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J Biol Chem, Vol. 273, Issue 22, 13645-13651, May 29, 1998
From the Laboratory of Kidney and Electrolyte Metabolism, NHLBI,
National Institutes of Health, Bethesda, Maryland 20892-1603
Murine kidney cells of the inner medullary
collecting duct (mIMCD) were exposed to either isosmotic (300 mosmol/kg) or hyperosmotic medium (isosmotic medium + 150 mM NaCl) after seeding. We determined cell numbers,
total nucleic acid, DNA, and RNA contents in both groups every day for
a total period of 7 days. Based on all 4 parameters it was evident that
growth of mIMCD3 cells is arrested for ~18 h following onset of
hyperosmolality. However, none of the parameters measured indicated
cell death because of hyperosmolality. Growth curves of hyperosmotic
samples were shifted compared with isosmotic samples showing a gap of
18 h but had the same shape otherwise. We demonstrated that at 24 and 48 h after onset of hyperosmolality, but not in isosmotic
controls, growth arrest and DNA
damage-inducible (GADD) proteins GADD45 and GADD153 are strongly induced. This result is consistent with growth arrest observed
in hyperosmotic medium. We tested if mitogen- and stress-activated protein kinase (SAPK) cascades are involved in osmosignaling that leads
to GADD45 and GADD153 induction. Using phosphospecific antibodies we
showed that extracellular signal-regulated kinases 1 and 2 (ERK), SAPK1
(JNK), and SAPK2 (p38) are hyperosmotically activated in mIMCD cells.
Hyperosmotic GADD45 induction was significantly decreased by 37.5%
following inhibition of the SAPK2 pathway, whereas it was significantly
increased (65.2%) after inhibition of the ERK pathway. We observed
similar, although less pronounced effects of SAPK2 and ERK inhibition
on hyperosmotic GADD153 induction. In conclusion, we demonstrate that
mIMCD cells arrest growth following hyperosmotic shock, that this
causes strong induction of GADD45 and GADD153, that GADD induction is
partially dependent on osmosignaling via SAPK2 and ERK, and that SAPK2
and ERK pathways have opposite effects on GADD expression.
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O. Nahm, S. K. Woo, J. S. Handler, and H. M. Kwon Involvement of multiple kinase pathways in stimulation of gene transcription by hypertonicity Am J Physiol Cell Physiol, January 1, 2002; 282(1): C49 - C58. [Abstract] [Full Text] [PDF]
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