Characterization of the promoter of SF-1, an orphan nuclear receptor required for adrenal and gonadal development.

Steroidogenic factor 1 (SF-1) is a transcription factor shown to be critical for regulation of adrenal and gonadal development and function. To dissect the mechanisms that direct expression of this regulator, we have studied the promoter of the SF-1 gene and have identified cis-acting elements that recognize a basic-helix-loop-helix transcription factor; the CAAT binding factor; and Sp1. We demonstrate in Y1 adrenocortical cells that a 90-bp proximal promoter fragment is sufficient to direct steroidogenic-specific expression and that all three elements are required for activity of the SF-1 promoter. Functional analysis of the binding sites on a heterologous TATA box-containing promoter demonstrates that the CAAT box and Sp1 site are not essential for promoter activity when a TATA box is present, whereas the E box is absolutely required for gene expression and is most likely the steroidogenic cell-specific element. We also demonstrate that SF-1 itself does not significantly affect the transcription of its own gene, and thus conclude that the E box, CAAT box, and Sp1 site of the proximal promoter direct expression of the SF-1 gene.

Association between GSTM1*0 and squamous dysplasia of the esophagus in the high risk region of Linxian, China.

Individuals with specific phase I and phase II enzyme polymorphisms may be at increased risk for squamous cell carcinoma of the esophagus. However, to our knowledge there has been only one previous report that evaluates a potential role for these polymorphisms in increasing risk for preneoplastic squamous lesions of the esophagus. To explore this further, we examined polymorphisms in CYP1A1, CYP2E1, GSTM1 and GSTT1, both independently and in combination, for potential associations with the risk of biopsy-proven squamous dysplasia of the esophagus in asymptomatic adults from Linxian, a high risk region in China. Cases consisted of 56 individuals from an esophageal cancer screening study with an endoscopic biopsy diagnosis of mild or moderate squamous dysplasia. Each case was matched on age (+/- 1 year) and gender to a control. Controls were defined as screening study participants with an endoscopic biopsy diagnosis of normal mucosa or esophagitis. DNA was extracted from frozen cell samples obtained by cytologic balloon examination and genotyped using standard methods. Individuals who were GSTM1 null (homozygous for GSTM1*0) were found to have a tendency for an increased risk of esophageal squamous dysplasia (odds ratio=2.6, 95% CI, 0.9-7.4). No excess risks were observed for inheritance of other putative at risk genotypes CYP1A1*2B, CYP2E1*6 or GSTT1*0. The risk associated with the inheritance of combined genotypes was not significantly different than the risk estimates from the univariate analysis. These results are consistent with the notion that exposure to environmental carcinogens that are detoxified by GSTM1, such as polycyclic aromatic hydrocarbons, may contribute to the etiology of esophageal cancer in Linxian.

Identification of a unique epigenetic sub-microenvironment in prostate cancer.

The glutathione S-transferase P1 (GSTP1) gene promoter is methylated in tumour cells in more than 90% of prostate carcinomas. Recently, GSTP1 promoter methylation was identified in tumour-associated stromal cells in addition to the tumour epithelium. To define the extent and location of stromal methylation, epigenetic mapping using pyrosequencing quantification of GSTP1 promoter methylation and an anatomical three-dimensional reconstruction of an entire human prostate specimen with cancer were performed. Normal epithelium and stroma, tumour epithelium, and tumour-associated stromal cells were laser capture-microdissected from multiple locations throughout the gland. As expected, the GSTP1 promoter in both normal epithelium and normal stromal cells distant from the tumour was not methylated and the tumour epithelium showed consistently high levels of promoter methylation throughout. However, tumour-associated stromal cells were found to be methylated only in a localized and distinct anatomical sub-field of the tumour, revealing the presence of an epigenetically unique microenvironment within the cancer. Morphologically, the sub-field consisted of typical, non-reactive stroma, representing a genomic alteration in cells that appeared otherwise histologically normal. Similar epigenetic anatomical mapping of a control prostate gland without cancer showed low background methylation levels in all cell types throughout the specimen. These data suggest that stromal cell methylation can occur in a distinct sub-region of prostate cancer and may have implications for understanding tumour biology and clinical intervention.

Role of steroidogenic-factor 1 in basal and 3',5'-cyclic adenosine monophosphate-mediated regulation of cytochrome P450 side-chain cleavage enzyme in the mouse.

The orphan receptor steroidogenic factor-1 (SF-1) plays a major role in adrenal and gonadal development and sexual differentiation, and it has been shown to interact with shared promotor elements to increase the expression of cytochrome P450 side-chain cleavage (P450scc) and other steroid hydroxylases in vitro. We took a step-wise approach to define the role of SF-1 in regulation of hormone-induced steroidogenesis. In a mouse Leydig cell line (MA-10) we show that hCG and forskolin are effective inducers of progesterone production and P450scc expression. In contrast, endogenous SF-1 expression was not increased by either hCG or forskolin. Similarly, these agents did not enhance the activity of SF-1 promoter transfected into MA-10 cells. The transcriptional activity of SF-1, measured by induction of an SF-1 synthetic reporter, was only minimally increased by forskolin. Within the context of the rat P450SCC promoter, mutation of the two SF-1-binding sites caused a dramatic decrease in constitutive activity of this promoter, but the degree of induction by 8-bromo-cAMP was only reduced from 7.9-fold to 5.9-fold. We conclude that SF-1 is required for the constitutive activity of P450scc, but that it does not play a direct role in the early induction of steroidogenesis by hCG or forskolin in MA-10 cells.

Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) is expressed in the adrenal cortex and gonads and regulates the expression of several P450 steroid hydroxylases in vitro. We examined the role of SF-1 in the adrenal glands and gonads in vivo by a targeted disruption of the mouse SF-1 gene. All SF-1-deficient mice died shortly after delivery. Their adrenal glands and gonads were absent, and persistent Mullerian structures were found in all genotypic males. While serum levels of corticosterone in SF-1-deficient mice were diminished, levels of adrenocorticotropic hormone (ACTH) were elevated, consistent with intact pituitary corticotrophs. Intrauterine survival of SF-1-deficient mice appeared normal, and they had normal serum level of corticosterone and ACTH, probably reflecting transplacental passage of maternal steroids. We tested whether SF-1 is required for P450 side-chain-cleavage enzyme (P450scc) expression in the placenta, which expresses both SF-1 and P450scc, and found that in contrast to its strong activation of the P450scc gene promoter in vitro, the absence of SF-1 had no effect on P450scc mRNA levels in vivo. Although the region targeted by our disruption is shared by SF-1 and by embryonal long terminal repeat-binding protein (ELP), a hypothesized alternatively spliced product, we believe that the observed phenotype reflects absent SF-1 alone, as PCR analysis failed to detect ELP transcripts in any mouse tissue, and sequences corresponding to ELP are not conserved across species. These results confirm that SF-1 is an important regulator of adrenal and gonadal development, but its regulation of steroid hydroxylase expression in vivo remains to be established.