Collagen gel overlay induces two phases of apoptosis in MDCK cells.
We previously demonstrated that collagen gel overlay induced cell remodeling to form lumen and apoptosis in Madin-Darby canine kidney cells. In the present study, we established that collagen gel overlay-induced apoptosis was initiated at areas exclusive of cell remodeling within 24 h (first phase) and extended into areas of cell remodeling within 48 h (second phase). Collagen gel overlay-induced apoptosis was accompanied by selective proteolysis of focal adhesion kinase (FAK), talin, p130(cas), and c-src. Upon collagen gel overlay, FAK was initially degraded into a 90-kDa product during the first phase and subsequently into a 80-kDa product during the second phase. Collagen gel overlay-induced apoptosis of focal adhesion complex proteins and apoptosis of the first phase could be blocked only by a protease inhibitor cocktail. In addition, we found that both DEVD-fmk and ZVAD-fmk inhibited secondary proteolysis of FAK, but only ZVAD-fmk blocked collagen gel overlay-induced apoptosis of the second phase. Finally, collagen gel overlay-induced apoptosis and proteolysis of focal adhesion complex proteins were completely inhibited by the combination of protease inhibitor cocktail and ZVAD-fmk. Taken together, collagen gel overlay induces two phases of apoptosis; the first phase is dependent on proteolysis of focal adhesion complex proteins, and the second phase on activation of caspases.
Wang
Y K
YK
Department of Physiology, National Cheng-Kung University Medical College, Tainan, Taiwan 70101, Republic of China.
Lin
H H
HH
Tang
M J
MJ
D000185:Actinin / Q000378:metabolism; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D003094:Collagen / Q000494:pharmacology*; D050719:Crk-Associated Substrate Protein; D003513:Cycloheximide / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology* / Q000201:enzymology; D051416:Focal Adhesion Protein-Tyrosine Kinases; D007668:Kidney / Q000166:cytology; D009842:Oligopeptides / Q000494:pharmacology; D010436:Pepstatins / Q000494:pharmacology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D050717:Retinoblastoma-Like Protein p130; D016608:Talin / Q000378:metabolism; D016596:Vinculin / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D050719:Crk-Associated Substrate Protein; D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; D010436:Pepstatins; D010750:Phosphoproteins; D011480:Protease Inhibitors; D011500:Protein Synthesis Inhibitors; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D016608:Talin; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C118632:tyrosyl-valyl-alanyl-aspartal; D000185:Actinin; C014195:Streptomyces pepsin inhibitor; D016596:Vinculin; D010664:Phenylmethylsulfonyl Fluoride; D003094:Collagen; D003513:Cycloheximide; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); C031375:pepstatin