NOS2 Regulation of NF-κB by S-Nitrosylation of p65

Signal transduction in the NF-κB transcription factor pathway is inhibited by inducible nitric oxide synthase (NOS2) activity, although the molecular mechanism(s) are incompletely understood. We have previously shown that nitric oxide (NO), derived from NOS2 consequent upon cytokine stimulation, attenuates NF-κB p50-p65 heterodimer DNA binding and have identified the p50 monomer as a locus for inhibitory S-nitrosylation. We now show that the binding partner of p50, NF-κB p65, is also targeted by NO following cytokine stimulation of respiratory epithelial cells and macrophages and identify a conserved cysteine within the Rel homology domain that is the site for S-nitrosylation. S-Nitrosylation of p65 inhibits NF-κB-dependent gene transcription, and nuclear levels of S-nitrosylated p65 correlate with decreased DNA binding of the p50-p65 heterodimer. NOS2 regulates cytokine-induced S-nitrosylation of p65, resulting in decreased NF-κB binding to the NOS2 promoter, thereby inhibiting further NOS2 expression. Collectively, these findings delineate a mechanism by which NOS2 modulates NF-κB activity and regulates gene expression in inflammation. Signal transduction in the NF-κB transcription factor pathway is inhibited by inducible nitric oxide synthase (NOS2) activity, although the molecular mechanism(s) are incompletely understood. We have previously shown that nitric oxide (NO), derived from NOS2 consequent upon cytokine stimulation, attenuates NF-κB p50-p65 heterodimer DNA binding and have identified the p50 monomer as a locus for inhibitory S-nitrosylation. We now show that the binding partner of p50, NF-κB p65, is also targeted by NO following cytokine stimulation of respiratory epithelial cells and macrophages and identify a conserved cysteine within the Rel homology domain that is the site for S-nitrosylation. S-Nitrosylation of p65 inhibits NF-κB-dependent gene transcription, and nuclear levels of S-nitrosylated p65 correlate with decreased DNA binding of the p50-p65 heterodimer. NOS2 regulates cytokine-induced S-nitrosylation of p65, resulting in decreased NF-κB binding to the NOS2 promoter, thereby inhibiting further NOS2 expression. Collectively, these findings delineate a mechanism by which NOS2 modulates NF-κB activity and regulates gene expression in inflammation. The transcription factor NF-κB controls the expression of many genes involved in the inflammatory response (1Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Crossref PubMed Scopus (3353) Google Scholar). One of these genes is the inducible nitric oxide synthase (NOS2) 2The abbreviations used are: NO, nitric oxide; NOS, nitric oxide synthase; SNO, S-nitrosothiol; SNOC, S-nitrosocysteine; RHD, Rel homology domain; HEK, human embryonic kidney; LPS, lipopolysaccharide; KO, knockout; WT, wild type; ChIP, chromatin immunoprecipitation; IP, immunoprecipitate; COX-2, cyclooxygenase-2. whose activity impacts the cellular response to acute injury (2Kolios G. Valatas V. Ward S.G. Immunology. 2004; 113: 427-437Crossref PubMed Scopus (390) Google Scholar). The product of NOS2, nitric oxide (NO), is known to modulate NF-κB activity at multiple steps in the signal transduction pathway (3Marshall H.E. Merchant K. Stamler J.S. FASEB J. 2000; 14: 1889-1900Crossref PubMed Scopus (375) Google Scholar). The primary molecular mechanism by which NO alters NF-κB signaling is via S-nitrosylation, with several different NF-κB proteins including IκB kinase β and p50 regulated by this post-translational modification (4Marshall H.E. Stamler J.S. Biochemistry. 2001; 40: 1688-1693Crossref PubMed Scopus (337) Google Scholar, 5Reynaert N.L. Ckless K. Korn S.H. Vos N. Guala A.S. Wouters E.F.M. van der Vliet A. Janssen-Heininger Y.M.W. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8945-8950Crossref PubMed Scopus (319) Google Scholar). Particularly, we have shown that p50 is S-nitrosylated under conditions of nitrosative stress and is associated with a decrease in NF-κB (p50-p65) DNA binding (4Marshall H.E. Stamler J.S. Biochemistry. 