Bharat Joshi

University of South Florida, Tampa, Florida, United States

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Publications (10)59.08 Total impact

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    ABSTRACT: Atherosclerosis involves a specialized inflammatory process regulated by an intricate network of cytokine and chemokine signaling. Atherosclerotic lesions lead to the release of cytokines that can have multiple affects on various vascular cell functions either promoting lesion expansion or alternatively retard progression. Tumor necrosis factor-α (TNF-α) is one such cytokine that can activate both cell survival and cell death mechanisms simultaneously. Here we show that TNF-α induces apoptosis in human aortic endothelial cells (HAECs), while it promotes the proliferation of vascular smooth muscle cells (VSMCs). Both events involved the activation of the Rb-E2F1 transcriptional regulatory pathway. Stimulation of HAECs with TNF-α led to an increased expression of p73 protein and a reduction in the levels of p53. This involved apoptosis signal-regulating kinase 1 (ASK1)- mediated inactivation of Rb and its dissociation from the p73 promoter. In contrast, TNF-α stimulation of VSMCs enhanced the association of E2F1 with proliferative promoters like thymidylate synthase and cdc25A, while Rb was dissociated. ASK1 kinase has a critical role in the apoptotic process, as its depletion or dissociation from Rb reduced TNF-α-induced apoptosis. These results show that the cytokine TNF-α can elicit diametrically opposite responses in vascular endothelial cells and VSMCs, utilizing the Rb-E2F pathway.
    Cell death and differentiation 07/2011; 19(2):274-83. · 8.24 Impact Factor
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    ABSTRACT: Recently, we have shown that RhoB suppresses EGFR-, ErbB2-, Ras- and Akt-mediated malignant transformation and metastasis. In this paper, we demonstrate that the novel antitumor agents farnesyltransferase inhibitors (FTIs) and geranylgeranyltransferase I inhibitors (GGTIs) upregulate RhoB expression in a wide spectrum of human cancer cells including those from pancreatic, breast, lung, colon, bladder and brain cancers. RhoB induction by FTI-277 and GGTI-298 occurs at the transcriptional level and is blocked by actinomycin D. Reverse transcription-PCR experiments documented that the increase in RhoB protein levels is due to an increase in RhoB transcription. Furthermore, treatment with FTIs and GGTIs of cancer cells results in HDAC1 dissociation, HAT association and histone acetylation of the RhoB promoter. Thus, promoter acetylation is a novel mechanism by which RhoB expression levels are regulated following treatment with the anticancer agents FTIs and GGTIs.
    Oncogene 03/2007; 26(5):633-40. · 7.36 Impact Factor
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    ABSTRACT: Prohibitin is a 30 kDa growth suppressive protein that has pleiotropic functions in the cell. Although prohibitin has been demonstrated to have potent transcriptional regulatory functions, it has also been proposed to facilitate protein folding in the mitochondria and promote cell migration in association with Raf-1. Our previous studies have shown that prohibitin physically interacts with the marked-box domain of E2F family members and represses their transcriptional activity; in contrast, prohibitin could bind to and enhance the transcriptional activity of p53. Here, we show that promoters of human YY1 (Yin and Yang 1) as well as caspase 7 genes are modulated by prohibitin. YY1 promoter activity was reduced upon overexpression of prohibitin, while it was enhanced when prohibitin was depleted by small interfering RNA techniques. The repressive effects of prohibitin on the YY1 promoter were mediated through E2F binding sites, as seen by mutational analysis and chromatin immunoprecipitation assays. Further, depletion of E2F1 prevented prohibitin from repressing the YY1 promoter. In contrast with YY1, prohibitin overexpression led to enhanced levels of caspase 7, whereas depletion of prohibitin reduced it. Interestingly, the caspase 7 promoter was found to have p53-binding sites and prohibitin activated this promoter through p53. These studies show that prohibitin can have diverse effects on the expression of different genes and the activity of various cellular promoters is affected by prohibitin. Further, it appears very likely that prohibitin carries out many of its cellular functions by affecting the transcription of different genes.
