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HIV-1 Nef Inhibits Lipopolysaccharide-induced IL-12p40 Expression by Inhibiting JNK-activated NFκB in Human Monocytic Cells

2008; Elsevier BV; Volume: 284; Issue: 12 Linguagem: Inglês

10.1074/jbc.m710013200

ISSN

1083-351X

Autores

Wei Ma, Sasmita Mishra, Niranjala Gajanayaka, Jonathan B. Angel, Ashok Kumar,

Tópico(s)

interferon and immune responses

Resumo

Impaired cellular immunity caused by decreased production of Th1-type cytokines, including interleukin-12 (IL-12) is a major feature of HIV-1-associated immunodeficiency and acquired immunodeficiency syndrome. IL-12p40, an inducible subunit shared between IL-12 and IL-23, plays a critical role in the development of cellular immunity, and its production is significantly decreased during HIV infection. The mechanism by which HIV induces loss of IL-12p40 production remains poorly understood. We have previously shown that lipopolysaccharide (LPS)-induced IL-12p40 production in monocytic cells is regulated by NFκB and AP-1 transcription factors through the activation of two distinct upstream signaling pathways, namely the c-Jun-N-terminal kinase (JNK) and the calmodulin-dependent protein kinase-II-activated pathways. Herein, we show that intracellular nef expressed through transduction of primary monocytes and promonocytic THP-1 cells with retroviral-mediated nef gene inhibited LPS-induced IL-12p40 transcription by inhibiting the JNK mitogen-activated protein kinases without affecting the calmodulin-dependent protein kinase-II-activated pathway. In addition, nef inhibited JNK-activated NFκB without affecting the AP-1 activity. Overall, our results suggest for the first time that intracellular nef inhibited LPS-activated JNK, which may cause inhibition of IL-12p40 expression in human monocytic cells by selectively inhibiting NFκB activity. Impaired cellular immunity caused by decreased production of Th1-type cytokines, including interleukin-12 (IL-12) is a major feature of HIV-1-associated immunodeficiency and acquired immunodeficiency syndrome. IL-12p40, an inducible subunit shared between IL-12 and IL-23, plays a critical role in the development of cellular immunity, and its production is significantly decreased during HIV infection. The mechanism by which HIV induces loss of IL-12p40 production remains poorly understood. We have previously shown that lipopolysaccharide (LPS)-induced IL-12p40 production in monocytic cells is regulated by NFκB and AP-1 transcription factors through the activation of two distinct upstream signaling pathways, namely the c-Jun-N-terminal kinase (JNK) and the calmodulin-dependent protein kinase-II-activated pathways. Herein, we show that intracellular nef expressed through transduction of primary monocytes and promonocytic THP-1 cells with retroviral-mediated nef gene inhibited LPS-induced IL-12p40 transcription by inhibiting the JNK mitogen-activated protein kinases without affecting the calmodulin-dependent protein kinase-II-activated pathway. In addition, nef inhibited JNK-activated NFκB without affecting the AP-1 activity. Overall, our results suggest for the first time that intracellular nef inhibited LPS-activated JNK, which may cause inhibition of IL-12p40 expression in human monocytic cells by selectively inhibiting NFκB activity. HIV-1 Nef inhibits lipopolysaccharide-induced IL-12p40 expression by inhibiting JNK-activated NFκB in human monocytic cells.Journal of Biological ChemistryVol. 