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• W2099080968 abstract "Human immunodeficiency virus type 1 (HIV-1) infection is known to cause neuronal injury and dementia in a significant proportion of patients. However, the mechanism by which HIV-1 mediates its deleterious effects in the brain is poorly defined. The present study was undertaken to investigate the effect of the HIV-1tat gene on the expression of inducible nitric-oxide synthase (iNOS) in human U373MG astroglial cells and primary astroglia. Expression of the tat gene as RSV-tat but not that of the CAT gene as RSV-CAT in U373MG astroglial cells led to the induction of NO production and the expression of iNOS protein and mRNA. Induction of NO production by recombinant HIV-1 Tat protein and inhibition of RSV-tat-induced NO production by anti-Tat antibodies suggest that RSV-tat-induced production of NO is dependent on Tat and that Tat is secreted from RSV-tat-transfected astroglia. Similar to U373MG astroglial cells, RSV-tat also induced the production of NO in human primary astroglia. The induction of human iNOS promoter-derived luciferase activity by the expression of RSV-tat suggests that RSV-tat induces the transcription of iNOS. To understand the mechanism of induction of iNOS, we investigated the role of NF-κB and C/EBPβ, transcription factors responsible for the induction of iNOS. Activation of NF-κB as well as C/EBPβ by RSV-tat, stimulation of RSV-tat-induced production of NO by the wild type of p65 and C/EBPβ, and inhibition of RSV-tat-induced production of NO by Δp65, a dominant-negative mutant of p65, and ΔC/EBPβ, a dominant-negative mutant of C/EBPβ, suggest that RSV-tat induces iNOS through the activation of NF-κB and C/EBPβ. In addition, we show that extracellular signal-regulated kinase (ERK) but not that p38 mitogen-activated protein kinase (MAPK) is involved in RSV-tat induced production of NO. Interestingly, PD98059, an inhibitor of the ERK pathway, and ΔERK2, a dominant-negative mutant of ERK2, inhibited RSV-tat-induced production of NO through the inhibition of C/EBPβ but not that of NF-κB. This study illustrates a novel role for HIV-1 tat in inducing the expression of iNOS in human astrocytes that may participate in the pathogenesis of HIV-associated dementia. Human immunodeficiency virus type 1 (HIV-1) infection is known to cause neuronal injury and dementia in a significant proportion of patients. However, the mechanism by which HIV-1 mediates its deleterious effects in the brain is poorly defined. The present study was undertaken to investigate the effect of the HIV-1tat gene on the expression of inducible nitric-oxide synthase (iNOS) in human U373MG astroglial cells and primary astroglia. Expression of the tat gene as RSV-tat but not that of the CAT gene as RSV-CAT in U373MG astroglial cells led to the induction of NO production and the expression of iNOS protein and mRNA. Induction of NO production by recombinant HIV-1 Tat protein and inhibition of RSV-tat-induced NO production by anti-Tat antibodies suggest that RSV-tat-induced production of NO is dependent on Tat and that Tat is secreted from RSV-tat-transfected astroglia. Similar to U373MG astroglial cells, RSV-tat also induced the production of NO in human primary astroglia. The induction of human iNOS promoter-derived luciferase activity by the expression of RSV-tat suggests that RSV-tat induces the transcription of iNOS. To understand the mechanism of induction of iNOS, we investigated the role of NF-κB and C/EBPβ, transcription factors responsible for the induction of iNOS. Activation of NF-κB as well as C/EBPβ by RSV-tat, stimulation of RSV-tat-induced production of NO by the wild type of p65 and C/EBPβ, and inhibition of RSV-tat-induced production of NO by Δp65, a dominant-negative mutant of p65, and ΔC/EBPβ, a dominant-negative mutant of C/EBPβ, suggest that RSV-tat induces iNOS through the activation of NF-κB and C/EBPβ. In addition, we show that extracellular signal-regulated kinase (ERK) but not that p38 mitogen-activated protein kinase (MAPK) is involved in RSV-tat induced production of NO. Interestingly, PD98059, an inhibitor of the ERK pathway, and ΔERK2, a dominant-negative mutant of ERK2, inhibited RSV-tat-induced production of NO through the inhibition of C/EBPβ but not that of NF-κB. This study illustrates a novel role for HIV-1 tat in inducing the expression of iNOS in human astrocytes that may participate in the pathogenesis of HIV-associated dementia. HIV-1-associated dementia human immunodeficiency virus inducible nitric-oxide synthase l-N G-monomethylarginine d-N G-monomethylarginine extracellular signal-regulated kinase mitogen-activated protein kinase central nervous system chloramphenicol acetyltransferase Rous sarcoma virus CCAAT enhancer-binding protein IκB kinase liver inhibitory protein HIV-1-associated dementia (HAD)1 is a severe form of neurological disability, observed in 20–30% of patients with acquired immunodeficiency syndrome (AIDS) (1Kolson D.L. Gonzalez-Scarano F. J. Clin. Invest. 2000; 106: 11-13Crossref PubMed Scopus (51) Google Scholar). The histopathological signs of HAD include infiltration of inflammatory cells, astrogliosis, pallor of myelin sheaths, abnormalities of dendritic processes, and neuronal apoptotic death. Productive HIV-1 infection in the brain occurs predominantly in macrophages, microglia, and multinucleated giant cells (2Bagasra O. Lavi E. Bobroski L. Khalili K. Pestaner J.B. Pomerantz R.J. AIDS. 1996; 10: 573-585Crossref PubMed Scopus (386) Google Scholar, 3Wiley C.A. Schrier R.D. Nelson J.A. Lampert P.W. Oldstone M.B.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7089-7093Crossref PubMed Scopus (1051) Google Scholar). Infection of astrocytes may also occur with restricted virus replication, affirming that the effects of HIV on astrocytes may be indirect (4Glass J.D. Johnson R.T. Annu. Rev. Neurosci. 1996; 19: 1-26Crossref PubMed Scopus (94) Google Scholar, 5Tornatore C. Chandra R. Berger J.R. Major E.O. Neurology. 1994; 44: 481-487Crossref PubMed Google Scholar, 6Ranki A. Nyberg M. Ovod V. Haltia M. Elovaara I. Raininko R. Haapasalo H. Krohn K. AIDS. 1995; 9: 1001-1008Crossref PubMed Scopus (252) Google Scholar). Neurons also are not infected. The correlation between the disease severity and the viral load is unconvincing, and the neurotoxicity of the virus itself is controversial (4Glass J.D. Johnson R.T. Annu. Rev. Neurosci. 1996; 19: 1-26Crossref PubMed Scopus (94) Google Scholar, 7Bernton E.W. Bryant H.U. Decoster M.A. Orenstein J.M. Ribas J.L. Meltzer M.S. Gendelman H.E. AIDS Res. Hum. Retroviruses. 1992; 8: 495-503Crossref PubMed Scopus (44) Google Scholar, 8Johnson R.T. Glass J.D. McArthur J.C. Chesebro B.W. Ann. Neurol. 1996; 39: 392-395Crossref PubMed Scopus (106) Google Scholar). Furthermore, little or no virus has been found in AIDS-related vacuolar myelopathy (9Petito C.K. Vecchio D. Chen Y.-T. J. Neuropathol. Exp. Neurol. 1994; 53: 86-94Crossref PubMed Scopus (53) Google Scholar). Taken together, these findings suggest that indirect mechanisms possibly play an important role in the observed neuronal loss in HAD. One means by which indirect effects may be exerted upon neural cells is via nitric oxide (NO) production. NO, a diffusible gas, plays an important role in many physiological and diverse pathophysiological conditions (10Nathan C. FASEB J. 1992; 6: 3051-3064Crossref PubMed Scopus (4129) Google Scholar, 11Jaffrey S.R. Snyder S.