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J. Cell Biol.,
Volume 143, Number 5, November 30, 1998 1361-1373

* Centre de recherche en cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, Québec, G1R 2J6, Canada; and In endothelial cells, H2O2 induces the rapid
formation of focal adhesion complexes at the ventral
face of the cells and a major reorganization of the actin
cytoskeleton into dense transcytoplasmic stress fibers.
This change in actin dynamics results from the activation of the mitogen-activated protein (MAP) kinase
stress-activated protein kinase-2/p38 (SAPK2/p38),
which, via MAP kinase-activated protein (MAPKAP)
kinase-2/3, leads to the phosphorylation of the actin
polymerization modulator heat shock protein of 27 kD
(HSP27). Here we show that the concomitant activation of the extracellular signal-regulated kinase (ERK)
MAP kinase pathway by H2O2 accomplishes an essential survival function during this process. When the activation of ERK was blocked with PD098059, the focal
adhesion complexes formed under the plasma membrane, and the actin polymerization activity led to a
rapid and intense membrane blebbing. The blebs were
delimited by a thin F-actin ring and contained enhanced levels of HSP27. Later, the cells displayed hallmarks of apoptosis, such as DEVD protease activities
and internucleosomal DNA fragmentation. Bleb formation but not apoptosis was blocked by extremely low concentrations of the actin polymerization inhibitor cytochalasin D or by the SAPK2 inhibitor SB203580, indicating that the two processes are not in the same linear cascade. The role of HSP27 in mediating membrane
blebbing was assessed in fibroblastic cells. In control fibroblasts expressing a low level of endogenous HSP27 or in fibroblasts expressing a high level of a nonphosphorylatable HSP27, H2O2 did not induce F-actin
accumulation, nor did it generate membrane blebbing
activity in the presence or absence of PD098059. In
contrast, in fibroblasts that expressed wild-type HSP27
to a level similar to that found in endothelial cells,
H2O2 induced accumulation of F-actin and caused bleb
formation when the ERK pathway was inhibited. Cis-platinum, which activated SAPK2 but induced little
ERK activity, also induced membrane blebbing that was dependent on the expression of HSP27. In these
cells, membrane blebbing was not followed by caspase
activation or DNA fragmentation. We conclude that
the HSP27-dependent actin polymerization-generating activity of SAPK2 associated with a misassembly of
the focal adhesions is responsible for induction of membrane blebbing by stressing agents.
Laboratoire de recherche sur le cancer de la peau CHUL, RC-9700, Sainte-Foy, Québec, Canada
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