287Issue 1PreviewVOLUME 284 (2009) PAGES 7578–7587 Full-Text PDF Open Access HIV 5The abbreviations used are: HIV, human immunodeficiency virus; IL-12, interleukin-12; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cell; ERK, extracellular signal-regulated kinase; ELISA, enzyme-linked immunosorbent assay; RT, reverse transcription; CaM, calmodulin; CaMK-II, calmodulin-dependent kinase-II; JNK, c-Jun-N-terminal kinase; MAPK, mitogen-activated protein kinase; pSRα, the retrovirus containing the empty vector; pSRα-Nef, pSRα containing the Nef gene; SEK1, stress-activated protein kinase/extra-cellular signal-regulated kinase 1. infection results in a progressive loss of general and HIV-specific cellular immunity by inhibiting the production of Th1-type cytokines such as IL-12 (1Fauci A.S. Nat. 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Kostman J.R. Montaner L.J. Trinchieri G. Blood. 1999; 94: 1003-1011Crossref PubMed Google Scholar, 6Langrish C.L. McKenzie B.S. Wilson N.J. de Waal M.R. Kastelein R.A. Cua D.J. Immunol. Rev. 2004; 202: 96-105Crossref PubMed Scopus (616) Google Scholar, 7Ma X. Trinchieri G. Adv. Immunol. 2001; 79: 55-92Crossref PubMed Scopus (172) Google Scholar). It promotes Th1-type cell-mediated immune responses by inducing interferon-γ from NK and T cells and enhances their cytotoxicity (7Ma X. Trinchieri G. Adv. Immunol. 2001; 79: 55-92Crossref PubMed Scopus (172) Google Scholar, 8Cooper A.M. Khader S.A. Trends Immunol. 2007; 28: 33-38Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). IL-12 is produced by monocytic and dendritic cells and B cells (7Ma X. Trinchieri G. Adv. Immunol. 2001; 79: 55-92Crossref PubMed Scopus (172) Google Scholar, 8Cooper A.M. Khader S.A. Trends Immunol. 2007; 28: 33-38Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). It is a 70-kDa heterodimer composed of p35 and p40 subunits that are disulfide-linked together to form biologically active IL-12 (9Gubler U. Chua A.O. Schoenhaut D.S. Dwyer C.M. McComas W. Motyka R. Nabavi N. Wolitzky A.G. Quinn P.M. Familletti P.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4143-4147Crossref PubMed Scopus (565) Google Scholar, 10Aste-Amezaga M. Ma X. Sartori A. Trinchieri G. J. Immunol. 1998; 160: 5936-5944PubMed Google Scholar). The p35 and p40 subunits are encoded by two distinct and differentially regulated genes: the p40 gene is tightly regulated at transcriptional level and detected only in IL-12-producing cells, whereas the p35 gene is constitutively expressed in various cell types (9Gubler U. Chua A.O. Schoenhaut D.S. Dwyer C.M. McComas W. Motyka R. Nabavi N. Wolitzky A.G. Quinn P.M. Familletti P.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4143-4147Crossref PubMed Scopus (565) Google Scholar, 10Aste-Amezaga M. Ma X. Sartori A. Trinchieri G. J. Immunol. 1998; 160: 5936-5944PubMed Google Scholar). IL-12p40, therefore, constitutes an indicator for IL-12 production. Moreover, IL-12p40 subunit is also shared by another Th1 cytokine IL-23, which makes it highly significant for determination of cell-mediated immune response (6Langrish C.L. McKenzie B.S. Wilson N.J. de Waal M.R. Kastelein R.A. Cua D.J. Immunol. Rev. 2004; 202: 96-105Crossref PubMed Scopus (616) Google Scholar). The signaling pathways involved in the regulation of IL-12p40 synthesis in monocytic cells following LPS stimulation have been investigated. Multiple transcription factors, including NFκB, Ets-2, AP-1, and C/EBP, PU.1, and interferon-γ regulatory factors and their complexes have been suggested to regulate IL-12p40 transcription in LPS-stimulated murine and human monocytic cells (7Ma X. Trinchieri G. Adv. Immunol. 