9 research outputs found
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
Deletion analysis of the <i>CYP11A1</i> promoter in normal and PCOS theca cells.
<p><b>A)</b> Theca cells were transiently transfected with pGL3 luciferase constructs containing β2327, β1676, β660, β160, or β90 to +49 bp of the 5β²-flanking sequence of the <i>CYP11A1</i> gene. All constructs contain the endogenous TATA box and transcriptional start site. <b>B)</b> Normal and PCOS theca cells were transiently transfected with the above constructs described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048963#s2" target="_blank">Materials and Methods</a>. Following transfection, cells were cultured in transfection medium alone or with forskolin (20 Β΅M) for 48 h. Data are presented as relative luciferase (LUC) activity that was normalized with Ξ²-galactosidase activity, and represent the mean Β± SEM of independent experiments in five normal and five PCOS theca cell cultures. <i>CYP11A1</i> promoter activity was increased in PCOS theca cells, under basal (<i>a</i>, <i>P</i><0.01) and forskolin-stimulated conditions (<i>b</i>, <i>P</i><0.01), as compared to normal theca cells for individual promoter constructs.</p
Quantification of <i>CYP11A1</i> mRNA abundance in normal and PCOS theca cells.
<p>The time course of CYP11A1 mRNA accumulation was examined in <b>A)</b> normal or <b>B)</b> PCOS theca cells treated in serum free medium for 0, 4, 8, 16, 24, or 48 h in the presence and absence of 20 Β΅M forskolin. <b>C)</b> CYP11A1 mRNA accumulation in normal and PCOS theca cells following 24 h treatment with and without 20 Β΅M forskolin serum free medium. CYP11A1 mRNA was measured using quantitative real-time PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048963#s2" target="_blank">Materials and Methods</a>. The data presented in <b>Panel A</b> and <b>Panel B</b> are data obtained from two normal and two PCOS patients, that are representative of data collected from theca cells from 5 normal and 5 PCOS patients. The data presented in <b>Panel C</b> are the results from independent analyses of theca cells isolated from 5 normal and 5 PCOS women. CYP11A1 mRNA accumulation was increased in PCOS theca cells as compared to normal theca cells under both control (<i>a</i>, <i>P</i><0.01) and forskolin-stimulated conditions (<i>b</i>, <i>P</i><0.01). Forskolin- treatment significantly increased CYP11A1 mRNA accumulation in both normal and PCOS theca cells (*, <i>P<0.01</i>).</p
The 5β²UTR of CYP11A1 mRNA confers increased stability in PCOS theca cells under basal conditions.
<p><b>A)</b> The individual half-lives of various CYP11A1 RNA probes were determined using RNA <i>in vitro</i> degradation assays. Biotinylated CYP11A1 mRNA transcripts (i.e., the full-length transcript, 5β²UTR+coding region, coding region alone, and coding region +3β²UTR) were incubated with cytoplasmic extracts isolated from either normal or PCOS theca cells. The stability of each transcript over time (half-life) is presented as the mean Β± SEM of five independent assays. The results of these experiments demonstrated that the stability of CYP11A1 RNA transcripts containing either the full-length (<i>a, P</i><0.01) or 5β²+coding region (<i>b, P</i><0.01), were significantly increased in assays using PCOS extracts, compared with normal extracts. The coding transcript+3β²-UTR was markedly reduced in normal (<i>*</i>, <i>P<0.01</i>) and PCOS (<i>**</i>, <i>P<0.01</i>) theca cells as compared to the 5β²UTR+coding transcript, and were similar in normal and PCOS cells. <b>B)</b> To examine functional differences in 5β²UTR of CYP11A1 in normal and PCOS theca cells, both cell types were transiently transfected with a luciferase (LUC) construct containing the 5β² UTR of CYP11A1 mRNA and incubated in the absence (untreated) or presence of forskolin (20 Β΅M) for 48 h. Data are presented as relative luciferase (LUC) activity following normalization by Γ-galactosidase and represent the mean Β± SEM from transfections performed in triplicate in 4 independent normal and 4 independent PCOS theca cells cultures. Luciferase expression of the 5β² UTR construct was significantly higher in PCOS theca cells as compared to normal cells (<i>a, P</i><0.05). Forskolin treatment had no effect on CYP11A1 stability in normal or PCOS theca cells.</p
Differential regulation of the minimal β160/β90 bp <i>CYP11A1</i> promoter region in normal and PCOS theca cells.
