title: Apoptosis may determine the release of skeletal alkaline phosphatase activity from human osteoblast-line cells.
abstract: Although quantitative measurement of skeletal alkaline phosphatase (sALP) activity in serum can provide an index of the rate of bone formation, the metabolic process that determines the release of sALP - from the surface of osteoblasts, into circulation-is unknown. The current studies were intended to examine the hypothesis that the release of sALP from human osteoblasts is a consequence of apoptotic cell death. We measured the release of sALP activity from human osteosarcoma (SaOS-2) cells and normal human bone cells, under basal conditions and in response to agents that increased apoptosis (TNF-a, okadiac acid) and agents that inhibit apoptosis (IGF-I, calpain, and caspase inhibitors). Apoptosis was determined by the presence of nucleosomes (histone-associated DNA) in the cytoplasm of the cells by using a commercial kit. The results of these studies showed that TNF-a and okadiac acid caused dose- and time-dependent increases in apoptosis in the SaOS-2 cells (r = 0.78 for doses of TNF-a and r = 0.93 for doses of okadiac acid, P <0.005 for each), with associated decreases in cell layer protein (P <0.05 for each) and concomitant increases in the release of sALP activity (e.g., r = 0.89 for TNF-a and r = 0.75 for okadiac acid, P <0.001 for each). In contrast, caspase and calpain inhibitors reduced apoptosis, increased cell layer protein, and decreased the release of sALP activity (P <0.05 for each). Exposure to IGF-I also decreased apoptosis, in a time- and dose-dependent manner (e.g., r = 0.93, P <0.001 for IGF-I doses), with associated proportional effects to increase cell layer protein (P <0.001) and decrease the release of sALP activity (P <0.001). IGF-I also inhibited the actions of TNF-a and okadiac acid to increase apoptosis and sALP release. The associations between apoptosis and sALP release were not unique to osteosarcoma (i.e., SaOS-2) cells, but also seen with osteoblast-line cells derived from normal human bone. Together, these data demonstrate that the release of sALP activity from human osteoblast-line cells in vitro is associated with, and may be a consequence of, apoptotic cell death. These findings are consistent with the general hypothesis that the appearance of sALP activity in serum may reflect the turnover of osteoblast-line cells.
authors.affiliation: Departments of Medicine and Biochemistry, Loma Linda University, and Jerry L. Pettis Memorial Veterans Medical Center, Loma Linda, California 92357, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Farley
authors.forename: B
authors.initials: B
authors.lastname: Stilt-Coffing
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001859:Bone Neoplasms / Q000201:enzymology; D002154:Calpain / Q000494:pharmacology; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D009707:Nucleosomes / Q000187:drug effects / Q000235:genetics; D019319:Okadaic Acid / Q000494:pharmacology; D010006:Osteoblasts / Q000187:drug effects / Q000201:enzymology*; D010012:Osteogenesis / Q000502:physiology*; D012516:Osteosarcoma / Q000201:enzymology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D009707:Nucleosomes; D014409:Tumor Necrosis Factor-alpha; D019319:Okadaic Acid; D007334:Insulin-Like Growth Factor I; D000469:Alkaline Phosphatase; D002154:Calpain