Prohibitin is an evolutionarily conserved gene that has antiproliferative activity, is ubiquitously expressed, and appears to be essential for cell survival (Dell'Orco et al., 1996).The gene codes for a 30 kD, post-synthetically modified protein located primarily in the mitochondria. It functionally inhibits cell cycle progression and DNA synthesis, but its mechanism of action is presently unknown. Prohibitin is proposed to be a member of a new class of tumor suppressor genes whose inhibitory activity plays a role in the dominant senescent phenotype. Its involvement in senescence has been postulated from results obtained from such diverse systems as yeast and human diploid fibroblasts.The emerging roles of prohibitins in folliculogenesis. Chowdhury I et al. 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.///////Prohibitin (PHB) roles in granulosa cell physiology. Chowdhury I et al. (2015) Ovarian granulosa cells (GC) play an important role in the growth and development of the follicle in the process known as folliculogenesis. In the present review, we focus on recent developments in prohibitin (PHB) research in relation to GC physiological functions. PHB is a member of a highly conserved eukaryotic protein family containing the repressor of estrogen activity (REA)/stomatin/PHB/flotillin/HflK/C (SPFH) domain (also known as the PHB domain) found in diverse species from prokaryotes to eukaryotes. PHB is ubiquitously expressed in a circulating free form or is present in multiple cellular compartments including mitochondria, nucleus and plasma membrane. In mitochondria, PHB is anchored to the mitochondrial inner membrane and forms complexes with the ATPases associated with proteases having diverse cellular activities. PHB continuously shuttles between the mitochondria, cytosol and nucleus. In the nucleus, PHB interacts with various transcription factors and modulates transcriptional activity directly or through interactions with chromatin remodeling proteins. Many functions have been attributed to the mitochondrial and nuclear PHB complexes such as cellular differentiation, anti-proliferation, morphogenesis and maintenance of the functional integrity of the mitochondria. However, to date, the regulation of PHB expression patterns and GC physiological functions are not completely understood.//////////////////
NCBI Summary:
This gene is evolutionarily conserved, and its product is proposed to play a role in human cellular senescence and tumor suppression. Antiproliferative activity is reported to be localized to the 3' UTR, which is proposed to function as a trans-acting regulatory RNA. Several pseudogenes of this gene have been identified. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jul 2013]
Prohibitin (PHB) inhibits apoptosis in rat granulosa cells (GCs) through the extracellular signal-regulated kinase 1/2 (ERK1/2) and the Bcl family of proteins. Chowdhury I 2013 et al.
Mammalian ovarian follicular development is tightly regulated by crosstalk between cell death and survival signals, which include both endocrine and intra-ovarian regulators. Whether the follicle ultimately ovulates or undergoes atresia is dependent on the expression and actions of factors promoting follicular cell proliferation, differentiation or apoptosis. Prohibitin (PHB) is a highly conserved, ubiquitous protein that is abundantly expressed in granulosa cells (GCs) and associated with GC differentiation and apoptosis. The current study was designed to characterize the regulation of anti-apoptotic and pro-apoptotic factors in undifferentiated rat GCs (gonadotropin independent phase) governed by PHB. Microarray technology was initially employed to identify potential apoptosis-related genes, whose expression levels within GCs were altered by either staurosporine (STS) alone or STS in presence of ectopically over-expressed PHB. Next, immunoblot studies were performed to examine the expression patterns of selective Bcl-2 family members identified by the microarray analysis, which are commonly regulated in the intrinsic-apoptotic pathway. These studies were designed to measure protein levels of Bcl2 family in relation to expression of the acidic isoform (phosphorylated) PHB and the components of MEK-Erk1/2 pathway. These studies indicated that over-expression of PHB in undifferentiated GCs inhibit apoptosis which concomitantly results in an increased level of the anti-apoptotic proteins Bcl2 and Bclxl, reduced release of cytochrome c from mitochondria and inhibition of caspase-3 activity. In contrast, silencing of PHB expression resulted in change of mitochondrial morphology from the regular reticular network to a fragmented form, which enhanced sensitization of these GCs to the induction of apoptosis. Collectively, these studies have provided new insights on the PHB-mediated anti-apoptotic mechanism, which occurs in undifferentiated GCs through a PHB?Mek-Erk1/2?Bcl/Bcl-xL pathway and may have important clinical implications.
