Abstract
In this study, we report that the cytoplasmic domain of the integrin beta3 subunit is a target for limited proteolysis during apoptosis of human umbilical vein endothelial cells. Calpain inhibitors inhibited the cleavage of the beta3 cytoplasmic domain, indicating that calpain is required. Calpain-mediated proteolysis of fodrin was also detected, indicating that calpain is activated during endothelial cell apoptosis. A phosphatase inhibitor, sodium orthovanadate, inhibited endothelial cell apoptosis and cleavage beta3, suggesting that protein dephosphorylation preceded integrin cleavage in the apoptosis signaling pathway. beta3 cleavage was observed in cells that were viable, suggesting that it is an early event and not the consequence of post-death proteolysis. The extent of beta3 cleavage correlated with a loss in the capacity of cells to reattach to matrix proteins. Loss of reattachment capacity during apoptosis was significantly retarded by a calpain inhibitor. As the beta3 cytoplasmic domain is required for integrin signaling and interaction with the cytoskeleton, our results suggest that cleavage in the beta3 cytoplasmic domain by calpain or a calpain-like protease negatively regulates integrin-mediated adhesion, signaling, and cytoskeleton association.
Highlights
Programmed cell death or apoptosis is a cell suicide pathway involved in a variety of physiological and pathological events such as tissue morphogenesis, development, cancer, and neurodegenerative disorders
To determine if and how integrins are regulated during apoptosis, we investigated the possible relationship between intracellular proteases and integrins
We and others have shown previously that endothelial cells undergo apoptosis when cultured in suspension which is characterized by DNA fragmentation and apoptotic morphology [17, 18]
Summary
Materials—Rabbit anti-peptide antibody, anti-3C, is directed against the C-terminal 20 amino acid residues of the 3 cytoplasmic domain. Rabbit antibody anti-3 was produced using purified 3 subunit as an immunogen and is directed against the extracellular domain. These antibodies have been described previously [14]. The cell-permeable calpain inhibitor E64d was purchased from Sigma, and the antibody specific for calpain-cleaved fodrin was described previously [12]. HUVECs were serum-starved by incubation in serum-free medium containing M199 (Life Technologies, Inc.), 0.1% bovine serum albumin (nuclease- and protease-free; Calbiochem), and insulin, selenium, and transferrin (GMS-G; Life Technologies, Inc.). For incubation in suspension culture, cells were detached with 0.01 M Na2HPO4, 0.15 M NaCl, 2.5 mM EDTA, pH 7.4 (PBS/EDTA) and resuspended in EGM containing.
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