The Na+/H+ Exchanger, NHE1, Differentially Regulates Mitogen-Activated Protein Kinase Subfamilies after Osmotic Shrinkage in Ehrlich Lettre Ascites Cells

Abstract

Osmotic stress modulates mitogen activated protein kinase (MAPK) activities, leading to altered gene transcription and cell death/survival balance, however, the mechanisms involved are incompletely elucidated. Here, we show, using a combination of biochemical and molecular biology approaches, that three MAPKs exhibit unique interrelationships with the Na⁺/H⁺ exchanger, NHE1, after osmotic cell shrinkage: Extracellular Signal Regulated Kinase (ERK1/2) is inhibited in an NHE1-dependent, pH_i-independent manner, c-Jun N-terminal kinase (JNK1/2) is stimulated, in part through NHE1-mediated intracellular alkalinization, and p38 MAPK is activated in an NHE1-independent manner, and contributes to NHE1 activation and ERK inhibition. Shrinkage-induced ERK1/2 inhibition was attenuated in Ehrlich Lettre Ascites cells by NHE1 inhibitors (EIPA, cariporide) or removal of extracellular Na⁺, and mimicked by human (h) NHE1 expression in cells lacking endogenous NHE1 activity. The effect of NHE1 on ERK1/2 was pH_i-independent and upstream of MEK1/2. Shrinkage-activation of JNK1/2 was attenuated by EIPA, augmented by hNHE1 expression, and abolished in the presence of HCO₃^-. Basal JNK activity was augmented at alkaline pH_i. Shrinkage-activation of p38 MAPK was NHE1-independent, and p38 MAPK inhibition (SB203580) attenuated NHE1 activation and ERK1/2 inhibition. Long-term shrinkage elicited caspase-3 activation and a loss of cell viability, which was augmented by ERK1/2 or JNK1/2 inhibition, and attenuated by p38 MAPK inhibition.