The Microtubule Plus End Tracking Protein TIP150 Interacts with Cortactin to Steer Directional Cell Migration.

Cell migration is orchestrated by dynamic interactions of microtubules with the plasma membrane cortex. How these interactions facilitate these dynamic processes is still being actively investigated. TIP150 is a newly characterized microtubule plus end tracking protein essential for mitosis and entosis (Ward, T., Wang, M., Liu, X., Wang, Z., Xia, P., Chu, Y., Wang, X., Liu, L., Jiang, K., Yu, H., Yan, M., Wang, J., Hill, D. L., Huang, Y., Zhu, T., and Yao, X. (2013) Regulation of a dynamic interaction between two microtubule-binding proteins, EB1 and TIP150, by the mitotic p300/CBP-associated factor (PCAF) orchestrates kinetochore microtubule plasticity and chromosome stability during mitosis. J. Biol. Chem. 288, 15771-15785; Xia, P., Zhou, J., Song, X., Wu, B., Liu, X., Li, D., Zhang, S., Wang, Z., Yu, H., Ward, T., Zhang, J., Li, Y., Wang, X., Chen, Y., Guo, Z., and Yao, X. (2014) Aurora A orchestrates entosis by regulating a dynamic MCAK-TIP150 interaction. J. Mol. Cell Biol. 6, 240-254). Here we show that TIP150 links dynamic microtubules to steer cell migration by interacting with cortactin. Mechanistically, TIP150 binds to cortactin via its C-terminal tail. Interestingly, the C-terminal TIP150 proline-rich region (CT150) binds to the Src homology 3 domain of cortactin specifically, and such an interaction is negatively regulated by EGF-elicited tyrosine phosphorylation of cortactin. Importantly, suppression of TIP150 or overexpression of phospho-mimicking cortactin inhibits polarized cell migration. In addition, CT150 disrupts the biochemical interaction between TIP150 and cortactin in vitro, and perturbation of the TIP150-cortactin interaction in vivo using a membrane-permeable TAT-CT150 peptide results in an inhibition of directional cell migration. We reason that a dynamic TIP150-cortactin interaction orchestrates directional cell migration via coupling dynamic microtubule plus ends to the cortical cytoskeleton.

Prohibitin regulates the FSH signaling pathway in rat granulosa cell differentiation.

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 rat primary 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-knockdown studies were carried out in undifferentiated GCs using adenoviral (Ad)-mediated RNA interference methodology. Knockdown of PHB in GCs resulted in the suppression of the key steroidogenic enzymes including steroidogenic acute regulatory protein (StAR), p450 cholesterol side-chain cleavage enzyme (p450scc), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and aromatase (Cyp19a1); and decreased E2 and P4 synthesis and secretions 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 Y249, T258, and Y259 were significantly low in GCs in the absence of FSH stimulation. In addition, during GC differentiation FSH-induced expression of phospho-PHB (pPHB) requires the activation of MEK1-ERK1/2 signaling pathway. Taken together, these studies provide new evidence supporting FSH-dependent PHB/pPHB upregulation in GCs is required to sustain the differentiated state of GCs.

Prohibitin( PHB) roles in granulosa cell physiology.

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.

The spatiotemporal dynamics of chromatin protein HP1α is essential for accurate chromosome segregation during cell division.

