Overexpression of inducible heat shock protein 70 in Cos-1 cells fails to protect from cytotoxicity of oxidized ldls.
Oxidized low density lipoproteins (OxLDLs) are believed to play a central role in atherogenesis and to possess a wide variety of biological properties; among them, OxLDLs are cytotoxic to cultured vascular cells in that they induce necrosis and apoptosis. Moreover, OxLDLs are known to induce the expression of heat shock protein 70 (Hsp70), a protein that protects cells from several cytotoxic stimuli. To determine whether Hsp70 can protect cells against OxLDL-induced cytotoxicity, COS-1 cells were transfected with a construct containing human Hsp70. A number of cell lines permanently expressing Hsp70 were obtained, 1 of which (cos-Hsp70/10, with high Hsp70 expression) was selected for further studies. Hsp70 overexpression protected cells from toxic stimuli, such as H(2)O(2), UV irradiation, and heat shock, suggesting that the overexpressed protein was functional. When incubated with OxLDLs, however, the clone overexpressing Hsp70 showed a significant decrease in viability, as determined by the [(3)H]adenine release assay (319.8+/-3.16% of control for transfected cells versus 217.6+/-6.08% for control cells exposed to 100 microgram protein/mL of OxLDL), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (12.5+/-0.9% versus 28.9+/-1.99% of control, respectively), and LDH release (48.4+/-0.04% versus 15.2+/-0.06% of control cells). The increased expression of BAX and the decreased expression of Bcl-2 (a proapoptotic and an antiapoptotic protein, respectively) in cos-Hsp70/10 cells and in control cells on incubation with OxLDLs suggested that overexpression of Hsp70 did not confer protection against apoptosis induced by OxLDLs. The analysis of nucleosome content and the nuclear staining with Hoechst 33258 confirmed this finding. These data suggest that overexpression of Hsp70 not only fails to protect COS-1 cells against OxLDL-induced apoptosis but rather confers a higher sensitivity to the cytotoxic action of these lipoproteins. Thus, the Hsp70 response, although induced by OxLDLs, cannot protect cells from lipoprotein toxicity.
Institute of Pharmacological Sciences, University of Milano, Italy.
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Pirillo
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Norata
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Zanelli
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D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects* / Q000528:radiation effects; D005786:Gene Expression Regulation; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008077:Lipoproteins, LDL / Q000633:toxicity*; D009707:Nucleosomes / Q000187:drug effects / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000187:drug effects / Q000378:metabolism; D014162:Transfection; D014466:Ultraviolet Rays; D051028:bcl-2-Associated X Protein
D018840:HSP70 Heat-Shock Proteins; D008077:Lipoproteins, LDL; D009707:Nucleosomes; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C099252:oxidized low density lipoprotein; D006861:Hydrogen Peroxide