FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2109332684> ?p ?o ?g. }

• W2109332684 endingPage "26390" @default.
• W2109332684 startingPage "26380" @default.
• W2109332684 abstract "Polycystic ovary syndrome (PCOS) affects 5% of reproductive aged women and is the leading cause of anovulatory infertility. A hallmark of PCOS is excessive theca cell androgen secretion, which is directly linked to the symptoms of PCOS. Our previous studies demonstrated that theca cells from PCOS ovaries maintained in long term culture persistently secrete significantly greater amounts of androgens than normal theca cells, suggesting an intrinsic abnormality. Furthermore, previous studies suggested that ovarian hyperandrogenemia is inherited as an autosomal dominant trait. However, the genes responsible for ovarian hyperandrogenemia of PCOS have not been identified. In this present study, we carried out microarray analysis to define the gene networks involved in excess androgen synthesis by the PCOS theca cells in order to identify candidate PCOS genes. Our analysis revealed that PCOS theca cells have a gene expression profile that is distinct from normal theca cells. Included in the cohort of genes with increased mRNA abundance in PCOS theca cells were aldehyde dehydrogenase 6 and retinol dehydrogenase 2, which play a role in all-trans-retinoic acid biosynthesis and the transcription factor GATA6. We demonstrated that retinoic acid and GATA6 increased the expression of 17α-hydroxylase, providing a functional link between altered gene expression and intrinsic abnormalities in PCOS theca cells. Thus, our analyses have 1) defined a stable molecular phenotype of PCOS theca cells, 2) suggested new mechanisms for excess androgen synthesis by PCOS theca cells, and 3) identified new candidate genes that may be involved in the genetic etiology of PCOS. Polycystic ovary syndrome (PCOS) affects 5% of reproductive aged women and is the leading cause of anovulatory infertility. A hallmark of PCOS is excessive theca cell androgen secretion, which is directly linked to the symptoms of PCOS. Our previous studies demonstrated that theca cells from PCOS ovaries maintained in long term culture persistently secrete significantly greater amounts of androgens than normal theca cells, suggesting an intrinsic abnormality. Furthermore, previous studies suggested that ovarian hyperandrogenemia is inherited as an autosomal dominant trait. However, the genes responsible for ovarian hyperandrogenemia of PCOS have not been identified. In this present study, we carried out microarray analysis to define the gene networks involved in excess androgen synthesis by the PCOS theca cells in order to identify candidate PCOS genes. Our analysis revealed that PCOS theca cells have a gene expression profile that is distinct from normal theca cells. Included in the cohort of genes with increased mRNA abundance in PCOS theca cells were aldehyde dehydrogenase 6 and retinol dehydrogenase 2, which play a role in all-trans-retinoic acid biosynthesis and the transcription factor GATA6. We demonstrated that retinoic acid and GATA6 increased the expression of 17α-hydroxylase, providing a functional link between altered gene expression and intrinsic abnormalities in PCOS theca cells. Thus, our analyses have 1) defined a stable molecular phenotype of PCOS theca cells, 2) suggested new mechanisms for excess androgen synthesis by PCOS theca cells, and 3) identified new candidate genes that may be involved in the genetic etiology of PCOS. Polycystic ovary syndrome (PCOS) 1The abbreviations used are: PCOS, polycystic ovary syndrome;P450scc, P450 side chain cleavage; StAR, steroidogenic acute regulatory protein; LH, luteinizing hormone; CYP17, 17α-hydroxylase/17,20-lyase; RT, reverse transcriptase; atRA, all-trans-retinoic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RoDH2 and -4, retinol dehydrogenase 2 and 4, respectively; ALDH6, aldehyde dehydrogenase 6; TGF, transforming growth factor; TIG1, tazarotene-induced gene 1; IGFBP3, insulin-like growth factor-binding protein 3; FSH, follicle stimulating hormone; DHEA, dehydroepiandosterone.1The abbreviations used are: PCOS, polycystic ovary syndrome;P450scc, P450 side chain cleavage; StAR, steroidogenic acute regulatory protein; LH, luteinizing hormone; CYP17, 17α-hydroxylase/17,20-lyase; RT, reverse