SAPK2/p38-dependent F-actin reorganization regulates early membrane blebbing during stress-induced apoptosis

In endothelial cells, H2O2 induces the rapid formation of focal adhesion co mplexes at the ventral face of the cells and a major reorganization of the actin cytoskeleton into dense transcytoplasmic stress fibers. This change i n actin dynamics results from the activation of the mitogen-activated prote in (MAP) kinase stress-activated protein kinase-2/p38 (SAPK2/p38), which, v ia MAP kinase-activated protein (MAPKAP) kinase-2/3, leads to the phosphory lation of the actin polymerization modulator heat shock protein of 27 kD (H SP27). Here we show that the concomitant activation of the extracellular si gnal-regulated kinase (ERK) MAP kinase pathway by H2O2 accomplishes an esse ntial survival function during this process. When the activation of ERK was blocked with PD098059, the focal adhesion complexes formed under the plasm a membrane, and the actin polymerization activity led to a rapid and intens e membrane blebbing. The blebs were delimited by a thin F-actin ring and co ntained enhanced levels of HSP27. Later, the cells displayed hallmarks of a poptosis, such as DEVD protease activities and internucleosomal DNA fragmen tation. Bleb formation but not apoptosis was blocked by extremely low conce ntrations of the actin polymerization inhibitor cytochalasin D or by the SA PK2 inhibitor SB203580, indicating that the two processes are not in the sa me linear cascade. The role of HSP27 in mediating membrane blebbing was ass essed in fibroblastic cells. In control fibroblasts expressing a low level of endogenous HSP27 or in fibroblasts expressing a high level of a nonphosp horylatable HSP27, H2O2 did not induce F-actin accumulation, nor did it gen erate 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 a nd caused bleb formation when the ERK pathway was inhibited. Cis-platinum, which activated SAPK2 but induced little ERK activity, also induced membran e blebbing that was dependent on the expression of HSP27. In these cells, m embrane blebbing was not followed by caspase activation or DNA fragmentatio n. We conclude that the HSP27-dependent actin polymerization-generating act ivity of SAPK2 associated with a misassembly of the focal adhesions is resp onsible for induction of membrane blebbing by stressing agents.