title: Cell death in cultured human Saos2 osteoblasts exposed to low-density lipoprotein.
abstract: Osteoporosis (OP) and atherosclerotic-cardiovascular diseases (and possibly dementia) constitute emerging age-related co-morbidity states that might share risk factors. Blood-born lipids, like LDL involved in atherosclerosis and apolipoprotein-E4 (ApoE4) involved in dementia, may also be implicated in development of OP. We examined osteoblast cell lines as a culture model for OP by exposure to lipoproteins. ApoE expression in Saos2 and U2OS osteoblasts was confirmed by PCR. ApoE4 did decrease cell counts relatively to ApoE3, especially in Saos2 cells in which it was less selective for cells with higher alkaline phosphatase (ALP, an osteoblast marker) activity than ApoE3. This associates with ApoE4, being a risk factor for both dementia and OP. Saos2, but not U2OS, showed a decrease in cell counts after 48 h exposure to native LDL (NLDL). Both cell lines had decreased cell counts already after 24 h when exposed to oxidized-LDL (OxLDL) for which Saos2 also showed a higher sensitivity than U2OS. Exposure of Saos2 to both, OxLDL at low concentration (5 microg/ml) and NLDL revealed a shrunken size cell fraction of 17-23% on the fluorescence-activated cell sorter (FACS) analysis. Such shrunken cell fraction was not seen when Saos2 cells were exposed to 50 microg/ml of OxLDL or to OxLDL combined with 10 nM dexamethasone (DEX, a stimulator of osteoprogenitor differentiation). DEX treatment has lysed the cells earlier than 24 h post exposure and has selected more resistant cells that did not show apoptotic shrinkage in the FACS analysis done after 24 h. We interpret this as a failure to detect the apoptotic cell fraction due to their lysis prior to the FACS analysis. Western blots performed at different time points (10 min, 30 min, 4 h, 24 h, and 48 h) under OxLDL + DEX revealed a fall in the positive regulator of pp60Src-kinase phosphotyrosine (pY)418 relative to the DEX controls during the first 4 h. This is consistent with DEX osteogenic induction, known to be negatively regulated by c-Src, although the pY418/pY529 ratios (negative/positive kinase regulation) fell only at the 10 min time point. Contrarily the pY418/pY529 ratio increased, relative to untreated controls, under 5 microg/ml and 50 microg/ml of NLDL at the 4 h time point and under 50 microg/ml NLDL only at the 10 min time point, being consistent with the ability of a higher dose of LDL to antagonize osteoblast differentiation. This could be even more acceptable if the NLDL would have become minimally oxidized during its long purification procedure. Under NLDL, the Bcl-2/Bax ratio was pro-apoptotic at 10 min, 30 min, and 4 h only under 50 microg/ml, whereas under OxLDL + DEX it was pro-apoptotic only after 4 h suggesting that additional pathways contribute to cell death. These results indicate that lipid effects on human osteoblast lines in culture may be used as a model to identify molecular targets shared between OP and atherosclerosis for intervention in this co-morbidity.
authors.affiliation: Laboratory of Experimental Surgery, Hadassah University Hospital, Jerusalem 91120, Israel. byklein@md.huji.ac.il
authors.forename: Benjamin Y
authors.initials: BY
authors.lastname: Klein
authors.forename: N
authors.initials: N
authors.lastname: Rojansky
authors.forename: A
authors.initials: A
authors.lastname: Ben-Yehuda
authors.forename: I
authors.initials: I
authors.lastname: Abou-Atta
authors.forename: S
authors.initials: S
authors.lastname: Abedat
authors.forename: G
authors.initials: G
authors.lastname: Friedman
mesh_terms: D001057:Apolipoproteins E / Q000235:genetics / Q000378:metabolism; D001161:Arteriosclerosis / Q000378:metabolism; D016923:Cell Death; D002460:Cell Line; D003907:Dexamethasone / Q000494:pharmacology; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism / Q000494:pharmacology*; D010006:Osteoblasts / Q000187:drug effects* / Q000502:physiology*; D010024:Osteoporosis / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism; D051028:bcl-2-Associated X Protein; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D001057:Apolipoproteins E; C494826:BAX protein, human; D005938:Glucocorticoids; D008077:Lipoproteins, LDL; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011973:Receptors, LDL; D051028:bcl-2-Associated X Protein; C099252:oxidized low density lipoprotein; D003907:Dexamethasone; D019061:src-Family Kinases