Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, 1901 Perdido Street, New Orleans, LA 70112, USA.
J Immunol. 2010 Aug 1;185(3):1894-902. doi: 10.4049/jimmunol.1000646. Epub 2010 Jul 7.
The role of NF-kappaB 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-kappaB. However, the mechanism by which PARP-1 affects NF-kappaB activation has been elusive. In this study, we show that PARP-1 inhibition by gene knockout, knockdown, or pharmacologic blockade prevented p65 NF-kappaB nuclear translocation in smooth muscle cells upon TLR4 stimulation, NF-kappaB 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 IkappaBalpha phosphorylation and subsequent degradation but was required for p65 NF-kappaB phosphorylation. A perinuclear p65 NF-kappaB 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 alpha3 and importin alpha4 and their cytosolic localization, the cytosolic levels of exportin (Crm)-1 were increased. Crm1 inhibition promoted p65 NF-kappaB nuclear accumulation as well as reversed LPS-induced p65 NF-kappaB phosphorylation and inducible NO synthase and ICAM-1 expression. Interestingly, p65 NF-kappaB poly(ADP-ribosyl)ation decreased its interaction with Crm1 in vitro. Pharmacologic inhibition of PARP-1 increased p65 NF-kappaB-Crm1 interaction in LPS-treated smooth muscle cells. These results suggest that p65 NF-kappaB 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-kappaB nuclear retention, which ultimately can influence NF-kappaB-dependent gene regulation.
NF-κB 在炎症基因的表达及其在慢性疾病和急性组织损伤的整体炎症过程中的作用已得到充分证实。我们和其他人已经证明，多聚（ADP-核糖）聚合酶（PARP）-1 在炎症中具有关键作用，部分原因是它与 NF-κB 的关系。然而，PARP-1 影响 NF-κB 激活的机制尚不清楚。在这项研究中，我们表明，通过基因敲除、敲低或药理学阻断 PARP-1 的抑制作用，可以防止 TLR4 刺激时平滑肌细胞中 p65 NF-κB 的核易位、NF-κB 的 DNA 结合活性以及随后诱导型一氧化氮合酶和 ICAM-1 的表达。这种缺陷可以通过 PARP-1 表达的重建来逆转。PARP-1 对于 LPS 诱导的 IκBα磷酸化及其随后的降解不是必需的，但对于 p65 NF-κB 的磷酸化是必需的。在 LPS 处理的 PARP-1（-/-）细胞中，p65 NF-κB 的核周定位与出口而不是进口缺陷有关。事实上，尽管 PARP-1 缺乏并没有改变输入蛋白 α3 和输入蛋白 α4 的表达及其在细胞质中的定位，但细胞质中 CRM1（exportin）的水平增加了。CRM1 抑制促进了 p65 NF-κB 的核积累，以及逆转了 LPS 诱导的 p65 NF-κB 的磷酸化和诱导型一氧化氮合酶和 ICAM-1 的表达。有趣的是，p65 NF-κB 的聚（ADP-核糖）化降低了其与 CRM1 在体外的相互作用。PARP-1 的药理学抑制作用增加了 LPS 处理的平滑肌细胞中 p65 NF-κB-CRM1 的相互作用。这些结果表明，p65 NF-κB 的聚（ADP-核糖）化可能是其与 CRM1 相互作用及其在 TLR4 刺激时核保留的关键决定因素。这些结果为 PARP-1 促进 NF-κB 核保留的机制提供了新的见解，这最终可以影响 NF-κB 依赖性基因调控。
