Regulation of sterol regulatory element-binding transcription factor 1a by human chorionic gonadotropin and insulin in cultured rat theca-interstitial cells

Abstract

Theca-interstitial (T-I) cells of the ovary synthesize androgens in response to luteinizing hormone (LH). In pathological conditions such as polycystic ovarian syndrome, T-I cells are hyperactive in androgen production in response to LH and insulin. Because cholesterol is an essential substrate for androgen production, we examined the effect of human chorionic gonadotropin (hCG) and insulin on signaling pathways that are known to increase cholesterol accumulation in steroidogenic cells. Specifically, the effect of hCG and insulin on sterol regulatory element-binding transcription factor 1a (SREBF1a) required for cholesterol biosynthesis and uptake was examined. Primary cultures of T-I cells isolated from 25-day-old rat ovaries responded to hCG and insulin to increase the active/processed form of SREBF1a. The hCG and insulin significantly reduced insulin-induced gene 1 (INSIG1) protein, a negative regulator of SREBF processing. Furthermore, an increase in the expression of selected SREBF target genes, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr) and mevalonate kinase (Mvk), was also observed. Protein kinase A (PRKA) inhibitor completely abolished the hCG-induced increase in SREBF1a, while increasing INSIG1. Although the hCG-induced depletion of total and free cholesterol was abolished by aminoglutethimide, the stimulatory effect on SREBF1a was not totally suppressed. Treatment with 25-hydroxycholesterol abrogated the effect of hCG on SREBF1a. Inhibition of the phosphatidylinositol 3-kinase pathway did not block the insulin-induced increase in SREBF1a, whereas mitogen-activated protein kinase inhibition reduced the insulin response. These results suggest that the increased androgen biosynthesis by T-I cells in response to hCG and insulin is regulated, at least in part, by increasing the expression of sterol response element-responsive genes by increasing SREBF1a.

FIG. 1.

Immunofluorescence staining of CYP17A1 in T-I cells. Cells were harvested, plated, and allowed to attach for 24 h. Cells were then washed, fixed, and immunostained using a CYP17A1 primary antibody and a Texas red-conjugated secondary antibody. Cells were examined by fluorescence microscopy. A) Representative area of control T-I cells incubated without CYP17A1 antibody (original magnification ×20). B) Representative area of cells stained with CYP17A1 antibody (original magnification ×20).

FIG. 2.

The hCG-mediated regulation of SREBF1a and its target genes. Ovaries were collected from 25-day-old Sprague-Dawley rats. The T-I cells were isolated by collagenase digestion, and 3 × 10⁶ cells were plated with McCoy 5A medium. After 24-h attachment, cells were treated with different concentrations of hCG (0, 25, and 50 ng/ml) for 4 h. Whole-cell lysates were then analyzed for the expression of the active form of SREBF1a (A) and INSIG1 (B) by Western blot (top panels). The graphs of the densitometric scans are shown in the bottom panels of A and B. Protein loading was monitored by stripping and reprobing the same blot with antibody for β-tubulin. Blots are representative of one experiment, and the graphs represent the mean of three experiments. Error bars represent the mean ± SE. C and D) Cells were treated with hCG (50 ng/ml) for 4 h. Total RNA was reverse transcribed, and the resulting cDNA was subjected to real-time PCR using predesigned primers and probes for rat Hmgcr and Mvk as described in Materials and Methods. C) The graph shows the change in Hmgcr mRNA expression normalized for 18S rRNA. D) The graph shows the change in Mvk mRNA expression normalized for 18S rRNA. Error bars represent the mean ± SE of two independent experiments, triplicate determinations in each, n = 6. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. control. a, Represents significant differences (P < 0.01) compared with hCG 25 ng/ml.

FIG. 3.

Effect of hCG and PKA inhibitor on SREBF1a and INSIG1 proteins in T-I cells. Cells were pretreated with the PRKA inhibitor H89 (10 μM) for 1 h, followed by hCG (50 ng/ml) for 4 h. Whole-cell lysates were analyzed for the expression of the active form of SREBF1a (A) and INSIG1 (B) by Western blot analysis (top panels). The graphs of the densitometric scans are shown in the bottom panels. Protein loading was monitored by stripping and reprobing the same blot with antibody for β-tubulin. Blots are representative of one experiment, and the graphs represent the mean of three experiments. Error bars represent the mean ± SE. * P < 0.05 and *** P < 0.001 vs. control. a, Represents significant differences (P < 0.001) compared with hCG. b, Represents significant differences (P < 0.01) compared with hCG.

FIG. 4.

