Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-kappa B - PubMed (original) (raw)

Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-kappa B

R W Ganster et al. Proc Natl Acad Sci U S A. 2001.

Abstract

The human inducible nitric oxide synthase (hiNOS) gene is expressed in several disease states and is also important in the normal immune response. Previously, we described a cytokine-responsive enhancer between -5.2 and -6.1 kb in the 5'-flanking hiNOS promoter DNA, which contains multiple nuclear factor kappa beta (NF-kappa B) elements. Here, we describe the role of the IFN-Jak kinase-Stat (signal transducer and activator of transcription) 1 pathway for regulation of hiNOS gene transcription. In A549 human lung epithelial cells, a combination of cytokines tumor necrosis factor-alpha, interleukin-1 beta, and IFN-gamma (TNF-alpha, IL-1 beta, and IFN-gamma) function synergistically for induction of hiNOS transcription. Pharmacological inhibitors of Jak2 kinase inhibit cytokine-induced Stat 1 DNA-binding and hiNOS gene expression. Expression of a dominant-negative mutant Stat 1 inhibits cytokine-induced hiNOS reporter expression. Site-directed mutagenesis of a cis-acting DNA element at -5.8 kb in the hiNOS promoter identifies a bifunctional NF-kappa B/Stat 1 motif. In contrast, gel shift assays indicate that only Stat 1 binds to the DNA element at -5.2 kb in the hiNOS promoter. Interestingly, Stat 1 is repressive to basal and stimulated iNOS mRNA expression in 2fTGH human fibroblasts, which are refractory to iNOS induction. Overexpression of NF-kappa B activates hiNOS promoter-reporter expression in Stat 1 mutant fibroblasts, but not in the wild type, suggesting that Stat 1 inhibits NF-kappa B function in these cells. These results indicate that both Stat 1 and NF-kappa B are important in the regulation of hiNOS transcription by cytokines in a complex and cell type-specific manner.

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Figures

Figure 1

Figure 1

Overlapping NF-κB and Stat 1 elements at −5.2 kb and −5.8 kb in the hiNOS promoter.

Figure 2

Figure 2

The effect of Jak kinase inhibitors on hiNOS expression and on cytokine induction of nuclear NF-κB or Stat 1 DNA-binding. (A) Northern and Western blot analysis of hiNOS mRNA and protein induced by CM. The Jak kinase inhibitor (tyrophostin A25) inhibits cytokine-induced iNOS mRNA and protein expression in a dose-dependent fashion. (B) The gel shift experiment shows the effects of the Jak 2 kinase inhibitor tyrophostin B42 on the nuclear DNA-binding activities of both NF-κB and Stat 1. Nuclear protein extracts were prepared from cells exposed for 2 h to a cytokine mixture in the absence or presence of tyrophostin B42 as indicated. The nuclear proteins were subjected to EMSA, using the consensus NF-κB or hSIE oligonucleotide probes. Blots shown are representative of three similar experiments.

Figure 3

Figure 3

The cis-acting transcription element at −5.8 kb in the hiNOS promoter is a bifunctional, composite NF-κB/GAS element. The graph depicts the cytokine-induced expression of the −7.2-kb hiNOS promoter–luciferase reporter and various mutant derivatives shown in Fig. 1. Mutation of both the NF-κB and the Stat 1 elements eliminate cytokine induction of the hiNOS reporter plasmid, whereas mutation of the NF-κB or Stat 1 sequence individually had no effect on hiNOS promoter activity (n = 4–6 transfections per condition).

Figure 4

Figure 4

Inflammatory cytokines induce distinct NF-κB or Stat 1–DNA complexes at the −5.8 kb hiNOS promoter element. The figure is a gel shift assay analyzing the induction of nuclear DNA-binding proteins in response to either TNF-α, IFN-γ, or a combination in nuclear extracts from human A549 lung epithelium. Antibody supershift assays indicate that TNF-α induces a protein–DNA complex containing NF-κB protein, whereas IFN-γ induces a Stat 1–DNA complex. Blots shown are representative of two similar experiments.

Figure 5

Figure 5

The cytokine responsive element at −5.2 kb in the hiNOS promoter is a functional Stat 1 DNA-binding sequence. (A) Only IFN-γ alone or as part of CM induces a protein/DNA-binding complex with the −5.2-kb element. Mutation of the NF-κB domain does not alter binding, but mutation of the Stat 1 site abolishes all binding. (B) Antibody supershift shows that the protein–DNA complex at −5.2 kb is recognized exclusively by Stat 1 antibody. (C) Purified recombinant NF-κB protein binds to a consensus NF-κB oligo, but not to the −5.2 kb or to a consensus Stat 1 (hSIE) element. Blots shown are representative of three similar experiments.

Figure 6

Figure 6

Interferons and Stat 1 repress hiNOS mRNA and NF-κB-mediated hiNOS transcription in a human fibroblast cell line. (A) The Northern blot shows hiNOS mRNA expression in response to various combinations of cytokines as labeled. IFN-β or IFN-γ inhibit basal and stimulated hiNOS mRNA expression in the wild-type 2fTGH human fibroblasts, but not in the Stat 1-null U3A cells. (B) Analysis of hiNOS promoter–luciferase reporter plasmids in human 2fTGH and U3A fibroblasts indicate that Stat 1 can function as a repressor of NF-κB-induced iNOS transcription. The figure illustrates the fold induction of a hiNOS–luciferase reporter in cells that were cotransfected with empty vector or vectors that express either p50 + p65 NF-κB protein, wild-type Stat 1, or dominant-negative mutant Stat 1 protein. Notice that NF-κB overexpression will induce a significant hiNOS expression only in the Stat 1 mutant U3A cells. Blots shown are representative of three similar experiments.

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