Non-Metabolic Role of PKM2 in Regulation of the HIV-1 LTR.

Identification of cellular proteins, in addition to already known transcription factors such as NF-κB, Sp1, C-EBPß, NFAT, ATF/CREB, and LEF-1, which interact with the HIV-1 LTR, is critical in understanding the mechanism of HIV-1 replication in monocytes/macrophages. Our studies demonstrate upregulation of pyruvate kinase isoform M2 (PKM2) expression during HIV-1SF162 infection of monocyte/macrophages and reactivation of HIV-1 in U1 cells, a macrophage model of latency. We observed that HIV-1SF162 infection of monocyte/macrophages and reactivation of HIV-1 in U1 cells by PMA resulted in increased levels of nuclear PKM2 compared to PMA-induced U937 cells. Furthermore, there was a significant increase in the nuclear dimeric form of PKM2 in the PMA-induced U1 cells in comparison to PMA-induced U937 cells. We focused on understanding the potential role of PKM2 in HIV-1 LTR transactivation. Chromatin immunoprecipitation (ChIP) analysis in PMA-activated U1 and TZM-bl cells demonstrated the interaction of PKM2 with the HIV-1 LTR. Our studies show that overexpression of PKM2 results in transactivation of HIV-1 LTR-luciferase reporter in U937, U-87 MG, and TZM-bl cells. Using various truncated constructs of the HIV-1 LTR, we mapped the region spanning -120 bp to -80 bp to be essential for PKM2-mediated transactivation. This region contains the NF-κB binding site and deletion of this site attenuated PKM2-mediated activation of HIV-1 LTR. Immunoprecipitation experiments using U1 cell lysates demonstrated a physical interaction between PKM2 and the p65 subunit of NF-κB. These observations demonstrate for the first time that PKM2 is a transcriptional co-activator of HIV-1 LTR. J. Cell. Physiol. 232: 517-525, 2017. © 2016 Wiley Periodicals, Inc.

Regulation of the HIV-1 promoter by HIF-1α and Vpr proteins.

We previously demonstrated the ability of HIV-1 Vpr protein to activate the oxidative stress pathway, thus leading to the induction of the hypoxia inducible factor 1 alpha (HIF-1α). Therefore, we sought to examine the interplay between the two proteins and the impact of HIF-1α activation on HIV-1 transcription. Using transient transfection assays, we identified the optimal concentration of HIF-1α necessary for the activation of the HIV-1 promoter as well as the domain within HIF-1α responsible for this activation. Our findings indicated that activation of the HIV-1 LTR by Vpr is HIF-1α dependent. Furthermore, we showed that both Vpr and HIF-1α activate the HIV-1 promoter through the GC-rich binding domain within the LTR. Taken together, these data shed more light on the mechanisms used by Vpr to activate the HIV-1 promoter and placed HIF-1α as a major participant in this activation.

High-performance capillary electrophoresis for determining HIV-1 Tat protein in neurons.

The HIV-1 protein, Tat has been implicated in AIDS pathogenesis however, the amount of circulating Tat is believed to be very low and its quantification has been difficult. We performed the quantification of Tat released from infected cells and taken up by neurons using high performance capillary electrophoresis. This is the first report to successfully measure the amount of Tat in neurons and places Tat as a key player involved in HIV-associated neurocognitive disorders.

IKK{gamma} protein is a target of BAG3 regulatory activity in human tumor growth.

BAG3, a member of the BAG family of heat shock protein (HSP) 70 cochaperones, is expressed in response to stressful stimuli in a number of normal cell types and constitutively in a variety of tumors, including pancreas carcinomas, lymphocytic and myeloblastic leukemias, and thyroid carcinomas. Down-regulation of BAG3 results in cell death, but the underlying molecular mechanisms are still elusive. Here, we investigated the molecular mechanism of BAG3-dependent survival in human osteosarcoma (SAOS-2) and melanoma (M14) cells. We show that bag3 overexpression in tumors promotes survival through the NF-kappaB pathway. Indeed, we demonstrate that BAG3 alters the interaction between HSP70 and IKKgamma, increasing availability of IKKgamma and protecting it from proteasome-dependent degradation; this, in turn, results in increased NF-kappaB activity and survival. These results identify bag3 as a potential target for anticancer therapies in those tumors in which this gene is constitutively expressed. As a proof of principle, we show that treatment of a mouse xenograft tumor model with bag3siRNA-adenovirus that down-regulates bag3 results in reduced tumor growth and increased animal survival.

