Localized activation of m-calpain in human umbilical vein endothelial cells upon hypoxia.
Bleb formation is an early event of cellular damage observed in a variety of cell types upon hypoxia. Although we previously found the appearance of the localized cytoplasmic ionized Ca(2+) concentration ([Ca(2+)](i)) rise before bleb formation at the same loci of human umbilical vein endothelial cell (HUVEC) upon hypoxia, the mode of [Ca(2+)](i)-rise-induced cytoskeletal alteration remains ill-defined. The aim of this study is to clarify the mechanisms causing bleb formation after localized [Ca(2+)](i) rise. We studied the activation of m-calpain associated with the alteration of cytoskeleton-related proteins, F-actin, mu-actin, or ezrin by employing specific antibodies in conjunction with a confocal laser scanning microscopy (CLSM). Specific antibodies against 80-kDa-preactivated and 78-kDa-activated m-calpain clearly demonstrated redistribution of 80-kDa m-calpain followed by autoproteolytic activation of m-calpain to the 78-kDa form at the same loci of [Ca(2+)](i) rise in hypoxia-treated HUVECs, which was associated with the decrease of ezrin and the localized appearance of beta-actin at the same loci. In conclusion, hypoxia-induced localized [Ca(2+)](i) rise causes bleb formation at the same loci through m-calpain-catalyzed destruction of cross-linking between plasma membrane and actin filaments.
Aono
Y
Y
Department of Surgery and Clinical Oncology, Graduate School of Medicine, Osaka University, Japan.
Ariyoshi
H
H
Tsuji
Y
Y
Ueda
A
A
Tokunaga
M
M
Sakon
M
M
Monden
M
M
D000199:Actins / Q000378:metabolism; D000332:Aerobiosis; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D015687:Cell Hypoxia / Q000502:physiology*; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology / Q000502:physiology*; D004789:Enzyme Activation; D006801:Humans; D018613:Microscopy, Confocal; D010750:Phosphoproteins / Q000378:metabolism; D021381:Protein Transport; D014471:Umbilical Veins; D000099149:Ezrin
D000199:Actins; D002118:Calcium; D002154:Calpain; D003598:Cytoskeletal Proteins; D010750:Phosphoproteins; D000099149:Ezrin