The emerging roles of prohibitins in folliculogenesis

Abstract

Prohibitins are members of a highly conserved eukaryotic protein family containing the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain (also known as the prohibitin (PHB) domain) found in divergent species from prokaryotes to eukaryotes. Prohibitins are found in unicellular eukaryotes, fungi, plants, animals and humans. Prohibitins are ubiquitously expressed and present in multiple cellular compartments including the mitochondria, nucleus, and the plasma membrane, and shuttles between the mitochondria, cytosol and nucleus. Multiple functions have been attributed to the mitochondrial and nuclear prohibitins, including cellular differentiation, anti-proliferation, and morphogenesis. In the present review, we focus on the recent developments in prohibitins research related to folliculogenesis. Based on current research findings, the data suggest that these molecules play important roles in modulating specific responses of granulose cells to follicle stimulating hormone (FSH) by acting at multiple levels of the FSH signal transduction pathway. Understanding the molecular mechanisms by which the intracellular signaling pathways utilize prohibitins in governing folliculogenesis is likely to result in development of strategies to overcome fertility disorders and suppress ovarian cancer growth.

Figure 1

A. Schematic domain representation of human prohibitin (hPHB) protein. B. Schematic domain representation of human repressor of estrogen activity (REA/hPHB2) protein. N - amino terminal; C - carboxy terminal.

Figure 2

A. Localization by immunofluorescence of REA (left panel, green) and PHB (right panel, green) in GCs treated with FSH+T. B. GST-pull down assays for REA and PHB complexes. Mitochondrial and nuclear fractions of GCs treated with FSH+T (24h) were incubated with GST fusion proteins of REA and PHB, pre-conjugated to glutathione-Sepharose beads, and pull down, electrophoresed, blotted and probed with PHB and REA antibodies.

Figure 3

PHB and REA form a complex in primary GCs treated with FSH+T and their stability is interdependent. A. GCs were infected with Ad-scrambled (control), Ad-shPHB-GFP or Ad-shREA-GFP vectors (MOI=10) for 48h and the media replaced with serum-free medium for 24h. Semiquantitative RT-PCR revealed that PHB and REA mRNAs expression levels were efficiently and significantly reduced when compared to the scrambled control group. B. Forty-eight hours after infections as above, GCs were treated with FSH+T for 24h. Cell lysates (30 micro gram) were subjected to 12% SDS-PAGE and Western blot analysis with antibodies directed toward PHB, REA, and cyclophilin A as indicated. C. Live cell photographs taken under an inverted microscope uncovered that down-regulation of PHB in the presence of FSH+T results in a change from a polygonal to elongated morphology of GCs indicative of proliferation. Results are representative of 3 independent experiments.

Figure 4

Schematic representation of involvement of the prohibitins complex (PHB and REA/PHB2) in folliculogenesis in response to differentiation and stress inducing factors.