Nitric Oxide-dependent Negative Feedback of PARP-1 trans-Activation of the Inducible Nitric-oxide Synthase Gene

Zhiyuan Yu,Teresa Kuncewicz,William P Dubinsky,Bruce C Kone

doi:10.1074/jbc.m511049200

Abstract

Nitric oxide (NO) participates in a variety of physiologic and pathophysiologic processes in diverse tissues, including the kidney. Although mechanisms for cytokine induction of inducible nitric-oxide synthase (iNOS) have been increasingly clarified, the controls for termination of NO production remain unclear. Because excessive NO production can be cytotoxic to host cells, feedback inhibition of iNOS transcription would represent a means of cytoprotection. Many of the cGMP-independent functions of NO are mediated by S-nitrosylation of cysteine thiols of target proteins. We hypothesized that NO-mediated S-nitrosylation of transcription factors might serve to feedback inhibit their trans-activation potential and deactivate iNOS gene transcription. Transient transfection of murine mesangial cells with iNOS promoter deletion-luciferase constructs revealed the region -915 to -849 to be NO sensitive with respect to IL-1beta-induced promoter activity. In vitro DNase I footprinting identified a footprint at -865/-842 in the absence of NO, but not in the presence of endogenous or exogenously delivered NO. Southwestern blotting using this probe coupled with partial peptide sequencing of the protein bands revealed that poly(ADP-ribose) polymerase isoform 1 (PARP-1) bound the probe in a sequence-specific manner. Gel shift/supershift experiments and chromatin immunoprecipitation assay analysis confirmed this binding in vitro and in vivo. Functionally, mutation of the -859/-850 site to prevent PARP-1 binding or PARP-1 knockdown by RNA interference relieved the inhibitory effects of NO on iNOS promoter activity. Biotin-switch assays and co-immunoprecipitation with an anti-nitrocysteine antibody indicated that PARP-1 was S-nitrosylated. We conclude that NO feedback inhibits iNOS gene transcription by S-nitrosylating the trans-activator PARP-1 and decreasing its binding and/or action at the iNOS promoter.

Highlights

Inducible nitric-oxide synthase2 is expressed in numerous cell types in mammals after induction by cytokines and/or lipopolysaccharide, and once expressed, it is active at resting levels of intracellular Ca2ϩ [1]
The cells were subsequently treated with vehicle, IL-1␤, or IL-1␤ ϩ L-NAME, and the fold induction of Inducible nitric-oxide synthase (iNOS) promoter-driven luciferase activity was measured
Because of the potent biological actions of Nitric oxide (NO) in the kidney and other tissues, considerable effort has been directed toward identifying the mechanisms that activate and limit iNOS gene expression

Summary

Introduction

Inducible nitric-oxide synthase (iNOS) is expressed in numerous cell types in mammals after induction by cytokines and/or lipopolysaccharide, and once expressed, it is active at resting levels of intracellular Ca2ϩ [1]. Reaction of NO with superoxide forms peroxynitrite with resultant protein thiol nitrosylation, tyrosine nitration, DNA damage, and excessive activation of poly(ADP-ribose) polymerase (PARP) [2]. Activation of PARP-1 plays an important role in the up-regulation of inflammatory cascades via a functional association with mitogen-activated protein kinases [8] and several transcription factors (9 –12), including NF-␬B [13,14,15,16], or its direct binding to gene-regulating DNA sequence [17], augmenting production of pro-inflammatory mediators. For example NF-␬B, a key transactivator of the iNOS gene, is subject to NO-mediated S-nitrosylation, which inhibits its ability to bind the iNOS promoter and results in down-regulation of iNOS gene transcription [24]. PARP-1 Regulates iNOS Gene Transcription activator of cytokine-induced iNOS transcription in glomerular mesangial cells and that NO-mediated S-nitrosylation of PARP-1 occurs and may account for feedback inhibition of the iNOS promoter

Methods

Results

Conclusion