MAPK mediation of hypertonicity-stimulated cyclooxygenase-2 expression in renal medullary collecting duct cells

We have previously shown that hypertonicity stimulates cyclooxygenase-2 (CO X-2) expression in cultured medullary epithelial cells. The aims of the pre sent study were (i) to examine the role of cytoplasmic signaling through MA PK pathways in tonicity regulation of COX-2 expression in collecting duct c ells 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 in duced a marked increase in COX-2 protein expression, The stimulation was re duced significantly by inhibition of MEK1 (PD-98059, 5-50 mu M and p38 (SB- 203580, 5-100 mu M) 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 th ree JNKs (JNK-1, -2, and -3) were used. Hypertonicity-stimulated COX-2 prot ein expression was significantly reduced in dominant-negative JNK-2-express ing cells and was unchanged in dominant-negative JNK-1- and JNK-3-expressin g cells compared with controls. The reduction of COX-2 expression was assoc iated 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 mu M), an inhibitor of Src kinases, r educed 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 mu M) and SC-58236 (10-20 mu M) significantly re duced viability of mIMCD-K2 cells subjected to prolonged hypertonic treatme nt. We conclude that 1) all three members of the MAPK family (ERK, JNK-2, a nd p38) as well as Src kinases are required for tonicity-stimulated COX-2 e xpression in mouse collecting duct cells and that 2) COX-2 may play a role in cell survival of medullary cells under hypertonic conditions.