Critical role of the automodification of poly(ADP-ribose) polymerase-1 in nuclear factor-kappaB-dependent gene expression in primary cultured mouse 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-kappaB (NF-kappaB)-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-kappaB-dependent gene expression. PARP-1 inhibitors and the antisense RNA for PARP-1 mRNA suppressed lipopolysaccharide (LPS)-induced expression of tumor necrosis factor-alpha 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-kappaB. Non-modified PARP-1 also reduced the DNA binding of NF-kappaB via its physical association with NF-kappaB, 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-kappaB. Furthermore, in in vitro transcription assay, the addition of AM-PARP or NAD+ to nuclear extracts promoted NF-kappaB p50-dependent transcription. These results indicate that automodification of PARP-1 positively up-regulates formation of the NF-kappaB.DNA complex and enhances transcriptional activation. Therefore, AM-PARP may be critical for the NF-kappaB-dependent gene expression of some inflammatory mediators in glial cells.
Pharmaceutical Research Center, Meiji Seika Kaisha Limited, 760 Moro-oka-cho, Kohoku-ku, Yokohama 222-8567, Japan.
Hidemitsu
H
Nakajima
Hiroshi
H
Nagaso
Nobukazu
N
Kakui
Midori
M
Ishikawa
Toyokazu
T
Hiranuma
Shigeru
S
Hoshiko
D000818:Animals; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D047468:Immunoprecipitation; D007249:Inflammation; D008070:Lipopolysaccharides / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D016328:NF-kappa B / Q000737:chemistry / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D009457:Neuroglia / Q000166:cytology / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D021381:Protein Transport; D016372:RNA, Antisense; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015854:Up-Regulation
D016207:Cytokines; D008070:Lipopolysaccharides; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D009573:Nitrites; D016376:Oligonucleotides, Antisense; D016372:RNA, Antisense; D012333:RNA, Messenger; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; C073701:Nfkb1 protein, mouse; D004247:DNA; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D011065:Poly(ADP-ribose) Polymerases