HO and Tumor Necrosis Factor- Activate Intercellular Adhesion Molecule 1 (ICAM-1) Gene Transcription through Distinct cis-Regulatory Elements within the ICAM-1 Promoter

doi:10.1074/jbc.270.32.18966

HO and Tumor Necrosis Factor- Activate Intercellular Adhesion Molecule 1 (ICAM-1) Gene Transcription through Distinct cis-Regulatory Elements within the ICAM-1 Promoter (*)

From the (1) Departments of Pharmacology and
(2) Immunology/Microbiology, Rush-Presbyterian-St. Luke's Medical Center/Rush Medical College, Chicago, Illinois 60612 and the
(3) Department of Physiology and Cell Biology, Albany Medical College, Albany, New York 12208

^¶ To whom correspondence and reprint requests should be addressed:
Dept. of Immunology/Microbiology and Pharmacology, Rush Medical College, 1750 W. Harrison St., Chicago, IL 60612.
Tel.: 312-942-6259; Fax: 312-942-2808.

Abstract

We investigated the mechanisms by which H₂O₂ increases intercellular adhesion molecule 1 (ICAM-1; CD54) expression in endothelial cells. The H₂O₂-induced increase in ICAM-1 mRNA was inhibited by actinomycin D, by the antioxidant N-acetylcysteine, and by 3-aminobenzamide (which blocks oxidant-induced AP-1 activity), but not by pyrrolidine dithiocarbamate (which blocks oxidant-induced NF-κB activity). Nuclear run-on and transient transfections of ICAM-1 promoter constructs indicated that H₂O₂ stimulated ICAM-1 gene transcription by activation of a distinct region of the ICAM-1 promoter. The H₂O₂-responsive element was localized to sequences between −981 and −769 (relative to the start codon). Located within this region are two 16-base pair repeats, each containing binding sites for the transcription factors AP-1 and Ets. A similar composite AP-1/Ets element isolated from the macrophage scavenger receptor gene conferred H₂O₂ responsiveness to a minimal promoter. Mutation of the 16-base pair repeats within the ICAM-1 promoter prevented H₂O₂-induced DNA binding activity, and their deletion abrogated the H₂O₂-induced transcriptional activity. In contrast, TNFα induced ICAM-1 transcription via activation of promoter sequences between −393 and −176, a region with C/EBP and NF-κB binding sites. The results indicate that H₂O₂ activates ICAM-1 transcription through AP-1/Ets elements within the ICAM-1 promoter, which are distinct from NF-κB-mediated ICAM-1 expression induced by TNFα.

Footnotes

↵^§ The first and second authors contributed equally to this work.
↵* This work was supported by a grant-in-aid from the American Heart Association, American Cancer Society Grants IRG-195 and 94-10, and National Institutes of Health Grants HL27016, HL46350, and HL45638. 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:

PMN

polymorphonuclear leukocytes

ICAM-1

intercellular adhesion molecule 1

LUC

luciferase

HUVEC

human umbilical vein endothelial cell

TNFα

tumor necrosis factor-α

N-Cys(Ac)

N-acetylcysteine

PDTC

pyrrolidine dithiocarbamate

3-AB

3-aminobenzamide

AP-1

activator protein-1

NF-κB

nuclear factor κB

ARE

anti-oxidant response element

MSR

macrophage scavenger receptor

bp

base pair(s)

kb

kilobase(s)

DMEM

Dulbecco's modified Eagle's medium

MOPS

4-morpholinepropanesulfonic acid

PMSF

phenylmethylsulfonyl fluoride

PBS

phosphate-buffered saline

DTT

dithiothreitol

GST

glutathione S-transferase.
↵²K. A. Roebuck, unpublished result.
- Received May 9, 1995.
- Revision received June 7, 1995.