Oxidized low-density lipoprotein induces calpain-dependent cell death and ubiquitination of caspase 3 in HMEC-1 endothelial cells.
Oxidized low-density lipoprotein (oxLDL) is known to induce apoptosis in endothelial cells, and this is believed to contribute to the progression of atherosclerosis. In the present study we made the novel observation that oxLDL-induced death of HMEC-1 cells is accompanied by activation of calpain. The mu-calpain inhibitor PD 151746 decreased oxLDL-induced cytotoxicity, whereas the general caspase inhibitor BAF (t-butoxycarbonyl-Asp-methoxyfluoromethylketone) had no effect. Also, oxLDL provoked calpain-dependent proteolysis of cytoskeletal alpha-fodrin in the HMEC-1 cells. Our observation of an autoproteolytic cleavage of the 80 kDa subunit of mu-calpain provided further evidence for an oxLDL-induced stimulation of calpain activity. The Bcl-2 protein Bid was also cleaved during oxLDL-elicited cell death, and this was prevented by calpain inhibitors, but not by inhibitors of cathepsin B and caspases. Treating the HMEC-1 cells with oxLDL did not result in detectable activation of procaspase 3 or cleavage of PARP [poly(ADP-ribose) polymerase], but it did cause polyubiquitination of caspase 3, indicating inactivation and possible degradation of this protease. Despite the lack of caspase 3 activation, oxLDL treatment led to the formation of nucleosomal DNA fragments characteristic of apoptosis. These novel results show that oxLDL initiates a calpain-mediated death-signalling pathway in endothelial cells.
Pörn-Ares
M Isabella
MI
Division of Experimental Pathology, Department of Laboratory Medicine, Lund University, University Hospital MAS, S-20502 Malmö, Sweden. isabella.ares@exppat.mas.lu.se
Saido
Takaomi C
TC
Andersson
Tommy
T
Ares
Mikko P S
MP
D017209:Apoptosis / Q000187:drug effects; D051023:BH3 Interacting Domain Death Agonist Protein; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D053938:DNA Fragmentation / Q000502:physiology; D010450:Endopeptidases / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology* / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects; D006131:Growth Inhibitors / Q000633:toxicity*; D006801:Humans; D006868:Hydrolysis; D007424:Intracellular Fluid / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000633:toxicity*; D008840:Microfilament Proteins / Q000378:metabolism; D010084:Oxidation-Reduction; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D013049:Spectrin
D051023:BH3 Interacting Domain Death Agonist Protein; D002118:Calcium; D002154:Calpain; D002352:Carrier Proteins; D053148:Caspase 3; D020169:Caspases; D010450:Endopeptidases; D006131:Growth Inhibitors; D008077:Lipoproteins, LDL; D008840:Microfilament Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D025801:Ubiquitin; D013049:Spectrin; C494814:BID protein, human; C099252:oxidized low density lipoprotein; C505407:CASP3 protein, human