Critical role of the automodification of poly(ADP-ribose) polymerase-1 in nuclear factor-κB-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.