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

Abstract

The role of NF-κB 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-κB. However, the mechanism by which PARP-1 affects NF-κB activation has been elusive. In this study, we show that PARP-1 inhibition by gene knockout, knockdown, or pharmacologic blockade prevented p65 NF-κB nuclear translocation in smooth muscle cells upon TLR4 stimulation, NF-κB 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 IκBα phosphorylation and subsequent degradation but was required for p65 NF-κB phosphorylation. A perinuclear p65 NF-κB 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 α3 and importin α4 and their cytosolic localization, the cytosolic levels of exportin (Crm)-1 were increased. Crm1 inhibition promoted p65 NF-κB nuclear accumulation as well as reversed LPS-induced p65 NF-κB phosphorylation and inducible NO synthase and ICAM-1 expression. Interestingly, p65 NF-κB poly(ADP-ribosyl)ation decreased its interaction with Crm1 in vitro. Pharmacologic inhibition of PARP-1 increased p65 NF-κB–Crm1 interaction in LPS-treated smooth muscle cells. These results suggest that p65 NF-κB 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-κB nuclear retention, which ultimately can influence NF-κB–dependent gene regulation.