Critical Role of the Automodification of Poly(ADP-ribose) Polymerase-1 in Nuclear Factor-κB-dependent Gene Expression in Primary Cultured Mouse Glial Cells
2004; Elsevier BV; Volume: 279; Issue: 41 Linguagem: Inglês
10.1074/jbc.m407923200
ISSN1083-351X
AutoresHidemitsu Nakajima, Hiroshi Nagaso, Nobukazu Kakui, Midori Ishikawa, Toyokazu Hiranuma, Shigeru Hoshiko,
Tópico(s)Cell death mechanisms and regulation
ResumoSynthesis of ADP-ribose polymers catalyzed by poly-(ADP-ribose) polymerase-1 (PARP-1) has been implicated in transcriptional regulation. Recent studies with PARP-1 null mice and PARP-1 inhibitors have also demonstrated that PARP-1 has an essential role in nuclear factor-κB (NF-κB)-dependent gene expression induced by various inflammatory stimuli. In this study, we used primary cultured mouse glial cells to investigate the role of poly(ADP-ribosyl)ation by PARP-1 in NF-κB-dependent gene expression. PARP-1 inhibitors and the antisense RNA for PARP-1 mRNA suppressed lipopolysaccharide (LPS)-induced expression of tumor necrosis factor-α and inducible nitric-oxide synthase, suggesting that PARP-1 activity has a critical role in synthesis. Western blotting with anti-poly(ADP-ribose) antibody revealed that PARP-1 itself was mainly poly(ADP-ribosyl)ated in glial cells, i.e. automodified PARP-1 (AM-PARP). The amounts of AM-PARP were not affected by LPS treatment, but were decreased by PARP-1 inhibitors. Electrophoretic mobility shift assay revealed that PARP-1 inhibitors and the antisense RNA for PARP-1 mRNA reduced the LPS-induced DNA binding of NF-κB. Non-modified PARP-1 also reduced the DNA binding of NF-κB via its physical association with NF-κB, whereas AM-PARP had no effect. On the other hand, enhancement of the automodification of PARP-1 by the addition of NAD+, its substrate, promoted the DNA binding of NF-κB. Furthermore, in in vitro transcription assay, the addition of AM-PARP or NAD+ to nuclear extracts promoted NF-κB p50-dependent transcription. These results indicate that automodification of PARP-1 positively up-regulates formation of the NF-κB·DNA complex and enhances transcriptional activation. Therefore, AM-PARP may be critical for the NF-κB-dependent gene expression of some inflammatory mediators in glial cells. Synthesis of ADP-ribose polymers catalyzed by poly-(ADP-ribose) polymerase-1 (PARP-1) has been implicated in transcriptional regulation. Recent studies with PARP-1 null mice and PARP-1 inhibitors have also demonstrated that PARP-1 has an essential role in nuclear factor-κB (NF-κB)-dependent gene expression induced by various inflammatory stimuli. In this study, we used primary cultured mouse glial cells to investigate the role of poly(ADP-ribosyl)ation by PARP-1 in NF-κB-dependent gene expression. PARP-1 inhibitors and the antisense RNA for PARP-1 mRNA suppressed lipopolysaccharide (LPS)-induced expression of tumor necrosis factor-α and inducible nitric-oxide synthase, suggesting that PARP-1 activity has a critical role in synthesis. Western blotting with anti-poly(ADP-ribose) antibody revealed that PARP-1 itself was mainly poly(ADP-ribosyl)ated in glial cells, i.e. automodified PARP-1 (AM-PARP). The amounts of AM-PARP were not affected by LPS treatment, but were decreased by PARP-1 inhibitors. Electrophoretic mobility shift assay revealed that PARP-1 inhibitors and the antisense RNA for PARP-1 mRNA reduced the LPS-induced DNA binding of NF-κB. Non-modified PARP-1 also reduced the DNA binding of NF-κB via its physical association with NF-κB, whereas AM-PARP had no effect. On the other hand, enhancement of the automodification of PARP-1 by the addition of NAD+, its substrate, promoted the DNA binding of NF-κB. Furthermore, in in vitro