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 417-440Crossref PubMed Scopus (304) Google Scholar). At low concentration, NO has been shown to play a unique role in neurotransmission and vasodilation, whereas at higher concentrations it is neurotoxic (10Nathan C. FASEB J. 1992; 6: 3051-3064Crossref PubMed Scopus (4129) Google Scholar, 11Jaffrey S.R. Snyder S.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 417-440Crossref PubMed Scopus (304) Google Scholar). Consistently, NO, derived in excessive amount from the activation of inducible nitric-oxide synthase (iNOS) in glial cells (astroglia and microglia) and macrophages, is assumed to contribute to neuronal abnormalities in HAD (12Adamson D.C. McArthur J.C. Dawson T.M. Dawson V.L. Mol. Med. 1999; 5: 98-109Crossref PubMed Google Scholar, 13Adamson D.C. Wildemann B. Sasaki M. Glass J.D. McArthur J.C. Christov V.I. Dawson T.M. Dawson V.L. Science. 1996; 274: 1917-1921Crossref PubMed Scopus (391) Google Scholar, 14Hori K. Burd P.R. Furuke K. Kutza J. Weih K.A. Clouse K.A. Blood. 1999; 93: 1843-1850Crossref PubMed Google Scholar). By immunocytochemical analysis, Zhao et al. (15Zhao M.L. Kim M.O. Morgello S. Lee S.C. J. Neuroimmunol. 2001; 115: 182-191Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) have shown that iNOS expression is present in all of the HAD cases tested and that iNOS immunoreactivity is localized primarily to reactive astrocytes. Analysis of cerebrospinal fluid and serum from HAD patients has shown increased levels of nitrite and nitrate compared with non-HIV infected patients (16Giovannoni G. Miller R.F. Heales S.J. Land J.M. Harrison M.J. Thompson E.J. J. Neurol. Sci. 1998; 156: 53-58Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The reaction of NO with O2− forms peroxynitrite, ONOO−, a strong nitrosating agent capable of nitrosating tyrosine residues of a protein to nitrotyrosine. Consistently increasing levels of nitrotyrosine have been found in brains of demented, but not in nondemented, AIDS patients (17Boven L.A. Gomes L. Hery C. Gray F. Verhoef J. Portegies P. Tardieu M. Nottet H.S. J. Immunol. 1999; 162: 4319-4327PubMed Google Scholar). Subsequently, reverse transcription-PCR and Western blot analysis of normal and HAD brains also show markedly higher expression of iNOS mRNA and protein in HAD brains than in normal brains (13Adamson D.C. Wildemann B. Sasaki M. Glass J.D. McArthur J.C. Christov V.I. Dawson T.M. Dawson V.L. Science. 1996; 274: 1917-1921Crossref PubMed Scopus (391) Google Scholar). However, the mechanism by which NO is produced in the brains of HAD patients is unclear. The HIV-1 regulatory protein, Tat, is a potent transactivator of viral and cellular gene expression that is produced in the early phase of infection and actively secreted into the extracellular environment, from where it can act in an autocrine or a paracrine manner (18Chang H.K. Gallo R.C. Ensoli B. J. Biomed. Sci. 1995; 2: 189-202Crossref PubMed Scopus (91) Google Scholar). We report herein that the HIV-1 tatgene induces the production of NO and the expression of iNOS through the activation of NF-κB and C/EBPβ in human astrocytes. Fetal bovine serum, Hanks' balanced salt solution, and Dulbecco's modified Eagle's medium/F-12 were from Invitrogen.l-N G-Monomethylarginine (l-NMA),d-N G-monomethylarginine (d-NMA), PD98059, and SB203580 were purchased from Biomol. Arginase was purchased from Sigma. Antibodies against mouse macrophage iNOS were obtained from Calbiochem. Recombinant Tat protein and anti-Tat monoclonal antibodies were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health. HIV-Tat was from Dr. J. Brady, Tat monoclonal antibodies from Dr. K. Krohn. 