Involvement of the transcription factor STAT1 in the regulation of porcine ovarian granulosa cell functions treated and not treated with ghrelin

Abstract

The aim of our in vitro experiments was to study the role of the transcription factor STAT1 and the hormone ghrelin in controlling porcine ovarian function. The effects of treatment with ghrelin (0, 1, 10, 100 ng/ml), transfection-induced overexpression of transcription factor STAT1, and their combination on apoptosis (expression of apoptosis-related peptides caspase-3, BAX and anti-apoptotic peptide BCL2), proliferation (expression of proliferating cell nuclear antigene PCNA, proliferation-associated protein kinase MAPK/ERK1,2) and release of the hormones progesterone (P 4 ), prostaglandin F (PGF) and oxytocin (OXT) in cultured porcine ovarian granulosa cells was evaluated using RIA, immunocytochemistry and SDS-PAGE-western immunoblotting. It was found that ghrelin, when given alone, increased the expression of proliferation-associated PCNA and MAPK/ERK1,2, decreased the accumulation of apoptosis-related substances caspase-3, BAX, BCL2, decreased P 4 , and increased PGF and OXT release. Ghrelin tended to promote accumulation of STAT1 in both control and transfected cells, although in transfected cells ghrelin at 1 ng/ml decreased STAT1 accumulation. Transfection of porcine granulosa cells by a gene construct encoding STAT1 promoted the expression of STAT1 and apoptosis-related-BAX but the expression of BCL2 did not, and decreased the accumulation of proliferation-associated MAPK/ERK1,2 but not that of PCNA. It also promoted PGF and OXT but not P 4 release. Overexpression of STAT1 reversed the effect of ghrelin on STAT1, PCNA, PGF, OXT (from stimulatory to inhibitory), BCL2, P 4 (from inhibitory to stimulatory), prevented ghrelin effect on caspase-3 and BAX, but did not affect ghrelin's effect on MAPK/ERK1,2 expression. These results suggest that ghrelin directly affects porcine ovarian cells function -stimulates proliferation, inhibits apoptosis and affects secretory activity. Furthermore, they demonstrated the involvement of the transcription factor STAT1 in controlling these functions, the promotion of some markers of apoptosis (BAX), inhibition of some markers of proliferation (MAPK/ERK1,2) and stimulation of PGF release. Finally, the obtained data failed to demonstrate that STAT1 is involved in mediating the action of ghrelin on ovarian cell functions.

Capsaicin Protects Against Cisplatin Ototoxicity by Changing the STAT3/STAT1 Ratio and Activating Cannabinoid (CB2) Receptors in the Cochlea

“…These findings support our hypotheses that capsaicin protects from cisplatin-induced hearing loss not only by inhibiting the pro-inflammatory pathway but also, by stabilizing the p-STAT3/p-STAT1 ratio and tilting the delicate balance towards cell survival. We next monitored two genes, Bax and Bcl-2, whose expressions are regulated by STAT1 26,27 and STAT3 28,29 , respectively. In UB/OC1 cells, Bax/Bcl-2 ratios were 1 ± 0.2 for control, 2.9 ± 0.1 for cisplatin, 1.31 ± 0.1 capsaicin + cisplatin, and 1.1 ± 0.2 for capsaicin-treated cells, respectively (graph not shown).…”

Quantitative bioluminescence imaging of transgene expression in intact porcine antral follicles in vitro

“…However, these methods do not provide information regarding whether functional specific genes are present or active since proteins often require activation (e.g., phosphorylation and methylation), and mRNA is often degraded during the RNA processing and transport prior to its translation into protein [5]. With recent advances in gene transfer methods, many laboratories have used reporter gene technologies to study functional gene activity and transcription factor interactions in signal transduction pathways involved in follicular atresia and steroidogenesis in cultured granulosa cells in vitro [6-9]. Although these studies have offered the opportunity to elucidate gene functions and cellular pathways within cultured granulosa cells, it does not take into account major interactions between the oocyte and the follicular components in the context of the whole follicle.…”

Transcription factors and ovarian functions

“…Transcription factor p53 inhibits proliferation in normal (Sirotkin et al, 2008) and cancer (Naidu et al, 2007) ovarian cells. Overexpression of the signal transducer and activator of transcription 1 (STAT‐1) decreased the accumulation of proliferation‐associated MAPK/ERK1,2 but not that of PCNA (Benčo et al, 2009; Fig. 1).…”

“… The transfection‐induced overexpression of STAT‐1 affects basal and ghrelin‐induced expression of marker of apoptosis bax (immunocytochemistry) (a), proliferation marker MAPK/ERK1,2 (immunocytochemistry) (b), and release of oxytocin (RIA) (c) by cultured porcine granulosa cells. (a) Significant ( P < 0.05) difference between corresponding groups of transfected and non‐transfected cells; (b) significant ( P < 0.05) differences between ghrelin (1, 10, or 100 ng/ml) treated and control (0 ng/ml) cells (Benčo et al, 2009). …”

“…Analysis of FOXL2 mutations demonstrated the involvement of this transcription factor in the regulation of apoptosis, reactive oxygen species detoxification, and inflammation processes (Moumné et al, 2008). Transfection of porcine granulosa cells by a gene construct encoding STAT1 promoted the expression of apoptosis‐related marker Bax, but not of anti‐apoptotic Bcl‐2 (Benčo et al, 2009; Fig. 1).…”