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.

Physiology of Cell Volume Regulation in Vertebrates

“…In some cell types, the shrinkage-induced decrease in ERK1/2 activity is dependent on the concomitant increase in p38 MAPK activity (249,1139). This was also the case in ELA cells, pointing to a necessary role of both NHE1 and p38 MAPK (788). It is interesting to note that the suppressors of ERK signaling, Sprouty (Spry) and Spred, are targeted to the plasma membrane by PtdIns(4,5)P 2 after Rac activation and that this is essential for their inhibition of ERK signaling (569).…”

“…The specific pattern of MAPK modulation is highly cell type and condition dependent. This is especially true for ERK1/2 activity, which in some cells is increased (255,483,1007,1133), and in others decreased (249,708,788) by cell shrinkage, whereas cell swelling is generally associated with ERK1/2 stimulation (e.g., Refs. 446, 708, 874) (Fig.…”

“…The signal initiating the formation of this scaffold is unknown, but a role for the actin cytoskeleton as part of the osmosensing system was pro-posed (1014). On the other hand, in other cell types, endogenous MKK3/6 activity was not detectably increased by shrinkage in spite of robust p38 MAPK activation, possibly due to negative autoregulation (757,788).…”

“…In ELA cells in interphase (which exhibit a high basal ERK1/2 activity) (788) and in NIH3T3 cells (249), ERK was strongly inhibited within 5 min of osmotic shrinkage and recovered to normal levels within ϳ1 h. In ELA cells, the inhibitory effect of shrinkage appeared to occur at or upstream of the level of Raf, since both MEK1/2 (788) and Raf (802) activity were also rapidly inhibited under these conditions. Notably, shrinkage-induced inhibition of ERK1/2 was strongly dependent on NHE1 activity (788), in a manner which was at least in part upstream of or at the level of Raf (802).…”

“…Notably, shrinkage-induced inhibition of ERK1/2 was strongly dependent on NHE1 activity (788), in a manner which was at least in part upstream of or at the level of Raf (802). This points to a major role for NHE1 in regulation of ERK activity under these conditions, in accordance with the role of this transporter in regulation of MAPK activity after a variety of other physiological and pathophysiological stimuli (789).…”