We have previously shown that hypertonicity stimulates cyclooxygenase-2 (COX-2) expression in cultured medullary epithelial cells. The aims of the present study were (i) to examine the role of cytoplasmic signaling through MAPK pathways in tonicity regulation of COX-2 expression in collecting duct cells and (ii) to assess the possible contribution of COX-2 to the survival of inner medullary collecting duct (IMCD) cells under hypertonic conditions. In mIMCD-K2 cells, a cell line derived from mouse IMCDs, hypertonicity induced a marked increase in COX-2 protein expression. The stimulation was reduced significantly by inhibition of MEK1 (PD-98059, 5-50 microm) and p38 (SB-203580, 5-100 microm) and was almost abolished by the combination of the two compounds. To study the role of JNK in tonicity-stimulated COX-2 expression, IMCD-3 cell lines stably transfected with dominant-negative mutants of three JNKs (JNK-1, -2, and -3) were used. Hypertonicity-stimulated COX-2 protein expression was significantly reduced in dominant-negative JNK-2-expressing cells and was unchanged in dominant-negative JNK-1- and JNK-3-expressing cells compared with controls. The reduction of COX-2 expression was associated with greatly reduced viability of dominant-negative JNK-2-expressing cells during hypertonicity treatment. 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) (2-8 microm), an inhibitor of Src kinases, reduced the tonicity-stimulated COX-2 expression in a dose-dependent manner, whereas PP3, an inactive analog of PP2, had no effect. Inhibition of COX-2 activity by NS-398 (30-90 microm) and SC-58236 (10-20 microm) significantly reduced viability of mIMCD-K2 cells subjected to prolonged hypertonic treatment. We conclude that 1) all three members of the MAPK family (ERK, JNK-2, and p38) as well as Src kinases are required for tonicity-stimulated COX-2 expression in mouse collecting duct cells and that 2) COX-2 may play a role in cell survival of medullary cells under hypertonic conditions.