Roles of ERK and P38 Mitogen-activated Protein Kinases in Phorbol Ester-induced NF-kappaB 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-kappaB. In an attempt to link the NF-kappaB 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-kappaB 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-kappaB DNA binding with maximal activation observed at 30 min. Increased DNA binding of NF-kappaB 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 2h and 4h, respectively. TPA treatment caused an increase in the production of prostaglandin E(2). Treatment of cells with the NF-kappaB 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-kappaB 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-kappaB signaling, which in turn induces COX-2 expression in TPA-stimulated human mammary epithelial cells.