transcriptase; atRA, all-trans-retinoic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RoDH2 and -4, retinol dehydrogenase 2 and 4, respectively; ALDH6, aldehyde dehydrogenase 6; TGF, transforming growth factor; TIG1, tazarotene-induced gene 1; IGFBP3, insulin-like growth factor-binding protein 3; FSH, follicle stimulating hormone; DHEA, dehydroepiandosterone. is characterized by failure of ovulation, excessive ovarian androgen production, and, consequently, infertility (1Ehrmann D.A. Barnes R.B. Rosenfield R.L. Endocr. Rev. 1995; 16: 322-353Crossref PubMed Scopus (507) Google Scholar, 2Legro R.S. Spielman R. Urbanek M. Driscoll D. Strauss III, J.F. Dunaif A. Recent Prog. Horm. Res. 1998; 53: 217-256PubMed Google Scholar). The PCOS ovaries are enlarged bilaterally and contain follicles arrested at a size no larger than 10 mm embedded in a dense stroma (3Franks S. N. Engl. J. Med. 1995; 333: 853-861Crossref PubMed Scopus (1778) Google Scholar). The theca layers of these follicles are prominent and represent the major source of the increased circulating androgens in PCOS women (2Legro R.S. Spielman R. Urbanek M. Driscoll D. Strauss III, J.F. Dunaif A. Recent Prog. Horm. Res. 1998; 53: 217-256PubMed Google Scholar). Previous studies have suggested that the ovarian hyperandrogenemia associated with PCOS clusters in families and appears to be inherited as an autosomal dominant trait (2Legro R.S. Spielman R. Urbanek M. Driscoll D. Strauss III, J.F. Dunaif A. Recent Prog. Horm. Res. 1998; 53: 217-256PubMed Google Scholar). However, the genetic etiology of PCOS has not been defined. Studies using freshly isolated and short and long term cultures of theca cells have demonstrated that androgen synthesis is increased in PCOS compared with normal theca cells, suggesting that this PCOS phenotype is an intrinsic property of the theca cell (1Ehrmann D.A. Barnes R.B. Rosenfield R.L. Endocr. Rev. 1995; 16: 322-353Crossref PubMed Scopus (507) Google Scholar, 4Axelrod L. Goldzieher J. J. Clin. Endocrinol. Metab. 1962; 22: 431-440Crossref PubMed Scopus (56) Google Scholar, 5Gilling-Smith C. Willis D.S. Beard R.W. Franks S. J. Clin. Endocrinol. Metab. 1994; 79: 1158-1165Crossref PubMed Scopus (285) Google Scholar, 6Nelson V.L. Legro R.S. Strauss III, J.F. McAllister J.M. Mol. Endocrinol. 1999; 13: 946-957Crossref PubMed Google Scholar). Increased theca cell steroidogenesis has been attributed to increased activity of the 17α-hydroxylase/17,20-lyase (CYP17) and 3β-hydroxysteroid dehydrogenase type II enzymes (1Ehrmann D.A. Barnes R.B. Rosenfield R.L. Endocr. Rev. 1995; 16: 322-353Crossref PubMed Scopus (507) Google Scholar, 5Gilling-Smith C. Willis D.S. Beard R.W. Franks S. J. Clin. Endocrinol. Metab. 1994; 79: 1158-1165Crossref PubMed Scopus (285) Google Scholar, 6Nelson V.L. Legro R.S. Strauss III, J.F. McAllister J.M. Mol. Endocrinol. 1999; 13: 946-957Crossref PubMed Google Scholar) and increased expression of the P450 side chain cleavage (P450scc) and CYP17 mRNAs. Furthermore, CYP17 promoter activity is increased in PCOS compared with normal theca cells (6Nelson V.L. Legro R.S. Strauss III, J.F. McAllister J.M. Mol. Endocrinol. 1999; 13: 946-957Crossref PubMed Google Scholar, 7Wickenheisser J.K. Quinn P.G. Nelson V.L. Legro R.S. Strauss III, J.F. McAllister J.M. J. Clin. Endocrinol. Metab. 2000; 85: 2304-2311Crossref PubMed Scopus (162) Google Scholar). In contrast, the abundance of the mRNAs for steroidogenic acute regulatory protein (StAR), which controls the rate-limiting step in steroidogenesis (8Christenson L.K. Strauss III, J.F. Biochim. Biophys. Acta. 2000; 1529: 175-187Crossref PubMed Scopus (164) Google Scholar), and 17β-hydroxysteroid dehydrogenase type V, which is the theca cell enzyme that is thought to be responsible for the reduction of androsterone into testosterone (9Luu-The V. Dufort I. Pelletier G. Labrie F. Mol. Cell. Endocrinol. 2001; 171: 77-82Crossref PubMed Scopus (97) Google Scholar) as well as the transcriptional activity of the StAR promoter, are not different between normal and PCOS theca cells (6Nelson V.L. Legro R.S. Strauss III, J.F. McAllister J.M. Mol. Endocrinol. 1999; 13: 946-957Crossref PubMed Google Scholar, 7Wickenheisser J.K. Quinn P.G. Nelson V.L. Legro R.S. Strauss III, J.F. McAllister J.M. J. Clin. Endocrinol. Metab. 