We monitored cyclooxygenase-2 (COX-2) expression in the insulin-like growth factor-II (IGF-II) treated human keratinocytes and explored the IGF-II signaling pathways with respect to the expression of COX-2. IGF-II induced COX-2 mRNA and protein levels, and the up-regulation of COX-2 expression by IGF-II was reduced by pretreatment with inhibitors of tyrosine kinase, Src and PI3-kinase. The inhibition of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) 1 also reduced the increased expression of COX-2 by IGF-II, but the inhibition of p38 did not. To further examine the roles of these mitogen-activated protein kinases (MAPKs) in IGF-II-induced COX-2 expression, we performed COX-2 promoter analysis using dominant negative plasmids of MEK1 (DN-MEK1), p38 (DN-p38) and JNK1 (DN-JNK1). Although IGF-II increased COX-2 promoter activity approximately 2.5-fold, this increase was blocked by cotransfection with DN-MEK1 or DN-JNK1. However, DN-p38 did not block the IGF-II-induced COX-2 promoter activity. In addition, inhibition of ERK or JNK1 reduced the increase of IGF-II-induced prostaglandin E(2) synthesis or cell proliferation. These results suggest that IGF-II induces COX-2 expression through the tyrosine kinase-Src-ERK and tyrosine kinase-PI3-kinase pathways, but not via p38 MAPK pathway, and that the basal JNK activity is required for the upregulation of COX-2 by IGF-II, as well.