Role of vaspin in porcine ovary: effect on signaling pathways and steroid synthesis via GRP78 receptor and protein kinase A†

Abstract

Vaspin, visceral-adipose-tissue-derived serine protease inhibitor, is involved in the development of obesity, insulin resistance, inflammation, and energy metabolism. Our previous study showed vaspin expression and its regulation in the ovary; however, the role of this adipokine in ovarian cells has never been studied. Here, we studied the in vitro effect of vaspin on various kinase-signaling pathways: mitogen-activated kinase (MAP3/1), serine/threonine kinase (AKT), signal transducer and activator of transcription 3 (STAT3) protein kinase AMP (PRKAA1), protein kinase A (PKA), and on expression of nuclear factor kappa B (NFKB2) as well as on steroid synthesis by porcine ovarian cells. By using western blot, we found that vaspin (1 ng/ml), in a time-dependent manner, increased phosphorylation of MAP3/1, AKT, STAT3, PRKAA1, and PKA, while it decreased the expression of NFKB2. We observed that vaspin, in a dose-dependent manner, increased the basal steroid hormone secretion (progesterone and estradiol), mRNA and protein expression of steroid enzymes using real-time PCR and western blot, respectively, and the mRNA of gonadotropins (FSHR, LHCGR) and steroids (PGR, ESR2) receptors. The stimulatory effect of vaspin on basal steroidogenesis was reversed when ovarian cells were cultured in the presence of a PKA pharmacological inhibitor (KT5720) and when GRP78 receptor was knocked down (siRNA). However, in the presence of insulin-like growth factor type 1 and gonadotropins, vaspin reduced steroidogenesis. Thus, vaspin, by activation of various signaling pathways and stimulation of basal steroid production via GRP78 receptor and PKA, could be a new regulator of porcine ovarian function.

Keywords: kinases; ovary; pig; steroid hormones; vaspin.

Figure 1

Time-dependent effect of vaspin on signaling pathways of MAP3/1, AKT, STAT3, PRKAA1, PKA, and expression of NFKB2 in coculture of Gc plus Tc and monoculture of Gc or Tc. Representative blots from five independent experiments are shown (A). The protein levels were shown as the ratio of phosphorylated protein to total protein for kinases, and the protein level in relation to actin for NFKB2 (B). Western blotting experiments were performed independently and repeated five times (n = 5). Data are plotted as the mean ± SEM. Significance between control and vaspin treatments is indicated by different letters (P < 0.05) from all five experiments.

Figure 2

Dose-dependent effect of vaspin added alone (A–D) or in combination with IGF1, or FSH or LH (E–H) on P4 secretion (A, E) and HSD3B mRNA (B, F) and protein (C, G) as well CYP17A1 protein (D, H) expression. ELISA, real-time PCR, and western blotting experiments were performed independently and repeated five times (n = 5). Data are plotted as the mean ± SEM. Significance between control and treatments is indicated by ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001; or by different letters (P < 0.05), C: control.

Figure 3

Dose-dependent effect of vaspin added alone (A–D) or in combination with IGF1, or FSH or LH (E–H) on STAR and CYP1A1 mRNA and expression. Real-time PCR experiments were performed independently and repeated five times (n = 5). Data are plotted as the mean ± SEM. Significance between control and treatments is indicated by ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001; or by different letters (P < 0.05), C: control.

Figure 4

Dose-dependent effect of vaspin added alone (A–C) or in combination with IGF1, or FSH or LH (D-F) on E2 secretion (A, D) and CYP19A1 mRNA (B, E), and protein (C, F) expression. ELISA, real-time PCR, and western blotting experiments were performed independently and repeated five times (n = 5). Data are plotted as the mean ± SEM. Significance between control and treatments is indicated by ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001; or by different letters (P < 0.05), C: control.

Figure 5

Dose-dependent effect of vaspin on mRNA expression levels of FSHR (A), LHCGR (B), PGR (C), and ESR2 (D). Real-time PCR experiments were performed independently and repeated five times (n = 5). Data are plotted as the mean ± SEM. Significance between control and treatments is indicated by ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001; C: control.

Figure 6

mRNA (A) and protein (B) expression of GRP78 in ovarian follicles: small (SF), medium (MF), and large (LF) and in vitro effect of vaspin on GRP78 level (C) as well GRP78 receptor silencing (D). Real-time PCR experiments were performed independently and repeated five times (n = 5). Representative blots from five independent experiments are shown. Data are plotted as the mean ± SEM. Significance between control and treatments is indicated by ^***P < 0.001; C: control.

Figure 7

Involvement of GRP78 receptor and kinases MAP3/1 and PKA in vaspin action on P4 (A) and E2 (C) secretion as well as HSD3B (B) and CYP19A1 (D) protein expression. Representative blots from five independent experiments are shown. ELISA and Western blotting experiments were performed independently and repeated five times (n = 5). Data are plotted as the mean ± SEM. Significance between control and treatments is indicated by ^***P < 0.001; or by different letters (P < 0.05), C: control.

Figure 8

Proposed intracellular mechanism of GRP78 and protein kinase A (PKA) signaling pathway activation by vaspin on basal and IGF-1 and gonadotropin-induced steroid synthesis in ovarian cells.