2001; 79: 55-92Crossref PubMed Scopus (172) Google Scholar, 11Plevy S.E. Gemberling J.H. Hsu S. Dorner A.J. Smale S.T. Mol. Cell Biol. 1997; 17: 4572-4588Crossref PubMed Scopus (273) Google Scholar, 12Ma X. Neurath M. Gri G. Trinchieri G. J. Biol. Chem. 1997; 272: 10389-10395Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 13Ma W. Gee K. Lim W. Chambers K. Angel J.B. Kozlowski M. Kumar A. J. Immunol. 2004; 172: 318-330Crossref PubMed Scopus (118) Google Scholar, 14Zhu C. Rao K. Xiong H. Gagnidze K. Li F. Horvath C. Plevy S. J. Biol. Chem. 2003; 278: 39372-39382Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). We and others have demonstrated that c-Jun-N-terminal kinase (JNK) plays a key role in the regulation of IL-12p40 production in LPS-stimulated human monocytic cells (13Ma W. Gee K. Lim W. Chambers K. Angel J.B. Kozlowski M. Kumar A. J. Immunol. 2004; 172: 318-330Crossref PubMed Scopus (118) Google Scholar, 15Utsugi M. Dobashi K. Ishizuka T. Endou K. Hamuro J. Murata Y. Nakazawa T. Mori M. J. Immunol. 2003; 171: 628-635Crossref PubMed Scopus (99) Google Scholar). Recently, we have also shown that LPS-induced IL-12p40 production is regulated by another distinct pathway, the calmodulin/CaM-activated protein kinase (CaMK-II)-activated phosphatidylinositol-3-kinase pathway (16Ma W. Mishra S. Gee K. Mishra J.P. Nandan D. Reiner N.E. Angel J.B. Kumar A. J. Biol. Chem. 2007; 282: 13351-13362Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Interestingly, both pathways regulated IL-12p40 production through the NFκB and AP-1 transcription factors (13Ma W. Gee K. Lim W. Chambers K. Angel J.B. Kozlowski M. Kumar A. J. Immunol. 2004; 172: 318-330Crossref PubMed Scopus (118) Google Scholar, 16Ma W. Mishra S. Gee K. Mishra J.P. Nandan D. Reiner N.E. Angel J.B. Kumar A. J. Biol. Chem. 2007; 282: 13351-13362Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Monocytic cells play a key role in HIV pathogenesis and serve as long-term reservoirs in chronically infected patients (2Kedzierska K. Crowe S.M. Curr. Med. Chem. 2002; 9: 1893-1903Crossref PubMed Scopus (167) Google Scholar, 17Collman R.G. Perno C.F. Crowe S.M. Stevenson M. Montaner L.J. J. Leukoc. Biol. 2003; 74: 631-634Crossref PubMed Scopus (55) Google Scholar). IL-12 and in particular IL-12p40 production is impaired in HIV-infected patients and in monocytic cells infected in vitro with HIV (3Chehimi J. Starr S.E. Frank I. D'Andrea A. Ma X. MacGregor R.R. Sennelier J. Trinchieri G. J. Exp. Med. 1994; 179: 1361-1366Crossref PubMed Scopus (416) Google Scholar, 4Marshall J.D. Chehimi J. Gri G. Kostman J.R. Montaner L.J. Trinchieri G. Blood. 1999; 94: 1003-1011Crossref PubMed Google Scholar, 5Chambers K.A. Parato K.G. Angel J.B. Cell Immunol. 2002; 215: 120-132Crossref PubMed Scopus (15) Google Scholar). Active cellular infection and HIV replication inhibited IL-12p40 transcription and its synthesis in monocytic cells (5Chambers K.A. Parato K.G. Angel J.B. Cell Immunol. 