Modulation of JC virus transcription by C/EBPbeta.

The polyomavirus JC (JCV) causes the demyelinating disease progressive multifocal leukoencephalopathy (PML). Infection by JCV is very common in childhood after which the virus enters a latent state, which is poorly understood. Under conditions of severe immunosuppression, especially AIDS, JCV may reactivate to cause PML. Expression of JC viral proteins is regulated by the JCV non-coding control region (NCCR), which contains an NF-kappaB binding site previously shown to activate transcription. We now report that C/EBPbeta inhibits basal and NF-kappaB-stimulated JCV transcription via the same site. Gel shift analysis showed C/EBPbeta bound to this region in vitro and ChIP assays confirmed this binding in vivo. Further, a ternary complex of NF-kappaB/p65, C/EBPbeta-LIP and JCV DNA could be detected in co-immunoprecipitation experiments. Mutagenesis analysis of the JCV NCCR indicated p65 and C/EBPbeta-LIP bound to adjacent but distinct sites and that both sites regulate basal and p65-stimulated transcription. Thus C/EBPbeta negatively regulates JCV, which together with NF-kappaB activation, may control the balance between JCV latency and activation leading to PML. This balance may be regulated by proinflammatory cytokines in the brain.

Monocyte chemoattractant protein-1 (MCP-1): an overview.

Chemokines constitute a family of chemoattractant cytokines and are subdivided into four families on the basis of the number and spacing of the conserved cysteine residues in the N-terminus of the protein. Chemokines play a major role in selectively recruiting monocytes, neutrophils, and lymphocytes, as well as in inducing chemotaxis through the activation of G-protein-coupled receptors. Monocyte chemoattractant protein-1 (MCP-1/CCL2) is one of the key chemokines that regulate migration and infiltration of monocytes/macrophages. Both CCL2 and its receptor CCR2 have been demonstrated to be induced and involved in various diseases. Migration of monocytes from the blood stream across the vascular endothelium is required for routine immunological surveillance of tissues, as well as in response to inflammation. This review will discuss these biological processes and the structure and function of CCL2.

HIV-1 Vpr deregulates calcium secretion in neural cells.

The lack of productive infection of neurons by HIV-1 suggests that the neuronal damage seen in AIDS patients with cognitive disorders is caused indirectly via viral and cellular proteins with neurotoxic activity. Among HIV-1 proteins, Vpr has been shown to deregulate expression of various important cytokines and inflammatory proteins in infected and uninfected cells. However, the mechanisms underlying these changes remain unclear. Here, we demonstrate that neurons can take up Vpr that is released into the supernatant of HIV-infected microglia. We also found that administration of recombinant Vpr (rVpr) to human neurons resulted in a slow but sustained elevation of intracellular calcium [Ca(2+)]i. Interestingly, our data also show that [Ca(2+)]i elevation by Vpr leads to ROS production and impairs glutamate signaling in neuronal cells. Vpr disturbs calcium homeostasis through downregulation of endogenous PMCA. Finally, we found that the permeability of the plasma membrane increases in neurons treated with Vpr. Therefore, we conclude that soluble Vpr is a major viral factor that causes a disturbance in neuronal communication leading to neuronal dysfunction. The outcome of these studies will advance the understanding of HIV-1 pathogenesis and will help in the development of new therapeutic approaches.

Activation of the oxidative stress pathway by HIV-1 Vpr leads to induction of hypoxia-inducible factor 1alpha expression.