transcription assay, the addition of AM-PARP or NAD+ to nuclear extracts promoted NF-κB p50-dependent transcription. These results indicate that automodification of PARP-1 positively up-regulates formation of the NF-κB·DNA complex and enhances transcriptional activation. Therefore, AM-PARP may be critical for the NF-κB-dependent gene expression of some inflammatory mediators in glial cells. Poly(ADP-ribose) polymerase-1 (PARP-1 1The abbreviations used are: PARP-1, poly(ADP-ribose) polymerase-1; PAR, poly(ADP-ribose); NF-κB, nuclear factor-κB; TNF-α, tumor necrosis factor-α; IL, interleukin; LPS, lipopolysaccharide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mAb, monoclonal antibody; pAb, polyclonal antibody; iNOS, inducible nitric-oxide synthase; AM-PARP, automodified PARP-1; NM-PARP, non-modified PARP-1; EMSA, electrophoretic mobility shift assay. 1The abbreviations used are: PARP-1, poly(ADP-ribose) polymerase-1; PAR, poly(ADP-ribose); NF-κB, nuclear factor-κB; TNF-α, tumor necrosis factor-α; IL, interleukin; LPS, lipopolysaccharide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mAb, monoclonal antibody; pAb, polyclonal antibody; iNOS, inducible nitric-oxide synthase; AM-PARP, automodified PARP-1; NM-PARP, non-modified PARP-1; EMSA, electrophoretic mobility shift assay.; EC 2.4.2.30) is an abundant nuclear protein that is activated by DNA strand breakage and that catalyzes the covalent attachment of poly-(ADP-ribose) (PAR) from NAD+ to numerous nuclear proteins and transcription factors, including histones; DNA polymerase α and β; p53; and PARP-1, itself being the major target, via its automodification domain (1de Murcia G. de Murcia J.M. Trends Biochem. Sci. 1994; 19: 172-176Abstract Full Text PDF PubMed Scopus (761) Google Scholar, 2D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar). Besides PARP-1, another six PARPs have been identified: short PARP, PARP-2, PARP-3, tankylase-1/2, and vault PARP (2D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar, 3Chiarugi A. Trends Pharmacol. Sci. 2002; 23: 122-129Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). However, the physiological roles of poly(ADP-ribosyl)ation of nuclear proteins and transcription factors induced by PARPs are not completely understood. The initially identified subtype of the enzyme, PARP-1, has been thought to play a central role in the process of poly(ADP-ribosyl)ation because poly(ADP-ribosyl)ation is markedly reduced in most tissues of PARP-1 null mice (4Shieh W.M. Ame J.C. Wilson M.V. Wang Z.-Q. Koh D.W. Jacobson M.K. Jacobson E.L. J. Biol. Chem. 1998; 273: 30069-30072Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Transient poly(ADP-ribosyl)ation by PARP-1 can be induced by a wide variety of environmental stimuli, including reactive oxygen, ionizing radiation, and genotoxic stress (1de Murcia G. de Murcia J.M. Trends Biochem. Sci. 1994; 19: 172-176Abstract Full Text PDF PubMed Scopus (761) Google Scholar, 2D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar). Thus, PARP-1 has been suggested to regulate DNA repair (5Dantzer F. Schreiber V. Niedergang C. Trucco C. Flatter E. de la Rubia G. Oliver J. Rolli V. de Murcia J.M. de Murcia G. Biochimie (Paris). 1999; 81: 69-75Crossref PubMed Scopus (304) Google Scholar). On the other hand, overactivation of PARP-1 by massively damaged DNA consumes NAD+ and consequently ATP, resulting in necrotic cell death by energy failure (3Chiarugi A. Trends Pharmacol. Sci. 2002; 23: 122-129Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 6Pieper A.A. Verma A. Zhang J. Snyder S.H. Trends Pharmacol. Sci. 1999; 20: 171-181Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). There are many reports suggesting that PARP-1 is also involved in regulation of gene expression at the transcriptional step (2D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar, 3Chiarugi A. Trends Pharmacol. Sci. 2002; 23: 122-129Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). PARP-1 seems to play dual roles in transcription. Poly(ADP-ribosyl)ation of transcription factors such as Yin-Yang 1 (7Oei S.L. Shi Y. Biochem. Biophys. Res. Commun. 