HDAC6 mediates HIV-1 tat-induced proinflammatory responses by regulating MAPK-NF-kappaB/AP-1 pathways in astrocytes.

Human immunodeficiency virus (HIV)-1 transactivator of transcription (Tat) is a viral protein that induces extensive neuroinflammation by up-regulating proinflammatory mediators, including cytokines, chemokines, and adhesion molecules. Histone deacetylase 6 (HDAC6) has been implicated in the transcriptional regulation of inflammatory genes. In this study, we investigated the possible role of HDAC6 in HIV-1 Tat-induced up-regulation of proinflammatory mediators in astrocytes. HIV-1 Tat augmented HDAC6 expression, which was correlated with a reduction in acetylated α-tubulin in CRT-MG human astroglioma cells and primary mouse astrocytes. Knockdown and pharmacological inhibition of HDAC6 significantly inhibited HIV-1 Tat-induced expression of CCL2, CXCL8, and CXCL10 chemokines; adhesion molecules; and subsequent adhesion of monocytes to astrocytes. HDAC6 knockdown attenuated HIV-1 Tat-induced activation of mitogen-activated protein kinase species, including ERK, JNK, and p38. Furthermore, HDAC6 knockdown suppressed HIV-1 Tat-induced activation of NF-κB and AP-1. Thus, HDAC6 is involved in HIV-1 Tat-induced expression of proinflammatory genes by regulating mitogen-activated protein kinase-NF-κB/AP-1 pathways and serves as a molecular target for HIV-1 Tat-mediated neuroinflammation GLIA 2015;63:1953-1965.

Celastrol ameliorates HIV-1 Tat-induced inflammatory responses via NF-kappaB and AP-1 inhibition and heme oxygenase-1 induction in astrocytes.

HIV-1 Tat causes extensive neuroinflammation that may progress to AIDS-related encephalitis and dementia. Celastrol possesses various biological activities such as anti-oxidant, anti-tumor, and anti-inflammatory activities. In this study, we investigated the modulatory effects of celastrol on HIV-1 Tat-induced inflammatory responses and the molecular mechanisms underlying its action in astrocytes. Pre-treatment of CRT-MG human astroglioma cells with celastrol significantly inhibited HIV-1 Tat-induced expression of ICAM-1/VCAM-1 and subsequent monocyte adhesiveness in CRT-MG cells. In addition, celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory chemokines, such as CXCL10, IL-8, and MCP-1. Celastrol decreased HIV-1 Tat-induced activation of JNK MAPK, AP-1, and NF-κB. Furthermore, celastrol induced mRNA and protein expression of HO-1 as well as Nrf2 activation. Blockage of HO-1 expression using siRNA reversed the inhibitory effect of celastrol on HIV-1 Tat-induced inflammatory responses. These results suggest that celastrol has regulatory effects on HIV-1 Tat-induced inflammatory responses by blocking the JNK MAPK-AP-1/NF-κB signaling pathways and inducing HO-1 expression in astrocytes.

Casuarinin suppresses TARC/CCL17 and MDC/CCL22 production via blockade of NF-κB and STAT1 activation in HaCaT cells.

Casuarinin is a naturally occurring tannin that is isolated from the leaves of Hippophae rhamnoides. It has been shown to have anti-oxidant, anti-cancer, anti-viral, and anti-inflammatory activities. The aim of this study was to investigate the possible mechanism by which casuarinin inhibits TNF-α/IFN-γ-induced Th2 chemokines expression in the human keratinocytes cell line HaCaT. We found that casuarinin suppressed TNF-α/IFN-γ-induced expression of TARC and MDC mRNA and protein in HaCaT cells. Casuarinin significantly inhibited TNF-α/IFN-γ-induced activation of NF-κB, STAT1, and p38 MAPK. Furthermore, we observed that p38 MAPK contributes to inhibition of TNF-α/IFN-γ-induced TARC and MDC production by blocking NF-κB and STAT1 activation in HaCaT cells. Taken together, these results suggest that casuarinin may exert anti-inflammatory responses by suppressing TNF-α/IFN-γ-induced expression of TARC and MDC via blockage of p38 MAPK activation and subsequent activation of NF-κB and STAT1. We propose that it could therefore be used as a therapeutic agent against inflammatory skin diseases.

