Dynamic Regulation of Cyclooxygenase-2 Promoter Activity by Isoforms of CCAAT/Enhancer-binding Proteins

Wu-Guo Deng,Kenneth K Wu,Howard Yeh,Ying Zhu,Michael A Saunders

doi:10.1074/jbc.m108075200

Abstract

To elucidate the mechanism by which isoforms of CCAAT/enhancer-binding proteins regulate cyclooxygenase-2 expression, we determined by a novel technique binding of six isoforms of this transactivator to two sequence-specific CCAAT/enhancer-binding protein (-132/-125) and cyclic AMP (-59/-53) regulatory elements in human foreskin fibroblasts treated with phorbol 12-myristate 13-acetate for 4 h. The delta isoform bound to these two elements at basal state, which was displaced by full-length as well as two truncated beta isoforms, a 41-kDa liver-enriched activating protein and a 16-kDa liver-enriched inhibitory protein, after phorbol ester stimulation. Kinetic analysis shows time-dependent changes in beta and delta binding that were concordant with time-dependent increase in cyclooxygenase-2 induction. Overexpression of the 16-kDa beta isoform blocked the promoter activity and protein level induced by phorbol ester. Paradoxically, it increased binding of beta isoforms to the sequence-specific promoter DNA but suppressed cyclooxygenase-2 promoter activation by p300 cotransfection. These findings provide new insight into the regulation of cyclooxygenase-2 promoter by an interplay between two opposite beta isoforms and p300 co-activator.

Highlights

Cyclooxygenase-2 (COX-2)1 plays diverse pathophysiological roles notably in inflammation, tissue damage, and tumorigenesis [1,2,3]
Results from this study indicate that phorbol 12-myristate 13-acetate (PMA) induces a dynamic switch of CCAAT/enhancer binding protein (C/EBP) isoform binding to C/EBP and CRE sites on the 5Ј-untranslated region of COX-2 promoter, thereby stimulating COX-2 promoter activity
Increased binding of C/EBP␤-liver-enriched transcription activating protein (LAP) to these two regulatory elements plays a key role in transactivation of COX-2 promoter, while increased C/EBP␤-liver-enriched transcription inhibitory protein (LIP) binding provides a negative regulation of COX-2 promoter activity

Summary

Introduction

Cyclooxygenase-2 (COX-2)1 plays diverse pathophysiological roles notably in inflammation, tissue damage, and tumorigenesis [1,2,3]. 1 The abbreviations used are: COX-2, cyclooxygenase-2; C/EBP, CCAAT/enhancer-binding protein; PMA, phorbol 12-myristate 13-acetate; LAP, liver-enriched activating protein; LIP, liver-enriched inhibitory protein; HFF, human foreskin fibroblasts; CRE, cyclic AMP responsive element; C/EBP␤-FL, full-length C/EBP␤. Our results show that PMA increased binding of full-length C/EBP␤ (C/EBP␤FL), C/EBP␤-LAP, and C/EBP␤-LIP to C/EBP and CRE sites while it reduced C/EBP␦ level and its binding to the C/EBP site.

Results

Conclusion