Effect of hCG and AGM on SREBF1a protein expression and androstenedione production in T-I cells. Cells were treated with AGM (5 μg/ml) for 1 h, followed by hCG (50 ng/ml) for 4 h. A) Whole-cell lysates were analyzed for the active form of SREBF1a by Western blot analysis (top panel). The graph of the densitometric scans is shown in the bottom panel. Protein loading was monitored by stripping and reprobing the same blot with antibody for β-tubulin. B) The media were collected, and androstenedione was extracted and measured by RIA as described in Materials and Methods. The blot in A is representative of one experiment, and both graphs represent the mean of three experiments. Error bars represent the mean ± SE. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. control. a, Represents significant differences (P < 0.01) compared with hCG. b, Represents significant differences (P < 0.001) compared with hCG.

FIG. 5.

Effect of hCG and AGM on cellular total (A) and free (B) cholesterol levels in T-I cells. Cells were treated with AGM (5 μg/ml) for 1 h, followed by hCG (50 ng/ml) for 4 h, and lysed with 1% Triton X-100 in choloroform. Total and free cholesterol contents of the cell lysates were determined using the cholesterol/cholesteryl ester quantitation kit as described in Materials and Methods. The graph represents the mean of three experiments. Data are expressed as the mean ± SE. * P < 0.05 vs. control.

FIG. 6.

Effect of hCG and 25-OHC on SREBF1a protein expression. Cells were pretreated with 25-OHC (10 μg/ml) for 1 h, followed by hCG (50 ng/ml) for 4 h. Whole-cell lysates were analyzed for the active form of SREBF1a by Western blot analysis (top panel). The graph of the densitometric scans is shown in the bottom panel. Protein loading was monitored by stripping and reprobing the same blot with antibody for β-tubulin. The blot is representative of one experiment, and the graph represents the mean of three experiments. Error bars represent the mean ± SE. *** P < 0.001 vs. control. a, Represents significant differences (P < 0.001) compared with hCG.

FIG. 7.

Insulin-mediated regulation of SREBF1a and its target genes. Cells were treated with different concentrations of insulin (0, 0.5, and 1 μg/ml) for 4 h. Whole-cell lysates were analyzed for the expression of the active form of SREBF1a (A) and INSIG1 (B) by Western blot analysis (top panels). The graphs of the densitometric scans are shown in the bottom panels of A and B. Protein loading was monitored by stripping and reprobing the same blot with antibody for β-tubulin. Blots are representative of one experiment, and the graphs represent the mean of three experiments. Error bars represent the mean ± SE. C and D) The T-I cells were treated with insulin (1 μg/ml) for 4 h. Total RNA was reverse transcribed, and the resulting cDNA was subjected to real-time PCR using predesigned primers and probes for rat Hmgcr and Mvk as described in Materials and Methods. C) The graph represents the change in Hmgcr mRNA expression normalized for 18S rRNA. D) The graph represents the change in Mvk mRNA expression normalized for 18S rRNA. Error bars represent the mean ± SE of two independent experiments, triplicate determinations in each, n = 6. * P < 0.05 and ** P < 0.01 vs. control. a, Represents significant differences (P < 0.05) compared with insulin (INS) 0.5 μg/ml. b, Represents significant differences (P < 0.01) compared with INS 0.5 μg/ml.

FIG. 8.

Effect of insulin and 25-OHC on SREBF1a protein expression. Cells were pretreated with 25-OHC (10 μg/ml) for 1 h, followed by insulin (1 μg/ml) for 4 h. Whole-cell lysates were analyzed for the active form of SREBF1a by Western blot analysis (top panel). The graph of the densitometric scans is shown in the bottom panel. Protein loading was monitored by stripping and reprobing the same blot with antibody for β-tubulin. The blot is representative of one experiment, and the graph represents the mean of three experiments. Error bars represent the mean ± SE. ** P < 0.01 vs. control. INS, insulin.

FIG. 9.

Effect of PIK3 and MAP kinase inhibitors on SREBF1a protein expression in T-I cells. Cells were pretreated with the PIK3 inhibitor wortmannin (100 nM) for 30 min or the MAP2K inhibitor U0126 (10 μM) for 1 h, followed by insulin (1 μg/ml) for 4 h. Whole-cell lysates were analyzed for the expression of the active form of SREBF1a using Western blot analysis. The graph of the densitometric scans is shown in A. Protein loading was monitored by stripping and reprobing the same blot with antibody for β-tubulin. The blot is representative of one experiment, and the graph represents the mean of three experiments. Error bars represent the mean ± SE. ** P < 0.01 vs. control. a, Represents significant differences (P < 0.05) compared with insulin (INS). b, Represents significant differences (P < 0.05) compared with INS + wortmannin (W). B) The T-I cells were pretreated with the PIK3 inhibitor wortmannin (100 nM) for 30 min, followed by insulin (1 μg/ml) for 30 min. Western blot analysis was performed with antibody for phosphorylated AKT, and the same blot was stripped and reprobed with antibody for total MAPK1 to normalize for protein loading. The blot represents one of two independent experiments.