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- Kappa B (NF- Kappa B)-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- Kappa B-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- Kappa B. Non-modified PARP-1 also reduced the DNA binding of NF- Kappa B via its physical association with NF- Kappa B, whereas AM-PARP had no effect. On the other hand, enhancement of the automodification of PARP-1 by the addition of NAD super(+), its substrate, promoted the DNA binding of NF- Kappa B. Furthermore, in in vitro transcription assay, the addition of AM-PARP or NAD super(+) to nuclear extracts promoted NF- Kappa B p50-dependent transcription. These results indicate that automodification of PARP-1 positively up-regulates formation of the NF- Kappa B.DNA complex and enhances transcriptional activation. Therefore, AM-PARP may be critical for the NF- Kappa B-dependent gene expression of some inflammatory mediators in glial cells.