Apoptosis may determine the release of skeletal alkaline phosphatase activity from human osteoblast-line cells

Although quantitative measurement of skeletal alkaline phosphatase (sALP) a ctivity in serum can provide an index of the rate of bone formation, the me tabolic process that determines the release of sALP - from the surface of o steoblasts, into circulation-is unknown. The current studies were intended to examine the hypothesis that the release of sALP from human osteoblasts i s a consequence of apoptotic cell death. We measured the release of sALP ac tivity from human osteosarcoma (SaOS-2) cells and normal human bone cells, under basal conditions and in response to agents that increased apoptosis ( TNF-alpha, okadiac acid) and agents that inhibit apoptosis (IGF-I, calpain, and caspase inhibitors). Apoptosis was determined by the presence of nucle osomes (histone-associated DNA) in the cytoplasm of the cells by using a co mmercial kit. The results of these studies showed that TNF-alpha and okadia c acid caused dose- and time-dependent increases in apoptosis in the SaOS-2 cells (r = 0.78 for doses of TNF-alpha and r = 0.93 for doses of okadiac a cid, 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 activit y (e.g., r = 0.89 for TNF-alpha and r = 0.75 for okadiac acid, P < 0.001 fo r each). In contrast, caspase and calpain inhibitors reduced apoptosis, inc reased 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 ass ociated proportional effects to increase cell layer protein (P < 0.001) and decrease the release of sALP activity (P < 0.001). TGF-I also inhibited th e actions of TNF-a and okadiac acid to increase apoptosis and sALP release. The associations between apoptosis and sALP re lease were not unique to os teosarcoma (i.e., SaOS-2) cells, but also seen with osteoblast-line cells d erived from normal human bone. Together, these data demonstrate that the re lease of sALP activity from human osteoblast-line cells in vitro is associa ted with, and may be a consequence of, apoptotic cell death. These findings are consistent with the general hypothesis that the appearance of sALP act ivity in serum may reflect the turnover of osteoblast-line cells.