Poly(ADP-Ribose) Polymerase-1 Is a Determining Factor in Crm1-Mediated Nuclear Export and Retention of p65 NF-κB upon TLR4 Stimulation

The role of NF-kappa B in the expression of inflammatory genes and its participation in the overall inflammatory process of chronic diseases and acute tissue injury are well established. We and others have demonstrated a critical involvement of poly(ADP-ribose) polymerase (PARP)-1 during inflammation, in part, through its relationship with NF-kappa B. However, the mechanism by which PARP-1 affects NF-kappa B activation has been elusive. In this study, we show that PARP-1 inhibition by gene knockout, knockdown, or pharmacologic blockade prevented p65 NF-kappa B nuclear translocation in smooth muscle cells upon TLR4 stimulation, NF-kappa B DNA-binding activity, and subsequent inducible NO synthase and ICAM-1 expression. Such defects were reversed by reconstitution of PARP-1 expression. PARP-1 was dispensable for LPS-induced I kappa B alpha phosphorylation and subsequent degradation but was required for p65 NF-kappa B phosphorylation. A perinuclear p65 NF-kappa B localization in LPS-treated PARP-1(-/-) cells was associated with an export rather an import defect. Indeed, whereas PARP-1 deficiency did not alter expression of importin alpha 3 and importin alpha 4 and their cytosolic localization, the cytosolic levels of exportin (Crm)-1 were increased. Crm1 inhibition promoted p65 NF-kappa B nuclear accumulation as well as reversed LPS-induced p65 NF-kappa B phosphorylation and inducible NO synthase and ICAM-1 expression. Interestingly, p65 NF-kappa B poly(ADP-ribosyl)ation decreased its interaction with Crm1 in vitro. Pharmacologic inhibition of PARP-1 increased p65 NF-kappa B-Crm1 interaction in LPS-treated smooth muscle cells. These results suggest that p65 NF-kappa B poly(ADP-ribosyl)ation may be a critical determinant for the interaction with Crm1 and its nuclear retention upon TLR4 stimulation. These results provide novel insights into the mechanism by which PARP-1 promotes NF-kappa B nuclear retention, which ultimately can influence NF-kappa B-dependent gene regulation. The Journal of Immunology, 2010, 185: 1894-1902.