Vasoactive intestinal peptide (VIP)-mediated expression and function of steroidogenic acute regulatory protein (StAR) in granulosa cells

Abstract

VIP is a peptide hormone capable of activating the cAMP/PKA pathway and modifying gonadal steroidogenic capacity. Less is known about the molecular mechanisms of VIP-mediated steroidogenesis and its role in regulating the steroidogenic acute regulatory protein (STAR). We examined the impact of VIP on STAR expression and function in immortalized (KK1) and primary mouse granulosa cells, where VIP strongly upregulated STAR expression and steroidogenesis. Inhibitors of the PKA and PKC pathways suggested that both are activated by VIP. VIP did not efficiently phosphorylate STAR (P-STAR); however, VIP together with cAMP-analogs that activate Type II PKA increased P-STAR and further increased steroidogenesis. Our results suggest that VIP-induced STAR expression and function in granulosa cells result from the preferential activation of Type I PKA. Furthermore, the PKA and PKC pathways appear to converge at regulating VIP-mediated Star transcription and translation.

Figure 1

Expression of VIPR1 and VIPR2 and concentration-dependent increase in STAR expression and steroidogenic output in immortalized KK1 mouse granulosa cells treated with vasoactive intestinal peptide (VIP) against the background PKA activity. Cells were cultured in serum-free DMEM/F12 (1:1) medium with increasing concentrations of VIP with or without 0.3 mM dbcAMP for 6 h. A) Expression of VIPR1 and VIPR2 in KK1 control cells. B) Progesterone production in the collected media was determined by radioimmunoassay C) Cells were collected and homogenized, 20 µg of the lysate was used in western blot analysis of STAR (30 kDa), phospho (P)-STAR (30 kDa), 3β-hydroxysteroid dehydrogenase (3βHSD) (42 kDa), P450scc (45 kDa) and GAPDH (37 kDa). Protein expression was normalised against GAPDH; the average integrated optical density (IOD) for STAR and 3βHSD is shown as fold changes relative to the untreated control.

Figure 2

Concentration-dependent increase in Star mRNA expression in immortalized KK1 mouse granulosa cells treated with vasoactive intestinal peptide (VIP) against the background PKA activity. A) Star mRNA expression as determined by Real Time (TaqMan) PCR normalised against the GAPDH. One-way ANOVA with p < 0.0001 (A,B,C) followed by Dunnett’s multiple comparison test was applied; all samples were compared against the untreated control. (**) indicates p < 0.01 and (***) inicates p < 0.05. B) VIP increases Star promoter activity in immortalized KK1 mouse granulosa cells. Cells were transfected with −151/−1 bp fragment of Star promoter subcloned to pGL2 vector containing Firefly luciferase as a reporter. Transfection efficiency was normalized by cotransfecting pRL-SV40 vector constitutively expressing Renilla luciferase. Cells were treated for 6 h. One-way ANOVA with p < 0.0025 and Dunnett’s multiple comparison test were applied; all samples were compared against the untreated control. Bars with (**) differ at p < 0.01.

Figure 3

Vasoactive intestinal peptide (VIP) increases Star gene expression and steroidogenesis in primary mouse granulosa cells. Mature female CD-1 strain mice were stimulated with 8 IE pregnant mare serum gonadotropin (PMSG). After isolation cells were cultivated for 24h in culture medium and subsequently stimulated for 6 h in serum free media containing either 1mM dbcAMP or 1µM VIP. A) Progesterone production in the collected media was determined by radioimmunoassay B) Star mRNA expression as determined by Real Time (TaqMan) PCR normalised against the GAPDH. One-way ANOVA with p < 0.0001 (A,B) followed by Dunnett’s multiple comparison test was applied; all samples were compared against the untreated control. (**) indicates p < 0.01.