2000; 85: 2304-2311Crossref PubMed Scopus (162) Google Scholar, 10Nelson V.L. Qin Kn K.N. Rosenfield R.L. Wood J.R. Penning T.M. Legro R.S. Strauss III, J.F. McAllister J.M. J. Clin. Endocrinol. Metab. 2001; 86: 5925-5933Crossref PubMed Scopus (239) Google Scholar). Thus, these collective studies have defined a stable steroidogenic phenotype for the PCOS theca cell that includes altered expression of a subset of proteins that are important for androgen synthesis. Steroidogenesis in the ovarian theca cell is primarily regulated by luteinizing hormone (LH), which upon binding to the LH receptor promotes increased steroid production through activation of cAMP-dependent signal transduction cascades (11Leung P.C. Steele G.L. Endocr. Rev. 1992; 13: 476-498Crossref PubMed Scopus (216) Google Scholar). Women with PCOS tend to have elevated levels of LH (3Franks S. N. Engl. J. Med. 1995; 333: 853-861Crossref PubMed Scopus (1778) Google Scholar). Furthermore, there is evidence that PCOS theca cells may be hypersensitive to LH action (1Ehrmann D.A. Barnes R.B. Rosenfield R.L. Endocr. Rev. 1995; 16: 322-353Crossref PubMed Scopus (507) Google Scholar). Interestingly, the differences in CYP17 and P450scc mRNA abundance and CYP17 promoter activity in PCOS theca cells are enhanced when cells are treated with the adenylate cyclase activator forskolin (6Nelson V.L. Legro R.S. Strauss III, J.F. McAllister J.M. Mol. Endocrinol. 1999; 13: 946-957Crossref PubMed Google Scholar, 7Wickenheisser J.K. Quinn P.G. Nelson V.L. Legro R.S. Strauss III, J.F. McAllister J.M. J. Clin. Endocrinol. Metab. 2000; 85: 2304-2311Crossref PubMed Scopus (162) Google Scholar), which mimics LH-dependent signal transduction in theca cells through production of the second messenger cAMP. However, the transcription of the StAR gene and other genes encoding steroidogenic proteins is also regulated by LH and cAMP (12Christenson L.K. Johnson P.F. McAllister J.M. Strauss III, J.F. J. Biol. Chem. 1999; 274: 26591-26598Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 13Strauss III, J.F. Kallen C.B. Christenson L.K. Watari H. Devoto L. Arakane F. Kiriakidou M. Sugawara T. Recent Prog. Horm. Res. 1999; 54: 369-394PubMed Google Scholar), suggesting that a downstream component of the cAMP-dependent signal transduction cascade that affects CYP17 and P450scc but not StAR gene transcription is affected in PCOS theca cells. Although the studies described above have identified important correlations between increased steroidogenic enzyme gene expression and increased androgen biosynthesis in PCOS theca cells, they have not disclosed the upstream genes that are important for increased transcription. Furthermore, the global changes in theca cell gene expression or the alterations in gene networks or signal transduction cascades that may play an important role in the manifestation of other PCOS theca cell phenotypes, which may contribute to arrested follicular growth, have not been defined. In order to define the genes that are differentially expressed in PCOS theca cells and to identify new candidate genes that may contribute to the etiology of PCOS, we compared gene expression profiles of normal and PCOS theca cells using Affymetrix oligonucleotide microarray chips (Affymetrix, Santa Clara, CA). Theca Cell Culturing and RNA Isolation—Theca cells were isolated from 3–5-mm follicles from the ovaries of four normal women and five PCOS patients, and independent cultures were established using the cells that were isolated from each woman as previously described (6Nelson V.L. Legro R.S. Strauss III, J.F. McAllister J.M. Mol. Endocrinol. 1999; 13: 946-957Crossref PubMed Google Scholar, 7Wickenheisser J.K. Quinn P.G. Nelson V.L. Legro R.S. Strauss III, J.F. McAllister J.M. J. Clin. Endocrinol. Metab. 2000; 85: 2304-2311Crossref PubMed Scopus (162) Google Scholar). The diagnosis of PCOS and the steroidogenic capacity of each sample was determined as previously described (6Nelson V.L. Legro R.S. Strauss III, J.F. McAllister J.M. Mol. Endocrinol. 1999; 13: 946-957Crossref PubMed Google Scholar, 7Wickenheisser J.K. Quinn P.G. Nelson V.L. Legro R.S. Strauss III, J.F. McAllister J.M. J. Clin. Endocrinol. Metab. 