Osmotic stimulation of the Na+/H+ exchanger NHE1: relationship to the activation of three MAPK pathways.
The Na+/H+ exchanger (NHE) becomes activated by hyperosmolar stress, thereby contributing to cell volume regulation. The signaling pathway(s) responsible for the shrinkage-induced activation of NHE, however, remain unknown. A family of mitogen-activated protein kinases (MAPK), encompassing p42/p44 Erk, p38 MAPK and SAPK, has been implicated in a variety of cellular responses to changes in osmolarity. We therefore investigated whether these kinases similarly signal the hyperosmotic activation of NHE. The time course and osmolyte concentration dependence of hypertonic activation of NHE and of the three sub-families of MAPK were compared in U937 cells. The temporal course and dependence on osmolarity of Erk and p38 MAPK activation were found to be similar to that of NHE stimulation. However, while pretreatment of U937 cells with the kinase inhibitors PD98059 and SB203580 abrogated the osmotic activation of Erk and p38 MAPK, respectively, it did not prevent the associated stimulation of NHE. Thus, Erk1/2 and/or p38 MAPK are unlikely to mediate the osmotic regulation of NHE. The kinetics of NHE activation by hyperosmolarity appeared to precede SAPK activation. In addition, hyperosmotic activation of NHE persisted in mouse embryonic fibroblasts lacking SEK1/MKK4, an upstream activator of SAPK. Moreover, shrinkage-induced activation of NHE still occurred in COS-7 cells that were transiently transfected with a dominant-negative form of SEK1/MKK4 (SEK1/MKK4-A/L) that is expected to inhibit other isoforms of SEK as well. Together, these results demonstrate that the stimulation of NHE and the activation of Erk, p38 MAPK and SAPK are parallel but independent events.
Gillis
D
D
Programme in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
Shrode
L D
LD
Krump
E
E
Howard
C M
CM
Rubie
E A
EA
Tibbles
L A
LA
Woodgett
J
J
Grinstein
S
S
D000818:Animals; D019556:COS Cells; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005347:Fibroblasts; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006982:Hypertonic Solutions; D015151:Immunoblotting; D048369:MAP Kinase Kinase 1; D048670:MAP Kinase Kinase 4; D020935:MAP Kinase Signaling System; D051379:Mice; D048055:Mitogen-Activated Protein Kinase 8; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009994:Osmolar Concentration; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D014162:Transfection; D020298:U937 Cells; D048051:p38 Mitogen-Activated Protein Kinases
D006982:Hypertonic Solutions; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1; D017346:Protein Serine-Threonine Kinases; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D048670:MAP Kinase Kinase 4; C482125:MAP2K1 protein, human; C482559:MAP2K4 protein, human; C482127:Map2k1 protein, mouse; C482562:Map2k4 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases