Osmotic stress activates Rac and Cdc42 in neutrophils: role in hypertonicity-induced actin polymerization.
Hypertonicity inhibits a variety of neutrophil functions through poorly defined mechanisms. Our earlier studies suggest that osmotically induced actin polymerization and cytoskeleton remodeling is a key component in the hypertonic block of exocytosis and cell movement. To gain insight into the signaling mechanisms underlying the hyperosmotic F-actin response, we investigated whether hypertonicity stimulates Rac and Cdc42 and, if so, whether their activation contributes to the hypertonic rise in F-actin. Using a recently developed pull-down assay that specifically captures the active forms of these small GTPases, we found that hypertonicity caused an ~2.5- and ~7.2-fold activation of Rac and Cdc42, respectively. This response was rapid and sustained. Small GTPase activation was not mediated by the osmotic stimulation of Src kinases, heterotrimeric G proteins, or phosphatidylinositol 3-kinase. Interestingly, an increase in intracellular ionic strength was sufficient to activate Rac even in the absence of cell shrinkage. Inhibition of Rac and Cdc42 by Clostridium difficile toxin B substantially reduced but did not abolish the hypertonicity-induced F-actin response. Thus hypertonicity is a potent activator of Rac and Cdc42, and this effect seems to play an important but not exclusive role in the hyperosmolarity-triggered cytoskeleton remodeling.
Lewis
Alison
A
Department of Surgery, The Toronto General Hospital University Health Network, and University of Toronto, Toronto, Ontario, Canada.
Di Ciano
Caterina
C
Rotstein
Ori D
OD
Kapus
András
A
D000199:Actins / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000502:physiology; D006801:Humans; D006982:Hypertonic Solutions / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000378:metabolism; D007477:Ions / Q000378:metabolism; D009997:Osmotic Pressure; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D010766:Phosphorylation; D011108:Polymers / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000502:physiology*; D020744:rac GTP-Binding Proteins / Q000502:physiology*; D019061:src-Family Kinases / Q000502:physiology
D000199:Actins; D006982:Hypertonic Solutions; D007477:Ions; D011108:Polymers; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D019061:src-Family Kinases; D020962:Heterotrimeric GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins