Prasun K. Datta

HIV-1 Vpr modulates macrophage metabolic pathways: a SILAC-based quantitative analysis.

Human immunodeficiency virus type 1 encoded viral protein Vpr is essential for infection of macrophages by HIV-1. Furthermore, these macrophages are resistant to cell death and are viral reservoir. However, the impact of Vpr on the macrophage proteome is yet to be comprehended. The goal of the present study was to use a stable-isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry-based proteomics approach to characterize the Vpr response in macrophages. Cultured human monocytic cells, U937, were differentiated into macrophages and transduced with adenovirus construct harboring the Vpr gene. More than 600 proteins were quantified in SILAC coupled with LC-MS/MS approach, among which 136 were significantly altered upon Vpr overexpression in macrophages. Quantified proteins were selected and clustered by biological functions, pathway and network analysis using Ingenuity computational pathway analysis. The proteomic data illustrating increase in abundance of enzymes in the glycolytic pathway (pentose phosphate and pyruvate metabolism) was further validated by western blot analysis. In addition, the proteomic data demonstrate down regulation of some key mitochondrial enzymes such as glutamate dehydrogenase 2 (GLUD2), adenylate kinase 2 (AK2) and transketolase (TKT). Based on these observations we postulate that HIV-1 hijacks the macrophage glucose metabolism pathway via the Vpr-hypoxia inducible factor 1 alpha (HIF-1 alpha) axis to induce expression of hexokinase (HK), glucose-6-phosphate dehyrogenase (G6PD) and pyruvate kinase muscle type 2 (PKM2) that facilitates viral replication and biogenesis, and long-term survival of macrophages. Furthermore, dysregulation of mitochondrial glutamate metabolism in macrophages can contribute to neurodegeneration via neuroexcitotoxic mechanisms in the context of NeuroAIDS.

Interleukin-19 (IL-19) Induces Heme Oxygenase-1 (HO-1) Expression and Decreases Reactive Oxygen Species in Human Vascular Smooth Muscle Cells

Background: IL-19 is a Th2 interleukin recently recognized to have anti-inflammatory activity in vascular cells. Results: IL-19 induced HO-1 expression, decreased apoptosis, and ROS abundance in vascular cells in an HO-1-dependent fashion. Conclusion: IL-19 represents a link for two previously unassociated protective processes; Th2 cytokine mediated anti-inflammation and ROS reduction. Significance: IL-19 may represent a potential therapeutic for vascular diseases. Heme oxygenase-1 (HO-1) has potent anti-inflammatory activity and recognized vascular protective effects. We have recently described the expression and vascular protective effects of an anti-inflammatory interleukin (IL-19), in vascular smooth muscle cells (VSMC) and injured arteries. The objective of this study was to link the anti-inflammatory effects of IL-19 with HO-1 expression in resident vascular cells. IL-19 induced HO-1 mRNA and protein in cultured human VSMC, as assayed by quantitative RT-PCR, immunoblot, and ELISA. IL-19 does not induce HO-1 mRNA or protein in human endothelial cells. IL-19 activates STAT3 in VSMC, and IL-19-induced HO-1 expression is significantly reduced by transfection of VSMC with STAT3 siRNA or mutation of the consensus STAT binding site in the HO-1 promoter. IL-19 treatment can significantly reduce ROS-induced apoptosis, as assayed by Annexin V flow cytometry. IL-19 significantly reduced ROS concentrations in cultured VSMC. The IL-19-induced reduction in ROS concentration is attenuated when HO-1 is reduced by siRNA, indicating that the IL-19-driven decrease in ROS is mediated by HO-1 expression. IL-19 reduces vascular ROS in vivo in mice treated with TNFα. This points to IL-19 as a potential therapeutic for vascular inflammatory diseases and a link for two previously unassociated protective processes: Th2 cytokine-induced anti-inflammation and ROS reduction.

