Cell shrinkage as a signal to apoptosis in NIH 3T3 fibroblasts.
Cell shrinkage is a hallmark of the apoptotic mode of programmed cell death, but it is as yet unclear whether a reduction in cell volume is a primary activation signal of apoptosis. Here we studied the effect of an acute elevation of osmolarity (NaCl or sucrose additions, final osmolarity 687 mosmol l(-1)) on NIH 3T3 fibroblasts to identify components involved in the signal transduction from shrinkage to apoptosis. After 1.5 h the activity of caspase-3 started to increase followed after 3 h by the appearance of many apoptotic-like bodies. The caspase-3 activity increase was greatly enhanced in cells expressing a constitutively active G protein, Rac (RacV12A3 cell), indicating that Rac acts upstream to caspase-3 activation. The stress-activated protein kinase, p38, was significantly activated by phosphorylation within 30 min after induction of osmotic shrinkage, the phosphorylation being accelerated in fibroblasts overexpressing Rac. Conversely, the activation of the extracellular signal-regulated kinase (Erk1/2) was initially significantly decreased. Subsequent to activation of p38, p53 was activated through serine-15 phosphorylation, and active p53 was translocated from the cytosol to the nucleus. Inhibition of p38 in Rac cells reduced the activation of both p53 and caspase-3. After 60 min in hypertonic medium the rate constants for K+ and taurine efflux were increased, particular in Rac cells. We suggest the following sequence of events in the cell shrinkage-induced apoptotic response: cellular shrinkage activates Rac, with activation of p38, followed by phosphorylation and nuclear translocation of p53, resulting in permeability increases and caspase-3 activation.
Department of Biochemistry, Institute of Molecular Biology and Physiology, The August Krogh Building, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen, Denmark.
Martin B
MB
Friis
Christel R
CR
Friborg
Linda
L
Schneider
Maj-Britt
MB
Nielsen
Ian H
IH
Lambert
Søren T
ST
Christensen
Else K
EK
Hoffmann
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000502:physiology; D048429:Cell Size; D019204:GTP-Binding Proteins / Q000378:metabolism*; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D041681:NIH 3T3 Cells; D009997:Osmotic Pressure; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D014882:Water-Electrolyte Balance / Q000502:physiology*
D016159:Tumor Suppressor Protein p53; D020928:Mitogen-Activated Protein Kinases; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D019204:GTP-Binding Proteins