Casuarinin suppresses TNF-α-induced ICAM-1 expression via blockade of NF-κB activation in HaCaT cells.

Hippophae rhamnoides has been extensively used in oriental traditional medicines for treatment of asthma, skin diseases, gastric ulcers, and lung disorders. In this study, we isolated casuarinin from the leaves of H.rhamnoides and examined the effect of casuarinin on the TNF-α-induced ICAM-1 expression in a human keratinocytes cell line HaCaT. Pretreatment with casuarinin inhibited TNF-α-induced protein and mRNA expression of ICAM-1 and subsequent monocyte adhesiveness in HaCaT cells. Casuarinin significantly inhibited TNF-α-induced NF-κB activation. In addition, casuarinin inhibited activation of ERK and p38 MAPK in a dose-dependent manner. Furthermore, pretreatment with casuarinin decreased TNF-α-induced pro-inflammatory mediators, such as IL-1ß, IL-6, IL-8, and MCP-1. These results demonstrated that casuarinin exerts its anti-inflammatory activity by suppressing TNF-α-induced expression of ICAM-1 and pro-inflammatory cytokines/chemokines via blockage of activation of NF-κB and ERK/p38 MAPK and can be used as a therapeutic agent against inflammatory skin diseases.

Celastrol induces expression of heme oxygenase-1 through ROS/Nrf2/ARE signaling in the HaCaT cells.

We previously demonstrated that celastrol, a quinone methide triterpenoid derived from the medicinal plant Tripterygium wilfordii, exerts its anti-inflammatory activity through up-regulation of heme oxygenase-1 (HO-1) expression in the keratinocytes. In this study, we examined the signaling pathways that lead to the up-regulation of HO-1 expression by celastrol. In HaCaT cells, celastrol-induced HO-1 expression was dependent on ROS generation. ERK and p38 MAPK were major MAPK pathways responsible for celastrol-induced HO-1 expression. Celastrol induced Nrf2 activation. Nrf2 knockdown using small interfering RNA (siRNA) inhibited celastrol-induced HO-1 expression. Treatment with celastrol resulted in a marked increase in antioxidant response element (ARE)-driven transcriptional activity, which was dependent on ROS generation and activation of ERK and p38 MAPK. Furthermore, Nrf2 siRNA significantly reversed the inhibitory effect of celastrol on IFN-γ-induced expression of ICAM-1 in the keratinocytes. Taken together, our results indicate that celastrol can activate the ROS-ERK/p38-Nrf2-ARE signaling cascades leading to the up-regulation of HO-1 which is partly responsible for its anti-inflammatory activity in the keratinocytes.

Celastrol suppresses IFN-gamma-induced ICAM-1 expression and subsequent monocyte adhesiveness via the induction of heme oxygenase-1 in the HaCaT cells.

Celastrol, a quinone methide triterpenoid derived from the medicinal plant Tripterygium wilfordii, possesses various biological activities such as anti-oxidant, anti-tumor, and anti-inflammatory activities. In this study, we examined the suppressive effect of celastrol on IFN-gamma-induced expression of ICAM-1 and the molecular mechanism responsible for these activities. We found that celastrol induced mRNA and protein expression of heme oxygenase-1 (HO-1) in the human keratinocyte cell line HaCaT. Treatment of HaCaT cells with tin protoporphyrin IX (SnPP), a specific inhibitor of HO-1, reversed the suppressive effect of celastrol on IFN-gamma-induced protein and mRNA expression of ICAM-1. HO-1 knockdown using small interfering RNA (siRNA) led to reverse inhibition of IFN-gamma-induced up-regulation of ICAM-1 by celastrol. In addition, SnPP reversed suppression of IFN-gamma-induced promoter activity of ICAM-1 by celastrol. Furthermore, blockage of HO-1 activity by SnPP and HO-1 siRNA reversed the inhibitory effect of celastrol on IFN-gamma-induced adhesion of monocytes to keratinocytes. These results suggest that celastrol may exert anti-inflammatory responses by suppressing IFN-gamma-induced expression of ICAM-1 and subsequent monocyte adhesion via expression of HO-1 in the keratinocytes.

Suppression of thymus- and activation-regulated chemokine (TARC/CCL17) production by 1,2,3,4,6-penta-O-galloyl-beta-D-glucose via blockade of NF-kappaB and STAT1 activation in the HaCaT cells.

Keratinocytes, one of major cell types in the skin, can be induced by TNF-alpha and IFN-gamma to express thymus- and activation-regulated chemokine (TARC/CCL17), which is considered to be a pivotal mediator in the inflammatory responses during the development of inflammatory skin diseases, such as atopic dermatitis (AD). In this study, we examined the effect of 1,2,3,4,6-penta-O-galloyl-beta-d-glucose (PGG), isolated from the barks of Juglans mandshurica, on TNF-alpha/IFN-gamma induced CCL17 expression in the human keratinocyte cell line HaCaT. Pretreatment of HaCaT cells with PGG suppressed TNF-alpha/IFN-gamma-induced protein and mRNA expression of CCL17. PGG significantly inhibited TNF-alpha/IFN-gamma-induced NF-kappaB activation as well as STAT1 activation. Furthermore, pretreatment with PGG resulted in significant reduction in expression of CXCL9, 10, and 11 in the HaCaT cells treated with IFN-gamma. These results suggest that PGG may exert anti-inflammatory responses by suppressing TNF-alpha and/or IFN-gamma-induced activation of NF-kappaB and STAT1 in the keratinocytes and might be a useful tool in therapy of skin inflammatory diseases.

Extracellular HIV-1 Tat up-regulates expression of matrix metalloproteinase-9 via a MAPK-NF-kappaB dependent pathway in human astrocytes.

The infiltration of monocytes into the CNS represents one of the early steps to inflammatory events in AIDS-related encephalitis and dementia. Increased activity of selected matrix metalloproteinases (MMPs) such as MMP-9 impairs the integrity of blood-brain barrier leading to enhanced monocyte infiltration into the CNS. In this study, we examined the effect of HIV-1 Tat on the expression of MMP-9 in CRT-MG human astroglioma cells. Treatment of CRT-MG cells with HIV-1 Tat protein significantly increased protein levels of MMP-9, as measured by Western blot analysis, zymography and an ELISA. Treatment of CRT-MG cells with HIV-1 Tat protein markedly increased mRNA levels of MMP-9, as analyzed by RT-PCR. Pretreatment of CRT-MG cells with NF-kappaB inhibitors led to decrease in Tat-induced protein and mRNA expression of MMP-9. Pretreatment of CRT-MG cells with MAPK inhibitors suppressed Tat-induced MMP-9 expression. Furthermore, HIV-1 Tat-induced expression of MMP-9 was significantly inhibited by neutralization of TNF-alpha, but not IL-1beta and IL-6. Taken together, our results indicate that HIV-1 Tat can up-regulate expression of MMP-9 via MAPK-NF-kappaB-dependent mechanisms as well as Tat-induced TNF-alpha production in astrocytes.

Suppression of HIV-1 Tat-induced monocyte adhesiveness by a cell-permeable superoxide dismutase in astrocytes.

HIV-1 Tat is considered to be one of key players to facilitate monocyte entry into the CNS, which is characteristic feature of AIDS-related encephalitis and dementia. This study was performed to determine the regulatory function of superoxide dismutase (SOD) on the HIV-1 Tat-induced signaling pathways leading to NF-kappaB activation, expression of adhesion molecules, and monocyte adhesion in CRT-MG human astroglioma cells by using cell-permeable SOD. When cell-permeable SOD was added to the culture medium of CRT-MG cells, it rapidly entered the cells in dose- and time-dependent manners. Treatment of astrocytes with cell-permeable SOD led to decrease in Tat-induced ROS generation as well as NF-kappaB activation. Cell-permeable SOD inhibited the activation of MAP kinases including ERK, JNK and p38 by HIV-1 Tat. Treatment of CRT-MG cells with cell-permeable SOD significantly inhibited protein and mRNA levels of ICAM-1 and VCAM-1 up-regulated by HIV-1 Tat, as measured by Western blot analysis and RT-PCR. Furthermore, enhanced adhesiveness of monocyte to astrocyte by HIV-1 Tat was significantly abrogated by pretreatment with cell-permeable SOD fusion proteins. These data indicate that SOD has a regulatory function for HIV-1 Tat-induced NF-kappaB activation in astrocytes and suggest that cell-permeable SOD can be used as a feasible therapeutic agent for regulation of ROS-related neurological diseases.

Extracellular HIV-1 Tat enhances monocyte adhesion by up-regulation of ICAM-1 and VCAM-1 gene expression via ROS-dependent NF-kappaB activation in astrocytes.

One of characteristic features of AIDS-related encephalitis and dementia is the infiltration of monocytes into the CNS. HIV-1 Tat was demonstrated to facilitate monocyte entry into the CNS. In this study, we examined the effect of HIV-1 Tat on the expression of adhesion molecules, generation of reactive oxygen species (ROS) and NF-kappaB activation in CRT-MG human astroglioma cells. Treatment of CRT-MG cells with HIV-1 Tat protein significantly increased protein and mRNA levels of ICAM-1 and VCAM-1, as measured by Western blot analysis and RT-PCR, indicating that Tat increases these protein levels at an mRNA level. In addition, Tat induced the activation of NF-kappaB in astrocytes. Treatment of CRT-MG with NF-kappaB inhibitors led to decrease in Tat-induced protein and mRNA expression of ICAM-1 and VCAM-1. Furthermore, HIV-1 Tat protein increased ROS generation. Inhibition of Tat-induced ROS generation by N-acetyl cysteine, vitamin C and diphenyl iodonium suppressed Tat-induced NF-kappaB activation, ICAM-1 and VCAM-1 expression, and monocyte adhesion in CRT-MG. These data indicate that HIV-1 Tat can modulate monocyte adhesiveness by increasing expression of adhesion molecules such as ICAM-1 and VCAM-1 via ROS- and NF-kappaB-dependent mechanisms in astrocytes.

The enhanced monocyte adhesiveness after UVB exposure requires ROS and NF-kappaB signaling in human keratinocyte.

The infiltration of both monocyte and activated T cells in the skin is one of critical steps in the development of UVB-induced inflammation. Upregulation of adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) on the surface of keratinocytes plays an important role in this process. In this study, we examined the molecular mechanism responsible for UVB-induced expression of ICAM-1 and subsequent monocyte adhesion by keratinocyte. We observed that (1) UVB induced protein and mRNA expression of ICAM-1 in a dose- and time-dependent manner in human keratinocyte cell HaCaT; (2) UVB induced the translocation of NF-kappaB and inhibition of NF-kappaB by NF-kappaB inhibitors suppressed UVB-induced mRNA and protein expression of ICAM-1; (3) UVB increased the intracellular level of reactive oxygen species (ROS) by HaCaT cells; (4) UVB-induced increase of intracellular ROS level was suppressed by pretreatment with diphenyl iodonium (DPI) and N-acetyl cysteine (NAC); and (5) inhibition of UVB-induced ROS production by DPI or NAC suppressed UVB-mediated translocation of NF-kappaB, expression of ICAM-1 and subsequent monocyte adhesion in HaCaT cells. These results suggest that UVB-induced ROS is involved in the translocation of NF-kappaB which is responsible for expression of ICAM-1 and subsequent increased monocyte adhesion in human keratinocyte.

Celastrol ameliorates cytokine toxicity and pro-inflammatory immune responses by suppressing NF-κB activation in RINm5F beta cells.

Upregulation of pro-inflammatory mediators contributes to ß-cell destruction and enhanced infiltration of immune cells into pancreatic islets during development of type 1 diabetes mellitus. In this study, we examined the regulatory effects and the mechanisms of action of celastrol against cytotoxicity and pro-inflammatory immune responses in the RINm5F rat pancreatic ß-cell line stimulated with a combination of interleukin-1 beta, tumor necrosis factor-alpha, and interferon-γ. Celastrol significantly restored cytokine-induced cell death and significantly inhibited cytokine-induced nitric oxide production. In addition, the protective effect of celastrol was correlated with a reduction in pro-inflammatory mediators, such as inducible nitric oxide synthase, cyclooxygenase-2, and CC chemokine ligand 2. Furthermore, celastrol significantly suppressed cytokine- induced signaling cascades leading to nuclear factor kappa B (NF-κB) activation, including IκB-kinase (IKK) activation, IκB degradation, p65 phosphorylation, and p65 DNA binding activity. These results suggest that celastrol may exert its cytoprotective activity by suppressing cytokine-induced expression of pro-inflammatory mediators by inhibiting activation of NF-κB in RINm5F cells.

Development of a monoclonal antibody-based ELISA system for glyceraldehyde-derived advanced glycation end products.

We have previously found that glyceraldehyde-derived advanced glycation end products (glycer-AGEs) elicit oxidative stress generation and evoke inflammatory and thrombotic reactions through their higher binding affinity to RAGE (receptor for AGEs), thereby playing a role in vascular complications in diabetes. Furthermore, circulating levels of glycer-AGEs are elevated in diabetes. We characterized a monoclonal antibody (mAb) raised against glycer-AGEs and prepared its specific ELISA system in human serum. We developed here mAb reacted specifically with glycer-AGEs or glyceraldehyde-derived pyridinium, but not other structurally identified AGEs or AGE precursors. The mAb not only completely neutralized the deleterious effects of glycer-AGEs on endothelial cells, but also detected glycer-AGEs in the aorta of type 2 diabetic rats. Intra and inter-assay coefficient variations of the ELISA were 6 and 2.6%, respectively. ELISA linearity was shown intact within 5-fold dilution, and recovery ratio of added glycer-AGEs was 88-117%. Results of serum and plasma were comparable, and repeated freeze-thawing of samples did not affect the results (90.1-112.4%). Serum glycer-AGEs levels in 30 healthy subjects evaluated by the ELISA were strongly correlated with those by polyclonal Ab-based one (r=0.82). Our present study suggests the clinical utility of mAb for evaluating glycer-AGE levels in both tissue and serum.

Salicortin suppresses lipopolysaccharide-stimulated inflammatory responses via blockade of NF-κB and JNK activation in RAW 264.7 macrophages.

We isolated the phenolic glucoside salicortin from a Populus euramericana bark extract, and examined its ability to suppress inflammatory responses as well as the molecular mechanisms underlying these abilities, using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Salicortin inhibited iNOS expression and the subsequent production of NO in a dose-dependent manner in the LPS-stimulated RAW 264.7 cells. Salicortin significantly suppressed LPS-induced signal cascades of NF-κB activation, such as IKK activation, IκBα phosphorylation and p65 phosphorylation in RAW 264.7 cells. In addition, salicortin inhibited the LPS-induced activation of JNK, but not ERK or p38 MAPK. Furthermore, salicortin significantly inhibited production of pro-inflammatory cytokines, such as TNF-α, IL-1ß and IL-6 in the LPS-stimulated RAW 264.7 cells. These findings suggest that salicortin may show its anti-inflammatory activity by suppressing the LPS-induced expression of pro-inflammatory mediators through inhibition of NF-κB and JNK MAPK signaling cascades in macrophages.

Suppression of iNOS and COX-2 expression by flavokawain A via blockade of NF-κB and AP-1 activation in RAW 264.7 macrophages.

Flavokawain A, a major constituent of chalcones derived from kava extracts, exerts various biological activities such as anti-tumor activities. In this study, we examined the suppressive effect of flavokawain A on LPS-induced expression of pro-inflammatory mediators and the molecular mechanisms responsible for these activities in the murine macrophages. Flavokawain A significantly suppressed expression of iNOS and COX-2, as well as the subsequent production of NO and PGE2 in the LPS-stimulated RAW 264.7 cells. Flavokawain A significantly inhibited LPS-induced activation of NF-κB and AP-1 signaling pathways. In addition, flavokawain A inhibited activation of JNK and p38 MAPK which was responsible for expression of iNOS and COX-2 in the LPS-stimulated RAW 264.7 cells. Furthermore, flavokawain A suppressed LPS-induced expression of pro-inflammatory cytokines, such as TNF-α, IL-1ß and IL-6. These results suggest that flavokawain A may exert anti-inflammatory responses by suppressing LPS-induced expression of pro-inflammatory mediators via blockage of NF-κB-AP-1-JNK/p38 MAPK signaling pathways in the murine macrophages.

Curcumin ameliorates TNF-α-induced ICAM-1 expression and subsequent THP-1 adhesiveness via the induction of heme oxygenase-1 in the HaCaT cells.

Adhesion molecules such as ICAM-1 are important in the infiltration of leukocytes into the site of inflammation. In this study, we investigated the inhibitory effects of curcumin on ICAM-1 expression and monocyte adhesiveness as well as its underlying action mechanism in the TNF-α-stimulated keratinocytes. Curcumin induced expression of heme oxygenase-1 (HO-1) in the human keratinocyte cell line HaCaT. In addition, curcumin induced Nrf2 activation in dose- and time-dependent manners in the HaCaT cells. Curcumin suppressed TNF-α- induced ICAM-1 expression and subsequent monocyte adhesion, which were reversed by the addition of tin protoporphyrin IX (SnPP), a specific inhibitor of HO-1, or HO-1 knockdown using siRNA. Furthermore, Nrf2 knockdown using siRNA reversed the inhibitory effect of curcumin on the TNF-α-induced ICAM-1 expression and adhesion of monocytes to keratinocytes. These results suggest that curcumin may exert its anti-inflammatory activity by suppressing the TNF-α-induced ICAM-1 expression and subsequent monocyte adhesion via expression of HO-1 in the keratinocytes.

Extracellular HIV-1 Tat induces human beta-defensin-2 production via NF-kappaB/AP-1 dependent pathways in human B cells.

Defensins, a family of antimicrobial peptides, are one of the first lines of host defense. Human beta-defensins (hBD) such as hBD-2 and -3 have anti-HIV activity. Previous studies have shown that HIV-1 virion can induce the expression of hBD, although the exact components of HIV-1 virion that are responsible for hBD expression have not yet been elucidated. In this study, we examined the effect of HIV-1 Tat on the expression of hBD in B cells. Stimulation of B cells with HIV-1 Tat protein significantly increased the mRNA and protein levels of hBD-2. HIV-1 Tat also induced the activation of a reporter gene for hBD-2 in a dose-dependent manner in B cells. Pretreatment of B cells with a JNK inhibitor suppressed HIV-1 Tat-induced hBD-2 expression. Pretreatment of B cells with AP-1 inhibitors or NF-κB inhibitors led to a decrease in HIV-1 Tat-induced protein and mRNA expression of hBD-2. Taken together, our results indicate that HIV-1 Tat can up-regulate the expression of hBD-2 via JNK-NF-κB/AP-1-dependent pathways in human B cells.

Suppression of TNF-alpha-induced MMP-9 expression by a cell-permeable superoxide dismutase in keratinocytes.