Superoxide Dismutase Mimetic Preserves the Glomerular Capillary Permeability Barrier to Protein

Overproduction of superoxide (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document}) occurs in glomerular disease and may overwhelm the capacity of superoxide dismutase (SOD), thereby intensifying oxidant injury by \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} and related radical species that disrupt the glomerular capillary permeability barrier to protein. We examined the efficacy of the SOD mimetic tempol in preserving glomerular permeability to protein using 1) a rat model of glomerular immune injury induced by an antiglomerular basement membrane antibody (anti-GBM), and 2) isolated rat glomeruli in which injury was induced by the cytokine tumor necrosis factor-α (TNFα). To induce glomerular immune injury, rats received anti-GBM using a protocol that results in prominent infiltration of glomeruli by macrophages and in which macrophage-derived TNFα has been shown to mediate albuminuria. To increase glomerular capillary permeability to albumin (Palb) ex vivo, isolated glomeruli were incubated with TNFα at concentrations (0.5–4.0 μg/ml) known to stimulate \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} production. Increments in Palb were detected by measuring changes in glomerular volume in response to an applied oncotic gradient. Significant increases in the urine excretion of albumin and F2α-isoprostane were observed in rats with glomerular immune injury without a significant change in systolic blood pressure. Tempol treatment significantly reduced urine isoprostane and albumin excretion. In isolated glomeruli, TNFα increased Palb and tempol abrogated this effect, both in a dose-dependent manner. These observations indicate that SOD mimetics can preserve the glomerular permeability barrier to protein under conditions of oxidative stress from \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} production.

Induction of Heme Oxygenase 1 in Radiation Nephropathy: Role of Angiotensin II

Abstract Datta, P. K., Moulder, J. E., Fish, B. L., Cohen, E. P. and Lianos, E. A. Induction of Heme Oxygenase 1 in Radiation Nephropathy: Role of Angiotensin II. Radiat. Res. 155, 734–739 (2001). In a rat model of radiation-induced nephropathy, we investigated changes in expression of heme oxygenase 1 (Hmox1, also known as HO-1), an enzyme that catalyzes conversion of heme into biliverdin, carbon monoxide and iron. The study explored whether radiation induces Hmox1 expression in the irradiated kidney and whether angiotensin II (AII) mediates Hmox1 expression in glomeruli isolated from irradiated kidneys. To assess the effects of radiation on Hmox1 expression, rats received 20 Gy bilateral renal irradiation and were randomized to groups receiving an AII type 1 (AT1) receptor antagonist (L-158,809) or no treatment. Drug treatment began 9 days prior to bilateral renal irradiation and continued for the duration of the study. Estimation of Hmox1 levels in glomerular protein lysates assessed by Western blot analysis revealed a significant increase in Hmox1 protein at 50 and 65 days postirradiation. In animals treated with the AT1 receptor antagonist, there was no induction of Hmox1, suggesting that AII may be a mediator of Hmox1 induction. To confirm that AII stimulates Hmox1 expression, animals were infused with 200, 400 or 800 ng/kg min–1 of AII for 18–19 days, and Hmox1 protein levels in glomeruli were assessed. There was a significant induction of Hmox1 in glomeruli of animals infused with 800 ng/kg min–1 of AII. These studies demonstrate that glomerular Hmox1 expression is elevated in the middle phase of radiation nephropathy and that AII can increase glomerular Hmox1 levels.

Epigenetics of µ-opioid receptors: Intersection with HIV-1 infection of the central nervous system

The abuse of intravenous drugs, such as heroin, has become a major public health concern due to the increased risk of HIV‐1 infection. Opioids such as heroin were originally identified and subsequently abused for their analgesic effects. However, many investigations have found additional effects of opioids, including regulation of the immune system. As such, chronic opioid abuse has been shown to promote HIV‐1 pathogenesis and facilitate HIV‐1‐associated neurocognitive dysfunction. Clinical opioids, such as morphine and methadone, as well as illicit opioids, such as heroin, exert their effects primarily through interactions with the µ‐opioid receptor (MOR). However, the mechanisms by which opioids enhance neurocognitive dysfunction through MOR‐mediated signaling pathways are not completely understood. New findings in the regulation of MOR expression, particularly epigenetic and transcriptional regulation as well as alternative splicing, sheds new insights into possible mechanisms of HIV‐1 and opiate synergy. In this review, we identify mechanisms regulating MOR expression and propose novel mechanisms by which opioids and HIV‐1 may modulate this regulation. Additionally, we suggest that differential regulation of newly identified MOR isoforms by opioids and HIV‐1 has functional consequence in enhancing HIV‐1 neurocognitive dysfunction. J. Cell. Physiol. 227: 2832–2841, 2012.