2001; 285: 27-31Crossref PubMed Scopus (67) Google Scholar), RNA polymerase II-associated factors (8Oei S.L. Griesenbeck J. Ziegler M. Schweiger M. Biochemistry. 1998; 37: 1465-1469Crossref PubMed Scopus (57) Google Scholar), and p53 (9Mendoza-Alvarez H. Alvarez-Gonzalez R. J. Biol. Chem. 2001; 276: 36425-36430Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) results in reversible silencing of transcription by impairing the DNA binding of these proteins. In other instances, PARP-1 was found to have only one function, stimulating the DNA binding activity of transcription factors such as Oct-1 (10Nie J. Sakamoto S. Song D. Qu Z. Ota K. Taniguchi T. FEBS Lett. 1998; 424: 27-32Crossref PubMed Scopus (84) Google Scholar) and B-Myb (11Cervellera M.N. Sala A. J. Biol. Chem. 2000; 275: 10692-10696Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Recent reports have also shown that PARP-1 is required for specific nuclear factor-κB (NF-κB)-dependent gene expression and acts as a coactivator for NF-κB in vitro (13Oliver F.J. de Murcia J.M. Nacci C. Decker P. Andriantsitohaina R. Muller S. de la Rubia G. Stoclet J.C. de Murcia G. EMBO J. 1999; 18: 4446-4454Crossref PubMed Scopus (535) Google Scholar, 14Hassa P.O. Covic M. Hasan S. Imhof R. Hottiger M.O. J. Biol. Chem. 2001; 276: 45588-45597Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). Indeed, the NF-κB-dependent transcription of some inflammatory mediators in response to endotoxin (13Oliver F.J. de Murcia J.M. Nacci C. Decker P. Andriantsitohaina R. Muller S. de la Rubia G. Stoclet J.C. de Murcia G. EMBO J. 1999; 18: 4446-4454Crossref PubMed Scopus (535) Google Scholar) or pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) (12Hassa P.O. Hottiger M.O. Biol. Chem. 1999; 380: 953-959Crossref PubMed Scopus (263) Google Scholar, 13Oliver F.J. de Murcia J.M. Nacci C. Decker P. Andriantsitohaina R. Muller S. de la Rubia G. Stoclet J.C. de Murcia G. EMBO J. 1999; 18: 4446-4454Crossref PubMed Scopus (535) Google Scholar, 14Hassa P.O. Covic M. Hasan S. Imhof R. Hottiger M.O. J. Biol. Chem. 2001; 276: 45588-45597Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar) is almost completely abrogated in PARP-1 null mice. Thus, anti-inflammatory effects of PARP-1 inhibitors have been extensively discussed in relation to various inflammation-related diseases (15Hassa P.O. Hottiger M.O. CMLS Cell. Mol. Life Sci. 2002; 59: 1534-1553Crossref PubMed Scopus (345) Google Scholar, 16Hasko G. Mabley J.G. Nemeth Z.H. Pacher P. Deitch E.A. Szabo C. Mol. Med. (N. Y.). 2002; 8: 283-289Crossref PubMed Google Scholar). However, the exact biochemical mechanism by which PARP-1 regulates NF-κB-dependent transcription is obscure. To date, some groups have reported that the enzyme activity of PARP-1 might directly influence NF-κB-dependent transcription. Kameoka et al. (17Kameoka M. Ota K. Tetsuka T. Tanaka Y. Itaya A. Okamoto T. Yoshihara K. Biochem. J. 2000; 346: 641-649Crossref PubMed Scopus (105) Google Scholar) showed that poly(ADP-ribosyl)ation markedly suppresses the DNA binding activity of NF-κB via direct modification in vitro. Chang and Alvarez-Gonzalez (18Chang W.J. Alvarez-Gonzalez R. J. Biol. Chem. 2001; 276: 47664-47670Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) demonstrated that the DNA binding activity