The detection of biomarkers of oxidative stress in brain tissue and cerebrospinal fluid of patients with human immunodeficiency virus, type 1 (HIV)-associated dementia indicates the involvement of stress pathways in the neuropathogenesis of AIDS. Although the biological importance of oxidative stress on events involved in AIDS neuropathogenesis and the HIV-1 proteins responsible for oxidative stress remain to be elucidated, our results point to the activation of hypoxia-inducible factor 1 (HIF-1) upon HIV-1 infection and its elevation in brain cells of AIDS patients with dementia. HIF-1 is a transcription factor that is responsive to oxygen. Under hypoxic conditions, HIF-1alpha becomes stable and translocates to the nucleus where it dimerizes with aryl hydrocarbon receptor nuclear translocator and modulates gene transcription. Activation of HIF-1 can also be mediated by the HIV-1 accessory protein Vpr. In addition, cellular components, including reactive oxygen species, contribute to the induction of HIF-1alpha. Our results show that Vpr induces reactive oxygen species by increasing H(2)O(2) production, which can contribute to HIF-1alpha accumulation. Interestingly, increased levels of HIF-1alpha stimulated HIV-1 gene transcription through HIF-1 association with HIV-1 long terminal repeat. These observations point to the existence of a positive feedback interplay between HIF-1alpha and Vpr and that, by inducing oxidative stress via activation of HIF-1, Vpr can induce HIV-1 gene expression and dysregulate multiple host cellular pathways.

TNF alpha production in morphine-treated human neural cells is NF-kappaB-dependent.

The cytokine tumor necrosis factor alpha (TNFalpha) is a key factor in several inflammatory diseases and its levels increase in response to a variety of internal or external stimuli. The regulation of the TNFalpha promoter is mediated by several transcription factors including the nuclear factor kappa B protein (NF-kappaB). This study examines the role of NF-kappaB in the regulation of TNFalpha production by morphine in microglia. Using reverse transcriptase polymerase chain reaction, we demonstrated the presence of morphine receptors in these cells. We next demonstrated the ability of morphine to promote TNFalpha production and secretion by these cells using a cytokine array assay. Transient transfection experiments led to the identification of the region located between nucleotides -751 and -615 within the TNFalpha promoter as being responsive to morphine treatment. The DNA sequence of this region contains a motif indicative of a potential NF-kappaB binding site. The use of a small interfering RNA directed against p65, a subunit of NF-kappaB, demonstrated that TNFalpha induction by morphine is NF-kappaB-dependent. All of the effects of morphine were reversed by the morphine inhibitor, naloxone. These data provide important insights into the effects of morphine on microglia.

BAG3 protein regulates caspase-3 activation in HIV-1-infected human primary microglial cells.

BAG3, a member of the BAG co-chaperones family, is expressed in several cell types subjected to stressful conditions, such as exposure to high temperature, heavy metals, drugs. Furthermore, it is constitutively expressed in some tumors. Among the biological activities of the protein, there is apoptosis downmodulation; this appears to be exerted through BAG3 interaction with the heat shock protein (Hsp) 70, that influences cell apoptosis at several levels. We recently reported that BAG3 protein was detectable in the cytoplasm of reactive astrocytes in HIV-1-associated encephalopathy biopsies. Here we report that downmodulation of BAG3 protein levels allows caspase-3 activation by HIV-1 infection in human primary microglial cells. This is the first reported evidence of a role for BAG3 in the balance of death versus survival during viral infection.

Protective role of Puralpha to cisplatin.

BACKGROUND: The nucleic acid-binding protein Puralpha is involved at stalled DNA replication forks, in double-strand break (DSB) DNA repair and the cellular response to DNA replication stress. Puralpha also regulates homologous recombination-directed DNA repair (HRR). RESULTS: Cells lacking Puralpha showed enhanced sensitivity to cisplatin as evaluated by assays for cell viability and cell clonogenicity. This was seen both in Puralpha-negative MEFs and in human glioblastoma cells treated with siRNA directed against Puralpha. MEFs lacking Puralpha also showed enhanced H2AX phosphorylation in response to cisplatin. Repair of a reporter plasmid that had been treated with cisplatin was decreased in a reactivation assay using Puralpha-negative MEFs and the capacity of nuclear extracts from Puralpha-negative MEFs to perform non-homologous end-joining in vitro was also impaired. METHODS: We investigated the effects of the DNA damage-inducing cancer chemotherapeutic agent cisplatin on mouse embryo fibroblasts (MEFs) from PURA(-/-) knockout mice that lack Puralpha. CONCLUSIONS: Puralpha has a role in the cellular response to cisplatin-induced DNA damage and may provide new therapeutic modalities for cisplatin-resistant tumors.