Induction of metallothionein-I protects glomeruli from superoxide-mediated increase in albumin permeability.

Metallothioneins (MT) are low-molecular-weight, heat-stable, cysteine-rich proteins with four isoforms. MT-I and MT-II are ubiquitous and are induced by oxidative, physical, and chemical stress. MT-I is an efficient scavenger of superoxide (O2) and hydroxyl ion (OH–). We have demonstrated that O2 and hypohalous acid can cause an increase in glomerular albumin permeability (Palb) in vitro. The purpose of this study was to document the protective effect of MT gene product on the O2-mediated increase in Palb. Glomeruli from Sprague-Dawley rats in 4% BSA medium were incubated for 4 hr at 37°C in duplicate tubes. Each set contained glomeruli alone or with 5 μM Cd++, 0.3 mM Spermine-NONOate (NO donor), 0.3 mM Sulfo-NONOate (nitrous oxide donor), 0.6 mM SNP (nonspecific NO donor) and SNP + carboxy-PTIO (10 mg/ml). After incubation, one set of tubes was used to isolate total RNA for the measurement of the mRNA levels of MT-I by reverse transcriptase polymerase chain reaction (RT-PCR). Duplicate tubes were incubated for an additional 10 min with 10 nM of O2, and Palb was measured using video microscopy. RT-PCR of total RNA from Cd++ and Spermine-NONOate treated glomeruli revealed a 2-fold induction of MT-I expression at the mRNA level. O2 caused a significant increase in Palb (0.8 ± 0.06 vs. control 0.0 ± 0.12, P < 0.05) and induction of MT-I in glomeruli by Cd++ or by Spermine-NONOate blocked this effect (0.21 ± 0.12 and 0.24 ± 0.19, respectively, P < 0.05 vs. O2). In contrast, Sulfo-NONOate and SNP did not induce mRNA for MT-I in glomeruli and did not provide protection against O2-mediated increase in Palb We conclude that MT-I gene products may play an important role in protecting the glomerular filtration barrier from the injury induced by reactive oxygen species in immune and/or nonimmune renal diseases

Role of Hexokinase-1 in the survival of HIV-1-infected macrophages

Viruses have developed various strategies to protect infected cells from apoptosis. HIV-1 infected macrophages are long-lived and considered reservoirs for HIV-1. One significant deciding factor between cell survival and cell death is glucose metabolism. We hypothesized that HIV-1 protects infected macrophages from apoptosis in part by modulating the host glycolytic pathway specifically by regulating hexokinase-1 (HK-1) an enzyme that converts glucose to glucose-6-phosphate. Therefore, we analyzed the regulation of HK-1 in HIV-1 infected PBMCs, and in a chronically HIV-1 infected monocyte-like cell line, U1. Our results demonstrate that HIV-1 induces a robust increase in HK-1 expression. Surprisingly, hexokinase enzymatic activity was significantly inhibited in HIV-1 infected PBMCs and in PMA differentiated U1 cells. Interestingly, we observed increased levels of mitochondria-bound HK-1 in PMA induced U1 cells and in the HIV-1 accessory protein, viral protein R (Vpr) transduced U937 cell derived macrophages. Dissociation of HK-1 from mitochondria in U1 cells using a pharmacological agent, clotrimazole (CTZ) induced mitochondrial membrane depolarization and caspase-3/7 mediated apoptosis. Dissociation of HK-1 from mitochondria in Vpr transduced U937 also activated caspase-3/7 activity. These observations indicate that HK-1 plays a non-metabolic role in HIV-1 infected macrophages by binding to mitochondria thereby maintaining mitochondrial integrity. These results suggest that targeting the interaction of HK-1 with the mitochondria to induce apoptosis in persistently infected macrophages may prove beneficial in purging the macrophage HIV reservoir.