    Biochemical Journal 02/2007; 401(1):155-66. · 4.65 Impact Factor
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    ABSTRACT: Prohibitin is a growth regulatory gene that has pleiotropic functions in the nucleus, mitochondria, and cytoplasmic compartments. Earlier studies had proposed a role for prohibitin in modulating cellular senescence, but the underlying mechanisms remain unknown. Here we show that senescence induced by DNA-damaging agents causes the localization of prohibitin to specific heterochromatic foci. Prohibitin could bind to heterochromatin protein 1 (HP1) family proteins and colocalized with HP1gamma in senescence-associated heterochromatic foci. Further, HP1gamma could synergize with prohibitin to repress E2F1-mediated transcriptional activity. The depletion of prohibitin by small interfering RNA or antisense techniques led to a reduction in the senescent phenotype, correlating with a reduced expression of senescence-associated beta-galactosidase and fewer numbers of senescence-associated heterochromatic foci. Chromatin immunoprecipitation assays showed that prohibitin is needed for the recruitment of HP1gamma to E2F1-regulated proliferative promoters, leading to their repression. The ablation of prohibitin prevented the recruitment of HPIgamma, but not Suv39H, to the promoters upon senescence. Prohibitin-mediated recruitment of HP1gamma occurred in only senescent cells, not in quiescent cells; thus, there is a dichotomy in the recruitment of different corepressors by prohibitin, depending on the type of growth arrest. These studies show that prohibitin plays a vital role in inducing cellular senescence.
    Molecular and Cellular Biology 07/2006; 26(11):4161-71. · 5.37 Impact Factor
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    ABSTRACT: Non-small cell lung cancer (NSCLC) demonstrates a strong etiologic association with smoking. Although nicotine is not carcinogenic, it can induce cell proliferation and angiogenesis and suppress apoptosis induced by certain agents. Here we show that nicotine inhibits apoptosis induced by the drugs gemcitabine, cisplatin, and taxol, which are used to treat NSCLCs. This protection correlated with the induction of XIAP and survivin by nicotine in a panel of human NSCLC cell lines, and depletion of XIAP and survivin ablated the protective effects of nicotine. The antiapoptotic effects of nicotine were mediated by dihydro beta-erythroidine-sensitive alpha3-containing nicotinic acetylcholine receptors and required the Akt pathway. Chromatin immunoprecipitation assays demonstrated that nicotine stimulation caused an increased recruitment of E2F1 and concomitant dissociation of retinoblastoma tumor suppressor protein (Rb) from survivin promoter in A549 cells. Moreover, ablation of E2F1 levels caused abrogation of the protective effects of nicotine against cisplatin-induced apoptosis in A549 cells whereas ablation of signal transducer and activator of transcription 3 levels had no effect. These studies suggest that exposure to nicotine might negatively impact the apoptotic potential of chemotherapeutic drugs and that survivin and XIAP play a key role in the antiapoptotic activity of nicotine.
    Proceedings of the National Academy of Sciences 05/2006; 103(16):6332-7. · 9.74 Impact Factor
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    ABSTRACT: Prohibitin is a growth-suppressive protein that has multiple functions in the nucleus and the mitochondria. Our earlier studies had shown that prohibitin represses the activity of E2F transcription factors while enhancing p53-mediated transcription. At the same time, prohibitin has been implicated in mediating the proper folding of mitochondrial proteins. We had found that treatment of cells with camptothecin, a topoisomerase 1 inhibitor, led to the export of prohibitin and p53 from the nucleus to the mitochondria. Here we show that the camptothecin-induced export of prohibitin occurs preferentially in transformed cell lines, but not in untransformed or primary cells. Cells that did not display the translocation of prohibitin were refractive to the apoptotic effects of camptothecin. The translocation was mediated by a putative nuclear export signal at the C-terminal region of prohibitin; fusion of the nuclear export signal (NES) of prohibitin to green fluorescence protein led to its export from the nucleus. Leptomycin B could inhibit the nuclear export of prohibitin showing that it was a CRM-1-dependent event driven by Ran GTPase. Confirming this, prohibitin was found to physically interact with CRM-1, and this interaction was significantly higher in transformed cells. Delivery of a peptide corresponding to the NES of prohibitin prevented the export of prohibitin to cytoplasm and protected cells from apoptosis. These results suggest that the regulated translocation of prohibitin from the nucleus to the mitochondria facilitates its pleiotropic functions and might contribute to its anti-proliferative and tumor suppressive properties.
