Cyclooxygenase 2 (COX-2) and cytosolic phospholipase A(2) (cPLA(2)) are the crucial rate-limiting enzymes in prostaglandin (PG) metabolism that show increased expression in a number of human cancers, including cholangiocarcinomas; and treatment of cholangiocarcinoma cell lines with COX-2 inhibitors can decrease proliferation. Cholangiocarcinomas also produce and proliferate in response to nonneoplastic biliary epithelial cell mitogens, such as interleukin 6 (IL-6) and hepatocyte growth factor (HGF). This study was designed to determine whether there is any relationship between eicosanoid metabolism and growth stimulation by IL-6 and HGF, two important biliary epithelial cell and cholangiocarcinoma mitogens. Incubation of SG231, a well-characterized human cholangiocarcinoma cell line, with HGF, IL-6, PGE(2), or PGF(2)alpha resulted in significantly increased cell growth. HGF and IL-6 also induced a rapid release of arachidonic acid (AA) from SG231 and increased the synthesis of PGE(2) and PGF(2)alpha. The cPLA(2) inhibitor arachidonyl fluorophosphonate (MAFP) and the COX-2 inhibitor NS-398 significantly inhibited HGF- and IL-6-induced release of AA, PG synthesis, and proliferation in SG231 cells as well as two other human cholangiocarcinoma cell lines, HuCCT1 and CC-LP-1 cells. Thus, PGs alone can induce cholangiocarcinoma growth, and the HGF- and IL-6-induced proliferation is mediated, at least in part, by PGs. HGF and IL-6 also induced a rapid phosphorylation of cPLA(2) (within 1 minute) but did not alter cPLA(2) and COX-2 protein expression. The HGF- and IL-6-induced cPLA(2) phosphorylation was blocked by the inhibitors of p38 and p42/44 MAP kinases, protein kinase C, calmodulin kinase, and tyrosine kinase, showing that HGF- and IL-6-induced AA release and PG production are mediated by phosphorylation of cPLA(2). In conclusion, molecular pathways link classic biliary epithelial cell mitogens to PG metabolism constituents in cholangiocarcinoma growth, which may be exploited as potential therapeutic targets.