ROLE OF C/EBP-β, p38 MAPK and MKK6 IN IL-1β MEDIATED C3 GENE REGULATION IN ASTROCYTES

Complement component C3, the central player in the complement cascade and the pro‐inflammatory cytokine IL‐1β is expressed by activated glial cells and may contribute to neurodegeneration. This study examines the regulation of the expression of C3 by IL‐1β in astroglial cells focusing on the role of the upstream kinase MKK6, p38‐α MAPK, and C/EBP‐β isoforms (LAP1, LAP2, or LIP) in astroglial cells. Activation of human astroglial cell line, U373 with IL‐1β, led to the induction of C3 mRNA and protein expression as determined by real‐time RT‐PCR and Western blot analysis, respectively. This induction was suppressed by the pharmacological inhibitor of p38 MAPK (i.e., SB202190‐HCl), suggesting the involvement of p38 MAPK in C3 gene expression. IL‐1β also induced C3 promoter activity in U373 cells in a MAP kinase‐ and C/EBP‐β‐dependent manner. Cotransfection of C3 luciferase reporter construct with constitutively active form of the upstream kinase in the MAP kinase cascade, that is, MKK6 (the immediate upstream activator of p38 kinase) resulted in marked stimulation of the promoter activity, whereas overexpression of a dominant negative forms of MKK6 and p38α MAPK inhibited C3 promoter activity. Furthermore, a mutant form of C/EBP‐β, LAPT235A showed reduction in IL‐1β‐mediated C3 promoter activation. These results suggest that the p38α, MAPK, and MKK6 play prominent roles in IL‐1β and C/EBP‐β‐mediated C3 gene expression in astrocytes. J. Cell. Biochem. 112: 1168–1175, 2011.

HIV-1 Tat and Cocaine Impair Survival of Cultured Primary Neuronal Cells via a Mitochondrial Pathway

Addictive stimulant drugs, such as cocaine, are known to increase the risk of exposure to HIV-1 infection and hence predispose towards the development of AIDS. Previous findings suggested that the combined effect of chronic cocaine administration and HIV-1 infection enhances cell death. Neuronal survival is highly dependent on the health of mitochondria providing a rationale for assessing mitochondrial integrity and functionality following cocaine treatment, either alone or in combination with the HIV-1 viral protein Tat, by monitoring ATP release and mitochondrial membrane potential (ΔΨm). Our results indicate that exposing human and rat primary hippocampal neurons to cocaine and HIV-1 Tat synergistically decreased both mitochondrial membrane potential and ATP production. Additionally, since previous studies suggested HIV-1 infection alters autophagy in the CNS, we investigated how HIV-1 Tat and cocaine affect autophagy in neurons. The results indicated that Tat induces an increase in LC3-II levels and the formation of Parkin-ring-like structures surrounding damaged mitochondria, indicating the possible involvement of the Parkin/PINK1/DJ-1 (PPD) complex in neuronal degeneration. The importance of mitochondrial damage is also indicated by reductions in mitochondrial membrane potential and ATP content induced by HIV-1 Tat and cocaine.

Effect of nitric oxide synthase inhibition on proteinuria in glomerular immune injury

In glomerular immune injury, the inducible isoform of nitric oxide synthase (iNOS) becomes a major catalyst of NO production. Although iNOS-catalyzed NO production is sustained and can be cytotoxic, iNOS inhibition exacerbates the magnitude of proteinuria that accompanies immune injury. To investigate putative mechanisms of this effect, we assessed changes in glomerular permeability to albumin by using the following two approaches: (i) an in vivo rat model of glomerular immune injury induced by antibody against the glomerular basement membrane (GBM), in which urine albumin excretion was measured under conditions of iNOS Inhibition, and (ii) an ex vivo model of isolated rat glomeruli, in which changes in glomerular capillary permeability to albumin were assessed under conditions of NOS inhibition. In rats with anti-GBM antibody–induced glomerular injury, there was an increase in urine albumin excretion. Treatment with two structurally dissimilar iNOS inhibitors at doses sufficient to decrease urine nitrate and/or nitrite exacerbated proteinuria. In these animals, urine excretion of the isoprostane 8-iso-PGF2α (marker of oxidative stress) was increased. In isolated glomeruli incubated with the NOS inhibitor L-NMMA, the permeability to albumin increased. This effect was reversed by the NO donor DETA NONOate and by the superoxide dismutase mimetic Tempol. We conclude that NOS-catalyzed NO production is an important mechanism in regulating glomerular permeability to protein. This mechanism involves control of the bioavailability of superoxide.