125I-labeled protein A, [α-32P]dCTP, and [γ-32P]ATP were obtained from PerkinElmer Life Sciences. Dominant-negative mutants of ERK1, ERK2, and p38 were kindly provided by Dr. Jawed Alam (Alton Ochsner Medical Foundation, New Orleans, LA). The wild type p65, the wild type C/EBPβ, and the dominant-negative mutant of C/EBPβ were kindly provided by Dr. Sankar Ghosh (Yale University School of Medicine), Dr. Ormond A. Macdougald (University of Michigan Medical School), and Dr. Steve Smale (University of California at Los Angeles), respectively. Human CNS tissue was obtained from the Human Embryology Laboratory, University of Washington, Seattle. The CNS tissue from each specimen was processed separately and independently, as were subsequent cell cultures. There was no pooling of CNS tissue from distinct specimens. All of the experimental protocols were reviewed and approved by the Institutional Review Board (IRB 224-01-FB) of the University of Nebraska Medical Center. These cells were grown in a serum-free, defined medium (B16) enriched with 5 ng of basic fibroblast growth factor/ml for optimal growth of astrocytes and for the suppression of fibroblast growth (19McCarthy M. Wood C. Fedoseyeva L. Whittemore S. J. Neurovirol. 1995; 1: 275-285Crossref PubMed Scopus (32) Google Scholar). By immunofluorescence assay, these cultures homogeneously expressed glial fibrillary acidic protein (GFAP). Cells were trypsinized, subcultured, and stimulated with different cytokines in serum-free Dulbecco's modified Eagle's medium/F-12 medium. Human U373MG astrocytoma cells obtained from American Type Culture Collection (ATCC) were also maintained and induced with different stimuli as indicated above. The plasmid RSV-CAT expressing the chloramphenicol acetyltransferase enzyme under the control of the Rous sarcoma virus promoter was constructed as described (20Yamamoto T. Jay G. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 176-180Crossref PubMed Scopus (39) Google Scholar). The recombinant plasmid RSV-tat was constructed by replacing the CAT gene in the RSV-CAT construct with theSalI-KpnI fragment of HIV-1, which contains the exon I of the HIV tat gene. The construction of this plasmid was described in detail previously (21Jung M. Wood C. Life Sci. Adv. 1991; 10: 77-88Google Scholar). Cells at 50–60% confluence were transfected with different amounts of either RSV-CAT or RSV-tat construct by LipofectAMINE Plus (Invitrogen) following the manufacturer's protocol (22Pahan K. Sheikh F.G. Liu X. Hilger S. McKinney M. Petro T.M. J. Biol. Chem. 2001; 276: 7899-7905Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 23Pahan K. Liu X. Wood C. Raymond J.R. FEBS Lett. 2000; 472: 203-207Crossref PubMed Scopus (32) Google Scholar, 24Pahan K. Liu X. McKinney M.J. Wood C. Sheikh F.G. Raymond J.R. J. Neurochem. 2000; 74: 2288-2295Crossref PubMed Scopus (82) Google Scholar). Twenty-four hours after transfection, cells were incubated under serum-free conditions. After 24 h of incubation, culture supernatants were transferred to measure NO production. Synthesis of NO was determined by assay of culture supernatants for nitrite, a stable reaction product of NO with molecular oxygen. Briefly, 400 μl of culture supernatant was allowed to react with 200 μl of Griess reagent (25Feinstein D.L. Galea E. Roberts S. Berquist H. Wang H. Reis D.J. J. Neurochem. 