2002; 215: 120-132Crossref PubMed Scopus (15) Google Scholar). We and others have shown that IL-12 production is decreased in HIV-infected patients and treatment with anti-retroviral therapy enhanced IL-12 production (3Chehimi J. Starr S.E. Frank I. D'Andrea A. Ma X. MacGregor R.R. Sennelier J. Trinchieri G. J. Exp. Med. 1994; 179: 1361-1366Crossref PubMed Scopus (416) Google Scholar, 4Marshall J.D. Chehimi J. Gri G. Kostman J.R. Montaner L.J. Trinchieri G. Blood. 1999; 94: 1003-1011Crossref PubMed Google Scholar, 5Chambers K.A. Parato K.G. Angel J.B. Cell Immunol. 2002; 215: 120-132Crossref PubMed Scopus (15) Google Scholar, 18Landay A.L. Clerici M. Hashemi F. Kessler H. Berzofsky J.A. Shearer G.M. J. Infect. Dis. 1996; 173: 1085-1091Crossref PubMed Scopus (80) Google Scholar, 19Clerici M. Lucey D.R. Berzofsky J.A. Pinto L.A. Wynn T.A. Blatt S.P. Dolan M.J. Hendrix C.W. Wolf S.F. Shearer G.M. Science. 1993; 262: 1721-1724Crossref PubMed Scopus (383) Google Scholar, 20Angel J.B. Kumar A. Parato K. Filion L.G. Diaz-Mitoma F. Daftarian P. Pham B. Sun E. Leonard J.M. Cameron D.W. J. Infect. Dis. 1998; 177: 898-904Crossref PubMed Scopus (121) Google Scholar, 21Angel J.B. Parato K.G. Kumar A. Kravcik S. Badley A.D. Fex C. Ashby D. Sun E. Cameron D.W. J. Infect. Dis. 2001; 183: 546-554Crossref PubMed Scopus (54) Google Scholar, 22Daftarian M.P. Diaz-Mitoma F. Creery W.D. Cameron W. Kumar A. Clin. Diagn. Lab. Immunol. 1995; 2: 712-718Crossref PubMed Google Scholar). Furthermore, exogenous addition of IL-12 enhanced IL-2 production, cell proliferation, and the development of cell-mediated cytotoxicity of HIV antigen-stimulated PBMCs from HIV-infected individuals (3Chehimi J. Starr S.E. Frank I. D'Andrea A. Ma X. MacGregor R.R. Sennelier J. Trinchieri G. J. Exp. Med. 1994; 179: 1361-1366Crossref PubMed Scopus (416) Google Scholar, 4Marshall J.D. Chehimi J. Gri G. Kostman J.R. Montaner L.J. Trinchieri G. Blood. 1999; 94: 1003-1011Crossref PubMed Google Scholar, 18Landay A.L. Clerici M. Hashemi F. Kessler H. Berzofsky J.A. Shearer G.M. 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To understand the mechanism underlying the loss of cell-mediated immune response during HIV infection and development of AIDS, it is imperative to investigate the signaling pathways responsible for the loss of Th1 cytokines IL-12 and IL-23 and in particular the inducible IL-12p40 subunit shared between these two cytokines. At present, little is known regarding the regulation and expression of IL-12p40 in monocytic cells following HIV infection. There is evidence to suggest that HIV regulatory protein, nef, inhibits IL-12 synthesis. IL-12p40 production was shown to be suppressed in lymph nodes of macaques infected with simian immunodeficiency virus compared with those infected with the non-pathogenic nef-deleted strain, SIVmac239Δ nef (25Zou W. Lackner A.A. Simon M. Durand-Gasselin I. Galanaud P. Desrosiers R.C. Emilie D. J. Virol. 1997; 71: 1227-1236Crossref PubMed Google Scholar). However, the exact role of nef and the mechanism involved in the inhibition of IL-12p40 production in monocytic cells are not known. Nef is a 27-kDa myristoylated protein expressed early in HIV infection (26Geyer M. Munte C.E. Schorr J. Kellner R. Kalbitzer H.R. J. Mol. Biol. 1999; 289: 123-138Crossref PubMed Scopus (96) Google Scholar). In addition to the well known down-regulation of the cell surface CD4 and MHC-I receptors (27Piguet V. Wan L. Borel C. Mangasarian A. Demaurex N. Thomas G. Trono D. Nat. Cell Biol. 2000; 2: 163-167Crossref PubMed Scopus (248) Google Scholar, 28Piguet V. Schwartz O. Le Gall S. Trono D. Immunol. Rev. 1999; 168: 51-63Crossref PubMed Scopus (179) Google Scholar), nef uniquely can interact with a number of signaling molecules through its myristoyl moiety, leading to the dysregulation of host immune responses (29Olivetta E. Percario Z. Fiorucci G. Mattia G. Schiavoni I. Dennis C. Jager J. Harris M. Romeo G. Affabris E. Federico M. J. 