2000; 85: 2304-2311Crossref PubMed Scopus (162) Google Scholar, 14Zawadski J. Dunaif A. Dunaif A. Givens J. Haseltine F. Merriam G. Current Issues in Endocrinology and Metabolism. Blackwell Scientific Publications, Boston1992: 377-384Google Scholar). For microarray hybridizations and RT-PCR experiments, fourth passage cells from the four normal and the five PCOS theca cell samples were cultured for 48 h in serum-free medium, which contained no treatment (untreated), 20 μm forskolin (Sigma), and/or 5 μm all-trans-retinoic acid (Sigma). After treatment, the medium was removed, the cells were washed with phosphate-buffered saline, and RNA was isolated using TRIzol reagent (Invitrogen). Dehydroepiandosterone (DHEA) Radioimmunoassay—Fourth passage normal and PCOS theca cells were grown to 80% confluence in six-well tissue culture plates. The cells were transferred to serum-free medium and were untreated, treated with 20 μm forskolin, treated with 5 μm atRA, or treated with 5 μm atRA and 20 μm forskolin. After 72 h, the medium was collected, and DHEA levels were detected with the Coat-A-Count DHEA Radioimmunoassay kit (Diagonistic Products Corp., Los Angeles, CA). Microarray Hybridization—The Affymetrix GeneChip Human Genome U95A, U133A, and U133B microarray chips (Affymetrix) were hybridized at the University of Pennsylvania Microarray Core Facility. Briefly, biotin-labeled cRNA, which was generated from four different normal and five different PCOS theca cell samples that were untreated or forskolin-stimulated, was fragmented according to Affymetrix protocols. The fragmented cRNA from each sample was hybridized to individual Affymetrix U95A gene array chips using the GeneChip Fluidics Station 400 protocol (Affymetrix), the hybridized chips were scanned using the Agilent GeneArray Scanner (Affymetrix), and a scaling factor was applied to each chip using the Affymetrix Microarray Suite 5.0 software to normalize the mean raw fluorescence intensity for each chip to an average base-line fluorescence level. The same fragmented cRNA from each theca cell sample was subsequently hybridized to individual Affymetrix U133A and Affymetrix U133B gene array chips, and the hybridized chips were scanned and normalized as described. Gene Expression Analysis—Each transcript on the U95A, U133A, and U133B chip was determined to be present or absent in each theca cell sample using the statistical expression algorithm of the Affymetrix Microarray Suite 5.0 software package (15Affymetrix, Inc.Affymetrix Microarray Suite, Version 5.0. Affymetrix, Santa Clara, CA2001Google Scholar) and was identified as expressed in the normal or PCOS theca cell samples if it was called present in at least three samples in each group. The average normalized fluorescence intensity for each expressed transcript in the four normal or the five PCOS samples was determined using GeneSpring 4.2 (Silicon Genetics, Redwood City, CA) and expressed as a ratio of the mean normalized fluorescence intensity for the transcript in PCOS theca cells to the mean normalized fluorescence intensity for the transcript in normal theca cells (PCOS-to-normal ratio). The S.E. associated with each transcript's average normalized fluorescence intensity was determined using a cross-gene error model generated by the GeneSpring software program (16Silicon Genetics Inc. GeneSpring, Version 4.2. Silicon Genetics, Redwood City, CA2002Google Scholar). Statistically significant differences (p < 0.05) in the average normalized fluorescence intensity of each transcript between the normal and PCOS samples were determined by parametric testing, which used the cross-gene error model (16Silicon Genetics Inc. GeneSpring, Version 4.2. Silicon Genetics, Redwood City, CA2002Google Scholar). The gene associated with each differentially expressed transcript on the U95A, U133A, and U133B chips was identified, and the function of each of gene was determined. Reverse Transcription, PCR, and Quantitative RT-PCR—Total RNA (5 μg) which was isolated from the same theca cell samples that were used for the microarray hybridization was treated with DNase I (Promega, Madison, WI) and reverse transcribed with Moloney murine leukemia virus (Promega) as previously described (10Nelson V.L. Qin Kn K.N. Rosenfield R.L. Wood J.R. Penning T.M. Legro R.S. Strauss III, J.F. McAllister J.M. J. Clin. Endocrinol. Metab. 