Figure 1.
Quantification of CYP11A1 mRNA abundance in normal and PCOS theca cells.
The time course of CYP11A1 mRNA accumulation was examined in A) normal or 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 µM forskolin. C) CYP11A1 mRNA accumulation in normal and PCOS theca cells following 24 h treatment with and without 20 µM forskolin serum free medium. CYP11A1 mRNA was measured using quantitative real-time PCR as described in Materials and Methods. The data presented in Panel A and Panel 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 Panel C 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 (a, P<0.01) and forskolin-stimulated conditions (b, P<0.01). Forskolin- treatment significantly increased CYP11A1 mRNA accumulation in both normal and PCOS theca cells (*, P<0.01).
Figure 2.
Deletion analysis of the CYP11A1 promoter in normal and PCOS theca cells.
A) Theca cells were transiently transfected with pGL3 luciferase constructs containing −2327, −1676, −660, −160, or −90 to +49 bp of the 5′-flanking sequence of the CYP11A1 gene. All constructs contain the endogenous TATA box and transcriptional start site. B) Normal and PCOS theca cells were transiently transfected with the above constructs described in Materials and Methods. Following transfection, cells were cultured in transfection medium alone or with forskolin (20 µM) for 48 h. Data are presented as relative luciferase (LUC) activity that was normalized with β-galactosidase activity, and represent the mean ± SEM of independent experiments in five normal and five PCOS theca cell cultures. CYP11A1 promoter activity was increased in PCOS theca cells, under basal (a, P<0.01) and forskolin-stimulated conditions (b, P<0.01), as compared to normal theca cells for individual promoter constructs.
Figure 3.
Differential regulation of the minimal −160/−90 bp CYP11A1 promoter region in normal and PCOS theca cells.
A) The full-length −1676 CYP11A1 construct, the −1676 construct that lacks the −160/−90 bp region but retains sequences from −90 to +49 bp (−1676Δ−160/−90), a −1676 construct containing the sequence between −1676 to −1540 bp fused the minimal −90 CYP11A1 promoter construct (−1676Δ−1540/−90) containing a putative U-CRS element, and constructs containing the thymidine kinase promoter alone (TK) or with the −160/−90 bp upstream of the TK promoter (−160/−90 TK). B) Normal and PCOS theca cells were transiently transfected with the−1676, −1676Δ−160/−90, −1676Δ−1540/−90 promoter constructs as described in Materials and Methods. Following transfection, cells were cultured in transfection medium alone or with forskolin (20 µM) for 48 h C) Normal and PCOS theca cells were transfected with promoter constructs containing either TK, or with −160/−90 TK. Following transfection, cells were cultured in transfection medium for 24 h. Data are presented as relative luciferase (LUC) activity that was normalized with β-galactosidase activity, and represent the mean ± SEM of independent experiments in four normal and four PCOS theca cell cultures. Both basal (a, P<0.01) and forskolin (b, P<0.01) stimulated −1676 CYP11A1 promoter regulation is increased in PCOS theca cells. In normal theca cells, −1676Δ−1540/−90 CYP11A1 activity was significantly increased under basal conditions (*, P<0.01), and forskolin-treatment (**, P<0.01) conditions, as compared to the full-length −1676 CYP11A construct. In contrast, in PCOS theca cells −1676Δ−1540/−90 CYP11A1 activity was significantly increased under basal conditions (*, P<0.01), and forskolin stimulated (**, P<0.01) conditions, as compared to the full-length −1676 CYP11A1 construct (Fig. 3B). These data demonstrate that 70 bp sequence between −160/−90 bp of the start site of transcription of the CYP11A1 gene confers increased basal expression in PCOS theca cells. The U-CRS element between −1676 to 1540 of the promoter confers basal and cAMP-dependent regulation in both normal and PCOS theca cells.
Figure 4.
NF-1C2 regulation of the CYP11A1 promoter in theca cells.
A) The effect of human NF-1C2 on CYP11A1 promoter function was examined in normal and PCOS theca cells transiently transfected with the −1676 CYP11A1 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 µM forskolin (F) for 48 h. To determine the sequences in the CYP11A1 promoter that confer NF-1C2 regulation, cells were transfected with a B) pGL3 constructs containing −1676, −160, or −90 to +49 bp of the 5′-flanking sequence of the CYP11A1 gene, or C) the minimal −160/−90 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 β-galactosidase activity and represent the mean ± SEM of multiple independent experiments. These experiments demonstrate that NF-1C2 inhibits both basal (a, P<0.01) and forskolin (b, P<0.01) stimulated −1676 CYP11A1 promoter function in normal and PCOS theca cells, as well as basal −160 CYP11A1 promoter function (a, P<0.01) in PCOS theca cells. Moreover, sequences between −160/−90 bp of the CYP11A1 promoter confer NF-1C2 inhibition.
Figure 5.
Endogenous CYP11A1 mRNA half-life in normal and PCOS theca cells.
The stability of endogenous CYP11A1 mRNA was examined in normal and PCOS theca cells under untreated and forskolin (20 µM)-stimulated conditions following treatment with the transcriptional inhibitor DRB (75 µM) from 2–42 h. A) Graphical representation of the amount of CYP11A1 mRNA remaining at each time interval, determined by quantitative real-time PCR. B) The half-life of endogenous CYP11A1 mRNA is presented as the mean ± SEM from independent examinations in 5 normal and 5 PCOS theca cell cultures. CYP11A1 mRNA half-life was increased in PCOS theca, under both untreated (a, P<0.01) and forskolin-stimulated conditions (a, P<0.01). Forskolin treatment did not significantly alter CYP11A1 mRNA half-life in normal or PCOS theca cells.
Figure 6.
The 5′UTR of CYP11A1 mRNA confers increased stability in PCOS theca cells under basal conditions.
A) The individual half-lives of various CYP11A1 RNA probes were determined using RNA in vitro degradation assays. Biotinylated CYP11A1 mRNA transcripts (i.e., the full-length transcript, 5′UTR+coding region, coding region alone, and coding region +3′UTR) 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 ± SEM of five independent assays. The results of these experiments demonstrated that the stability of CYP11A1 RNA transcripts containing either the full-length (a, P<0.01) or 5′+coding region (b, P<0.01), were significantly increased in assays using PCOS extracts, compared with normal extracts. The coding transcript+3′-UTR was markedly reduced in normal (*, P<0.01) and PCOS (**, P<0.01) theca cells as compared to the 5′UTR+coding transcript, and were similar in normal and PCOS cells. B) To examine functional differences in 5′UTR of CYP11A1 in normal and PCOS theca cells, both cell types were transiently transfected with a luciferase (LUC) construct containing the 5′ UTR of CYP11A1 mRNA and incubated in the absence (untreated) or presence of forskolin (20 µM) for 48 h. Data are presented as relative luciferase (LUC) activity following normalization by ß-galactosidase and represent the mean ± SEM from transfections performed in triplicate in 4 independent normal and 4 independent PCOS theca cells cultures. Luciferase expression of the 5′ UTR construct was significantly higher in PCOS theca cells as compared to normal cells (a, P<0.05). Forskolin treatment had no effect on CYP11A1 stability in normal or PCOS theca cells.