Cholesterol Side-chain Cleavage Gene Research Articles

Cholesterol side-chain cleavage gene expression in theca cells: augmented transcriptional regulation and mRNA stability in polycystic ovary syndrome.

Hyperandrogenism is characteristic of women with polycystic ovary syndrome (PCOS). Ovarian theca cells isolated from PCOS follicles and maintained in long-term culture produce elevated levels of progestins and androgens compared to normal theca cells. Augmented steroid production in PCOS theca cells is associated with changes in the expression of genes for several steroidogenic enzymes, including CYP11A1, which encodes cytochrome P450 cholesterol side-chain cleavage. Here, we further examined CYP11A1 gene expression, at both the transcriptional and post-transcriptional level in normal and PCOS theca cells propagated in long-term culture utilizing quantitative RT-PCR, functional promoter analyses, and mRNA degradation studies. The minimal element(s) that conferred increased basal and cAMP-dependent CYP11A1 promoter function were determined. CYP11A1 mRNA half-life in normal and PCOS theca cells was compared. Results of these cumulative studies showed that basal and forskolin stimulated steady state CYP11A1 mRNA abundance and CYP11A1 promoter activity were increased in PCOS theca cells. Deletion analysis of the CYP11A1 promoter demonstrated that augmented promoter function in PCOS theca cells results from increased basal regulation conferred by a minimal sequence between −160 and −90 bp of the transcriptional start site. The transcription factor, nuclear factor 1C2, was observed to regulate basal activity of this minimal CYP11A1 element. Examination of mRNA stability in normal and PCOS theca cells demonstrated that CYP11A1 mRNA half-life increased >2-fold, from approximately 9.22+/−1.62 h in normal cells, to 22.38+/−0.92 h in PCOS cells. Forskolin treatment did not prolong CYP11A1 mRNA stability in either normal or PCOS theca cells. The 5′-UTR of CYP11A1 mRNA confers increased basal mRNA stability in PCOS cells. In conclusion, these studies show that elevated steady state CYP11A1 mRNA abundance in PCOS cells results from increased transactivation of the CYP11A1 promoter and increased CYP11A1 mRNA stability.

Effect of methotrexate and leucovorin on female reproductive tract of albino rats

Methotrexate (MTX) an antifolate drug and leucovorin its antidote, are used in the treatment of both neoplastic and non-neoplastic diseases in young women. We hypothesize that MTX treatment might comprise a deleterious effect on fast proliferating reproductive cells, an unavoidable and unwanted side effect. MTX given dose dependently to rats for 20 days prevented vaginal cyclicity and caused a reduction in serum progesterone and estradiol. External morphology of reproductive tract displayed thinning of organs and reduction in their weights. To reveal mechanism of MTX action, we examined the histology of ovary, oviduct, uterus, cervix and vagina. Results suggested that in a dose-dependent fashion MTX restrained preantral and antral follicular growth in ovary. Epithelium and stroma of oviduct, uterus, cervix and vagina were disrupted and lost their normal structures. Such alterations in ovarian function raised serum follicle stimulating hormone, luteinizing hormonal profiles. Expression of steroidogenic acute regulatory protein and P450 cholesterol side chain cleavage gene, which are both essential for steroidogenesis, markedly decreased in ovary upon MTX treatment. Total RNA, DNA and protein concentrations, glucose 6 phosphate dehydrogenase, lactate dehydrogenase and alkaline phosphatase enzyme activities in ovary were distinctly altered. Leucovorin supplementation and withdrawal of the treatment, improved MTX caused effects partially. These results for the first time indicate that the malfunction of female reproductive organs by MTX treatment in young women is not only correlated to the disrupted circulating levels of hormones and histoarchitecture of tissues but also discrepancies in steroidogenic genes and hormone regulated enzyme activities in ovary.

PTB-associated splicing factor regulates growth factor-stimulated gene expression in mammalian cells.