Up-regulation of selected matrix metalloproteinases (MMPs) such as MMP-9 contributes to inflammatory processes during the development of various skin diseases, such as atopic dermatitis. In this study, we examined the effect of a cell-permeable superoxide dismutase (Tat-SOD) on TNF-α-induced MMP-9 expression in human keratinocyte cells (HaCaT). When Tat-SOD was added to the culture medium of HaCaT cells, it rapidly entered the cells in dose- and time-dependent manners. Tat-SOD decreased TNF-α-induced reactive oxygen species (ROS) generation. Tat-SOD also inhibited TNF-α-induced NF-κB DNA binding activity. Treatment of HaCaT cells with Tat-SOD significantly inhibited TNF-α-induced mRNA and protein expression of MMP-9, as measured by RT-PCR and Western blot analysis. In addition, Tat-SOD suppressed TNF-α-induced gelatinolytic activity of MMP-9. Taken together, our results indicate that Tat-SOD can suppress TNF-α-induced MMP-9 expression via ROS-NF-κB-dependent mechanisms in keratinocytes, and therefore can be used as an immunomodulatory agent against inflammatory skin diseases related to oxidative stress.

Nox2-based NADPH oxidase mediates HIV-1 Tat-induced up-regulation of VCAM-1/ICAM-1 and subsequent monocyte adhesion in human astrocytes.

Up-regulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) by the HIV-1 transactivator of transcription (Tat) in activated microglia and astrocytes may play a pivotal role during the development of AIDS-related encephalitis and dementia. Previous studies demonstrated that HIV-1 Tat-induced up-regulation of adhesion molecules was mediated by reactive oxygen species (ROS), although the mechanisms underlying HIV-1 Tat-induced ROS generation are unknown. In this study, we examined the possible role of NADPH oxidase in HIV-1 Tat-induced up-regulation of adhesion molecules in astroglioma cell lines. HIV-1 Tat-induced up-regulation of VCAM-1/ICAM-1 and subsequent increased adhesion of monocytes to astrocytes were blocked by a general NADPH oxidase inhibitor, diphenylene iodonium, and a specific inhibitor of NADPH oxidase assembly, 9R3A-gp91ds. Nox2 knockdown using small interfering RNA (siRNA) inhibited HIV-1 Tat-induced up-regulation of adhesion molecules and subsequent increased adhesion of monocytes to astrocytes. Nox2 siRNA blocked HIV-1 Tat-induced ROS production, increase in NADPH oxidase activity, and Rac1 activation. Furthermore, Nox2 siRNA decreased HIV-1 Tat-induced NF-κB activation as well as activation of MAP kinases including ERK, JNK, and p38. These data indicate that Nox2-based NADPH oxidase is responsible for HIV-1 Tat-induced generation of ROS and plays an important role in the up-regulation of adhesion molecules such as VCAM-1/ICAM-1 and subsequent increased adhesion of monocytes to astrocytes and serves as a novel target for HIV-1 Tat-mediated neurological diseases.

Suppression of inducible nitric oxide synthase and cyclooxygenase-2 by cell-permeable superoxide dismutase in lipopolysaccharide-stimulated BV-2 microglial cells.

Oxidative stress plays a pivotal role in uncontrolled neuro-inflammation leading to many neurological diseases including Alzheimer's. One of the major antioxidant enzymes known to prevent deleterious effects due to oxidative stress is Cu,Zn-superoxide dismutase (SOD). In this study, we examined the regulatory function of SOD on the LPS-induced signaling pathways leading to NF-kappaB activation, expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in BV-2 cells using cell-permeable SOD. Treatment of BV-2 cells with cell-permeable SOD led to a decrease in LPS-induced reactive oxygen species (ROS) generation and significantly inhibited protein and mRNA levels of iNOS and COX-2 upregulated by LPS. Production of NO and PGE2 in LPS stimulated BV-2 cells was significantly abrogated by pretreatment with a cell-permeable SOD fusion protein. Furthermore, cell-permeable SOD inhibited LPS-induced NF-kappaB DNA-binding activity and activation of MAP kinases including ERK, JNK, and p38 in BV-2 cells. These data indicate that SOD has a regulatory function for LPS-induced NF-kappaB activation leading to expression of iNOS and COX-2 in BV-2 cells and suggest that cell-permeable SOD is a feasible therapeutic agent for regulation of ROS-related neurological diseases.