Abstract
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 approximately 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.
Highlights
Murine kidney cells of the inner medullary collecting duct were exposed to either isosmotic (300 mosmol/kg) or hyperosmotic medium after seeding
We tested if mitogen- and stress-activated protein kinase (SAPK) cascades are involved in osmosignaling that leads to GADD45 and GADD153 induction
Growth arrest of mIMCD cells persisting for 18 h appears immediately following exposure to hyperosmolality of 600 mosmol/kg water (ϩ300 mosmol of NaCl)
Summary
INDUCTION OF GADD45 AND GADD153 BY OSMOSENSING VIA STRESS-ACTIVATED PROTEIN KINASE 2*. Proteins that define structural and functional aspects of specialized cell types at different osmolalities and certain immediate early genes are tightly regulated by osmotic strength This is the case in epithelial cells of animals that maintain osmotic homeostasis of their extracellular body fluids (osmoregulators), e.g. mammalian kidney cells [5] or teleost gill cells [6, 7]. Our second objective was to test whether osmosensing mitogen-activated protein kinase (MAPK) signal transduction pathways are involved in osmotic regulation of GADD expression. We continued to search for possible targets of MAPK-osmosensing pathways and found that GADD protein expression is at least partially activated by SAPK2 phosphorylation and inhibited by extracellular signal-regulated kinase (ERK) phosphorylation. Working model explaining these results is presented and the implications of our findings for strategies of cellular adaptation to osmotic stress are discussed
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