Roles of ERK and p38 mitogen-activated protein kinases in phorbol ester-induced NF-κB activation and COX-2 expression in human breast epithelial cells

Inappropriate up-regulation of cyclooxygenase-2 (COX-2) has been implicated in pathogenesis of various types of human cancer. Thus, COX-2 has been recognized as an important target for the chemoprevention of several human malignancies including breast cancer. COX-2 expression is known to be regulated by the eukaryotic transcription factor NF-kappa B. In an attempt to link the NF-kappa B activation and COX-2 induction during mammary carcinogenesis, we have examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), a prototype tumor promoter and a mitogen, on NF-kappa B activation and COX-2 expression in the immortalized human breast epithelial cell line (MCF10A). Treatment of MCF10A cells with TPA resulted in transient induction of NF-kappa B DNA binding with maximal activation observed at 30 min. Increased DNA binding of NF-kappa B was accompanied by enhancement of its transcriptional activity as determined by the luciferase reporter gene assay. Under the same experimental conditions, expression of COX-2 mRNA and its protein product peaked at 2 h and 4 h, respectively. TPA treatment caused an increase in the production of prostaglandin E-2. Treatment of cells with the NF-kappa B inhibitor pyrrolidine dithiocarbamate resulted in significant suppression of TPA-induced COX-2 expression. TPA induced activation of ERK1/2 and p38 mitogen-activated protein kinases (MAPK) via phosphorylation. PD98059 (ERK inhibitor) and SB203580 (p38 MAPK inhibitor) down-regulated the COX-2 expression induced by TPA. Furthermore, TPA-induced COX-2 induction as well as NF-kappa B activation was blocked in MCF10A cells transfected with dominant negative mutant ERK1/2 or p38 MAPK. These results suggest that both p38 and ERK MAPKs activates NF-kappa B signaling, which in turn induces COX-2 expression in TPA-stimulated human mammary epithelial cells. (c) 2007 Elsevier Ireland Ltd. All rights reserved.