An insulin-like growth factor I (IGF-I) response element (IGFRE) in the porcine P-450 cholesterol side-chain cleavage gene (P450scc) binds two transcription factors, Sp1 and polypyrimidine tract-binding protein-associated splicing factor (PSF). In this study, we investigated expression of these transcription factors in mouse Y1 adrenal cells, a cell line that does not increase P450scc expression in response to IGF-I. Western blot analysis showed a greater expression of PSF in Y1 cells when compared with a mouse fibroblast cell line (NWTb3) in which IGF-I stimulates the P450scc IGFRE. The two cell lines expressed Sp1 equally, and IGF-I did not increase expression of either transcription factor. Chromatin immunoprecipitation analysis with Y1 chromatin confirmed that PSF and Sp1 bound to the IGFRE. When increasing amounts of Sp1 were expressed in Y1 cells, the IGFRE became responsive to IGF-I. Moreover, a mutant oligonucleotide IGFRE reporter construct that lacks PSF binding was responsive to IGF-I. In conclusion, Y1 adrenal cells are a physiological example of PSF repression of growth factor-stimulated (IGF-I) gene expression (P450scc). The dynamic nature of this repression is consistent with PSF functioning as a regulator of growth factor-stimulated gene expression in mammalian cells.

NH2 terminus of PTB-associated splicing factor binds to the porcine P450scc IGF-I response element.

An insulin-like growth factor (IGF) I response element (IGFRE) in the porcine P-450 cholesterol side-chain cleavage gene (P450scc) regulates transcription through the binding of two proteins, Sp1 and polypyrimidine tract-binding protein-associated splicing factor (PSF). PSF is a component of spliceosomes and contains RNA-binding domains. In this study, we localized the NH2-terminal amino acid residues necessary for binding of PSF to the IGFRE. Three COOH-terminal truncated proteins (aa 304, 214, and 134) of PSF were designed to empirically partition the NH2-terminal region while excluding the RNA-binding domains. Southwestern analysis showed that only the largest expressed truncated protein, P3, strongly bound the porcine P450scc IGFRE. Truncated PSF protein expression in Y1 adrenal cells showed that P3 repressed transcriptional activity of the IGFRE similar to full-length PSF, whereas P2 (minimal binding to the IGFRE) had no effect. In conclusion, the NH2-terminal region of PSF contains the amino acid residues necessary for binding to the porcine P450scc IGFRE and repressing the transcriptional activity of the element.

Effects of progestins on progesterone synthesis in a stable porcine granulosa cell line: control of transcriptional activity of the cytochrome p450 side-chain cleavage gene.

The purpose of the present study was to examine the effects of progestins on progesterone synthesis and expression of the cytochrome P450 cholesterol side-chain cleavage gene (P450(scc)) in a stable porcine granulosa cell line, the JC-410. Cells were incubated for 48 h with the synthetic progestogen-levornorgestrel with or without RU486 (progesterone and glucocorticoid receptor antagonist) or RWJ26819 (progesterone agonist without affinity to glucocorticoid receptors). Both levonorgestrel and RU486 enhanced progesterone accumulation in a dose-dependent manner. RU486 did not antagonize the effects of levonorgestrel, and RWJ26819 had no effect on progesterone production in cultured JC-410 cells. Progesterone and levonorgestrel increased steady state P450(scc) mRNA levels after 3-6 h of treatment. Progesterone and RU486 at 0.1, 1, and 10 microM increased the transcription rate of P450(scc) transiently expressed in JC-410 cells after 18 h of incubation; 30 microM had no effect, and 100 microM suppressed transcription. Levonorgestrel did not affect transcription of the P450(scc) gene, and RWJ26819 reduced its transcription. Progesterone and RU486 significantly decreased the number of cells and total protein content after 72 and 24 h of incubation, respectively. Levonorgestrel had no effect, whereas RWJ26819 increased (24 h) but subsequently reduced (72 h) cell number and protein content. The present results indicate that progestins are capable of directly modulating progesterone biosynthesis in porcine JC-410 granulosa cells. These effects may be exerted in part through the regulation of P450(scc) gene expression. Ostensible differences exist between progesterone and its synthetic analogues in the control of progesterone secretion in the stable porcine granulosa cell line in vitro.

In situ amplification of the cytochrome P-450 cholesterol side-chain cleavage enzyme mRNA in single porcine granulosa cells by IGF-1 and FSH acting alone or in concert.