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In this study, we show for the first time that intracellular expression of HIV-nef by retroviral infection of primary monocytes and promonocytic THP-1 cells and following stable transfection of THP-1 cells with HIV-nef gene resulted in the inhibition of LPS-induced IL-12p40 production. Studies conducted to understand the underlying mechanism revealed that nef inhibited IL-12p40 transcription by selectively inhibiting LPS-activated JNK without affecting the calcium signaling pathway. Moreover, nef inhibited JNK-activated NFκB without affecting the activity of AP-1 transcription factor. Cell Lines, Cell Culture, and Reagents-THP-1, a promonocytic cell line and 293T cells, the embryonic kidney epithelial cells, were obtained from the American Type Culture Collection (Manassas, VA). A retroviral packaging cell line, PT67, derived from NIH 3T3 cells and packaging virus with a polytropic envelope, was purchased from BD Biosciences Clontech (Mississauga, Ontario, Canada). Cells were cultured in Iscove's modified Dulbecco's medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Invitrogen), 100 units/ml penicillin, 100 μg/ml gentamicin, 10 mm HEPES, and 2 mm glutamine. The anti-mitogen-activated protein kinase (MAPKs), including extracellular signal regulated kinase (ERK1/2), p38, and JNK and anti-phospho MAPKs antibodies (Cell Signaling), anti-CaMK-II and phospho-CaMK-II antibodies (StressGen Biotechnologies, Victoria, British Columbia, Canada), the JNK inhibitor SP600125 (Biomol, Plymouth Meeting, PA), the calcium chelating agent EGTA (Sigma), SKF-96365 hydrochloride, an inhibitor of receptor-mediated calcium entry, W7 hydrochloride, a calmodulin antagonist, and KN-93, an inhibitor of CaMK-II (all from Calbiochem), were purchased. Isolation of Monocytes-Monocytes were purified from PBMCs by negative selection as described earlier (16Ma W. Mishra S. Gee K. Mishra J.P. Nandan D. Reiner N.E. Angel J.B. Kumar A. J. Biol. Chem. 2007; 282: 13351-13362Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 35Mishra S. Mishra J.P. Kumar A. J. Biol. Chem. 2007; 282: 4288-4300Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Briefly, PBMCs were incubated with magnetic polystyrene Dynabeads (Dynal Biotech, Oslo, Norway) coated with anti-CD2 (T cells) and anti-CD19 (B cells) antibodies for 30 min on ice for depletion of T and B cells, respectively. Cells were further incubated for 2 h at 37 °C to eliminate nonadherent cells. The monocytes obtained contained <1% T and B cells as determined by flow cytometry. Some experiments were confirmed by using monocytes isolated by automacs negative selection (Miltenyi Biotech Inc., Auburn, CA). Briefly, PBMCs were washed twice in phosphate-buffered saline containing 2% EDTA followed by incubation with automacs FcR blocking reagent along with biotin antibody mixture for 10 min at 4 °C. Following incubation, cells were treated with anti-biotin microbeads for 15 min at 4 °C. Cells were then washed once and subjected to automacs negative selection separation as per the manufacturer's instructions. Cell populations thus obtained contained more than 90% CD14+ monocytes. Production of nef Retroviruses-The retroviral vector pSRα-MSVtkneo containing nef gene (pSR-α-Nef) derived from HIV-1 SF2 strain (GenBank™ accession number K02007, nucleotides 8504–9573) was a gift from Dr. T. Smithgall (University of Pittsburgh) (33Trible R.P. Emert-Sedlak L. Smithgall T.E. J. Biol. Chem. 2006; 281: 27029-27038Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The control retroviruses were prepared by cleaving nef fragment by using Klenow Fragment (New England Biolabs). To produce high titer nef retrovirus stocks, PT67 cells (1.5 × 106) were transduced with 4 μgofnef retrovirus along with 12 μg of FuGENE 6 (Roche Applied Science) in 2 ml of Iscove's modified Dulbecco's medium-fetal calf serum (10%) containing 4 mg/ml polybrene. After 24 h, the culture medium was replaced with fresh Iscove's modified Dulbecco's medium, and cells were selected by 300 μg/ml G418 (Invitrogen). Seven days post-transfection, genomic DNA was analyzed to determine nef gene integration by PCR, and total proteins were analyzed for nef expression by immunoblotting with anti-nef monoclonal antibody, EH-1 (NIH AIDS Research and Reference Reagent Program, Rockville, MD). The nef-transfected PT67 cells were grown to collect nef virus-containing supernatants followed by their titration by employing NIH 3T3 cells according to the manufacturer's protocol (BD Bioscience Clontech). Infection of Monocytic Cells with nef Retrovirus and Measurement of IL-12p40 by ELISA-Briefly, cells were cultured in 3 ml of virus-containing supernatant collected from packaging PT67 cells for 24 h and infected a second time under identical conditions for another 24 h followed by stimulation with LPS for various times. Cells were harvested for protein and RNA extraction. The supernatants were collected for measurement of IL-12p40 production by ELISA (R & D Systems) as described earlier (13Ma W. Gee K. Lim W. Chambers K. Angel J.B. Kozlowski M. Kumar A. J. Immunol. 2004; 172: 318-330Crossref PubMed Scopus (118) Google Scholar, 16Ma W. Mishra S. Gee K. Mishra J.P. Nandan D. Reiner N.E. Angel J.B. Kumar A. J. Biol. Chem. 2007; 282: 13351-13362Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Infection of Monocytic Cells with Wild-type and nef-deleted HIV Mutants-Wild-type (HIV-1pNL4–3) and nef-deleted (Δnef-HIV-1pNL4–3) HIV molecular clones were provided by Dr E. Cohen, University of Montreal. 293T cells were transfected with HIV-1pNL4–3 or Δnef-HIV-1pNL4–3 plasmids for 2 days using FuGENE6 as transfecting reagent as described earlier (13Ma W. Gee K. Lim W. Chambers K. Angel J.B. Kozlowski M. Kumar A. J. Immunol. 2004; 172: 318-330Crossref PubMed Scopus (118) Google Scholar, 16Ma W. Mishra S. Gee K. Mishra J.P. Nandan D. Reiner N.E. Angel J.B. Kumar A. J. Biol. Chem. 2007; 282: 13351-13362Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 36Gee K. Angel J.B. Ma W. Mishra S. Gajanayaka N. Parato K. Kumar A. J. Biol. Chem. 2006; 281: 31647-31658Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Supernatants were collected after 3 days followed by measurement of p24 by ELISA (NIH). THP-1 cells were infected with 100 pg/ml each of HIV-1NL4–3 or Δnef-HIV-1NL4–3 for 2 days. Cells were stimulated with LPS for another 24 h followed by measurement of IL-12p40 by ELISA. Stable Transfection of THP-1 Cells