulated promoter activity. When the putative C/EBP response elements were mutated, individually or together, a pronounced decline in basal StAR promoter activity in human granulosa-lutein cells resulted, but the fold stimulation of promoter activity by 8-Br-cAMP was unaffected. Recombinant C/EBPalpha and -beta bound to the two identified sequences but not the mutated elements. Human granulosa-lutein cell nuclear extracts also bound these elements but not the mutated sequences. An antibody to C/EBPbeta, but not C/EBPalpha, supershifted the nuclear protein complex associated with the more distal element. The complex formed by nuclear extracts with the proximal element was not supershifted by either antibody. Western blot analysis revealed the presence of C/EBPalpha and C/EBPbeta in human granulosa-lutein cell nuclear extracts. C/EBPbeta levels were up-regulated 3-fold by 8-Br-cAMP treatment. Our studies demonstrate a role for C/EBPbeta as well as yet to be identified proteins, which can bind to C/EBP response elements, in the regulation of StAR gene expression and suggest a mechanism by which C/EBPbeta participates in the cAMP regulation of StAR gene transcription.

Oxysterol regulation of steroidogenic acute regulatory protein gene expression. Structural specificity and transcriptional and posttranscriptional actions.

Oxysterols exert a major influence over cellular cholesterol homeostasis. We examined the effects of oxysterols on the expression of steroidogenic acute regulatory protein (StAR), which increases the delivery of cholesterol to sterol-metabolizing P450s in the mitochondria. 22(R)-hydroxycholesterol (22(R)-OHC), 25-OHC, and 27-OHC each increased steroidogenic factor-1 (SF-1)-mediated StAR gene transactivation by approximately 2-fold in CV-1 cells. In contrast, cholesterol, progesterone, and the 27-OHC metabolites, 27-OHC-5beta-3-one and 7alpha,27-OHC, had no effect. Unlike our findings in CV-1 cells, SF-1-dependent StAR promoter activity was not augmented by 27-OHC in COS-1 cells, Y-1 cells, BeWo choriocarcinoma cells, Chinese hamster ovary (CHO) cells, and human granulosa cells. Studies examining the metabolism of 27-OHC indicated that CV-1 cells formed a single polar metabolite, 3beta-OH-5-cholestenoic acid from radiolabeled 27-OHC. However, this metabolite inhibited StAR promoter activity in CV-1, COS-1 and CHO cells. Because 7alpha,27-OHC was unable to increase SF-1-dependent StAR promoter activity, we examined 27-OHC 7alpha-hydroxylase in COS-1 and CHO cells. COS-1 cells contained high 7alpha-hydroxylase activity, whereas the enzyme was undetectable in CHO cells. The hypothesis that oxysterols act in CV-1 cells to increase StAR promoter activity by reducing nuclear levels of sterol regulatory element binding protein was tested. This notion was refuted when it was discovered that sterol regulatory element binding protein-1a is a potent activator of the StAR promoter in CV-1, COS-1, and human granulosa cells. Human granulosa and theca cells, which express endogenous SF-1, contained more than 5-fold more StAR protein following addition of 27-OHC, whereas StAR mRNA levels remained unchanged. We conclude that 1) there are cell-specific effects of oxysterols on SF-1-dependent transactivation; 2) the ability to increase transactivation is limited to certain oxysterols; 3) there are cell-specific pathways of oxysterol metabolism; and 4) oxysterols elevate StAR protein levels through posttranscriptional actions.

Unveiling the mechanism of action and regulation of the steroidogenic acute regulatory protein.

Stimulation of steroid-producing cells of the gonads and adrenals with trophic hormone (LH, and ACTH, respectively) produces a marked increase in steroid hormone synthesis within minutes. The rate-limiting step in this acute steroidogenic response is the transport of cholesterol from the outer to the inner mitochondrial membrane, where the first committed step in steroid synthesis is performed by the side-chain cleavage enzyme system (P450scc), resulting in the production of pregnenolone. This process of cholesterol translocation is blocked by inhibitors of protein synthesis (i.e. cycloheximide) indicating that the effect of trophic hormones, acting through the intermediacy of cAMP, most likely involves the de novo synthesis of a protein that is rapidly inactivated. The recently identified steroidogenic acute regulatory protein (StAR) appears to be the most likely candidate for the labile protein: (1) StAR is synthesized in response to cAMP and the StAR preprotein disappears rapidly in the presence of inhibitors of protein synthesis; (2) StAR has an N-terminal targeting sequence that directs the protein to the mitochondria; and (3) StAR protein is expressed almost exclusively in steroid-producing cells, its presence is correlated with steroid hormone production, and lack of functional StAR causes the autosomal recessive disease congenital lipoid adrenal hyperplasia (lipoid CAH), characterized by markedly impaired gonadal and adrenal steroid hormone synthesis. We have demonstrated that StAR is a target for serine phosphorylation mediated by protein kinase A (PKA), a process that is essential to maximizing StAR activity. StAR import by mitochondria is not essential to its steroidogenesis enhancing activity, and more likely, represents a means of rapidly inactivating StAR. Truncation mutations and site-directed mutations in StAR demonstrated that the C-terminus of the protein contains the functionally important domains. Further, we have demonstrated potent steroidogenic activity of recombinant StAR protein on isolated mitochondria from bovine corpus luteum using protein that lacks the mitochondrial targeting sequence. These observations confirm that StAR import is not essential for its steroidogenic activity and suggest that StAR acts directly on the outer mitochondrial membrane in the absence of intermediary cytosolic factors. More recently, we have found that StAR functions as a cholesterol transfer protein that does not require a protein receptor or co-factor, suggesting that StAR acts directly on lipids of the outer mitochondrial membrane to promote cholesterol translocation.