1994; 62: 315-321Crossref PubMed Scopus (150) Google Scholar, 26Pahan K. Namboodiri A.M.S. Sheikh F.G. Smith B.T. Singh I. J. Biol. Chem. 1997; 272: 7786-7791Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 27Pahan K. Sheikh F.G. Khan M. Namboodiri A.M.S. Singh I. J. Biol. Chem. 1998; 273: 2591-2600Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 28Pahan K. Raymond J.R. Singh I. J. Biol. Chem. 1999; 274: 7528-7536Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) and incubated at room temperature for 15 min. The optical density of the assay samples was measured spectrophotometrically at 570 nm. Fresh culture medium served as the blank in all experiments. Nitrite concentrations were calculated from a standard curve derived from the reaction of NaNO2 in the assay. Protein was measured by the procedure of Bradford (29Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). Immunoblot analysis for iNOS was carried out as described earlier (26Pahan K. Namboodiri A.M.S. Sheikh F.G. Smith B.T. Singh I. J. Biol. Chem. 1997; 272: 7786-7791Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 27Pahan K. Sheikh F.G. Khan M. Namboodiri A.M.S. Singh I. J. Biol. Chem. 1998; 273: 2591-2600Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 28Pahan K. Raymond J.R. Singh I. J. Biol. Chem. 1999; 274: 7528-7536Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Briefly, cells were detached by scraping, washed with Hanks' buffer, and homogenized in 50 mm Tris-HCl (pH 7.4) containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 5 μg/ml pepstatin A, and 5 μg/ml leupeptin). After electrophoresis the proteins were transferred onto a nitrocellulose membrane, and the iNOS band was visualized by immunoblotting with antibodies against mouse macrophage iNOS and 125I-labeled protein A. Cells were taken out of the culture dishes directly by adding Ultraspec-II RNA reagent (Biotecx Laboratories, Inc.), and total RNA was isolated according to the manufacturer's protocol. For Northern blot analyses, 20 μg of total RNA was electrophoresed on 1.2% denaturing formaldehyde-agarose gels, electrotransferred to Hybond nylon membrane (Amersham Biosciences), and hybridized at 68 °C with32P-labeled cDNA probe using Express Hyb hybridization solution (Clontech) as described by the manufacturer. The cDNA probe was made by polymerase chain reaction amplification using two primers (forward primer, 5′-CTC CTT CAA AGA GGC AAA AAT A-3′; reverse primer, 5′-CAC TTC CTC CAG GAT GTT GT-3′) (26Pahan K. Namboodiri A.M.S. Sheikh F.G. Smith B.T. Singh I. J. Biol. Chem. 1997; 272: 7786-7791Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 27Pahan K. Sheikh F.G. Khan M. Namboodiri A.M.S. Singh I. J. Biol. Chem. 1998; 273: 2591-2600Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 28Pahan K. Raymond J.R. Singh I. J. Biol. Chem. 1999; 274: 7528-7536Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). After hybridization, the filters were washed two or three times in solution I (2× SSC, 0.05% SDS) for 1 h at room temperature followed by solution II (0.1× SSC, 0.1% SDS) at 50 °C for another hour. The membranes were then dried and exposed to x-ray films (Kodak). The same amount of RNA was hybridized with probe for glyceraldehyde 3-phosphate dehydrogenase. Cells plated at 50–60% confluence in 6-well plates were cotransfected with 0.5 μg of phiNOS(7.2)Luc 2K. Pahan, X. Liu, B. S. Taylor, C. Wood, and S. M. Fischer, submitted for publication. and different amounts of either RSV-tat or RSV-CAT by LipofectAMINE Plus (Invitrogen) following the manufacturer's protocol (22Pahan K. Sheikh F.G. Liu X. Hilger S. McKinney M. Petro T.M. J. Biol. Chem. 2001; 276: 7899-7905Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 23Pahan K. Liu X. Wood C. Raymond J.R. FEBS Lett. 2000; 472: 203-207Crossref PubMed Scopus (32) Google Scholar, 24Pahan K. Liu X. McKinney M.J. Wood C. Sheikh F.G. Raymond J.R. J. Neurochem. 