To investigate the cellular mechanisms and cell-cell heterogeneity of the actions of insulin-like growth factor-1 (IGF-1) and follicle-stimulating hormone (FSH) exerted alone and in combination on ovarian cholesterol side-chain cleavage gene expression (P450scc mRNA) in (pig) granulosa cells, we implemented semiquantitative in situ molecular hybridization at the single target-cell level. To this end, a 1-kb cDNA specific to the catalytic region of porcine p450scc gene was subcloned into pGEM-3 and directionally transcribed in vitro in the presence of 35S-dUTP to yield radiolabeled antisense (and sense, negative control) cRNA hybridization probes. Swine granulosa cells harvested nonenzymatically from immature (1-5 mm) Graafian follicles were anchored on eight-chamber multiwell slides and treated with control solvent, human recombinant IGF-1 (10nM), ovine FSH (10nM), or both hormones, for 48 h to stimulate progestin biosynthesis maximally. After appropriate cellular permeabilization, cRNA hybridization, and solvent washes, granulosa cells were exposed to Kodak NTB-2 emulsion for 6 wk. Semiquantitative automated image analysis software (NIH IMAGE 1.5) was used to evaluate the number of silver grains deposited/20,000 square pixels. Specificity controls included labeled sense riboprobe, pretreatment with RNase, and 100-fold molar excess unlabeled cRNA. Grain counts and their distributions were examined by ANOVA and the Wilcoxon nonparametric test. The mean number of silver grains deposited per granulosa cell increased over control (reflecting specific P450scc mRNA expression) in granulosa cells pretreated with IGF-1, FSH, or IGF-1 + FSH (p < 0.05 by ANOVA). The rank order of abundance of expression of P450scc mRNA (grains/ovarian cell) was (IGF-1 + FSH) > FSH > IGF-1 > control treatment. Distributional analysis showed that each treatment introduced skewed distributions toward granulosa cells expressing more P450scc per cell than controls (p < 0.01). The median grain count of granulosa cells treated with FSH was significantly increased over that of IGF-1 treatment (p < 0.05). Treatment with both IGF-1 and FSH further shifted the grain count distribution per cell to favor granulosa cells expressing more P450scc mRNA compared to IGF-1 or FSH treatment alone (p < 0.05). Accordingly, a demonstrable mechanism of IGF-1 and FSH's regulation of specific P450scc gene expression at the single granulosa cell level is amplification in the number of target ovarian cells expressing this enzymatically rate-determining gene transcript. Interestingly, the induction of P450scc mRNA is not sufficient to explain fully the synergistic increases in progesterone accumulation driven by combined treatment with IGF-1 and FSH, thus suggesting that other steroidogenic control points are also targets of IGF-1/FSH action.

Insulin-like growth factor-I increases expression of the porcine P-450 cholesterol side chain cleavage gene through a GC-rich domain.

The promoter/regulatory region of the porcine P-450 cholesterol side chain cleavage (P450scc) gene was cloned from a porcine genomic library. This gene contains a GC-rich region (-130-100) that mediates insulin-like growth factor I (IGF-I) and cAMP stimulation of gene expression in porcine granulosa cells. Stimulation of gene expression by cAMP occurs in 6 h, while IGF-I stimulation occurs in 24-48 h. This region is also responsive to insulin but not fibroblast growth factor. The effects of IGF-I and cAMP on gene expression in porcine granulosa cells are additive. In Y1 adrenal cells, the same region is responsive to cAMP but not to IGF-I. Gel shift assays using an oligonucleotide of this region and nuclear extract protein from porcine granulosa and Y1 adrenal cells identified three DNA-protein complexes (C1-C3). The binding activity of the complexes did not change with IGF-I or forskolin treatment of granulosa cells. Mutational analysis results were consistent with IGF-I regulating gene expression through the C2 DNA protein complex. Moreover, this complex binds differently in gel shift assay to mutant oligonucleotides with porcine and Y1 and nuclear extract protein. We conclude that IGF-I stimulates porcine P450scc gene expression through a distinct, cell-specific protein complex binding to a GC-rich domain.