The Cytokine Responsive Vascular Smooth Muscle Cell Enhancer of Inducible Nitric Oxide Synthase

doi:10.1074/jbc.270.49.29541

The Cytokine Responsive Vascular Smooth Muscle Cell Enhancer of Inducible Nitric Oxide Synthase

ACTIVATION BY NUCLEAR FACTOR-κB (*)

From the Department of Infectious Diseases and Bacteriology, Royal Postgraduate Medical School, London W12 0NN, United Kingdom

^§To whom correspondence should be addressed:
Dept. of Infectious Diseases and Bacteriology, Royal Postgraduate Medical School, Du Cane Rd., London W12 0NN, UK.
Tel.: 44-181-740-3243; Fax: 44-181-40-3394; tevans{at}rpms.ac.uk.

Abstract

The production of inducible nitric oxide synthase (iNOS) within vascular smooth muscle (VSM) cells following exposure to proinflammatory cytokines is a major cause of the vasorelaxation and hypotension of septic shock. We have defined the cytokine-responsive element of the murine iNOS promoter, transfected into a VSM cell line, and the role of the NF-κB/Rel family of proteins in iNOS gene activation in these cells. The combination of interleukin-1, interferon-, and tumor necrosis factor-α stimulates promoter activity by a factor of 8.1-fold; single cytokines show little activity, while pairs of cytokines produce an intermediate effect. Using a series of promoter deletion mutants, we have defined the cytokine-responsive element from position −890 to −1002; this region contains an NF-κB-binding site as well as a number of interferon response elements. Nuclear proteins from cytokine-stimulated VSM cells which bind to an oligonucleotide containing this κB site are composed of p65 together with an unidentified protein of 50 kDa, which is not a known Rel family member. A promoter mutant with a 2-base pair change within this κB site, which abolishes NF-κB binding, has an activity of only approximately 34% (S.E. ± 1.5) of the wild-type promoter. In addition, protein binding to this site is abolished by a specific inhibitor of NF-κB activation, which also abrogates iNOS activity. Residual inducibility in such mutant promoters is attributable to the presence of an independently functioning downstream κB site (−85 to −75). The mechanism by which NF-κB activates the iNOS promoter in VSM cells in response to cytokines appears to be markedly different to that operative in macrophages in response to lipopolysaccharide.

Footnotes

↵(*) This work was supported in part by the award of a Medical Research Council Clinician/Scientist fellowship (to T. J. E.), and through a grant from the Biotech Program of the European Community. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

NO

nitric oxide

NOS

NO synthase

iNOS

inducible NOS

VSM

vascular smooth muscle

IL

interleukin

IFN

interferon

TNF

tumor necrosis factor

EMSA

electrophoretic mobility shift assay

PDTC

pyrrolidine dithiocarbamate

LPS

lipopolysaccharide

DMEM

Dulbecco's modified Eagle's medium

PCR

polymerase chain reaction

CAT

chloramphenicol acetyltransferase

ISRE

IFN stimulus response element

GAS

-IFN-activated site

IRF

IFN regulatory factor

NF

nuclear factor.
↵⁽²⁾L. Heerdt, personal communication.
↵⁽³⁾T. J. Evans, unpublished data.
↵⁽⁴⁾J. Spink, J. Cohen, and T. J. Evans, unpublished observations.
- Received August 31, 1995.
- Revision received October 5, 1995.