Apoptosis of Rat Granulosa Cells after Staurosporine and Serum Withdrawal Is Suppressed by Adenovirus-Directed Overexpression of Prohibitin

Abstract

Prohibitin (Phb1) is a highly conserved mitochondrial protein that is associated with granulosa cell differentiation, atresia, and luteolysis. Although prohibitin has been implicated in the suppression of apoptosis in mammalian cells, its specific role in programmed cell death in granulosa cells is unknown. In the present study, we examined the role of prohibitin in mediating staurosporine (STS) and serum withdrawal induced apoptosis in undifferentiated rat granulosa cells. Treatment of granulosa cells isolated from immature rat ovaries with STS and/or serum withdrawal induced a rapid decrease in the transmembrane potential of mitochondria, resulting in increased prohibitin content and induced apoptosis in a time- and dose-dependent manner. Infection of granulosa cells with a Phb1 adenoviral construct resulted in overexpression of prohibitin that markedly attenuated the ability of STS and serum withdrawal to induce apoptosis via the intrinsic apoptotic pathway. To determine the site of action of Phb1, granulosa cells were transfected with a prohibitin-eGFP fusion construct, and the fusion protein expression patterns were analyzed by fluorescence microscopy and Western blot analysis of cell fractionated samples. These studies indicated that the prohibitin-eGFP fusion protein moved from the cytoplasm into the mitochondria. However, no prohibitin-eGFP fusion protein was observed in the nucleus in response to the STS-induced apoptotic stimulus. This result was corroborated by Western blot analysis with green fluorescent protein-specific antibody. Furthermore, the prohibitin-eGFP fusion protein also inhibited programmed cell death. These results provide evidence that prohibitin could serve an antiapoptotic role in undifferentiated granulosa cells.

Prohibitin protects against hypoxia-induced H9c2 cardiomyocyte cell death

“…Knockdown experiments on a cellular level reveal essential functions of prohibitin for cell proliferation (10,25) and apoptosis (13). Overexpression of prohibitin induces cellular resistance to various stimuli via the mitochondrial apoptotic pathway (6,10,16,33). We found that overexpression of prohibitin resulted in suppression of hypoxia-induced cell death (Fig.…”

“…Our data indicate that the prohibitin overexpression decreases the relative expression of Bax/Bcl-2 in mitochondria and protects against cardiomyocyte cell death induced by hypoxia. Chowdhury et al (6) and Liu et al (16) reported that prohibitin inhibited cytochrome c release to cytosol from mitochondria in granulosa cells isolated from rat ovaries and cardiomyocytes, respectively. As both groups did not investigate the levels of Bcl-2, this is the first report of prohibitin function that retains the Bcl-2 levels in mitochondria.…”

Comparative Mitochondrial Proteomic Analysis of Raji Cells Exposed to Adriamycin

“…PHB is a highly conserved mitochondrial protein that is thought to play roles in cell-cycle control, differentiation, senescence, and antiproliferative activity (47). Former studies confirmed that PHB may defend against oxidant injury, suppress apoptosis in mammalian cells, and promote survival of cancer cells (48,49). Results of a study by Fusaro et al indicated that overexpression of PHB in a human lymphoma cell line blocked apoptosis induced by the topoisomerase I inhibitor camptothecin (50).…”

Rescue of paclitaxel sensitivity by repression of Prohibitin1 in drug-resistant cancer cells

“…It resides at the inner mitochondrial membrane where it serves as a protein chaperone (9,10). Recently, studies have shown that PHB1 has a role in the regulation of the apoptotic pathway (11)(12)(13). Additionally prohibitins have been found to reside on the cell surface of certain cells, including adipose endothelial cells (14,15).…”