2001; 40: 1688-1693Crossref PubMed Scopus (337) Google Scholar). However, the physiological significance of S-nitrosylation of the NF-κB p50-p65 heterodimer in the context of cytokine signaling and cellular NOS2 expression has not been established. NOS2 expression is dependent upon NF-κB activation, with the cytokine-responsive κB-binding site(s) identified in both the human and the murine NOS2 promoters (6Taylor 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 (370) Google Scholar, 7Xie Q.W. Kashiwabara Y. Nathan C. J. Biol. Chem. 1994; 269: 4705-4708Abstract Full Text PDF PubMed Google Scholar). Cytokine-stimulated NOS2 activity, in turn, inhibits NF-κB-dependent transcription, but the specific molecular target(s) of NOS2 in the NF-κB pathway have not been elucidated (8Connelly L. Palacios-Callender M. Ameixa C. Moncada S. Hobbs A.J. J. Immunol. 2001; 166: 3873-3881Crossref PubMed Scopus (290) Google Scholar). We have previously demonstrated that cytokine-induced NOS activity inhibits NF-κB DNA binding in a reversible manner, a mechanism consistent with S-nitrosylation of the p50-p65 heterodimer (4Marshall H.E. Stamler J.S. Biochemistry. 2001; 40: 1688-1693Crossref PubMed Scopus (337) Google Scholar). Moreover, evidence accumulated recently suggests a central role of S-nitrosylation by NOS2 in the regulation of inflammatory mediators (9Kim S.F. Huri D.A. Snyder S.H. Science. 2005; 310: 1966-1970Crossref PubMed Scopus (452) Google Scholar, 10Park H.-S. Huh S.-H. Kim M.-S. Lee S.H. Choi E.-J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14382-14387Crossref PubMed Scopus (220) Google Scholar). In the past, the p50 monomer was felt to be the probable target for NOS2-mediated S-nitrosylation of the p50-p65 heterodimer. This rationale was based on the initial identification of a single redox-sensitive cysteine (Cys-62) located in the DNA-binding region of p50 (11Matthews J.R. Wakasugi N. Virelizier J.L. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Crossref PubMed Scopus (729) Google Scholar). Interestingly, this cysteine is not only conserved throughout all Rel family members but is also found within a canonical SNO motif (12Hess D.T. Matsumoto A. Kim S.-O. Marshall H.E. Stamler J.S. Nat. Rev. Mol. Cell. Biol. 2005; 6: 150-166Crossref PubMed Scopus (1742) Google Scholar), suggesting the possibility of a common NO-responsive site that could function to universally regulate NF-κB Rel protein-DNA binding (Fig. 1). We now show that the binding partner of p50, p65 (or RelA), is S-nitrosylated in cytokine-stimulated respiratory epithelium and macrophages and identify this conserved cysteine (Cys-38) within the DNA-binding site of the Rel homology domain (RHD) as the site of S-nitrosylation. S-Nitrosylation of p65 is dependent upon NOS2 activity, and nuclear SNO-p65 levels are inversely correlated with NF-κB p50-p65 DNA binding and NF-κB-dependent transcription. These results suggest a pathway by which NOS2 might coordinate the inflammatory response by regulating the NF-κB-dependent transcription of critical response mediators. Cell Lines and Plasmids—HEK 293 (CRL-1573), RAW 264.7 (TIB-71), and A549 (CCL-185) cells were grown in their ATCC-designated media supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. All cultures were maintained in 95% air, 5% CO2 at 37 °C. Peritoneal macrophages were harvested from 8–12-week-old male C57BL/6 (wild type (WT)) or NOS2 knock-out (KO) (B6.129P2-Nos2tm1Lau; Jackson Laboratory, Bar Harbor, ME) mice 72 h after intraperitoneal instillation of thioglycollate broth. The peritoneum was lavaged with ice-cold sterile phosphate-buffered saline, cells were collected by centrifugation at 500 × g, and the cell pellet was resuspended in RPMI 1640 media supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Approximately 