of NF-κB p50 is NAD+-dependent and reversibly regulated by the automodification of PARP-1 under cell-free conditions. In contrast, Hassa et al. (14Hassa P.O. Covic M. Hasan S. Imhof R. Hottiger M.O. J. Biol. Chem. 2001; 276: 45588-45597Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar) concluded that neither the enzyme activity nor the DNA binding activity of PARP-1 is required for NF-κB-dependent transcription. Thus, there are contradictory results (15Hassa P.O. Hottiger M.O. CMLS Cell. Mol. Life Sci. 2002; 59: 1534-1553Crossref PubMed Scopus (345) Google Scholar). To further investigate the role of poly(ADP-ribosyl)ation by PARP-1 in NF-κB-dependent transcription in inflammation, we examined the effects of several PARP-1 inhibitors and the antisense RNA for PARP-1 mRNA on lipopolysaccharide (LPS)-induced expression of some inflammatory mediators in primary cultured mouse glial cells. Cultured glial cells, composed of macroglia (astrocytes and oligodendrocytes) and microglia, were found to be a good in vitro model for neuro-inflammatory diseases such as stroke (19Eliasson M.J. Sampei K. Mandir A.S. Hurn P.D. Traystman R.J. Bao J. Pieper A. Wang Z.-Q. Dawson T.M. Snyder S.H. Dawson V.L. Nat. Med. 1997; 3: 1089-1095Crossref PubMed Scopus (943) Google Scholar) and Parkinson's disease (20Mandir A.S. Przedborski S. Jackson-Lewis V. Wang Z.-Q. Simbulan-Rosenthal C.M. Smulson M.E. Hoffman B.E. Guastella D.B. Dawson V.L. Dawson T.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5774-5779Crossref PubMed Scopus (347) Google Scholar), which are implicated in the activation of PARP-1. Although these previous reports have focused on the activation of neuronal PARP-1, the pathophysiological significance of poly(ADP-ribosyl)ation catalyzed by PARP-1 in glial cells remains unclear. Here, we report that poly(ADP-ribosyl)ated PARP-1 itself, i.e. automodified PARP-1, has a critical role in NF-κB-dependent transcription and gene expression in glial cells. Isotopes—[adenine-2,8-3H]NAD+, [adenylate-32P]NAD+, [α-32P]GTP, and [γ-32P]ATP were purchased from PerkinElmer Life Sciences. Antibodies—The following antibodies were obtained from the indicated commercial sources: mouse anti-GAPDH monoclonal antibody (mAb) (MAB374, Chemicon International, Inc., Temecula, CA); mouse anti-PARP-1 mAb (SA-250) and rabbit anti-PAR polyclonal antibody (pAb) (SA-276) (BIOMOL Research Labs Inc., Plymouth Meeting, PA); rabbit anti-PARP-1 pAb (Roche Applied Science, Mannheim, Germany); mouse anti-PAR mAb (4335-MC) and guinea pig anti-PAR pAb (4336-PC) (Trevigen, Gaithersburg, MD); mouse anti-PAR mAb (10H, Alexis Biochemicals, Lausen, Switzerland); mouse anti-NF-κB p50 mAb (sc-8414), rabbit anti-p50 pAb (sc-7178), and rabbit anti-NF-κB p65 pAb (sc-109) (Santa Cruz Biotechnology, Santa Cruz, CA); and mouse anti-inducible nitric-oxide synthetase (iNOS) mAb (Transduction Laboratories, Lexington, KY). Other Materials—The following reagents were obtained from the indicated commercial sources: benzamide and nicotinamide (Nacalai Tesque, Kyoto, Japan); 3-aminobenzamide (Sigma); 1,5-dihydroxyisoquinoline (RBI, Natick, MA); pcDNA3, Dulbecco's modified Eagle's medium, and SuperScript II RNase H– reverse transcriptase (Invitrogen); cytokine enzyme-linked immunosorbent assay kits, protein A and G-Sepharose, and the enhanced chemiluminescence ECL detection system (Amersham Biosciences); the gel shift assay kit, the HeLaScribe® nuclear extract in vitro transcription system, the ϕX174 DNA/HinfI dephosphorylation marker, and the human recombinant p50 subunit of NF-κB (Promega, Madison, WI); LPS (Escherichia coli 0111:B4) (Difco); and Effectene transfection reagent and the RNeasy mini-kit (QIAGEN Japan, Tokyo, Japan). All other materials we used were of analytical grade. Cell Culture and Transfection—Primary mixed glial cultures were prepared from neonatal BALB/c mice (1–2 days old) as described previously (21Kong L.-Y. Lai C. Wilson B.C. Simpson J.N. Hong J.-S. Neurochem. Int. 1997; 30: 491-497Crossref PubMed Scopus (44) Google Scholar) with some modifications. Cells harvested by centrifugation at 800 × g for 5 min were resuspended; grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (JRH Biosciences, Lenexa, KS), 100 units/ml penicillin, and 100 μg/ml streptomycin; and seeded in 6- or 24-well culture plates. The cells were incubated at 37 °C in a humidified atmosphere of 5% CO2. The medium was changed twice each week. The cultures were used at 14–18 days after plating. Immunohistochemical analysis indicated that the cultures consisted of 72% astrocytes and 22% microglia (data not shown). For transfection of the antisense RNA expression vector for PARP-1 mRNA, microglial cells were mechanically isolated from the primary mixed glial culture as described previously (22Nakajima K. Hamanoue M. Shimojo M. Takei N. Kohsaka S. Biomed. Res. 1989; 10: 411-423Crossref Scopus (0) Google Scholar) and then seeded in a 24-well plate at 1–2 × 105 cells/well. The purity of the cultures was >95% as estimated by morphological criteria and by their reactivity toward MRC-OX42 (CD11b, a maker of microglia). A 3.0-kb full-length mouse PARP-1 cDNA was cloned in an antisense orientation in the expression vector pcDNA3 as described previously (23Simbulan-Rosenthal C.M. Rosenthal D.S. Iyer S. Boulares A.H. Smulson M.E. J. Biol. Chem. 1998; 273: 13703-13712Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). The resulting antisense or mock vector (pcDNA3) was transfected into microglia (1 h after plating) using the Effectene transfection reagent for 24 h according to the manufacturer's protocol. The efficacy of transfection was assessed by PARP catalytic activity (24Tanuma S. Kanai Y. J. Biol. Chem. 1982; 257: 6565-6570Abstract Full Text PDF PubMed Google Scholar). Treatment of the cells with the antisense vector down-regulated the resulting PARP activity to 62% versus the control; efficacy was calculated as 38% of the total cells (n = 4) (data not shown). Assessment of Cell Viability—The viability of glial cells was assessed by Alamar Blue™ (BIOSOURCE) according to the manufacturer's protocol. Measurement of Cytokines and Nitrite—The culture supernatants were collected at the indicated times, and the levels of TNF-α, IL-1β, and IL-6 were measured using the respective mouse enzyme-linked immunosorbent assay kits according to the manufacturer's protocol. The detection limit for assays was 50 pg/ml. The nitrite concentration was measured with a standard Griess reaction adapted to microplates as described previously (25Kong L.-Y. McMillian M.K. Maronpot R. Hong J.-S. Brain Res. 1997; 729: 102-109Crossref Scopus (63) Google Scholar). The sensitivity of this assay was ∼0.5 μm. Western Blotting—For sample preparation, the attached cells were washed twice with ice-cold phosphate-buffered saline, solubilized in lysis buffer (50 mm Tris-HCl, pH 7.2, 1 mm EDTA, 150 mm NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin), and sonicated on ice for 10 s. After centrifugation at 15,000 × g for 10 min at 4 °C, the cleared supernatants were obtained; an equal volume of SDS sample buffer (0.25 m Tris-HCl, pH 6.8, 2% SDS, 10% 2-mercaptoethanol, 30% glycerol, and 0.01% bromphenol blue) was added; and the sample was heated at 100 °C for 5 min. Protein concentrations of the samples were determined by the Bradford assay (Bio-Rad). Protein samples were separated by 5–20% SDS-PAGE (DRC, Tokyo) and transferred