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Prohibitin regulates the FSH signaling pathway in rat granulosa cell differentiation. Chowdhury I et al. (2016) Published results from our laboratory identified prohibitin (PHB), a gene product expressed in granulosa cells (GCs) that progressively increases during follicle maturation. Our current in vitro studies demonstrate that follicle stimulating hormone (FSH) stimulates Phb expression in primary rat GCs. The FSH dependent expression of PHB was primarily localized within mitochondria, and positively correlates with the morphological changes in GCs organelles, and synthesis and secretions of estradiol (E2) and progesterone (P4). In order to confirm that PHB plays a regulatory role in rat GC differentiation, endogenous PHB knock-down studies were performed in undifferentiated GCs using adenoviral-mediated RNA interference methodology. Knock-down of PHB in GCs resulted in suppression of the key steroidogenic enzymes including StAR, p450scc, 3β-HSD, and aromatase (cyp19a1). Decreased E2 and P4 synthesis and secretions were observed in the presence of FSH stimulation. Furthermore, these experimental studies also provided direct evidence that PHB within the mitochondrial fraction in GCs is phosphorylated at residues Y249, T258 and Y259 in response to FSH stimulation. The observed levels of phosphorylation of PHB at Y248, T258 and Y259 were significantly low in GCs in the absence of FSH stimulation. In addition, during GC differentiation FSH-induced expression of pPHB requires the activation of MEK1-ERK1/2 signaling pathway. Taken together, these studies provide new evidence supporting the hypothesis that FSH dependent PHB/pPHB up-regulation in GCs is required to sustain the differentiated state of GCs.//////////////////
Follicular stage-dependent regulation of apoptosis and steroidogenesis by prohibitin in rat granulosa cells. Wang Q et al. BACKGROUND: Follicular growth and atresia are tightly regulated processes, which involve the participation of endocrine, autocrine and paracrine factors at the cellular level. Prohibitin (PHB) is a multifunctional intracellular protein playing an important role in the regulation of proliferation, apoptosis and differentiation. Here we examined the expression of PHB and its regulation by FSH in vitro and studied the role of PHB in the regulation of apoptosis and steroidogenesis in response to the apoptosis inducer staurosporine (STS) and to FSH, respectively. METHODS: Undifferentiated and differentiated granulosa cells were collected from diethylstilbestrol (DES)- and equine chronic gonadotropin (eCG)-primed immature rats, respectively and then cultured with various treatments (FSH, adenovirus infection, STS) according to experimental design. The apoptosis rate, the production of estradiol and progesterone, and the expression of distinct proteins (PHB, caspase-3, phospho- and total Akt) were assessed. RESULTS: PHB is anti-apoptotic and its action is dependent on the differentiated state of the granulosa cells. Data from gain- and loss-of-function experiments demonstrate that PHB inhibited STS-induced caspase-3 cleavage and apoptosis in undifferentiated granulosa cells, but was ineffective in differentiated cells. In contrast, PHB suppresses FSH-induced steroidogenesis and this response is evident irrespective of the differentiated state of granulosa cells. CONCLUSION: These findings suggest that PHB regulates granulosa cell apoptosis and steroidogenesis in a follicular stage-dependent manner and that the dysregulation of PHB expression and action may be relevant to ovarian dysfunction.
