Nitric oxide-dependent negative feedback of PARP-1 trans-activation of the inducible nitric-oxide synthase gene.
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.
Yu
Zhiyuan
Z
Department of Internal Medicine, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas Health Science Center at Houston, Houston, Texas 77030, USA.
Kuncewicz
Teresa
T
Dubinsky
William P
WP
Kone
Bruce C
BC
D000595:Amino Acid Sequence; D000818:Animals; D018929:Cell Culture Techniques; D025461:Feedback, Physiological; D050527:Mesangial Cells; D051379:Mice; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000502:physiology*; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis* / Q000235:genetics*; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases