Dominant-negative c-Jun NH2-terminal kinase 2 sensitizes renal inner medullary collecting duct cells to hypertonicity-induced lethality independent of organic osmolyte transport.
The c-Jun NH2-terminal protein kinases (JNKs), as well as the extracellular signal-regulated protein kinases (ERKs) and p38 mitogen-activated protein kinase, are activated in renal cells in response to extracellular hypertonicity. To determine whether activation of JNKs by hypertonicity is isoform-specific, renal inner medullary collecting duct cells were stably transfected with cDNA's encoding hemagglutinin (HA)-tagged JNK1 and JNK2 isoforms, and the expressed kinases were immunoprecipitated with an anti-HA antibody. Whereas both recombinant kinases were equivalently expressed, only immunoprecipitates from the HA-JNK2 cells displayed hypertonicity-inducible JNK activity. Furthermore, expression of dominant-negative JNK2 (HA-JNK2-APF) in stable clones inhibited hypertonicity-induced JNK activation by 40-70%, whereas expression of dominant-negative JNK1 (HA-JNK1-APF) had no significant inhibitory effect. Independent HA-JNK2-APF (but not HA-JNK1-APF) clones displayed greatly reduced viability relative to neomycin controls after 16 h of exposure to 600 mosM/kg hypertonic medium with percent survival of 20.5 +/- 2.7 and 31.5 +/- 7.3 for two independent HA-JNK2-APF clones compared with 80.1 +/- 1.0 for neomycin controls (p < 0.001, n = 5, mean +/- S.E.). However, neither JNK mutant blocked either regulatory volume increase or hypertonicity-induced enhancement of uptake of inositol, an organic osmolyte putatively involved in long term adaptation to hypertonicity. These results define JNK2 as the primary hypertonicity-activated JNK isoform in IMCD-3 cells and demonstrate its central importance in cellular survival in a hypertonic environment by a mechanism independent of acute regulatory volume increase as well as regulation of organic osmolyte uptake.
Wojtaszek
P A
PA
Department of Medicine, University of Colorado School of Medicine, Denver, Colorado 80262, USA.
Heasley
L E
LE
Siriwardana
G
G
Berl
T
T
D000222:Adaptation, Physiological; D001692:Biological Transport; D002461:Cell Line, Transformed; D004789:Enzyme Activation; D007679:Kidney Medulla / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D048688:MAP Kinase Kinase 7; D020929:Mitogen-Activated Protein Kinase Kinases; D009994:Osmolar Concentration; D011494:Protein Kinases / Q000378:metabolism*
D011494:Protein Kinases; D048688:MAP Kinase Kinase 7; D020929:Mitogen-Activated Protein Kinase Kinases