1 × 107 cells were plated for use in the biotin switch assay. The p65 wild-type (p65wt) expression plasmid was constructed by inserting full-length human p65 cDNA (a gift from Dr. Albert Baldwin, University of North Carolina) into pDNR-CMV (Clontech) using a BamH1 restriction site. The plasmid expressing p65 with a cysteine → serine mutation at amino acid 38 (p65C38ΔS) was created utilizing a site-directed mutagenesis kit (QuikChange, Stratagene, La Jolla, CA). The pNFκB-Luc and pRL-CMV (Renilla luciferase) plasmids were purchased from Stratagene and Promega (Madison, WI), respectively. Cell Lysates—Preparation of A549 and RAW 264.7 cytoplasmic and nuclear extracts was done as outlined previously (13Marshall H.E. Stamler J.S. J. Biol. Chem. 2002; 277: 34223-34228Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Whole cell lysates of HEK 293 cells were prepared by resuspending the harvested cells in 1 volume of cold lysis buffer (50 mm HEPES pH 7.9, 150 mm NaCl, 1% Nonidet P-40, 0.5 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride), placing on ice for 20 min, and pelleting debris by centrifugation at 14,000 × g for 20 min. The protein concentration of the extracts was determined using the BCA method (Pierce Biotechnology), and extracts were used immediately or stored at -80 °C. NF-κB Reporter Assay—HEK 293 cells were grown to ∼75% confluence in 6-well plates. Cells were transfected with 1 μg of pNFκB-Luc and 0.5 μg of pRL-CMV ± 5 μg of p65wt or p65C38ΔS expression plasmids using Lipofectamine 2000 (Invitrogen). Cells were grown for 24 h after transfection prior to the indicated treatments. Luciferase activity was quantitated on a TD 20/20 luminometer using a Dual-Luciferase kit (Promega). NF-κB DNA Binding Assays—Nuclear protein binding to a consensus NF-κB oligonucleotide was determined using an enzyme-linked immunosorbent assay-based kit (TransAm p65, Active Motif, Carlsbad, CA). Absorbance was read at 450 nm with samples appropriately blanked. A commercially available kit was utilized for the NF-κB chromatin immunoprecipitation (ChIP) assay (Upstate Biotechnology, Charlottesville, VA). A rabbit antibody directed against NF-κB p65 (C-20, Santa Cruz Biotechnology, Santa Cruz, CA) was used for immunoprecipitation. DNA input was quantified using a 10-fold dilution of the cell lysate. DNA was purified from the NF-κB ChIP eluates as well as input lysate using phenol-chloroform extraction followed by ethanol precipitation. The purified DNA was resuspended in Tris-EDTA and subjected to PCR using primers flanking the cytokine-responsive κB site in the human NOS2 promoter (forward primer, 5′-GGGCTTATGTGGCCTAACCAA-3, and reverse primer, 5′-CCACCAGGGAACTTGAAAAA-3′) and mouse NOS2 promoter (forward primer, 5′-ACACGAGGCTGAGCTGACTT-3, and reverse primer, 5′-CATTCACACATGGCATGGA-3′). PCR products were separated on a 2% agarose gel and visualized by ethidium bromide. Reverse Transcription-PCR—Total RNA was extracted from cells using the RNeasy Kit (Qiagen, Valencia, CA). Residual genomic DNA was removed by treatment with RNase-free DNase I (Invitrogen). RNA was reverse-transcribed using the High Capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). Real-time PCR amplification of the cDNA was performed using NOS2 (forward, 5′-ACCTTGTTCAGCTACGCCTT-3′; reverse, 5′-CATTCCCAAATGTGCTTGTC-3′) or β-actin (forward, 5′-TCAAGATCATTGCTCCTCCTG-3′; reverse, 5′-CTGCTTGCTGATCCACATCTG-3′) primers and SYBR green PCR mix (iQ SYBR Green supermix, Bio-Rad Laboratories). Samples were amplified using a PCR thermocycler (iCycler, Bio-Rad), and the single color real-time PCR detection system (MyIQ, Bio-Rad). Using β-actin as a reference gene, changes in NOS2 mRNA levels between samples were determined. S-Nitrosothiol Detection Assay—A biotin switch assay for S-nitrosothiol detection was performed as described previously (14Jaffrey S.R. Erdjument-Bromage H. Ferris C.D. Tempst P. Snyder S.H. Nat. Cell Biol. 2001; 3: 193-197Crossref PubMed Scopus (1228) Google Scholar) with minor modifications. All steps were done with minimal light exposure. 100–250 μg of protein lysate was diluted in HEN buffer (250 mm HEPES-NaOH, pH 7.7, 1 mm EDTA, 0.1 mm neocuproine, 1% SDS) with free thiols blocked by the addition of 3 mm methyl methane thiosulfonate followed by heating to 50 °C for 30 min. S-Nitrosothiols were labeled by the addition of 5 mm ascorbic acid and N-(6-(Biotinamido)hexyl)-3′-(2′-pyridyldithio)propionamide (EZ-Link Biotin-HPDP, Pierce Biotechnology). Negative controls were prepared by the omission of ascorbic acid. Biotinylated proteins were isolated by incubating overnight (4 °C) with NeutrAvidin agarose beads (Pierce Biotechnology). After extensive washing, proteins were eluted from the beads by heating to 95 °C in Laemmli buffer. Proteins recovered by the biotin switch assay and the input protein lysate were separated by SDS-PAGE and transferred to nitrocellulose, and blots were probed with a rabbit polyclonal antibody to NF-κB p65 (C-20). Immunoreactivity was visualized by enhanced chemiluminescence. Detection of S-nitrosothiols in p65 immunoprecipitates (IP) by mercury-coupled, photolysis chemiluminescence was performed using a previously described method with minor variations (4Marshall H.E. Stamler J.S. Biochemistry. 2001; 40: 1688-1693Crossref PubMed Scopus (337) Google Scholar). 500 μg of protein lysate was incubated with an antibody to NF-κB p65 (C-20) for 4 h at 4 °C followed by extraction of the immunocomplexes using a commercial kit (Seize IP, Pierce Biotechnology). The IP eluates were kept on ice with limited light exposure prior to analysis of S-nitrosothiols by photolysis chemiluminescence. S-Nitrosylation of NF-κB p65—To ascertain whether the NF-κB p65 protein can be modified by S-nitrosylation in situ, we and HEK 293 cells with 500 for 1 h followed by of S-nitrosylated p65 levels in cell lysates using the biotin switch assay. SNO-p65 was by in 293 cells only after treatment (Fig. the HEK 293 cells p65wt biotin at the possibility of S-nitrosylation, but a in SNO-p65 was after In the of minimal biotin of p65 was in the the of the biotin switch method for that NF-κB p65 cellular S-nitrosylation was by mercury-coupled, photolysis chemiluminescence of SNO in p65 IP prepared from and HEK 293 SNO was in the p65 IP prepared from HEK 293 cells only after treatment (Fig. In the SNO levels in the p65 IP after further that NF-κB p65 is a target for S-nitrosylation. S-Nitrosylation of NF-κB p65 on has that the NF-κB p50 monomer S-nitrosylation at cysteine in the DNA-binding region of the J.R. M. Morris Hay R.T. Nucleic Acids Res. PubMed Scopus Google Scholar). this cysteine is conserved in the p65 monomer (Fig. we whether this is the target for S-nitrosylation in Using site-directed we created a p65 expression plasmid for a cysteine → serine at amino acid 38 We quantified SNO-p65 levels by biotin switch in and HEK 293 cells with or treatment a in SNO-p65 levels was in the and cells after in biotin was in the cells after These results that cysteine 38 is the site of S-nitrosylation in the NF-κB p65 protein (Fig. In addition to the DNA-binding NF-κB p65 also a domain that to NF-κB-dependent transcription are to target DNA H. M. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). whether S-nitrosylation of p65 results in a decrease in NF-κB-dependent gene transcription, we performed an NF-κB assay in HEK 293 cells the p65wt or the p65C38ΔS both p65wt and p65C38ΔS expression in an in NF-κB-dependent activity treatment to a decrease in NF-κB activity only in the cells activity (Fig. These results are consistent with the prior that a serine at the conserved cysteine in the for Rel protein DNA binding but a of binding H. M. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). S-nitrosylation at in the p65 protein to be a molecular for NO of NF-κB-dependent gene transcription. S-Nitrosylation of NF-κB p65 in Cytokine-stimulated have previously demonstrated of NF-κB (p50-p65) DNA binding in cytokine-stimulated A549 and RAW 264.7 consistent with S-nitrosylation of the p50-p65 heterodimer (4Marshall H.E. Stamler J.S. Biochemistry. 2001; 40: 1688-1693Crossref PubMed Scopus (337) Google Scholar, H.E. Stamler J.S. J. Biol. Chem. 2002; 277: 34223-34228Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Using the biotin switch we now the of S-nitrosylated NF-κB p65 in both the the of cytokine-stimulated A549 and RAW 264.7 In p65 is in the to with a of S-nitrosylation in the A549 cells (Fig. cytokine stimulation, NF-κB p65 nuclear associated with an in NF-κB p65 DNA binding (Fig. However, at h after stimulation, both the cytoplasmic and the nuclear p65 show S-nitrosylation in with the of cellular NOS2 expression (Fig. The in nuclear SNO-p65 at h after stimulation with a decrease in NF-κB DNA binding with 1 h after in the of nuclear p65, that cytoplasmic of NF-κB is not (Fig. S-nitrosylation of NF-κB p65 within the to be a mechanism by which NF-κB DNA binding is regulated in cytokine-stimulated NOS2 S-Nitrosylation of p65 and NF-κB DNA prior have shown that NOS2 activity inhibits NF-κB signaling in cytokine-stimulated cells by a mechanism consistent with S-nitrosylation of p50-p65 (4Marshall H.E. Stamler J.S. Biochemistry. 2001; 40: 1688-1693Crossref PubMed Scopus (337) Google Scholar). These with the that cytokine-induced NOS2 expression in A549 and RAW 264.7 cells with a in nuclear SNO-p65 levels (Fig. to whether SNO-p65 in these cells is Cells were with with or the addition of the A decrease in nuclear SNO-p65 was in both cell with at h after stimulation, that NOS2 activity SNO-p65 in cytokine-stimulated cells (Fig. whether NOS2 in cytokine-stimulated cells to a decrease in NF-κB binding to target promoter we utilized an NF-κB ChIP assay. We that cytokine stimulation of A549 and RAW 264.7 cells results in an in NF-κB binding to the cytokine-responsive NF-κB in the NOS2 promoter (Fig. the cytokine-stimulated cells are with NF-κB DNA binding to these κB promoter is further Moreover, the in NF-κB binding to the NOS2 promoter by NOS2 into cellular NOS2 mRNA transcription and protein expression (Fig. and further that NOS2 regulates SNO-p65 we quantified SNO-p65 levels in cell lysates prepared from macrophages that were harvested from or NOS2 SNO-p65 levels were in the NOS2 macrophages with both at and after cytokine stimulation, an in p65 expression in the NOS2 macrophages (Fig. The decrease in SNO-p65 in the NOS2 macrophages also correlated with an in NF-κB DNA binding (Fig. Collectively, these that NOS2 to S-nitrosylation of NF-κB p50-p65 in the The the NF-κB p65 monomer as a target of S-nitrosylation by NOS2 and the nuclear of SNO-p65 with of a of that are regulated by NOS2 S-nitrosylation. 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Stamler J.S. 2001; PubMed Scopus Google a role in the of cytoplasmic NF-κB p65 and IκB kinase β that is upon cytokine stimulation of A549 (Fig. and cells N.L. Ckless K. Korn S.H. Vos N. Guala A.S. Wouters E.F.M. van der Vliet A. Janssen-Heininger Y.M.W. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8945-8950Crossref PubMed Scopus (319) Google Scholar), respectively. However, NOS2 is to the S-nitrosylation of NF-κB proteins as is only in the cell after cytokine NOS in proteins for S-nitrosylation. NOS2 to in cytokine-stimulated macrophages to S-nitrosylation (9Kim S.F. Huri D.A. Snyder S.H. Science. 2005; 310: 1966-1970Crossref PubMed Scopus (452) Google Scholar). which is inhibited by S-nitrosylation, also to NOS2, in this in a A. L. Stamler J.S. Science. PubMed Scopus Google Scholar). and are regulated by S-nitrosylation in the context of protein with and G. K. Stamler J.S. Y. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Kim S.F. V. Snyder S.H. 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