    Journal of Biological Chemistry 03/2006; 281(5):2951-9. · 4.65 Impact Factor
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    ABSTRACT: The E2F transcription factors induce the expression of many genes in response to specific extracellular stimuli. Here, we show that human metallothionein 1G (hMT1G) promoter is upregulated by E2F1 upon VEGF stimulation of human aortic endothelial cells. Analysis of the hMT1G promoter showed the presence of many potential E2F-binding sites flanked by potential SP1 sites and metal response elements (MREs). hMT1G promoter could be induced by E2F1 in transient transfections; further, deletion analysis suggested that the region spanning the E2F-binding sites was necessary for VEGF-mediated induction. E2Fs 1-5 could bind to the hMT1G promoter in a chromatin immunoprecipitation assay. VEGF stimulation led to an increased binding of E2Fs 1-3 to the endogenous hMT1G promoter; at the same time, the binding of Rb, p107 and p130 to the promoter was abolished. VEGF stimulation also led to the increased acetylation E2F1 as well as the histones in the hMT1G promoter region. Stimulation with metals or VEGF led to dissociation of histone deacetylase 1 (HDAC1) from the promoter, leading to acetylation of histones. Induction of the hMT1G promoter upon exposure to heavy metals such as Zn and Cd is mediated by the MRE. Interestingly, mutation of MRE affected the metal response, but not the VEGF response of the hMT1G promoter. In contrast, deletion of the E2F-binding sites did not affect the metal response. Based on these findings, we conclude that induction of the hMT1G promoter by VEGF and heavy metals occurs through the utilization of different transcription factors.
    Oncogene 04/2005; 24(13):2204-17. · 7.36 Impact Factor
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    ABSTRACT: The retinoblastoma protein Rb has antiproliferative and antiapoptotic functions. Our previous studies have shown that certain apoptotic signals can inactivate Rb via the p38 pathway. Here we show that Rb associates with the apoptosis signal-regulating kinase ASK1 in response to specific apoptotic signals. An LXCXE motif on ASK1 was required for Rb binding; this correlated with increased E2F1 transcriptional activity and up-regulation of the proapoptotic protein p73. Overexpression of Rb inhibited ASK1-induced apoptosis; in addition, an ASK1 mutant incapable of binding Rb could not induce apoptosis, indicating that ASK1 has to overcome the antiapoptotic properties of Rb to kill cells. Chromatin immunoprecipitation assays show that in asynchronous cells the p73P1 promoter is occupied predominantly by E2F3; upon tumor necrosis factor (TNF)-alpha stimulation, E2F3 is dissociated from the promoter and replaced by E2F1. At the same time, TNF-alpha stimulation causes Rb to dissociate from the p73P1 promoter. These are promoter-specific events because Rb binds to the mitogenic cdc25A promoter upon TNF-alpha stimulation. These studies suggest that Rb acts as a link between apoptotic and proliferative pathways by interacting with distinct kinases and occupying different promoters.
    Journal of Biological Chemistry 10/2004; 279(37):38762-9. · 4.65 Impact Factor
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    ABSTRACT: Prohibitin is a potential tumor suppressor protein that can repress E2F-mediated transcription and arrest cell proliferation. We had shown previously that prohibitin could bind to the Rb protein as well as E2F and this binding was necessary to suppress cell proliferation. Here we show that the E2F1 binding domain of prohibitin has the potential to fold into a coiled-coil structure. This coiled-coil domain by itself could physically interact with E2F1 and block its transcriptional activity. Like full-length prohibitin, the coiled-coil domain also recruited histone deacetylase 1 to repress E2F1. The coiled-coil domain also exhibited growth suppressive properties and we observed a 64% reduction in colony numbers when transfected into T47D cells. Interestingly, a synthetic peptide corresponding to the coiled-coil domain induced apoptosis in four different human cell lines. It is possible that agents that can mimic this peptide would be of value in controlling proliferative disorders.
    Biochemical and Biophysical Research Communications 01/2004; 312(2):459-66. · 2.41 Impact Factor
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    ABSTRACT: Prohibitin, a potential tumor suppressor protein, has been shown to inhibit cell proliferation and repress E2F transcriptional activity. Though prohibitin has potent transcriptional functions in the nucleus, a mitochondrial role for prohibitin has also been proposed. Here we show that prohibitin is predominantly nuclear in two breast cancer cell lines where it co-localizes with E2F1 and p53. Upon apoptotic stimulation by camptothecin, prohibitin is exported to perinuclear regions where it localizes to mitochondria. The data presented here also show that prohibitin is capable of physically interacting with p53 in vivo and in vitro. Prohibitin was found to enhance p53-mediated transcriptional activity and cotransfection of an antisense prohibitin construct reduces p53-mediated transcriptional activation. Prohibitin appears to induce p53-mediated transcription by enhancing its recruitment to promoters, as detected by chromatin immunoprecipitation assays. These results suggest that prohibitin is capable of modulating Rb/E2F as well as p53 regulatory pathways.
    Journal of Biological Chemistry 12/2003; 278(48):47853-61. · 4.65 Impact Factor