Copyright © 2005, European Society of Cardiology
Endoplasmic reticulum Ca2+ depletion induces endothelial cell apoptosis independently of caspase-12
aDepartment of Internal Medicine III, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan
bDepartment of Clinical Pharmacology and Therapeutics, Hamamatsu University School of Medicine. 1-20-1 Handayama, Hamamatsu, 431-3192, Japan
* Corresponding author. Tel.: +81 53 435 2267; fax: +81 53 434 2910. Email address: hwat{at}hama-med.ac.jp
Objective: Apoptosis of endothelial cells is considered an initial step in the development of atherosclerosis. Recent studies have indicated that depletion of the endoplasmic reticulum (ER) Ca2+ content plays an important role in apoptosis. Caspase-12 is a key signal in ER stress-induced apoptosis. However, it is not known whether the depletion of ER Ca2+ is linked to caspase-12 signalling in endothelial cells. Here we have investigated the interaction of Ca2+ signalling and caspase-12 cleavage in apoptosis of endothelial cells.
Methods: Cytosolic Ca2+ concentration ([Ca2+]i) of primary porcine aortic endothelial cells was measured using fura-2/AM. Apoptosis was assessed by DNA fragmentation, and cleavage of caspase-12 using Western blotting techniques.
Results: Thapsigargin (5 µM), an inhibitor of the ER Ca2+-ATPase, depleted ER Ca 2+ content, increased [Ca2+]i, cleaved caspase-12, and induced apoptosis. Bradykinin (10 nM) also increased [Ca2+]i but did not cleave caspase-12 or induce apoptosis. However, when intracellular Ca2+ was chelated with BAPTA/AM (100 µM), bradykinin caused ER Ca2+ depletion and apoptosis without accompanying caspase-12 cleavage. A non-selective caspase inhibitor, z-VAD.fmk (100 µM), inhibited apoptosis and cleavage of caspase-12 stimulated by thapsigargin, while a calpain inhibitor, MDL 28170 (120 µM), inhibited caspase-12 cleavage but not apoptosis.
Conclusions: Thus, increases in intracellular Ca2+ concentration are not sufficient for the induction of apoptosis in endothelial cells, and ER Ca2+ depletion appears to induce apoptosis independently of caspase-12.
KEYWORDS Apoptosis; Calcium; Caspase-12; Endothelial cells
Time for primary review 32 days
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