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,
* Cell and Molecular Biology Program and
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824; and
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109
Infections involving LPS-bearing, Gram-negative bacteria can lead to acute inflammation and septic shock. Cyclooxygenase-2 (COX-2), the target of nonsteroidal anti-inflammatory drugs and selective COX-2 inhibitors, is importantly involved in these responses. We examined the dynamics of COX-2 gene expression in RAW264.7 murine macrophages treated with LPS as a model for COX-2 gene expression during inflammation. We established, using Northern blotting, nuclear run-on assays, and RT-PCR, that COX-2 transcriptional activation continues for at least 12 h after LPS treatment and involves at least three phases. Previous studies with murine macrophages identified an NF-
B site, a C/EBP site, and a cAMP response element-1 (CRE-1) as cis-acting elements in the COX-2 promoter. We identified three additional functional elements including a second CRE (CRE-2), an AP-1 site, and an E-box that overlaps CRE-1. The E-box mediates transcriptional repression whereas the other cis-elements are activating. Using electrophoretic mobility supershift and chromatin immunoprecipitation assays, we cataloged binding to each functional cis element and found them occupied to varying extents and by different transcription factors during the 12 h following LPS treatment. This suggests that the cis elements and their cognate transcription factors participate in a sequential, coordinated regulation of COX-2 gene expression during an inflammatory response. In support of this concept, we found, using inhibitors of Jun kinase and NF-B p50 nuclear localization, that COX-2 gene transcription was completely dependent on phospho-c-Jun plus p50 at 6 h after LPS treatment but was only partially dependent on the combination of these factors at later treatment times.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Grants DK22042 and GM68848 from the National Institutes of Health.
2 Y.-J.K. and B.A.W. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. William L. Smith, Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109. E-mail address: smithww{at}umich.edu
4 Abbreviations used in this paper: COX, cyclooxygenase; mCOX, murine COX; CRE, cAMP response element; USF, upstream stimulating factor; CBP, CREB-binding protein; ChIP, chromatin immunoprecipitation; WT, wild type.
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