to Immobilon P membranes (Millipore, Tokyo). The membranes were incubated for 1 h with 5% bovine serum albumin in phosphate-buffered saline containing 0.05% Tween 20 and 0.02% NaN3 to block nonspecific binding and then incubated overnight at 4 °C with anti-PAR pAb (1:500), anti-PAR mAb (1:1000), anti-PARP-1 mAb (1:5000), or anti-iNOS mAb (1:5000). Subsequently, the membranes were incubated with an affinity-purified peroxidase-conjugated secondary antibody (Zymed Laboratories Inc.). PAR-bound proteins, PARP-1, and iNOS were detected using the ECL detection system according to the manufacturer's protocol. The membranes were also reprobed with anti-GAPDH mAb (1:500). These band intensities were quantified with NIH Image Version 1.61. Measurement of Cellular PARP-1 Activity—Measurement of cellular PARP-1 activity was performed as described previously (26Szabo C. Cuzzocrea S. Zingarelli B. O'Connor M. Salzman A.L. J. Clin. Investig. 1997; 100: 723-735Crossref PubMed Scopus (353) Google Scholar). Briefly, cells were washed twice with ice-cold phosphate-buffered saline; resuspended in 0.5 ml of assay buffer containing 56 mm Hepes-KOH, pH 7.5, 28 mm KCl, 28 mm NaCl, 2 mm MgCl2, 30 μm digitonin, 125 μm NAD+, and 18 kBq/ml [adenine-2,8-3H]; and incubated for 10 min at 37 °C. After incubation, ice-cold 20% trichloroacetic acid was added, and the samples were further incubated for 30 min at 4 °C. The samples were then washed twice with ice-cold 10% trichloroacetic acid and solubilized overnight in 2% SDS and 0.1 n NaOH at 37 °C. The radioactivity of each sample was measured in a liquid scintillation counter. Reverse Transcription-PCR—Total RNA from glial cells was prepared using RNeasy. cDNA was synthesized by SuperScript II RNase H– reverse transcriptase according to the manufacturer's protocol. The sequences of the oligonucleotide primers used as a control for RNA isolation and reverse-transcription were as follows: for mouse TNF-α (354 bp), 5′-TTCTGTCTACTGAACTTCGGGGTAATCGGTCC-3′ (upstream) and 5′-GTATGAGATAGCAAATCGGCTGACGGTGTGGG-3′ (downstream); and for mouse GAPDH (558 bp), 5′-TGCTGAGTATGTCGTGGAGTCT-3′ (upstream) and 5′-AATGGGAGTTGCTGTTGAAGTC-3′ (downstream). One PCR cycle was run under the following conditions: DNA denaturation at 94 °C for 30 s, primer annealing at 60 °C for 1 min, and DNA extension at 72 °C for 1 min (28 cycles for TNF-α and 22 cycles for GAPDH). Semiquantification of mRNA levels of TNF-α and 18 S ribosomal RNA (used as a control) was carried out using a TaqMan cytokine gene expression plate and ABI PRISM 7700 (PerkinElmer Life Sciences) according to the manufacturer's protocol. Preparation of Automodified PARP-1 (AM-PARP) and Non-modified PARP-1 (NM-PARP)—Construction of recombinant baculovirus containing cDNA of human His-tagged PARP-1, its expression in insect cells, and protein purification are described elsewhere (27Giner H. Simonin F. de Murcia G. de Murcia J.M. Gene (Amst.). 1992; 114: 279-283Crossref PubMed Scopus (59) Google Scholar). AM-PARP was prepared by the following procedure. Purified His-PARP-1 was incubated in buffer containing 100 mm Tris-HCl, pH 8.0, 10 mm MgCl2, 1mm dithiothreitol, 1 μm NAD+, and 20 μg/ml synthetic octameric DNA (5′-GGAATTCC-3′) at 37 °C. After 5 min, the reactions were terminated by passing the mixture through a spin column (prepared with a 1-ml cylinder filled with Sephadex G-25 and equilibrated with 10 mm Tris-HCl, pH 7.2, and 1 mm EDTA) by centrifugation at 800 × g for 3 min. The concentration of the resulting proteins was measured, and the proteins were stored at –30 °C until used. NM-PARP was obtained by the same protocol as used for AM-PARP, except for incubation with 1 μm NAD+. Preparation of Nuclear