Heterochromatin protein 1α (HP1α) is involved in regulation of chromatin plasticity, DNA damage repair, and centromere dynamics. HP1α detects histone dimethylation and trimethylation of Lys-9 via its chromodomain. HP1α localizes to heterochromatin in interphase cells but is liberated from chromosomal arms at the onset of mitosis. However, the structural determinants required for HP1α localization in interphase and the regulation of HP1α dynamics have remained elusive. Here we show that centromeric localization of HP1α depends on histone H3 Lys-9 trimethyltransferase SUV39H1 activity in interphase but not in mitotic cells. Surprisingly, HP1α liberates from chromosome arms in early mitosis. To test the role of this dissociation, we engineered an HP1α construct that persistently localizes to chromosome arms. Interestingly, persistent localization of HP1α to chromosome arms perturbs accurate kinetochore-microtubule attachment due to an aberrant distribution of chromosome passenger complex and Sgo1 from centromeres to chromosome arms that prevents resolution of sister chromatids. Further analyses showed that Mis14 and perhaps other PXVXL-containing proteins are involved in directing localization of HP1α to the centromere in mitosis. Taken together, our data suggest a model in which spatiotemporal dynamics of HP1α localization to centromere is governed by two distinct structural determinants. These findings reveal a previously unrecognized but essential link between HP1α-interacting molecular dynamics and chromosome plasticity in promoting accurate cell division.

Prohibitins role in cellular survival through Ras-Raf-MEK-ERK pathway.

Prohibitins are members of a highly conserved protein family containing the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain (also known as the prohibitin [PHB] domain) found in unicellular eukaryotes, fungi, plants, animals, and humans. Two highly homologous members of prohibitins expressed in eukaryotes are prohibitin (PHB; B-cell receptor associated protein-32, BAP-32) and prohibitin 2/repressor of estrogen receptor activity (PHB2, REA, BAP-37). Both PHB and REA/PHB2 are ubiquitously expressed and are present in multiple cellular compartments including the mitochondria, nucleus, and the plasma membrane. Multiple functions have been attributed to the mitochondrial and nuclear PHB and PHB2/REA including cellular differentiation, anti-proliferation, and morphogenesis. One of the major functions of the prohibitins are in maintaining the functional integrity of the mitochondria and protecting cells from various stresses. In the present review, we focus on the recent research developments indicating that PHB and PHB2/REA are involved in maintaining cellular survival through the Ras-Raf-MEK-Erk pathway. Understanding the molecular mechanisms by which the intracellular signaling pathways utilize prohibitins in governing cellular survival is likely to result in development of therapeutic strategies to overcome various human pathological disorders such as diabetes, obesity, neurological diseases, inflammatory bowel disease, and cancer.

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.

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.

The emerging roles of prohibitins in folliculogenesis.

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.

Sperm GIRK2-containing K+ inward rectifying channels participate in sperm capacitation and fertilization.

The GIRK2-containing inward-rectifying K(+) ion channels have been implicated in mammalian spermatogenesis. While the Girk2 null mice are fertile, the male weaver transgenic mice carrying a gain-of-function mutation in the Girk2 gene are infertile. To establish the exact period of spermatogenesis affected by this mutation, we performed StaPut isolation and morphological characterization of the germ cells present in the weaver testis. Germ cells representing all periods of spermatogenesis were identified. However, no spermatozoa were present, suggesting that this mutation only affected the haploid phase of spermatogenesis. Real-time PCR studies performed on StaPut purified germ cells from wild-type mice indicated that the Girk2 transcripts were exclusively expressed in spermatids. Immunofluorescence studies of mouse and boar spermatids/spermatozoa localized the GIRK2 K(+) containing channels to the acrosomal region of the sperm plasma membrane. During porcine in vitro fertilization (IVF), GIRK2-containing channels remained associated with the acrosomal shroud following zona-induced acrosome reaction. Fertilization was blocked by tertiapin-Q (TQ), a specific inhibitor of GIRK channels, and by anti-GIRK2 antibodies. Altogether, studies in two different mammalian species point to a conserved mechanism by which the GIRK2 inward-rectifying K(+) ion channels support sperm function during fertilization.

Prohibitin (PHB) acts as a potent survival factor against ceramide induced apoptosis in rat granulosa cells.

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 µM) 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.

Developmental patterns of PPAR and RXR gene expression during spermatogenesis.

