HYPEROSMOLALITY CAUSES GROWTH ARREST OF MURINE KIDNEY-CELLS - INDUCTION OF GADD45 AND GADD153 BY OSMOSENSING VIA STRESS-ACTIVATED PROTEIN-KINASE

Murine kidney cells of the inner medullary collecting duct (mIMCD) wer e exposed to either isosmotic (300 mosmol/kg) or hyperosmotic medium ( isosmotic medium + 150 mM NaCl) after seeding. We determined cell numb ers, total nucleic acid, DNA, and RNA contents in both groups every da y for a total period of 7 days, Based on all 4 parameters it was evide nt that growth of mIMCD3 cells is arrested for similar to 18 h followi ng onset of hyperosmolality, However, none of the parameters measured indicated cell death because of hyperosmolality, Growth curves of hype rosmotic samples were shifted compared with isosmotic samples showing a gap of 18 h but had the same shape otherwise. We demonstrated that a t 24 and 48 h after onset of hyperosmolality, but not in isosmotic con trols, growth arrest and DNA damage-inducible (GADD) proteins GADD45 a nd GADD153 are strongly induced. This result is consistent with growth arrest observed in hyperosmotic medium. We tested if mitogen-and stre ss-activated protein kinase (SAPK) cascades are involved in osmosignal ing 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 decrea sed 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 demon strate that mIMCD cells arrest growth following hyperosmotic shock, th at this causes strong induction of GADD45 and GADD153, that GADD induc tion is partially dependent on osmosignaling via SAPK2 and ERK, and th at SAPK2 and ERK pathways have opposite effects on GADD expression.