Involvement of the p53 and p73 transcription factors in neuroAIDS.

HIV-associated dementia (HAD) is the most common AIDS-associated neurological disorder and is characterized by the development of synaptodendritic injury to neurons. To advance HAD therapy, it is crucial to identify the mechanisms and factors involved. The viral protein HIV-1 Tat is among those factors and is released by HIV-1-infected cells and can be taken up by adjacent neuronal cells leading to neurotoxic effects. Multiple cellular host proteins have been identified as Tat cofactors in causing neuronal injury. Interestingly, most of these factors function through activation of the p53 pathway. We have now examined the ability of Tat to activate the p53 pathway leading to the induction of endogenous p53 and p73 in neuronal cells. We found that Tat induced p53 and p73 levels in SH-SY5Y cells and that this induction caused retraction of neurites. In the absence of either p53 or p73, Tat failed to induce dendritic retraction or to activate the proapoptotic proteins, such as Bax. Further, we found that p53-accumulation in Tat-treated cells depends on the presence of p73. Therefore, we conclude that Tat contributes to neuronal degeneration through activation of a pathway involving p53 and p73. This information will be valuable for the development of therapeutic agents that affect these pathways to protect CNS neurons and prevent HAD.

St. John's Wort protein, p27SJ, regulates the MCP-1 promoter.

St. John's Wort is commonly known for its antiviral, antidepressant, and cytotoxic properties, but traditionally St. John's Wort has also been used to treat inflammation. In this study, we sought to characterize the mechanisms used by St. John's Wort to treat inflammation by examining the effect of the recently isolated protein from St. John's Wort, p27SJ on the expression of MCP-1. By employing an adenovirus expression vector, we demonstrate that a low concentration of p27SJ upregulates the MCP-1 promoter through the transcription factor C/EBPbeta. In addition, we found that C/EBPbeta-homologous protein (CHOP) or siRNA-C/EBPbeta significantly reduced the ability of p27SJ to activate MCP-1 gene expression. Results from protein-protein interaction studies illustrate the existence of a physical interaction between p27SJ and C/EBPbeta in microglial cells. The use of chromatin immunoprecipitation assay (ChIP) led to the identification of a new cis-element that is responsive to C/EBPbeta within the MCP-1 promoter. Association of C/EBPbeta with MCP-1 DNA was not affected by the presence of p27SJ. The biological activity of MCP-1 produced by cultures of adenovirus-p27SJ transduced cells was increased relative to controls as measured by the transmigration of human Jurkat cells. Thus, we conclude that at high concentration, p27SJ is a potential agent that may be developed as a modulator of MCP-1 leading to the inhibition of the cytokine-mediated inflammatory responses.

JC virus agnoprotein inhibits in vitro differentiation of oligodendrocytes and promotes apoptosis.

Productive infection of oligodendrocytes, which are responsible for the formation of myelin sheath in the central nervous system, with the human neurotropic virus JC virus (JCV) causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). In addition to encoding T antigen and the capsid proteins, which are produced at the early and late phases of the infection cycle, respectively, JCV encodes a small regulatory protein named agnoprotein that is important for successful completion of the virus life cycle. Here we used bipotential CG-4 cells to examine the impact of agnoprotein on oligodendrocyte differentiation and survival in the absence of JCV lytic infection. We demonstrate that the expression of agnoprotein delayed the formation of complex outgrowth networks of the cells during oligodendrocyte differentiation. These alterations were accompanied by high levels of DNA damage, induction of proapoptotic proteins, and suppression of prosurvival signaling. Accordingly, apoptosis was significantly increased upon the induction of CG-4 cells toward differentiation in cells expressing agnoprotein. These observations provide the first evidence for the possible involvement of agnoprotein, independent from its role in viral replication, in a series of biological events that may contribute to the pathological features seen in PML lesions.

C/EBPbeta regulates human immunodeficiency virus 1 gene expression through its association with cdk9.