2000; 74: 2288-2295Crossref PubMed Scopus (82) Google Scholar). All transfections also included 50 ng of pRL-TK (a plasmid encoding Renilla luciferase, used as transfection efficiency control; Promega)/μg of total DNA. Twenty-four hours after transfection, cells were incubated with serum-free medium for 24 h. Firefly and Renilla luciferase activities were obtained by analyzing total cell extract according to standard instructions provided in the Dual Luciferase Kit (Promega) in a TD-20/20 luminometer (Turner Designs). Relative luciferase activity of cell extracts was typically represented as the ratio of firefly luciferase value/Renilla luciferase value × 103. To assay the transcriptional activities of NF-κB and C/EBPβ, cells at 50–60% confluence were transfected with either pBIIX-Luc, an NF-κB-dependent reporter construct (31Jana M. Liu X. Koka S. Ghosh S. Petro T.M. Pahan K. J. Biol. Chem. 2001; 276: 44527-44533Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), or pC/EBPβ-Luc using the LipofectAMINE Plus method (Invitrogen) (22Pahan K. Sheikh F.G. Liu X. Hilger S. McKinney M. Petro T.M. J. Biol. Chem. 2001; 276: 7899-7905Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 23Pahan K. Liu X. Wood C. Raymond J.R. FEBS Lett. 2000; 472: 203-207Crossref PubMed Scopus (32) Google Scholar, 24Pahan K. Liu X. McKinney M.J. Wood C. Sheikh F.G. Raymond J.R. J. Neurochem. 2000; 74: 2288-2295Crossref PubMed Scopus (82) Google Scholar). Construction of pC/EBPβ-Luc has been described earlier (31Jana M. Liu X. Koka S. Ghosh S. Petro T.M. Pahan K. J. Biol. Chem. 2001; 276: 44527-44533Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). This C/EBPβ-sensitive promoter contains four consensus C/EBPβ-binding sites. All transfections included 50 ng/μg total DNA of pRL-TK (a plasmid encoding Renilla luciferase, used as transfection efficiency control; Promega). After 24 h of transfection, cells were treated with different stimuli for 6 h. Firefly and Renilla luciferase activities were analyzed as described above. U373MG astroglial cells (50–60% confluent) were transfected with different concentrations of either RSV-tat or RSV-CAT. Twenty-four hours after transfection, cells were incubated with serum-free medium. After 18 h of incubation, ERK and p38 MAPK activities were measured using assay kits obtained from Cell Signaling Technology. Briefly, cells were harvested under nondenaturing conditions at different time intervals, and cell lysates were prepared. Activated forms of ERK and p38 MAPK were pulled down from the cell lysate by immunoprecipitation using immobilized phospho-ERK and phospho-p38 MAPK monoclonal antibodies, respectively. The pellets were washed twice with kinase buffer and finally resuspended with 50 μl kinase buffer supplemented with 200 μm ATP and either ELK-1 fusion protein (for ERK) or ATF-2 fusion protein (for p38 MAPK). Following incubation at 30 °C for 30 min, samples were analyzed by Western blot using antibodies against phospho-ELK-1 or phospho-ATF-2. To study the effect of HIV-1 Tat on the expression of iNOS, we transfected human astroglial cells transiently with HIV-1 tat gene. The plasmid RSV-tat expressing the exon I of the HIV-1 tatgene under the control of the Rous sarcoma virus promoter was used to transfect human U373MG astroglial cells. Expression of RSV-tat but not of RSV-CAT induced the production of NO (Table I). The inhibition of NO production by arginase, an enzyme that degrades the substrate (l-arginine) of NOS, and l-NMA, a competitive inhibitor of NOS, but not by d-NMA, a negative control ofl-NMA, suggests that RSV-tat induced the production of NO in U373MG astroglial cells through NOS-mediated arginine metabolism (Table I). To study the dose dependence of RSV-tat on the induction of NO