Prohibitin (PHB) acts as a potent survival factor against ceramide induced apoptosis in rat granulosa cells. Chowdhury I et al. AIM: Ceramide is a key factor in inducing germ cell apoptosis by translocating from cumulus cells into the adjacent oocyte and lipid rafts through gap junctions. Therefore studies designed to elucidate the mechanistic pathways in ceramide induced granulosa cell (GC) apoptosis and follicular atresia may potentially lead to the development of novel lipid-based therapeutic strategies that will prevent infertility and premature menopause associated with chemo and/or radiation therapy in female cancer patients. Our previous studies have shown that Prohibitin (PHB) is intimately involved in GCs differentiation, atresia, and luteolysis. MAIN METHODS: In the present study, we have examined the functional effects of loss-/gain-of-function of PHB using adenoviral technology in delaying apoptosis induced by the physiological ligand ceramide in rat GCs. KEY FINDINGS: Under these experimental conditions, exogenous ceramide C-8 (50?M) augmented the expression of mitochondrial PHB and subsequently cause the physical destruction of GC by the release of mitochondrial cytochrome c and activation of caspase-3. In further studies, silencing of PHB expression by adenoviral small interfering RNA (shRNA) sensitized GCs to ceramide C8-induce apoptosis. In contrast, adenovirus (Ad) directed overexpression of PHB in GCs resulted in increased PHB content in mitochondria and delayed the onset of ceramide induced apoptosis in the infected GCs. SIGNIFICANCE: Taken together, these results provide novel evidences that a critical level of PHB expression within the mitochondria plays a key intra-molecular role in GC fate by mediating the inhibition of apoptosis and may therefore, contribute significantly to ceramide induced follicular atresia.
Thompson et al. (1999) reported in studies involving a GnRH-agonist, that increases in prohibitin protein expression correlate with the initial events of apoptosis. They propose a growth regulatory role for prohibitin within the rat ovary.
Gene whose expression is detected by cDNA array hybridization: oncogenes, tumor
suppressors, cell cycle regulators. Also, relative transcript level reproducibly decreases during IVM Rozenn Dalbi?Tran and Pascal Mermilloda
Apoptosis of Rat Granulosa Cells After Staurosporine And Serum Withdrawal Is Suppressed by Adenovirus Directed Overexpression of Prohibitin* Chowdhury I et al. 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 programed cell death (PCD) in granulosa cells is unknown. In the present study we examined the role of prohibitin in mediating staurosporine and serum withdrawal induced apoptosis in undifferentiated rat granulosa cells. Treatment of granulosa cells isolated from immature rat ovaries with staurosporine 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 staurosporine 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 staurosporine-induced apoptotic stimulus. This result was corroborated by Western blot analysis with GFP-specific antibody. Furthermore, the prohibitin-eGFP fusion protein also inhibited PCD. These results provide evidence that prohibitin could serve an anti-apoptotic role in undifferentiated granulosa cells.
Expression regulated by
FSH, LH, Steroids
Comment
Thompson et al. (1997) reported changes in the expression of p28/prohibitin during the ongoing differentiation of granulosa cells. Although prohibitin is constitutively expressed in cultured granulosa cells, there is an increase in the more acidic isoform of prohibitin when oestrogen concentrations are raised by increased production or exogenous addition. This increase in this acidic isoform of prohibitin is due to phosphorylation. It is possible that oestrogen induces phosphorylation of prohibitin and, thus, may be involved in the regulation of granulosa cell proliferation and the ontogeny of the ovarian follicle.
Ovarian localization
Oocyte, Granulosa, Theca, Luteal cells
Comment
Inhibitory Roles of Prohibitin and Chemerin in FSH-Induced Rat Granulosa Cell Steroidogenesis. Wang Q et al. Follicular differentiation is a tightly regulated process involving various endocrine, autocrine, and paracrine factors. The biosynthesis of progesterone and estradiol in response to FSH involves the regulation of multiple steroidogenic enzymes, such as p450 cholesterol side-chain cleavage enzyme and aromatase. Here we demonstrated that prohibitin (PHB), a multifunctional protein, inhibits FSH-induced progesterone and estradiol secretion in rat granulosa cells. The mRNA abundances of cyp11a (coding p450 cholesterol side-chain cleavage enzyme) and cyp19 (coding aromatase) were also suppressed by PHB in a time-dependent manner. It is known that a novel adipokine chemerin suppresses FSH-induced steroidogenesis in granulosa cells. Chemerin up-regulates the content of PHB, and PHB knockdown attenuates the suppressive role of chemerin on steroidogenesis. In addition, inhibition of phosphatidylinositol 3-kinase/Akt pathway enhances the suppressive action of PHB, whereas expression of constitutively active Akt attenuates this response. These findings suggest that PHB is a novel negative regulator of FSH-induced steroidogenesis, and its action with chemerin may contribute to the dysregulation of steroidogenesis in the pathogenesis of polycystic ovarian syndrome.