with pcDNA-Nef-The full-length HIV-1-nef gene plasmid derived from pclonsnefSN (NIH AIDS Research and Reference Reagent) was subcloned into the pcDNA3.1zeo+ expression vector (Invitrogen) at the EcoRI restriction site. THP-1 cells were transfected with pcDNA3.1zeo+ containing nef gene (pcDNA-Nef) by Lipofectamine reagent as per the manufacturer's instructions (Invitrogen). Briefly, 2 μg of the pcDNA-Nef and 8 μl of Lipofectamine were incubated with 200 μl of Opti-MEM-I Reduced Serum Medium (Invitrogen) for 30 min at room temperature to allow formation of DNA-liposome complexes. The resulting complexes were added to the cell culture for 48 h following which the medium was replaced with fresh Iscove's modified Dulbecco's medium containing 200 μg/ml of Zeocin. After 7 days, nef-mRNA and proteins were measured by RT-PCR and Western blotting, respectively. RNA Isolation and RT-PCR Analysis-Total RNA was extracted using the RNeasy Plus Mini Kit® (Qiagen) and reverse transcribed with Moloney murine leukemia virus reverse transcriptase (PerkinElmer Life Sciences) as described (13Ma W. Gee K. Lim W. Chambers K. Angel J.B. Kozlowski M. Kumar A. J. Immunol. 2004; 172: 318-330Crossref PubMed Scopus (118) Google Scholar, 16Ma W. Mishra S. Gee K. Mishra J.P. Nandan D. Reiner N.E. Angel J.B. Kumar A. J. Biol. Chem. 2007; 282: 13351-13362Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Aliquots (5 μl) of cDNA equivalent to 100 ng of RNA were amplified by PCR for IL-12p40, pSRα-Nef, pcDNA-Nef, and β-actin. The primers used were as follows: pSRα-Nef: sense, 5′-ATG GGT GGCA AGT GGT CAA AAC GTA-3′; antisense, 5′-GGA AAA CCC ACC TCT TCC TC-3′; pcDNA-Nef, sense, 5′-ACC ATG GGT GGC AAG TGG TCA AAA CG-3′; antisense, 5′-GCA GTC TTT GTA GTA CTC CGG ATG-3′. The primers for IL-12p40 and β-actin have been described before (13Ma W. Gee K. Lim W. Chambers K. Angel J.B. Kozlowski M. Kumar A. J. Immunol. 2004; 172: 318-330Crossref PubMed Scopus (118) Google Scholar, 16Ma W. Mishra S. Gee K. Mishra J.P. Nandan D. Reiner N.E. Angel J.B. Kumar A. J. Biol. Chem. 2007; 282: 13351-13362Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). For determination of equal infectivity of monocytic cells with nef retroviruses, RT-PCR analysis was performed for the neomycin gene present in the pSRα retroviruses containing nef gene. The primer sequence for the neomycin gene is as follows: Neo: sense, 5′-AGA GGC TAT TCG GCT ATG ACT G-3′; antisense, 5′-TTC GTC CAG ATC ATC CTG ATC-3′. The conditions for PCR amplification were essentially as described above for IL-12p40 (13Ma W. Gee K. Lim W. Chambers K. Angel J.B. Kozlowski M. Kumar A. J. Immunol. 2004; 172: 318-330Crossref PubMed Scopus (118) Google Scholar, 16Ma W. Mishra S. Gee K. Mishra J.P. Nandan D. Reiner N.E. Angel J.B. Kumar A. J. Biol. Chem. 2007; 282: 13351-13362Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Calcium Influx-Calcium influx was measured as described earlier (16Ma W. Mishra S. Gee K. Mishra J.P. Nandan D. Reiner N.E. Angel J.B. Kumar A. J. Biol. Chem. 2007; 282: 13351-13362Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 37Mishra S. Mishra J.P. Gee K. McManus D.C. Lacasse E.C. Kumar A. J. Biol. Chem. 2005; 280: 37536-37546Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Briefly, cells were washed and suspended in Buffer A (RPMI 1640 containing 20 mm HEPES, pH 7.4) containing 1 mm Fluo-3/AM (Molecular Probes, Eugene, OR). The reaction was stopped by adding an equal volume of Buffer B (Buffer A containing 5% fetal calf serum, pH 7.4) followed by incubation for 15 min at 37 °C. The cells were washed, and intracellular Ca2+ levels were measured by FACScan flow cytometer (BD Biosciences) equipped with CellQuest software, version 3.2.1fl. Western Blotting-Briefly, total proteins were subjected to Western immunoblotting as described earlier (13Ma W. Gee K. Lim W. Chambers K. Angel J.B. Kozlowski M. Kumar A. J. Immunol. 2004; 172: 318-330Crossref PubMed Scopus (118) Google Scholar, 16Ma W. Mishra S. Gee K. Mishra J.P. Nandan D. Reiner N.E. Angel J.B. Kumar A. J. Biol. Chem. 2007; 282: 13351-13362Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). All immunoblots were visualized by ECL (Santa Cruz Biotechnology, Santa Cruz, CA). The densitometry analysis was preformed by CHEMIGENIUS2 XE Bio Imaging System (PerkinElmer Life Sciences). Transient Transfection with IL-12p40 Promoter-luciferase Reporter and Luciferase Assay-A series of truncated hIL-12p40 promoter fragments (–880 to +108) were generated by PCR and subcloned into the NheI/NcoI sites of pGL3B luciferase reporter plasmid as described earlier (13Ma W. Gee K. Lim W. Chambers K. Angel J.B. Kozlowski M. Kumar A. J. Immunol. 2004; 172: 318-330Crossref PubMed Scopus (118) Google Scholar, 16Ma W. Mishra S. Gee K. Mishra J.P. Nandan D. Reiner N.E. Angel J.B. Kumar A. J. Biol. Chem. 2007; 282: 13351-13362Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). To generate mutations in key transcription factor binding sites, site-directed mutagenesis was performed by PCR using mutagenic primers. All the sequences were confirmed by the Biotechnology Research Institute, University of Ottawa. THP-1 cells were transiently transfected by FuGENE6 reagent (Roche Diagnostics) as described earlier (13Ma W. Gee K. Lim W. Chambers K. Angel J.B. Kozlowski M. Kumar A. J. Immunol. 2004; 172: 318-330Crossref PubMed Scopus (118) Google Scholar, 16Ma W. Mishra S. Gee K. Mishra J.P. Nandan D. Reiner N.E. Angel J.B. Kumar A. J. Biol. Chem. 2007; 282: 13351-13362Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Briefly, THP-1 cells infected with pSRα-Nef retroviruses were transfected with 1 μgof IL-12p40 promoter/luciferase constructs and 0.5 μg of the pSV-β-galactosidase (Promega, Madison, WI) by employing FuGENE6 reagent as described earlier (13Ma W. Gee K. Lim W. Chambers K. Angel J.B. Kozlowski M. Kumar A. J. Immunol. 2004; 172: 318-330Crossref PubMed Scopus (118) Google Scholar, 16Ma W. Mishra S. Gee K. Mishra J.P. Nandan D. Reiner N.E. Angel J.B. Kumar A. J. Biol. Chem. 2007; 282: 13351-13362Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). After 24 h, cells were stimulated with LPS for another 24 h following which cells were assayed for luciferase and β-galactosidase activity by using respective assay kits (Promega). IL-12p40 promoter luciferase activity was normalized by measuring β-galactosidase values. Electrophoretic Mobility Shift Assay-Electrophoretic mobility shift assays were performed as described earlier (13Ma W. Gee K. Lim W. Chambers K. Angel J.B. Kozlowski M. Kumar A. J. Immunol. 2004; 172: 318-330Crossref PubMed Scopus (118) Google Scholar, 16Ma W. Mishra S. Gee K. Mishra J.P. Nandan D. Reiner N.E. Angel J.B. Kumar A. J. Biol. Chem. 2007; 282: 13351-13362Abstract Full Text Full Text PDF PubMed Scopus (

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