Transcriptional regulation of the human immunodeficiency virus type 1 (HIV-1) is a complex event that requires the cooperative action of both viral (e.g. Tat) and cellular (e.g. C/EBPbeta, NF-kappaB) factors. The HIV-1 Tat protein recruits the human positive transcription elongation factor P-TEFb, consisting of cdk9 and cyclin T1, to the HIV-1 transactivation response (TAR) region. In the absence of TAR, Tat activates the HIV-1 long terminal repeat (LTR) through its association with several cellular factors including C/EBPbeta. C/EBPbeta is a member of the CCAAT/enhancer-binding protein family of transcription factors and has been shown to be a critical transcriptional regulator of HIV-1 LTR. We examined whether Tat-C/EBPbeta association requires the presence of the P-TEFb complex. Using immunoprecipitation followed by Western blot, we demonstrated that C/EBPbeta-cyclin T1 association requires the presence of cdk9. Further, due to its instability, cdk9 was unable to physically interact with C/EBPbeta in the absence of cyclin T1 or Tat. Using kinase assays, we demonstrated that cdk9, but not a cdk9 dominant-negative mutant (cdk9-dn), phosphorylates C/EBPbeta. Our functional data show that co-transfection of C/EBPbeta and cdk9 leads to an increase in HIV-1 gene expression when compared to C/EBPbeta alone. Addition of C/EBP homologous protein (CHOP) inhibits C/EBPbeta transcriptional activity in the presence and absence of cdk9 and causes a delay in HIV-1 replication in T-cells. Together, our data suggest that Tat-C/EBPbeta association is mediated through cdk9, and that phosphorylated C/EBPbeta may influence AIDS progression by increasing expression of HIV-1 genes.

MH2 domain of Smad3 reduces HIV-1 Tat-induction of cytokine secretion.

HIV-1 infection of the central nervous system (CNS) is associated with dysregulation of several important cytokines and chemokines, which are involved in inflammatory process. Earlier studies ascribed a critical role for Tat, a potent viral transcription activator, in this process by enhancing the expression of several immunomodulators including TGFbeta and MCP-1. Investigation of signaling pathways which are controlled by these cytokines led to identification of MH2 domain of Smad3, the downstream activator of TGFbeta pathway, as a modulator of MCP-1 promoter activity. The level of MCP-1 is increased in AIDS patients with neurologic problems, through recruitment of inflammatory cells, which can contribute to neuropathogenesis of AIDS. Therefore, we attempted to investigate the effect of MH2 on expression of MCP-1 and other immunolmodulators in CNS cells. By employing an adenovirus expression vector, we demonstrated that MH2 can decrease the levels of Tat-induced activation of MCP-1 and several other cytokines and chemokines in astrocytic cells. In addition, we showed that MH2 significantly reduced the activity of cytokines produced by cultures of adenovirus-MH2 transduced cells as measured by the transmigration of human PBMC cells. Thus, MH2 domain of Smad3 is a potential agent that may be developed as an inhibitor for the cytokine-mediated inflammatory responses in the brain and may have the potential to prevent transmigration of HIV-1-infected monocytes across the blood brain barrier in AIDS patients.

Analysis of a mutant p53 protein arising in a medulloblastoma from a mouse transgenic for the JC virus early region.

BACKGROUND: JC virus (JCV) is a polyomavirus that causes progressive multifocal leukoencephalopathy (PML) in humans and is highly oncogenic in experimental animals. Transgenic mice with JCV T-antigen develop cerebellar tumors, which resemble human medulloblastomas, containing two distinct cell subpopulations, T-antigen positive and negative. In T-negative clones, a novel mutant p53 was detected (p53(mt)). MATERIALS AND METHODS: We have compared p53(mt) to wild-type p53 (p53wt) in p53-null cells. RESULTS: p53(mt) had lost the transcriptional transactivation activity of p53(wt), and unlike p53(wt), partially localized to the cytoplasm. Unlike mutant p53 from many human cancers, p53(mt) did not show a gain of function or a dominant negative phenotype. Adenovirus expressing p53(wt) but not p53(mt) inhibited cell growth and induced apoptosis of p53-null cells. CONCLUSION: During the course of tumor evolution of the JCV T-antigen mouse medulloblastoma, a mutation occurred that inactivated p53 allowing tumor progression even in the absence of continued T-antigen expression.