Human Intestine 407 cells respond to hypo-osmotic stress with a rapid stimulation of compensatory ionic conductances accompanied by a transient increase in the activity of the extracellular-signal-regulated protein kinases Erk-1 and Erk-2. In this study, we examined the upstream regulators of hypotonicity-induced Erk-1/Erk-2 activation and their possible role in cell-volume regulation. The hypotonicity-provoked Erk-1/Erk-2 activation was greatly reduced in cells pretreated with the specific mitogen-activated/Erk-activating kinase inhibitor PD098059 and was preceded by a transient stimulation of Raf-1. Pretreatment of the cells with PMA, GF109203X, wortmannin or Clostridium botulinum C3 exoenzyme did not appreciably affect the hypotonicity-provoked Erk-1/Erk-2 stimulation, suggesting the osmosensitive signalling pathway to be largely independent of protein kinase C and p21(rho). In contrast, expression of dominant negative RasN17 completely abolished the hypotonicity-induced Erk-1/Erk-2 activation. Stimulation of the swelling-induced ion efflux was independent of activation of these mitogen-activated protein kinases, as revealed by hypotonicity-provoked isotope efflux from 125I-- and 86Rb+-loaded cells after pretreatment with PD098059 and after expression of RasN17. In addition, the epidermal-growth-factor-induced potentiation of the hypotonicity-provoked anionic response did not depend on the increase in Erk-1/Erk-2 activity but, instead, was found to depend on Ca2+ influx. Taken together, these results indicate that hypotonic stress induces Erk-1/Erk-2 activation through the Ras/Raf-signalling pathway, and argue against a direct role for this pathway in cell-volume control.

*Botulinum Toxins, *Osmotic Pressure, ADP Ribose Transferases/pharmacology, Androstadienes/pharmacology, Ca(2+)-Calmodulin Dependent Protein Kinase/*pharmacology, Cell Line, Cell Size/drug effects, Enzyme Activation/physiology, Epidermal Growth Factor/pharmacology, Flavonoids/pharmacology, Humans, Hypotonic Solutions/pharmacology, Indoles/pharmacology, Intestines/*enzymology, Iodine Radioisotopes/metabolism, Maleimides/pharmacology, Phosphorylation, Rubidium Radioisotopes/metabolism, Signal Transduction/*physiology, Tetradecanoylphorbol Acetate/pharmacology, ras Proteins/*physiology
hdl.handle.net/1765/8810
Biochemical Journal
Erasmus MC: University Medical Center Rotterdam

van der Wijk, Th, Dorrestijn, J, Narumiya, S, Maassen, J.A, de Jonge, H.R, & Tilly, B.C. (1998). Osmotic swelling-induced activation of the extracellular-signal-regulated protein kinases Erk-1 and Erk-2 in intestine 407 cells involves the Ras/Raf-signalling pathway. Biochemical Journal. Retrieved from http://hdl.handle.net/1765/8810