Members of the family of nuclear receptors that include peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors (RXRs) are important mediators of selective gene activation during development and cellular differentiation. In this study, developmentally-specific PPAR and RXR patterns of expression that occur in somatic and germ cell populations in the testis were determined using quantative real-time PCR (qRT-PCR) studies on RNAs that were isolated from StaPut purified mouse germ cells and primary rat Sertoli cells. These qRT-PCR studies indicate that transcripts encoding the PPAR-Alpha (α), -Beta (ß), and -Gamma (γ) and RXR -Alpha (α), -Beta (ß), and -Gamma (γ) are developmentally expressed in both differentiating germ and Sertoli cells. In further experiments aimed at deciphering the physiological role that PPAR-Gamma (γ) plays in Sertoli cells, 15-day primary rat Sertoli cells were infected with recombinant adenoviral vectors containing PPAR-Gamma (γ) cDNA and PPAR-Gamma (γ) RNAi constructs. Affymetrix microarray analysis and qRT-PCR validation studies using total RNA isolated from these transfected cells indicated that PPAR-Gamma regulates the pattern of expression of key lipid metabolic genes in Sertoli cells.

PinX1 is a novel microtubule-binding protein essential for accurate chromosome segregation.

Mitosis is an orchestration of dynamic interactions between spindle microtubules and chromosomes, which is mediated by protein structures that include the kinetochores, and other protein complexes present on chromosomes. PinX1 is a potent telomerase inhibitor in interphase; however, its function in mitosis is not well documented. Here we show that PinX1 is essential for faithful chromosome segregation. Deconvolution microscopic analyses show that PinX1 localizes to nucleoli and telomeres in interphase and relocates to the periphery of chromosomes and the outer plate of the kinetochores in mitosis. Our deletion analyses mapped the kinetochore localization domain of PinX1 to the central region and its chromosome periphery localization domain to the C terminus. Interestingly, the kinetochore localization of PinX1 is dependent on Hec1 and CENP-E. Our biochemical characterization revealed that PinX1 is a novel microtubule-binding protein. Our real time imaging analyses show that suppression of PinX1 by small interference RNA abrogates faithful chromosome segregation and results in anaphase chromatid bridges in mitosis and micronuclei in interphase, suggesting an essential role of PinX1 in chromosome stability. Taken together, the results indicate that PinX1 plays an important role in faithful chromosome segregation in mitosis.

Molecular and functional characterization of the murine ldh2 promoter region: Sp-binding GC-box domains are the key cis-elements regulating ldh2 gene expression during spermatogenesis.

The goal of the present study was to elucidate the specific transcriptional mechanisms that regulate ldh2 gene expression during the early stages of spermatogenesis. DNA sequence analysis of the 1.0-kb ldh2 promoter region directly upstream of the transcriptional start site indicated the presence of three SP-protein binding GC-box elements and the absence of TATA and CAAT boxes. Functional characterization studies of the mouse ldh2 promoter were performed in the SV40 transformed mouse spermatogonial cell line, GC-1 spg. Transfection/transient expression studies using full-length and truncated ldh2 promoter/luciferase reporter constructs revealed that all three of the SP-binding cis-regulatory GC-box elements are required for optimal ldh2 promoter activity. Additional site-directed mutagenesis studies indicated that the two most proximal GC-box sites play essential regulatory roles in mediating basal ldh2 promoter activity. These studies suggest that the expression of the ldh2 gene in spermatogonia and early spermatocytes are regulated by SP-mediated transcriptional mechanisms.

Prohibitin silencing reverses stabilization of mitochondrial integrity and chemoresistance in ovarian cancer cells by increasing their sensitivity to apoptosis.