Extracts—Nuclear extracts from unstimulated or LPS-stimulated glial cells (1–5 × 105 cells) were prepared by the method of Lukasiuk et al. (28Lukasiuk K. Kaczmarek L. Condorelli D.F. Neurochem. Int. 1995; 26: 173-178Crossref PubMed Scopus (13) Google Scholar) with slight modifications. Cells were washed twice with ice-cold phosphate-buffered saline; lysed in 400 μlof buffer containing 10 mm Hepes-KOH, pH 7.8, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, 1 mm sodium vanadate, 0.5 mm phenylmethylsulfonyl fluoride, 2.5 μg/ml aprotinin, 1 μg/ml pepstatin A, and 0.5 μg/ml leupeptin containing 0.1% Nonidet P-40 for 10 min on ice; vortexed vigorously for 15 s; and centrifuged at 12,000 × g for 3 min. The pelleted nuclei were resuspended in 100 μl of buffer containing 20 mm Hepes-KOH, pH 7.8, 0.4 m KCl, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 1 mm sodium vanadate, 0.5 mm phenylmethylsulfonyl fluoride, 2.5 μg/ml aprotinin, 1 μg/ml pepstatin A, and 0.5 μg/ml leupeptin; mixed vigorously for 15 min at 4 °C; and centrifuged at 15,000 × g for 30 min. Supernatants containing the nuclear proteins were stored at –80 °C. Electrophoretic Mobility Shift Assay (EMSA)—The nuclear extracts (10 μg) were preincubated in a total volume of 19 μl of gel shift binding buffer (10 mm Tris-HCl, pH 7.5, 1 mm MgCl2, 0.5 mm dithiothreitol, 0.5 mm EDTA, 50 mm NaCl, 5% glycerol, and 0.05 mg/ml poly(dI-dC)) for 10 min on ice. The sample was mixed with 1 μlof 32P-labeled NF-κB probe (5′-AGTTGAGGGGACTTTCCCAGGC-3′), which was end-labeled with [γ-32P]ATP using T4 DNA polynucleotide kinase; and the mixtures were then further incubated for 20 min at room temperature. For EMSA with purified proteins, purified recombinant NF-κB p50 (350 ng) was preincubated with purified His-PARP-1 (20, 100, or 200 ng; non-modified or automodified) for 20 min at room temperature in buffer containing 20 mm Tris-HCl, pH 8.0, 60 mm KCl, 5 mm MgCl2, 1 mm dithiothreitol, 0.05% Nonidet P-40, 10% glycerol, and 50 μg/ml bovine serum albumin (18Chang W.J. Alvarez-Gonzalez R. J. Biol. Chem. 2001; 276: 47664-47670Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and mixed with the 32P-labeled probe, and the mixtures were further incubated for 30 min at room temperature. Additionally, LPS-stimulated nuclear extract (10 μg) and purified His-PARP-1 (200 ng; non-modified or automodified) were incubated for 20 min at room temperature in gel shift binding buffer and mixed with the 32P-labeled probe, and the mixtures were then further incubated for 20 min at room temperature. After the reactions, the protein·DNA complexes were separated on a 6% nondenaturing polyacrylamide gel (DRC) using the gel shift assay kit according to the manufacturer's protocol. In the supershift assay, 2 μg of anti-p65 pAb were added, and the samples were incubated for 30 min at 4 °C before the addition of 32P-labeled probes. The competition experiments were performed by adding a 100-fold molar excess of unlabeled probes with the 32P-labeled probes. Poly(ADP-ribosyl)ation in Nuclear Extracts—Assays were performed as described by Chang and Alvarez-Gonzalez (18Chang W.J. Alvarez-Gonzalez R. J. Biol. Chem. 2001; 276: 47664-47670Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Each 10-μg sample in assay buffer containing 100 mm Tris-HCl, pH 8.0, 20 mm MgCl2, and 1 mm dithiothreitol was preincubated with 10 mm 3-aminobenzamide for 5 min at 37 °C before the addition of 10 μm NAD+ to start a reaction. Control assays were performed in the same manner, except that no 3-aminobenzamide was added. After 10 min, an equal volume of 2× gel shift binding buffer was added to each mixture, which was then incubated for 20 min at room temperature before