production, cells plated in 6-well plates were transfected with RSV-tat at different doses ranging from 0.05 to 0.5 μg. The induction of NO production started at 0.05 μg of RSV-tat, reached the maximum at 0.2 μg of RSV-tat, and decreased at higher doses (Fig.1 A). This decrease in NO production was due to the increase in astroglial cell death when transfected at higher doses of RSV-tat (data not shown). In contrast, cells transfected with different doses of RSV-CAT were unable to induce the production of NO (Fig. 1 A) suggesting that the induction of NO production is due to the expression of thetat gene. To understand the mechanism of NO production, we examined the effect of RSV-tat and RSV-CAT on protein and mRNA levels of iNOS. For Northern blot analysis, cell plated in 100-mm dishes were transfected with different doses of either RSV-CAT or RSV-tat (Fig. 1 C). Consistent with the induction of NO production, Western blot analysis with antibodies against murine macrophage iNOS and Northern blot analysis for iNOS mRNA clearly showed that expression of RSV-CAT alone did not induce the expression of iNOS protein and mRNA. However, marked expression of iNOS protein (Fig. 1 B) and mRNA (Fig.1 C) was observed in cells transfected with different doses of RSV-tat. Similar to the induction of NO production, the expression of iNOS protein and mRNA by RSV-tat was also dose-dependent. The induction of iNOS protein was maximal in cells transfected with 0.2 μg (per well of 6-well plate) of RSV-tat (Fig. 1 B), whereas the expression of iNOS mRNA was maximal in cells transfected with 0.8 μg (100-mm dish) of RSV-tat (Fig. 1 C).Table IExpression of RSV-tat induces the production of NO in human U373MG astroglial cellsTransfections and treatmentsNitriteμg/mg protein/24 hRSV-CAT (0.2 μg)4.9 ± 0.5RSV-tat (0.2 μg)36.2 ± 4.1RSV-tat (0.2 μg) + arginase6.7 ± 0.4RSV-tat (0.2 μg) +l-NMA6.2 ± 0.8RSV-tat (0.2 μg) +d-NMA35.7 ± 4.5Cells plated at 50–60% confluence in 6-well plates were transfected with 0.2 μg of either RSV-CAT or RSV-tat using LipofectAMINE Plus (Invitrogen) as described under “Materials and Methods.” After 24 h of transfection, cells were incubated under serum-free conditions in the presence or absence of l-NMA (0.1 mm), d-NMA (0.1 mm), and arginase (100 units/ml). After 24 h of incubation, the concentrations of nitrite were measured in the supernatants as described under “Materials and Methods.” Data are mean ± S.D. of three different experiments. Open table in a new tab Cells plated at 50–60% confluence in 6-well plates were transfected with 0.2 μg of either RSV-CAT or RSV-tat using LipofectAMINE Plus (Invitrogen) as described under “Materials and Methods.” After 24 h of transfection, cells were incubated under serum-free conditions in the presence or absence of l-NMA (0.1 mm), d-NMA (0.1 mm), and arginase (100 units/ml). After 24 h of incubation, the concentrations of nitrite were measured in the supernatants as described under “Materials and Methods.” Data are mean ± S.D. of three different experiments. In the CNS, only microglia is known to be infected productively by HIV-1 and to secrete the HIV-1 regulatory protein, Tat (2Bagasra O. Lavi E. Bobroski L. Khalili K. Pestaner J.B. Pomerantz R.J. AIDS. 1996; 10: 573-585Crossref PubMed Scopus (386) Google Scholar, 3Wiley C.A. Schrier R.D. Nelson J.A. Lampert P.W. Oldstone M.B.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7089-7093Crossref PubMed Scopus (1051) Google Scholar, 32Tardieu M. Hery C. Peudenier S. Boespflug O. Montagnier L. Ann. Neurol. 1992; 132: 11-17Crossref Scopus (136) Google Scholar, 33Ensoli B. Buonaguro L. Barillari G. Fiorelli V. Gendelman R. Morgan R. Wingfield P. Gallo R.C. J. Virol. 1993; 67: 277-287Crossref PubMed Google Scholar) which in turn may act on astroglia in a paracrine fashion (18Chang H.K. Gallo R.C. Ensoli B. J. Biomed. Sci. 1995; 2: 189-202Crossref PubMed Scopus (91) Google Scholar). Therefore, to understand whether Tat protein is secreted from RSV-tat-transfected astroglial cells and whether Tat protein is in fact responsible for the induction of iNOS, we incubated RSV-tat-transfected cells with anti-Tat antibodies. Although anti-Tat antibodies at a dose of 1 μg/ml was not very effective in blocking the induction of NO production, about 50% inhibition of NO production was observed when RSV-tat-transfected cells were incubated with 5 μg/ml anti-Tat antibodies (Fig.2 A). In contrast, control IgG had no effect on the induction of NO production. We next examined whether exogenously added recombinant HIV-1 Tat protein is able to induce the production of NO. Consistent with the induction of NO production by RSV-tat, recombinant Tat protein also dose-dependently induced the production of NO with the maximum induction observed at 100 ng/ml (Fig. 2 B). However, in contrast to the 8–9-fold induction of NO production by the expression of RSV-tat (Fig. 1 A), recombinant Tat protein induced the production of NO by about 4-fold (Fig.2 B). Taken together, these observations suggest that the induction of iNOS in RSV-tat-transfected astroglial cells is due to Tat and that Tat protein is secreted from RSV-tat-transfected astroglial cells. Human primary astrocytes have been shown to induce the expression of iNOS in the presence of different proinflammatory cytokines (23Pahan K. Liu X. Wood C. Raymond J.R. FEBS Lett. 2000; 472: 203-207Crossref PubMed Scopus (32) Google Scholar, 24Pahan K. Liu X. McKinney M.J. Wood C. Sheikh F.G. Raymond J.R. J. Neurochem. 2000; 74: 2288-2295Crossref PubMed Scopus (82) Google Scholar).2 Because HIV-1tat gene induced the production of NO in human U373MG astroglial cells, we examined whether HIV-1 tat gene was also able to induce the production of NO in human primary astrocytes (Fig. 3). Consistent with the induction of NO production in human U373MG astroglial cells, expression of RSV-tat but not RSV-CAT (the control plasmid) dose-dependently induced the production of NO in human primary astroglia. The induction of NO was maximum at 0.2 μg of RSV-tat and decreased at higher concentration (Fig.3). To understand the effect of thetat gene on the transcription of iNOS, U373MG glial cells were cotransfected with phiNOS(7.2)Luc, a construct containing the human iNOS promoter fused to the luciferase gene,2 and either RSV-tat or RSV-CAT. Activation of the iNOS promoter was measured after incubating the cells with serum-free media. It is evident from Fig. 4 that transfection of cells with different amounts of RSV-tat but not RSV-CAT led to the induction of iNOS promoter-derived luciferase activity. About 3.7-fold activation of iNOS promoter-derived luciferase activity was observed in cells transfected with 0.2 μg of RSV-tat (Fig.4). These results suggest that the induction rate of the human iNOS promoter construct is much lower than the induction rate of human iNOS mRNA expression (Fig. 1 C). We used a 7.2-kb human iNOS promoter for this study. Earlier, Taylor et al. (34Taylor B.S. de Vera M.E. Ganster R.W. Wang Q. Shapiro R.A. Morris S.M. Billiar T.R. Geller D.A. J. Biol. Chem. 1998; 273: 15148-15156Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar) showed a 4.1-fold induction of this human iNOS promoter in human AKN-1 liver cells. They have also shown that transfection of a 16-kb human iNOS promoter construct produced a 9-fold increase in lucif" @default.
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