Thompson et al. (1999) showed that prohibitin was localized within granulosa cells of infant and juvenile rat ovaries in a relatively heterogeneous staining pattern. The oocyte also exhibited robust prohibitin expression at all stages of follicular development. In addition, strong prohibitin expression was evident in the corpus luteum as well as in follicles undergoing atresia.
Follicle stages
Primordial, Secondary, Antral, Preovulatory
Comment
Regulation of Prohibitin Expression During Follicular Development and Atresia in the Mammalian Ovary.
Thompson WE,et al 2004 .
Prohibitin is a ubiquitous and highly conserved protein implicated as an important regulator in cell survival. Prohibitin content is inversely associated with cell proliferation, but increases during granulosa cell differentiation, and in earlier events of apoptosis in a temperature-sensitive granulosa cell line. In this study we have characterized the spatial expression patterns for prohibitin, using established in vivo models for the induction of follicular development and atresia in the mammalian ovary. Comparative western blot analyses of granulosa cell lysates from control, equine chorionic gonadotropin (eCG)-primed, and eCG plus anti-eCG treated ovaries (gonadotropin withdrawal) were conducted. Prohibitin was immunolocalized in rat ovarian sections probed either with antibodies against proliferating cell nuclear antigen (PCNA), cholesterol side-chain cleavage cytochrome P-450 (P-450scc), or in in situ terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin end labeling (TUNEL) sections. Additionally, porcine oocytes, zygotes and blastocyts were also immunolocalized with prohibitin antibody. Immunolocalization revealed the presence of prohibitin in granulosa cells, theca-interstitial cells and the oocyte. The results indicate that prohibitin protein expression in the gonadotropin-treated cells was upregulated. Immunoreactivity of prohibitin was inversely related to PCNA expression during follicular maturation and co-localized with P-450scc. Prohibitin appeared to be translocated from the cytoplasm to the nucleus in atretic follicles, germinal vesicle-stage oocytes, zygotes and blastocysts. These results suggest that prohibitin has several functional regulatory roles in granulosa and theca-interstitial cells, and in the ovum during follicular maturation and atresia. It is likely that prohibitin may play an important role in determining the fate of these cells and eventual follicular destiny.
Arraztoa JA, et al 2005 reported the identification of genes expressed in primate primordial oocytes.
Phenotypes
PCO (polycystic ovarian syndrome)
Mutations
1 mutations
Species: mouse
Mutation name: type: targeted overexpression fertility: subfertile Comment: Prohibitin-induced obesity leads to anovulation and polycystic ovary in mice. Ande SR et al. (2017) Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder and the most common cause of female infertility. However, the etiology of the disease and the mechanisms by which this disorder progress remain unclear. Here we report that a transgenic obese mouse (Mito-Ob) developed by overexpressing prohibitin in adipocytes develops polycystic ovaries. Initially, the female Mito-Ob mice were equally fertile to their wild-type littermates. Mito-Ob mice begin to gain weight after puberty, become significantly obese between 3-6 months of age, and roughly 25% of them become infertile by 9 months of age. Despite obesity, female Mito-Ob mice maintained glucose homeostasis and insulin sensitivity similar to their wild-type littermates. Mito-Ob mice showed morphologically distinct polycystic ovaries and elevated estradiol, but normal testosterone and insulin levels. Histological analysis of the ovaries showed signs of impaired follicular dynamics like preantral follicular arrest and reduced number, or absence of corpus luteum. The ovaries in infertile Mito-Ob mice were found closely surrounded by periovarian adipose tissue suggesting a potential role in anovulation. Collectively, these data suggest that elevated estradiol and obesity per se may lead to anovulation and polycystic ovaries independent of hyperinsulinemia and hyperandrogenism. As obesity often coexists with other abnormalities known to be involved in the development of PCOS such as insulin resistance, compensatory hyperinsulinemia, and hyperandrogenism, the precise role of these factors in the disease process remain unclear. The Mito-Ob mice provide an opportunity to study the effect of obesity on anovulation and ovarian cyst formation independent of the major drivers of obesity-linked PCOS.//////////////////