Current approaches to the treatment of ovarian cancer are limited because of the development of resistance to chemotherapy. Prohibitin (Phb1) is a possible candidate protein that contributes to development of drug resistance, which could be targeted in neoplastic cells. Phb1 is a highly conserved protein that is associated with a block in the G0/G1 phase of the cell cycle and also with cell survival. Our study was designed to determine the role of Phb1 in regulating cellular growth and apoptosis in ovarian cancer cells. Our results showed that Phb1 content is differentially overexpressed in papillary serous ovarian carcinoma and endometrioid ovarian adenocarcinoma when compared to normal ovarian epithelium and was inversely related to Ki67 expression. Immunofluorescence microscopy and Western analyses revealed that Phb1 is primarily associated with the mitochondria in ovarian cancer cells. Over-expression of Phb1 by adenoviral Phb1 infection resulted in an increase in the percentage of ovarian cancer cells accumulating at G0/G1 phase of the cell cycle. Treatment of ovarian cancer cells with staurosporine (STS) induced apoptosis in a time-dependent manner. Phb1 over-expression induced cellular resistance to STS via the intrinsic apoptotic pathway. In contrast, silencing of Phb1 expression by adenoviral small interfering RNA (siRNA) sensitized ovarian cancer cells to STS-induce apoptosis. Taken together, these results suggest that Phb1 induces block at G0/G1 phase of the cell cycle and promotes survival of cancer cells. Furthermore, silencing of the Phb1 gene expression may prove to be a valuable therapeutic approach for chemoresistant ovarian cancer by increasing sensitivity of cancer cells to apoptosis.

SP1 transcription factors in male germ cell development and differentiation.

Transcription factor SP1 is a zinc finger protein that has been implicated in regulating the expression of several genes involved in cellular differentiation and embryonic development. The zinc finger region of SP1 transcription factors binds to GC or GT-box elements present in the promoters of a number of male germ cell target genes that are developmentally expressed during spermatogenesis. The glutamine and serine/threonine-rich regions of the SP1 proteins recruit co-regulatory factors to the multi-protein preinitiation complex that are important for mediating transcriptional activation in male germ cells. Studies in our laboratory have identified several alternatively spliced transcripts encoding SP1 isoforms that display stage and cell-type-specific expression profiles in differentiating germ cells in the seminiferous epithelium of the testis. This review summarizes the expression patterns and functional significance of these SP1 transcription factor variants during spermatogenesis.

Apoptosis of rat granulosa cells after staurosporine and serum withdrawal is suppressed by adenovirus-directed overexpression of prohibitin.

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.

Identification, characterization, and functional analysis of sp1 transcript variants expressed in germ cells during mouse spermatogenesis.

The SP family of zinc-finger transcription factors are important mediators of selective gene activation during embryonic development and cellular differentiation. SP-binding GC-box domains are common cis-regulatory elements present in the promoters of several genes expressed in a developmentally specific manner in differentiating mouse germ cells. Four Sp1 cDNAs were isolated from a mouse pachytene spermatocyte cDNA library and characterized by DNA sequence analysis. Northern blot studies revealed that these cDNAs corresponded to 3 full-length Sp1 transcripts (4.1, 3.7, and 3.2 kilobases [kb]) and an additional 1.4-kb 5'-truncated Sp1 transcript that are temporally expressed during spermatogenesis. Quantitative real-time polymerase chain reaction studies verified that the highest levels of Sp1 transcript expression of 4.1, 3.7, and 3.2 kb occur in the primary spermatocytes. The spatial and temporal expression patterns of these Sp1 transcripts and their encoded 60-kDa and 90-kDa SP1 proteins were demonstrated using in situ hybridization and immunohistochemical analyses. To assess the transcriptional properties of these SP1 transcription factors, SP-deficient Drosophila SL2 cells were stably transfected with the respective Sp1 cDNA expression vectors and cotransfected with either Ldh2, Ldh3, or Creb promoter/luciferase reporter constructs. The levels of SP-mediated luciferase expression observed depended on the structure of the glutamine-rich transactivation domains and the number of GC-box elements present in the respective promoters. The alterations observed in germ cells in the patterns of expression of the Sp1 transcripts encoding the 60-kDa and 90-kDa SP1 isoforms suggest that these SP1 factors may be involved in mediating stage-specific and cell type-specific gene expression during mouse spermatogenesis.