Src protein and tyrosine-phosphorylated protein profiles in marrow stroma during osteogenic stimulation

Kerem²,

2006

Ther Apher Dial

Atherosclerosis is epidemiologically associated with postmenopausal osteoporosis presumably by common etiologic factors, reflecting a state of comorbidity in aging. Osteoblasts make a significant facet of this comorbidity state. The present study shows that LDL (native and oxidized) separated by conventional density ultracentrifugation induces osteoblast cell growth arrest in culture. Since the density unltracentrifugation is a tedious procedure we examined, in the present study, the option of LDL purification by ionic strength elution from LDL-apheresis cartridges. We tested the ability of LDL and oxidized LDL (oxLDL) from apheresis columns to induce apoptosis in human Saos2 osteoblasts. Isotonic NaCl effluent washed from LDL-apheresis columns (before starting elution of LDL) induced cell proliferation. In some of the effluent fractions that stimulated Saos2 osteoblasts, up to 15% of the stimulation levels could be significantly inhibited with antilipoprotein A antibodies. After the isotonic washing (150 mM NaCl), upon elution with high ionic strength, 0.2-0.3 M NaCl, some front-runner LDL eluate fractions also induced cell growth and others did not inhibit Saos2 cell growth. This indicates that these fractions might have been contaminated with apolipoprotein A or with other mitogenic compounds. In contrast, the late-to-elute (last 1/3) LDL portion, with a mean density of 1.042 g/mL, killed the cells as expected. This suggests that only the very last one third of LDL eluted by high ionic strength (0.3-0.5 M) is free of osteoblast-mitogenic compounds or lipoprotein-A containing particles. This approach to LDL purification might serve as a convenient and economic method for studying the composition of individual LDL particles and their interaction with cells in culture.

Section: Introductionmentioning

confidence: 99%

Preparation of Low Density Lipoprotein from Large Apheresis Cartridges for Induction of Cell Death in Saos2 Osteoblasts

Kerem²,

2006

Ther Apher Dial

Cell death in cultured human Saos2 osteoblasts exposed to low‐density lipoprotein

J of Cellular Biochemistry

Ben‐Yehuda

et al. 2003

Self Cite

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.

LDL induces Saos2 osteoblasts death via Akt pathways responsive to a neutral sphingomyelinase inhibitor

Kerem

J of Cellular Biochemistry

2006

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Atherosclerosis is epidemiologically associated with postmenopausal osteoporosis (OP) presumably by common etiologic factors, reflecting a state of co-morbidity in aging. Osteoblasts make a significant facet of this co-morbidity state. Since oxidized low-density lipoprotein (oxLDL) is a major factor in generation of vascular wall pathology, we examined the ability of native LDL (nLDL) and oxLDL to induce Saos2 osteoblasts growth arrest. OxLDL induced Saos2 cell death with morphological features of apoptosis that was inhibited mainly by caspase-9 and partially by caspase-3 but not by caspase-8 inhibitors. nLDL, like oxLDL, has induced cell death, where 60% (P = 0.00033) and 30% (P = 0.075, ns) of the cell death, respectively, could be inhibited by scyphostatin (a neutral sphingomyelinase [nSMase] inhibitor). Upon similar condition, nLDL inhibited the phosphorylation of Akt and two of its downstream targets, fork head receptor (FKHR) and glycogen synthase kinase-3 (GSK3). This is a pathway that stimulates cell survival and proliferation. nLDL has also induced an increase in the proapoptotic Bcl-Xs and it has diminished the potential antiapoptotic Src kinase activity. At the 4 h time-point, upon a substantial decrease in nLDL-induced Akt phosphorylation, scyphostatin has inhibited the reduction in FKHR and GSK3 phosphorylation but inexplicably not that of Akt. Scyphostatin has also corrected the reduction in Src kinase activity. Taken together, the results indicate that nLDL has induced apoptosis in Saos2 osteoblasts by inactivation of the pathway downstream to Akt using nSMase, and by involvement of Src kinase. Inferring that caspase-9 was the main executioner (rather than caspase-8 and-3) in Saos2 cell death, indicates that the nSMase-induced release of ceramide, directly activated the intrinsic mitochondrial apoptotic pathway. With regard to the Akt inactivation by nLDL, Saos2 osteoblasts responded in an opposite fashion to the response reported by others, in macrophages.