EMSA. Alternatively, the poly(ADP-ribosyl)ated proteins were resolved by Western blotting with rabbit anti-PAR pAb as described above. Co-immunoprecipitation Assay—In a cell-free system, the procedure reported previously (18Chang W.J. Alvarez-Gonzalez R. J. Biol. Chem. 2001; 276: 47664-47670Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) was used. NM- or AM-PARP (200 ng) and the p50 subunit (350 ng) were preincubated for 30 min at 4 °C in buffer containing 10 mm Tris-HCl, pH 8.0, 150 mm NaCl, and 0.1% Nonidet P-40. Anti-p50 pAb (2 μg) was added, and the mixture was incubated for 60 min at 4 °C. Protein G-Sepharose beads (1:1 slurry) were added next, and the samples were further incubated for 60 min at 4 °C with rocking. After centrifugation at 5000 × g for 1 min, both the first supernatants and the pellet of beads were collected. The beads were further washed five times, and then the bead-bound proteins were eluted by adding SDS sample buffer and heating at 100 °C for 5 min. The eluted proteins and the supernatants were submitted to Western blotting with anti-p50 mAb, anti-PARP-1 mAb, or anti-PAR mAb (10H) as described above. To assess the association of PARP-1 with the p50 subunit in vivo, glial nuclear extracts (1 mg) prepared as described above were initially incubated with both rabbit control IgG (5 μg) and protein G-Sepharose beads for 30 min at 4 °C with rocking to block nonspecific binding. After centrifugation, the supernatants were obtained, and the following procedure, which was similar to the procedure used in the cell-free system, was performed. Immunodepletion Assay—To remove AM-PARP in glial cells by immunodepletion (29Akiyama T. Takasawa S. Nata K. Kobayashi S. Abe M. Shervani N.J. Ikeda T. Nakagawa K. Unno M. Matsuno S. Okamoto H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 48-53PubMed Google Scholar), total cell extracts (100 μg/50 μl) or LPS-treated nuclear extracts (50 μg/25 μl) were preincubated for 60 min at 4 °C with one of the following antibodies (2 μg/assay): rabbit control IgG, anti-PARP-1 pAb, or rabbit anti-PAR pAb. Subsequently, protein A-Sepharose beads (1:1 slurry) were added, and the samples were further incubated for 45 min at 4 °C with rocking. After centrifugation at 5000 × g for 1 min, the supernatants were obtained, and free proteins were submitted to Western blotting with anti-PARP-1 mAb or rabbit anti-PAR pAb as described above. In Vitro Transcription Assay (Runoff Assay)—In vitro transcription reactions were performed as described by Dignam et al. (30Dignam D.J. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9150) Google Scholar) following the HeLaScribe® nuclear extract in vitro transcription system protocol provided by the manufacturer. Briefly, 25-μl reaction mixtures were prepared by combining HeLa nuclear extract (8 units/reaction) with ATP, UTP, and CTP (0.4 mm each) and GTP (0.012 mm) in buffer containing 3 mm MgCl2. [α-32P]GTP (3000 Ci/mmol, 10 mCi/ml) was used to label runoff transcripts. The standard reactions were performed in the presence of 50 ng of human recombinant NF-κB p50 or bovine serum albumin, and 30 ng of linearized pNF-κB-Luc plasmid (Stratagene) digested with BsrGI were used as a template. The commercially available plasmid has five NF-κB-binding sites (TGGGGACTTTCCGC) in the promoter region and a unique restriction site by BsrGI in the open reading frame of the luciferase gene (see Fig. 9A). The reactions were started by the addition of HeLa nuclear extract and incubated at 30 °C for 45 min. The reactions were terminated by the addition of 175 μl of stop solution containing of 0.3 m Tris-HCl, pH 7.4, 0.3 m sodium acetate, 0.5% SDS,
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