Release of Calcium from Inositol 1,4,5-Trisphosphate Receptor-Regulated Stores by HIV-1 Tat Regulates TNF- Production in Human Macrophages

Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Canada.
The Journal of Immunology (Impact Factor: 4.92). 07/2000; 164(12):6538-42. DOI: 10.4049/jimmunol.164.12.6538
Source: PubMed


HIV-1 protein Tat is neurotoxic and increases macrophage and microglia production of TNF-alpha, a cytopathic cytokine linked to the neuropathogenesis of HIV dementia. Others have shown that intracellular calcium regulates TNF-alpha production in macrophages, and we have shown that Tat releases calcium from inositol 1,4, 5-trisphosphate (IP3) receptor-regulated stores in neurons and astrocytes. Accordingly, we tested the hypothesis that Tat-induced TNF-alpha production was dependent on the release of intracellular calcium from IP3-regulated calcium stores in primary macrophages. We found that Tat transiently and dose-dependently increased levels of intracellular calcium and that this increase was blocked by xestospongin C, pertussis toxin, and by phospholipase C and type 1 protein kinase C inhibitors but not by protein kinase A or phospholipase A2 inhibitors. Xestospongin C, BAPTA-AM, U73122, and bisindolylmalemide significantly inhibited Tat-induced TNF-alpha production. These results demonstrate that in macrophages, Tat-induced release of calcium from IP3-sensitive intracellular stores and activation of nonconventional PKC isoforms play an important role in Tat-induced TNF-alpha production.

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    • "Tat has been found to be toxic to the mice when injected into the cerebroventricular region [6], [7]. The neurotoxicity of Tat is attributed to various mechanisms such as, over excitation of the neurons via N-methyl-D-aspartate receptor [8], [9], [10], [11] increasing intracellular calcium levels [12], [13], [14] and disrupting the normal function of electron transport chain [15]. In addition, Tat induces a bystander effect on neurons by producing neurotoxic substances such as pro-inflammatory cytokines/chemokines [16], [17], nitric oxide synthase [18], [19] and quinolinic acid from the adjacent astrocytes and microglia [20]. "
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    ABSTRACT: The incidence of HIV-associated neurological disorders (HAND) has increased during recent years even though the highly active antiretroviral therapy (HAART) has significantly curtailed the virus replication and increased the life expectancy among HIV-1 infected individuals. These neurological deficits have been attributed to HIV proteins including HIV-1 Tat. HIV-1 Tat is known to up-regulate CCL5 expression in mouse astrocytes, but the mechanism of up-regulation is not known. The present study was undertaken with the objective of determining the mechanism(s) underlying HIV-1 Tat-mediated expression of CCL5 in astrocytes. SVGA astrocytes were transiently transfected with a plasmid encoding Tat, and expression of CCL5 was studied at the mRNA and protein levels using real time RT-PCR and multiplex cytokine bead array, respectively. HIV-1 Tat showed a time-dependent increase in the CCL5 expression with peak mRNA and protein levels, observed at 1 h and 48 h post-transfection, respectively. In order to explore the mechanism(s), pharmacological inhibitors and siRNA against different pathway(s) were used. Pre-treatment with SC514 (NF-κB inhibitor), LY294002 (PI3K inhibitor), AG490 (JAK2 inhibitor) and Janex-1 (JAK3 inhibitor) showed partial reduction of the Tat-mediated induction of CCL5 suggesting involvement of JAK, PI3K/Akt and NF-κB in CCL5 expression. These results were further confirmed by knockdown of the respective genes using siRNA. Furthermore, p38 MAPK was found to be involved since the knockdown of p38δ but not other isoforms showed partial reduction in CCL5 induction. This was further confirmed at transcriptional level that AP-1, C/EBPα and C/EBPγ were involved in CCL5 up-regulation.
    PLoS ONE 11/2013; 8(11):e78855. DOI:10.1371/journal.pone.0078855 · 3.23 Impact Factor
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    • "Alcohol abuse also increases a person's susceptibility to secondary infections by suppressing immune responses, both innate and adaptive (Baum et al., 2010; Cook, 1998), which are already compromised in HIV-1 patients (Baum et al., 2010; Flora et al., 2005). HIV-1 viral proteins, in particular Tat, can increase the EtOH-mediated impairment of neutrophil function (Prakash et al., 1998), and act synergistically with EtOH to induce secretion of apoptotic factors (Acheampong et al., 2002) and pro-inflammatory cytokines in brain cells (Flora et al., 2005; Lawson et al., 2011; Mayne et al., 2000). We used the HIV-1 transgenic (HIV-1Tg) rodent model in this study. "
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    ABSTRACT: Background Binge drinking of high ethanol (EtOH) concentration beverages is common among young adults and can be a risk factor for exposure to sexually transmitted diseases, including HIV-1. We used a novel noninfectious HIV-1 transgenic (HIV-1Tg) rat model that mimics HIV-1 patients in terms of altered immune responses and deficits in cognitive learning and memory to investigate EtOH concentration-dependent effects on 48 alcohol-modulated genes during binge EtOH administration. Methods HIV-1Tg and control F344 rats were administered water, 8% EtOH, or 52% EtOH by gavage (i.g.) for 3 days (2.0 g/kg/d). Two hours after final treatment, blood, liver, and spleen were collected from each animal. Serum blood EtOH concentration (BEC) was measured, and gene expression in the liver and spleen was determined using a specifically designed PCR array. Results The BEC was significantly higher in the 52% EtOH-treated HIV-1Tg rats compared with the 8% EtOH group; however, the BEC was higher in the 8% EtOH-treated control rats compared with the 52% EtOH group. There was no change in expression of the EtOH metabolism-related genes, Adh1, Adh4, and Cyp2e1, in either the 8 or 52% EtOH-treated HIV-1Tg rats, whereas expression of those genes was significantly higher in the liver of the 52% EtOH control rats, but not in the 8% EtOH group. In the HIV-1Tg rats, expression of the GABAA, metabotropic glutamate, and dopamine neurotransmitter receptor genes was significantly increased in the spleen of the 52% EtOH group, but not in the 8% EtOH group, whereas no change was observed in those genes in either of the control groups. Conclusions Our data indicate that, in the presence of HIV-1 infection, EtOH concentration-dependent binge drinking can have significantly different molecular effects.
    Alcoholism Clinical and Experimental Research 02/2013; 37(7). DOI:10.1111/acer.12077 · 3.21 Impact Factor
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    • "To ascertain whether eluted pTAT-mcMyc protein (and pTAT control protein) can transduce to the nucleus of MEF, 100 nM of pTAT-mcMyc (or 100 nM control protein) was added to cultures of MEF grown on coverslips and incubated for 1 hour. A concentration of 100 nM was adopted from a survey of previous reports for TAT fusion protein delivery [27–30]. Fixed and permeabilized cells were labeled with antibody recognizing the 6xHistidine sequence. "
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    ABSTRACT: Exogenous expression of Oct4, Sox2, Klf4, and cMyc forces mammalian somatic cells to adopt molecular and phenotypic characteristics of embryonic stem cells, commencing with the required suppression of lineage-associated genes (e.g., Thy1 in mouse). Although omitting cMyc from the reprogramming cocktail minimizes risks of uncontrolled proliferation, its exclusion results in fold reductions in reprogramming efficiency. Thus, the feasibility of substituting cMyc transgene with (non-integrative) recombinant "pTAT-mcMyc" protein delivery was assessed, without compromising reprogramming efficiency or the pluripotent phenotype. Purification and delivery of semisoluble/particulate pTAT-mcMyc maintained Oct4-GFP(+) colony formation (i.e., reprogramming efficiency) whilst supporting pluripotency by various criteria. Differential repression of Thy1 by pTAT-mcMyc ± Oct4, Sox2, and Klf4 (OSK) suggested differential (and non-additive) mechanisms of repression. Extending these findings, attempts to enhance reprogramming efficiency through a staggered approach (prerepression of Thy1) failed to improve reprogramming efficiency. We consider protein delivery a useful tool to decipher temporal/molecular events characterizing somatic cell reprogramming.
    04/2012; 2012:541014. DOI:10.1155/2012/541014
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