2001; 86: 5925-5933Crossref PubMed Scopus (239) Google Scholar). The resulting cDNA was used to carry out PCR amplification of prostate short-chain dehydrogenase reductase (5′-CCACCTCTACTAAAAAATTGTGTATATCTTTG and 5′-TGTGGCTGTTTTGAACTTTGTGA), retinol dehydrogenase 4 (5′-GGCACCAATCCCACTCCTT and 5′-CCCGTTTTTCAGCTGCGTAA), cellular retinoic acid-binding protein 1 (5′-TGGCCTTGGTGCCTCTTG and 5′-TGACTTCGAAACCGTGCAAA), and cellular retinoic acid-binding protein 2 (5′-GGTCACTGGGATGCCTCTTG and 5′-GCTCTTGCAGCCATTCCTCTT). The cDNA was also subjected to quantitative PCR amplification for 28 different transcripts (Supplemental Table 3) that were identified as present in theca cell samples by the Affymetrix U133A or U133B chip. For each of the 28 transcripts, primers were designed using the Primer Express 1.5 software (PerkinElmer Life Sciences). Each primer set was tested empirically to determine the maximal concentration of primers that could be used to produce specific amplification of the target sequence in the absence of primer dimer amplification. For each of the 28 targets, quantitative PCRs were carried out using equivalent dilutions of each cDNA sample, the fluorescent indicator SYBR green, the empirically determined concentration of each primer, and the Applied Biosystems model 7700 sequence detector PCR machine (PerkinElmer Life Sciences) as previously described (10Nelson V.L. Qin Kn K.N. Rosenfield R.L. Wood J.R. Penning T.M. Legro R.S. Strauss III, J.F. McAllister J.M. J. Clin. Endocrinol. Metab. 2001; 86: 5925-5933Crossref PubMed Scopus (239) Google Scholar). To verify that only a single PCR product was generated for each amplified transcript, the multicomponent data for each sample was subsequently analyzed using the Dissociation Curves 1.0 program (PerkinElmer Life Sciences). To account for differences in starting material, quantitative PCR was also carried out for each cDNA sample using the Applied Biosystems human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 20× primer and probe reagent (PerkinElmer Life Sciences). These quantitative PCRs defined a threshold cycle (C t) of detection for the target or the GAPDH in each cDNA sample. In order to convert this C t value into a relative abundance of target and GAPDH in each cDNA sample, quantitative PCR for the target and for the GAPDH was also carried out using serial dilutions of theca cell cDNA. An arbitrary value of template was assigned to the highest standard, and corresponding values were assigned to the subsequent dilutions, and these relative values were plotted against the C t value determined for each dilution to generate a standard curve. The relative amount of target and GAPDH in each sample was then determined using the equation, relativeabundance=10(Ct-b)/m(Eq. 1) where b represents the y intercept, and m is the slope. The relative abundance of the target was divided by the relative abundance of GAPDH in each sample to generate a normalized abundance for each of the 28 transcripts tested. Analysis of variance was then used to determine the mean and S.E. of the normalized abundance of each target in normal and PCOS theca cells. The nonparametric, Wilcoxon (rank sums) test was carried out to determine whether differences in the normalized abundance for each target between normal and PCOS samples were statistically significant (p < 0.05). Enzyme Assay—Whole cell extracts from two independent normal and two independent PCOS theca cell samples were assayed for retinol dehydrogenase activity. Briefly, 100 μg of each protein sample was combined with 178 pmol of [11,12-3H]all-trans-vitamin A alcohol (PerkinElmer Life Sciences) and 100 μm NAD+ (Sigma) in buffer (100 mm Tris, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, and 0.02% sodium azide) containing protease inhibitors (50 μg/ml leupeptin, 5 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin, and 5 μg/ml aprotinin). Samples were incubated for 1 h at 37 °C. Reactions were stopped by the addition of 200 μl of chloroform/methanol (2:1). The retinoids were collected in the organic phase, which was evaporated using a stream of liquid nitrogen. Each sample was resuspended in 50 μl of ethanol. Each sample was applied to a Silica-Rapid-Platten Woelm F 254 thin layer chromatography plate. In addition, 2 μmols of retinaldehyde (Sigma) was also applied to the plate. The retinol and retinaldehyde were resolved on the plate using a petroleum ether/acetone (82:18) solvent, the plate was sprayed with