Activation of the Adenosine A1 Receptor Inhibits HIV-1 Tat-Induced Apoptosis by Reducing Nuclear Factor- B Activation and Inducible Nitric-Oxide Synthase

Human immunodeficiency virus dementia (HIV-D) is a nonfocal central nervous system manifestation characterized by cognitive, behavioral, and motor abnormalities. The pathophysiology of neuronal damage in HIV-D includes a direct toxic effect of viral proteins on neuronal cells and an indirect effect caused by the release of inflammatory mediators and neurotoxins by activated macrophages/microglia and astrocytes, culminating into neuronal apoptosis. Previous studies have documented that the nucleoside adenosine mediates neuroprotection by activating adenosine A1 receptor subtype (A1AR) linked to suppression of neuronal excitability. In this study, we show that A1AR activation protects against HIV-1 Tat-induced toxicity in primary cultures of rat cerebellar granule neurons and in rat pheochromocytoma (PC12) cell. In PC12 cells, HIV-1 Tat increased [Ca ]i levels, release of nitric oxide (NO), and expression of inducible nitric-oxide synthase (iNOS) and A1AR. Activation of A1AR suppressed Tat-mediated increases in [Ca 2 ]i and NO. Furthermore, A1AR agonists inhibited iNOS expression in a nuclear factorB (NFB)-dependent manner. It is noteworthy that activation of the A1AR or inhibition of NOS protected against Tat-induced apoptosis in PC12 cells and cerebellar granule cells. Moreover, activation of the A1AR-inhibited Tat-induced increases in the levels of proapoptotic proteins Bax and caspase-3. Taken together, our results demonstrate that the A1AR protects against HIV-1 toxicity by inhibiting NFB, thereby reducing the expression of iNOS and NO radicals and neuronal apoptosis. HIV dementia (HIV-D) is a syndrome of cognitive and motor dysfunction (Kaul et al., 2001) that occurs late in the course of HIV infection and progresses slowly over months. Its major clinical features include a decline in cognitive and behavioral functions and motor abnormalities. After the introduction of highly active antiretroviral therapy, there was a significant decrease in the incidence of HIV-D. However, because there has been an increase in the number of individuals living with HIV/AIDS due to effective therapy, the prevalence of HIV-D has actually increased. Even though HIV primarily targets the immune system and produces widespread immunodeficiency, it also affects This work was supported from the Southern Illinois University School of Medicine Excellence in Academic Medicine Award (to V.R.) and National Institutes of Health grants DC02396 (to L.P.R.), NS054578 (to S.B.M.), and T32-AI49105 (to L.F.S.). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.031427. ABBREVIATIONS: HIV-D, human immunodeficiency virus-dementia; A1AR, adenosine A1 receptor; iNOS, inducible nitric-oxide synthase; NFB, nuclear factor B; PC12 cells, pheochromocytoma 12 cells; CGN, cerebellar granule neuron; R-PIA, R-phenylisopropyladenosine; DPCPX, 8-cyclopentyl-1,3-di[2 ,3 -3H] propylxanthine; AR, adenosine receptor; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; RTPCR, reverse transcription-polymerase chain reaction; bp, base pair; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde phosphate dehydrogenase; AP-1, activator protein-1; FITC, fluorescein isothiocyanate; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; AM, acetoxymethyl ester; L-NAME, N-nitro-L-arginine methyl ester; SNAP, S-nitroso-N-acetylpenicillamine; C-PTIO, 2-(4carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; OCT-1, octamer transcription factor-1; DAF-2 DA, 4,5-diaminofluorescein diacetate; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1 propanesulfonate; ZM241385, 4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a] [1,3,5] triazin-5-ylamino]ethyl) phenol; MRS1220, N-(9-chloro-2-(2-furanyl)[1,2,4]-triazolol[1,5-c]quinazolin-5-benzeneacetamide; BAPTA, 1,2-bis(2aminophenoxy)ethane-N,N,N ,N -tetraacetic acid; CGS21680, 4-[2-[[6-amino-9-(N-ethyl-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid. 0026-895X/07/7204-856–867$20.00 MOLECULAR PHARMACOLOGY Vol. 72, No. 4 Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics 31427/3252852 Mol Pharmacol 72:856–867, 2007 Printed in U.S.A. 856 at A PE T Jornals on O cber 9, 2017 m oharm .aspeurnals.org D ow nladed from the central nervous system, resulting in HIV encephalopathy, the pathologic hallmark of HIV-D. There are different theories describing mechanisms of neuronal damage seen in HIV-D, including a direct toxic effect on neurons and an indirect effect mediated by immune cells (Kaul et al., 2001). Once it enters the central nervous system, HIV productively infects brain macrophages, microglia, and multinucleate giant cells (Kaul et al., 2001), the resident immune cells of the brain, leading to the production of viral proteins such as the envelope glycoprotein gp120, Tat, and the viral proteins Nef and Vpr. These proteins, in previous studies, produce direct neuronal damage by different mechanisms (Kruman et al., 1998; Nath et al., 2000). Infection of immune cells stimulates an inflammatory response, leading to neuronal damage by an indirect or the so-called “bystander” effect (Kaul et al., 2001). Neuronal damage seen in HIV-D probably involves a mixture of the direct and indirect mechanisms (González-Scarano and Martin-Garcia, 2005). The nucleoside adenosine exerts its major physiological effects by interacting with different subtypes of adenosine receptors (ARs), of which A1AR is the predominant subtype in the central nervous system (Dunwiddie and Masino, 2001). Previous reports strongly support a cytoprotective role of A1AR in the central nervous system (Dunwiddie and Masino, 2001; Ramkumar et al., 2001). To date, we are not aware of any study demonstrating a beneficial action of activation of the A1AR or other ARs against HIV-1 proteins in the central nervous system or in neuronal cultures. However, recent data show that adenosine, acting via A2AAR, inhibits Tatinduced tumor necrosis factorproduction in primary monocytes (Fotheringham et al., 2004). In this study, we determined whether adenosine analogs could confer protection against HIV-induced toxicity in rat pheochromocytoma (PC) 12 cells and in primary cultures of cerebellar granule neurons (CGNs), used previously to study HIV-1 Tat signaling in vitro (Wong et al., 2005; Sui et al., 2006). HIV-1 Tat was used because this protein can cross the blood-brain barrier (Schwarze et al., 1999), is elevated in patients with HIV-D (Nath et al., 2000), and injection in experimental animals can produce pathological findings similar to those observed in HIV-D (Jones et al., 1998). Our data indicate that activation of the A1AR protects PC12 cells and CGN cultures from HIV-1 Tat-mediated apoptosis, presumably by inhibiting inducible nitric-oxide synthase (iNOS) expression and NO release via inhibition of nuclear factor (NF)B activation. Materials and Methods Cell Cultures. Rat pheochromocytoma cells (PC12) were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in flasks coated with 0.1 mg/ml poly(D-lysine) (Invitrogen, Carlsbad, CA) and grown in RPMI 1640 supplemented with 10% horse serum and 5% fetal bovine serum (all supplies were from Invitrogen). Cells were cultured at 37°C, in the presence of 5% CO2 and 95% ambient air, and the medium was changed every 2 to 3