<p><b>A)</b> The full-length β1676 <i>CYP11A1</i> construct, the β1676 construct that lacks the β160/β90 bp region but retains sequences from β90 to +49 bp (β1676Ξβ160/β90), a β1676 construct containing the sequence between β1676 to β1540 bp fused the minimal β90 <i>CYP11A1</i> promoter construct (β1676Ξβ1540/β90) containing a putative <i>U-CRS</i> element, and constructs containing the thymidine kinase promoter alone (TK) or with the β160/β90 bp upstream of the TK promoter (β160/β90 TK). <b>B)</b> Normal and PCOS theca cells were transiently transfected with theβ1676, β1676Ξβ160/β90, β1676Ξβ1540/β90 promoter constructs as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048963#s2" target="_blank">Materials and Methods</a>. Following transfection, cells were cultured in transfection medium alone or with forskolin (20 Β΅M) for 48 h <b>C)</b> Normal and PCOS theca cells were transfected with promoter constructs containing either TK, or with β160/β90 TK. Following transfection, cells were cultured in transfection medium for 24 h. Data are presented as relative luciferase (LUC) activity that was normalized with Ξ²-galactosidase activity, and represent the mean Β± SEM of independent experiments in four normal and four PCOS theca cell cultures. Both basal (<i>a</i>, P<0.01) and forskolin (<i>b</i>, P<0.01) stimulated β1676 <i>CYP11A1</i> promoter regulation is increased in PCOS theca cells. In normal theca cells, β1676Ξβ1540/β90 <i>CYP11A1</i> activity was significantly increased under basal conditions (*, <i>P</i><0.01), and forskolin-treatment (**, <i>P</i><0.01) conditions, as compared to the full-length β<i>1676 CYP11A</i> construct. In contrast, in PCOS theca cells β1676Ξβ1540/β90 <i>CYP11A1</i> activity was significantly increased under basal conditions (*, <i>P</i><0.01), and forskolin stimulated (**, <i>P</i><0.01) conditions, as compared to the full-length β<i>1676 CYP11A1</i> construct (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048963#pone-0048963-g003" target="_blank">Fig. 3B</a>). These data demonstrate that 70 bp sequence between β160/β90 bp of the start site of transcription of the <i>CYP11A1</i> gene confers increased basal expression in PCOS theca cells. The U-CRS element between β1676 to 1540 of the promoter confers basal and cAMP-dependent regulation in both normal and PCOS theca cells.</p
NF-1C2 regulation of the <i>CYP11A1</i> promoter in theca cells.
<p><b>A)</b> The effect of human NF-1C2 on <i>CYP11A1</i> promoter function was examined in normal and PCOS theca cells transiently transfected with the β1676 <i>CYP11A1</i> luciferase construct and co-transfected with an empty pcDNA plasmid, or a pcDNA plasmid expressing NF-1C2. Following transfection, cells were cultured in transfection medium with and without 20 Β΅M forskolin (F) for 48 h. To determine the sequences in the <i>CYP11A1</i> promoter that confer NF-1C2 regulation, cells were transfected with a <b>B</b>) pGL3 constructs containing β1676, β160, or β90 to +49 bp of the 5β²-flanking sequence of the CYP11A1 gene, or <b>C)</b> the minimal β160/β90 TK and empty TK constructs, following co-transfection with pcDNA plasmid expressing NF-1C2 or an empty pcDNA plasmid, the cells were cultured in serum free medium for 48 h. All data are presented as relative luciferase (LUC) activity that was normalized with Ξ²-galactosidase activity and represent the mean Β± SEM of multiple independent experiments. These experiments demonstrate that NF-1C2 inhibits both basal (<i>a</i>, <i>P</i><0.01) and forskolin (<i>b</i>, <i>P</i><0.01) stimulated β1676 <i>CYP11A1</i> promoter function in normal and PCOS theca cells, as well as basal β160 <i>CYP11A1</i> promoter function (<i>a</i>, <i>P</i><0.01) in PCOS theca cells. Moreover, sequences between β160/β90 bp of the <i>CYP11A1</i> promoter confer NF-1C2 inhibition.</p