EN3HANCE spray surface autoradiography enhancer (PerkinElmer Life Sciences), and the retinaldehyde was detected by autoradiography. The radioactive retinaldehyde in each sample was counted, the background levels were subtracted, and the mean fmol of retinaldehyde/mg of protein/h of incubation was determined. Western Blot Analysis—Nuclear extracts from two independent normal and three independent PCOS theca cells samples, which were untreated or treated with 20 μm forskolin, were probed for the presence of GATA-6 protein. The GATA-6 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used as described by the manufacturer for immunoblot assays. The 64- and 52-kDa immunoreactive bands were detected using SuperSignal West Pico Sensitivity Substrate (Pierce). Plasmids—The promoter region of CYP17 was amplified from genomic DNA using the primers 5′-GAACGAGCAAGCCTTCATCG-3′ (–1876 to –1857) and 5′-GACAGCAGTGGAGTAGAAGAGC-3′ (+12 to +33). Likewise, the promoter region of P450scc was amplified from genomic DNA using the primers 5′-GGAATGTGGGGCTGCGTAGA-3′ (–1843 to –1824) and 5′-CAGCTGTGACTGTACCTGCT-3′ (+12 to +31). Each amplified promoter product was cloned into pCR2.1-TOPO (Invitrogen), excised using the restriction endonucleases KpnI and XhoI, and ligated into the pGL3-basic luciferase reporter vector (Promega) to generate pGL3.CYP17–1876 and pGL3.CYP11A-1843. Sequence integrity and insert orientation were confirmed by DNA sequencing. The pGL2.StAR-885 reporter plasmid has been previously described (17Sugawara T. Holt J.A. Kiriakidou M. Strauss III, J.F. Biochemistry. 1996; 35: 9052-9059Crossref PubMed Scopus (240) Google Scholar). pcDNAG6, encoding the full-length murine GATA-6 was a generous gift from Dr. Edward Morrisey (18Morrisey E.E. Ip H.S. Lu M.M. Parmacek M.S. Dev. Biol. 1996; 177: 309-322Crossref PubMed Scopus (388) Google Scholar). pcDNA3 (Invitrogen) and pRL-TK (Promega) were purchased. Transient Transfections and Reporter Gene Assays—HeLa cells, which do not express GATA-6 mRNA (19Bruno M.D. Korfhagen T.R. Liu C. Morrisey E.E. Whitsett J.A. J. Biol. Chem. 2000; 275: 1043-1049Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. For transient co-transfection experiments, 5.5 × 104 cells were seeded into each well of a 12-well plate. After 24 h, 500 ng of pGL3.CYP17–1876, CYP11A-1843, or pGL2.StAR-885; 25 ng of pcDNAG6 or pcDNA3; and 20 ng of pRL-TK were transiently transfected into cells using FuGENE6 transfection reagent (Roche Applied Science) per the manufacturer's protocol. After 48 h, the transfected cells were lysed, and the firefly and Renilla luciferase activities were determined using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. The firefly luciferase activity for each sample was normalized using the Renilla luciferase activity. Analysis of variance was used to calculate the mean and S.D. of the normalized luciferase activity for each experimental group in three independent experiments. The unpaired Student's t test was used to detect statistically significant differences (p value < 0.05) between pcDNAG6 and pcDNAG3-transfected cells. PCOS Theca Cells Exhibit a Distinct Gene Expression Profile Compared with Normal Theca Cells—Previous studies indicated that excess androgen synthesis is an intrinsic property of PCOS theca cells (6Nelson V.L. Legro R.S. Strauss III, J.F. McAllister J.M. Mol. Endocrinol. 1999; 13: 946-957Crossref PubMed Google Scholar, 7Wickenheisser J.K. Quinn P.G. Nelson V.L. Legro R.S. Strauss III, J.F. McAllister J.M. J. Clin. Endocrinol. Metab. 2000; 85: 2304-2311Crossref PubMed Scopus (162) Google Scholar). In agreement with these previous findings, the PCOS theca cell samples used in this study also consistently produced higher levels of DHEA than the normal theca cell samples (Fig. 1), consistent with the possibility that PCOS and normal theca cells have distinct molecular phenotypes. To assess the molecular phenotype of these normal and PCOS theca cells, we carried out gene expression profiling using Affymetrix GeneChip arrays. RNA was collected from four independent normal and five independent PCOS theca cell samples, was hybridized to individual Affymetrix U133 gene chips, and the gene expression profile of each sample was determined using the GeneSpring 4.2 data-mining software program. Of the 45,000 transcripts interrogated on the U133 chips, 15,267 (∼34%) transcripts were identified as present in either normal or PCOS theca cells, which is consistent with gene expression profiles of other tissues (20Brown V. Jin P. Ceman S. Darnell J.C. O'Donnell W.T. Tenenbaum S.A. Jin X. Feng Y. Wilkinson K.D. Keene J.D. Darnell R.B. Warren S.T. Cell. 2001; 107: 477-487Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar, 21Rus V. Atamas S.P. Shustova V. Luzina I.G. Selaru F. Magder L.S. Via C.S. Clin. Immunol. 2002; 102: 283-290Crossref PubMed Scopus (102) Google Scholar, 22Sreekumar R. Halvatsiotis P. Schimke J.C. Nair K.S. Diabetes. 2002; 51: 1913-1920Crossref PubMed Scopus (225) Google Scholar). When the gene expression profiles from the normal and PCOS theca cell samples were compared, 346 genes had a statistically significant difference in mRNA abundance between normal and PCOS theca cells (Fig. 2A). These 346 transcripts represented only a small percentage (2.3%) of genes expressed in theca cells. Furthermore, only 106 of the genes had greater than a 2-fold difference in gene expression, and only four of the genes had greater than a 5-fold difference in mRNA abundance between normal and PCOS theca cells, demonstrating that the magnitude of differential gene expression between normal and PCOS theca cells is modest. When the 346 genes were organized into functional categories, signal transduction molecules, genes associated with cellular metabolism, transcription factors, cell adhesion molecules, cell surface antigens, ion channels, and expressed sequence tags were among the groups of genes with altered mRNA abundance in PCOS theca cells (Supplemental Table 4). One of the transcription factors that exhibited a highly significant increase in mRNA abundance in the PCOS theca cells was GATA-6, which has been shown to regulate StAR expression in the porcine ovary (23Gillio-Meina C. Hui Y.Y. LaVoie H.A. Biol. Reprod. 2003; 68: 412-422Crossref PubMed Scopus (64) Google Scholar), suggesting a role for this transcription factor in increased PCOS theca cell steroidogenesis. Several of the differentially expressed genes could also be classified into specific signaling cascades or gene networks. For example, the mRNA levels of retinol dehydrogenase 2 (RoDH2) and aldehyde dehydrogenase 6 (ALDH6), which are involved in the conversion of retinol to atRA (24Rexer B.N. Zheng W.L. Ong D.E. Cancer Res. 2001; 61: 7065-7070PubMed Google Scholar, 25Chetyrkin S.V. Hu J. Gough W.H. Dumaual N. Kedishvili N.Y. Arch. Biochem. Biophys. 2001; 386: 1-10Crossref PubMed Scopus (55) Google Scholar, 26Napoli J.L. Mol. Cell. Endocrinol. 2001; 171: 103-109Crossref PubMed Scopus (53) Google Scholar), and tazarotene-induced gene 1 (TIG1), which is a target of atRA action (27Nagpal S. Patel S. Asano A.T. Johnson A.T. Duvic M. Chandraratna R.A. J. Invest. Dermatol. 1996; 106: 269-274Abstract Full Text PDF PubMed Scopus (109) Google Scholar), were increased in PCOS theca cells. In addition, inhibin βA, which is a member of the TGF-β superfamily of growth factors (28Gaddy-Kurten D. Tsuchida K. Vale W. Recent Prog. Horm. Res. 1995; 50: 109-129PubMed Google Scholar, 29Knight P.G. Front. Neuroendocrinol. 1996; 17: 476-509Crossref PubMed Scopus (141) Google Scholar) and Gremlin and PACE4, which modulate the biological activity of TGF-β growth factors (30Hsu D.R. Economides A.N. Wang X. Eimon P.M. Harland R.M. Mol. Cell. 1998; 1: 673-683Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar, 31Constam D.B. Robertson E.J. J. Cell Biol. 1999; 144: 139-149Crossref PubMed Scopus (261) Google Scholar), were also identified as differentially expressed in PCOS theca cells.Fig. 2Untreated and forskolin-stimulated PCOS theca cells exhibit altered gene expression compared with normal theca cells. The normalized fluorescence intensity fo" @default.
• W2109332684 created "2016-06-24" @default.
• W2109332684 creator A5008552363 @default.
• W2109332684 creator A5022469198 @default.
• W2109332684 creator A5024150199 @default.
• W2109332684 creator A5027974386 @default.
• W2109332684 creator A5048278292 @default.
• W2109332684 creator A5056686361 @default.
• W2109332684 creator A5078418528 @default.
• W2109332684 creator A5090491933 @default.
• W2109332684 creator A5091573028 @default.
• W2109332684 date "2003-07-01" @default.
• W2109332684 modified "2023-10-11" @default.