Figure 4

Time course of vasoactive intestinal peptide (VIP)– induced PKA, CREB and cJUN activation in immortalized KK1 mouse granulosa cells. The KK1 cells were incubated in the presence of 1µM VIP for the times indicated. A) The effect of 1µM VIP on PKA activity in cultured KK1 cells over a period of 4 h. One-way ANOVA with p < 0.0001 and Dunnett’s multiple comparison test were applied; all samples were compared against the control. (**) indicates p < 0.01. B) Representative immunoblots using antibodies against total and phospho (P)- CREB (43 kDa) and (P)- cJUN (48 kDa). The lower panels show the densitometric values (integrated optical density) normalized against the respective total-CREB and -cJUN. One-way ANOVA with p < 0.0001 (CREB; cJUN) followed by Dunnett’s multiple comparison test was applied; all samples were compared against the control indicating: 0’ min. vs 5’-120’ min p<0.01 and 0’ min vs. 180’ min p < 0.05 for P-cJUN and 0’ min vs. 10-240’ min p< 0.01 for P-CREB. C) KK1 cells were transfected with PathDetect cJUN trans-reporting system; 36h after transfection cells were treated with 1 µM VIP and/or 1mM dbcAMP for additional 6 h and luciferase activity in the cell lysate was determined. Lower case letters are used to designate groups that differ significantly (p < 0.01)

Figure 5

Role of PKA in vasoactive intestinal peptide (VIP) – mediated STAR expression and steroidogenesis in immortalized KK1 mouse granulosa cells. KK1 cells were treated for 6 h with 1.0 µM VIP with or without 0.3 mM dbcAMP. PKA activity was blocked with H89 (25 µM). Untreated cells were used as negative control. A) Star mRNA expression as determined by Real Time (TaqMan) PCR normalised against the GAPDH. B) STAR (30kDa), P-STAR (30 kDa) and GAPDH (37 kDa) levels were examined by Western blot analysis. The average integrated optical density (IOD) for STAR, normalized with GAPDH, is shown as fold changes relative to the untreated control. C) Progesterone production in the collected media was determined by radioimmunoassay. One-way ANOVA with p < 0.0001 (A,C) followed by Dunnett’s multiple comparison test was applied; all samples were compared against the untreated control. Bars with (**) differ at p < 0.01.

Figure 6

Role of PKC in vasoactive intestinal peptide (VIP) – mediated STAR expression and steroidogenesis in immortalized KK1 mouse granulosa cells. KK1 cells were treated as indicated for 6h. GFX (20µM) was used to specifically block the PKC activity. Untreated cells and cells treated with 0.3 mM dbcAMP were used as controls. A) Star mRNA expression as determined by Real Time (TaqMan) PCR normalized against the GAPDH. B) STAR (30kDa), P-STAR (30 kDa) and GAPDH (37 kDa) levels were examined by Western blot analysis. The average integrated optical density (IOD) for STAR, normalized with GAPDH, is shown as fold changes relative to the untreated control. C) Progesterone production was determined by radioimmunoassay. One-way ANOVA with p < 0.0001 (A,C) followed by Dunnett’s multiple comparison test was applied; all samples were compared against the untreated control. Bars with (**) differ at p < 0.01.

Figure 7

The induction of STAR phosphorylation in response to vasoactive intestinal peptide (VIP). KK1 cells were treated for 6 h with either 0.1 µM or 1.0 µM VIP with or without type II PKA analogs (25 µm MBC-cAMP together with 100µM PIP-cAMP) and PMA (20 nM). Untreated cells and cells treated with 0.3 mM dbcAMP were used as controls. A) Star mRNA expression as determined by Real Time (TaqMan) PCR normalised against the GAPDH. B) STAR (30kDa), P-STAR (30 kDa) and GAPDH (37 kDa) levels were examined by Western blot analysis. The average integrated optical density (IOD) for STAR, normalized with GAPDH, is shown as fold changes relative to the untreated control. C) Progesterone production was determined by radioimmunoassay. One-way ANOVA with p < 0.0001 (A,C) followed by Dunnett’s multiple comparison test was applied; all samples were compared against the untreated control. Bars with (**) differ at p < 0.01.

Figure 8

The role of Vasoactive Intestinal Peptide (VIP) in regulating STAR. VIP-mediated induction of PKC and type I PKA can induce StAR expression but results only in weak P-STAR and hence lower steroidogenic response. However, together with selective type II PKA activators, VIP increases P-STAR and enhances progesterone output.