Prostaglandin E2 stimulates p53 transactivational activity through specific serine 15 phosphorylation in human synovial fibroblasts ROLE IN SUPPRESSION OF c/EBP/NF-κB-MEDIATED MEKK1-INDUCED MMP-1 EXPRESSION

Abstract

Cyclooxygenase-2 (COX-2) overexpression has been linked to cell survival, transformation, and hyperproliferation. We examined the regulation of the tumor suppressor gene p53 and p53 target genes by prostaglandin E2 (PGE2) in human synovial fibroblasts (HSF). PGE2 induced a time-dependent increase in p53 Ser15 phosphorylation, with no discernible change in overall p53 levels. PGE2-dependent Ser15 phosphorylation was apparently mediated by activated p38 MAP kinase as SB202190, a p38 kinase inhibitor, blocked the response. Overexpression of a MKK3 construct, but not MKK1, stimulated SB202190-sensitive p53 Ser15 phosphorylation. PGE2-stimulated [phospho-Ser15]p53 transactivated a p53 response element (GADD45)-luciferase reporter in transiently transfected HSF (SN7); the effect was compromised by overexpression of a dominant-negative mutant (dnm) of p53 or excess p53S15A expression plasmid but mimicked by a constitutively active p53S15E expression construct. PGE2, wtp53 expression in the presence of PGE2, and p53S15E suppressed steady-state levels of MEKK1-induced MMP-1 mRNA, effects nullified with co-transfection of p53 dnm or p53S15A. MEKK1-induced MMP-1 promoter-driven luciferase activity was largely dependent on a c/EBPβ-NF-κB-like enhancer site at –2008 to –1972 bp, as judged by deletion and point mutation analyses. PGE2, overexpression of p53wt with PGE2, or p53S15E abolished the MEKK1-induced MMP-1 promoter luciferase activity. Gel-shift/super gel-shift analyses identified c/EBPβ dimers and c/EBPβ/NF-κB p65 heterodimers as binding species at the apparent site of MEKK1-dependent transactivation. PGE2-stimulated [phospho-Ser15]p53 abrogated the DNA binding of c/EBPβ dimers and c/EBPβ/NF-κB p65 heterodimers. Our data suggest that COX-2 prostaglandins may be implicated in p53 function and p53 target gene expression