• W2109332684 title "The Molecular Phenotype of Polycystic Ovary Syndrome (PCOS) Theca Cells and New Candidate PCOS Genes Defined by Microarray Analysis" @default.
• W2109332684 cites W1777605394 @default.
• W2109332684 cites W1780615788 @default.
• W2109332684 cites W1967246618 @default.
• W2109332684 cites W1969041157 @default.
• W2109332684 cites W1969062482 @default.
• W2109332684 cites W1977681402 @default.
• W2109332684 cites W1979098148 @default.
• W2109332684 cites W1983097922 @default.
• W2109332684 cites W1986515287 @default.
• W2109332684 cites W1986970734 @default.
• W2109332684 cites W1987417875 @default.
• W2109332684 cites W1995640088 @default.
• W2109332684 cites W1995994721 @default.
• W2109332684 cites W1999076607 @default.
• W2109332684 cites W2000209912 @default.
• W2109332684 cites W2000231383 @default.
• W2109332684 cites W2003432885 @default.
• W2109332684 cites W2009254582 @default.
• W2109332684 cites W2010143253 @default.
• W2109332684 cites W2011762849 @default.
• W2109332684 cites W2013200752 @default.
• W2109332684 cites W2019771407 @default.
• W2109332684 cites W2020747940 @default.
• W2109332684 cites W2021163527 @default.
• W2109332684 cites W2023002592 @default.
• W2109332684 cites W2031091378 @default.
• W2109332684 cites W2034976273 @default.
• W2109332684 cites W2043377295 @default.
• W2109332684 cites W2044384686 @default.
• W2109332684 cites W2054889904 @default.
• W2109332684 cites W2057645498 @default.
• W2109332684 cites W2061904534 @default.
• W2109332684 cites W2062389660 @default.
• W2109332684 cites W2064577293 @default.
• W2109332684 cites W2073701436 @default.
• W2109332684 cites W2078037416 @default.
• W2109332684 cites W2089260361 @default.
• W2109332684 cites W2089269914 @default.
• W2109332684 cites W2092406654 @default.
• W2109332684 cites W2093442882 @default.
• W2109332684 cites W2095040842 @default.
• W2109332684 cites W2096778432 @default.
• W2109332684 cites W2098756736 @default.
• W2109332684 cites W2104565219 @default.
• W2109332684 cites W2104916769 @default.
• W2109332684 cites W2105651402 @default.
• W2109332684 cites W2112777136 @default.
• W2109332684 cites W2126733133 @default.
• W2109332684 cites W2142454930 @default.
• W2109332684 cites W2154957337 @default.
• W2109332684 cites W2156720376 @default.
• W2109332684 cites W2157058383 @default.
• W2109332684 cites W2169009865 @default.
• W2109332684 cites W2170438849 @default.
• W2109332684 cites W4250005844 @default.
• W2109332684 doi "https://doi.org/10.1074/jbc.m300688200" @default.
• W2109332684 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12734205" @default.
• W2109332684 hasPublicationYear "2003" @default.
• W2109332684 type Work @default.
• W2109332684 sameAs 2109332684 @default.
• W2109332684 citedByCount "228" @default.
• W2109332684 countsByYear W21093326842012 @default.
• W2109332684 countsByYear W21093326842013 @default.
• W2109332684 countsByYear W21093326842014 @default.
• W2109332684 countsByYear W21093326842015 @default.
• W2109332684 countsByYear W21093326842016 @default.
• W2109332684 countsByYear W21093326842017 @default.
• W2109332684 countsByYear W21093326842018 @default.
• W2109332684 countsByYear W21093326842019 @default.
• W2109332684 countsByYear W21093326842020 @default.
• W2109332684 countsByYear W21093326842021 @default.
• W2109332684 countsByYear W21093326842022 @default.
• W2109332684 countsByYear W21093326842023 @default.
• W2109332684 crossrefType "journal-article" @default.
• W2109332684 hasAuthorship W2109332684A5008552363 @default.
• W2109332684 hasAuthorship W2109332684A5022469198 @default.
• W2109332684 hasAuthorship W2109332684A5024150199 @default.
• W2109332684 hasAuthorship W2109332684A5027974386 @default.
• W2109332684 hasAuthorship W2109332684A5048278292 @default.
• W2109332684 hasAuthorship W2109332684A5056686361 @default.
• W2109332684 hasAuthorship W2109332684A5078418528 @default.
• W2109332684 hasAuthorship W2109332684A5090491933 @default.
• W2109332684 hasAuthorship W2109332684A5091573028 @default.
• W2109332684 hasBestOaLocation W21093326841 @default.

• next