Previously, we noted that inorganic phosphate (P i ), a major component of bone extracellular matrix, induced osteoblast apoptosis (Meleti, Z., Shapiro, I. M., and Adams, C. S. (2000 Bone adapts to mechanical and physiological stress by a unique form of tissue replacement contained within discrete structures defined as basic multicellular units (2). Within each of these units, the actual process of bone removal is carried out by osteoclasts; replacement bone matrix is synthesized and mineralized by cells of stromal origin, osteoblasts. Examination of resorbing sites in developing skeletal tissues indicates that many of the cells are apoptotic (3). Thus, there is evidence of DNA fragmentation in osteoclasts, osteoblasts, and osteocytes (4, 5). In contrast, in mature skeletal tissues, only about 1-2% of all bone cells are dying or dead. In both the developing and mature skeleton, most of the apoptotic cells are confined to bone remodeling sites or locales of high bone turnover (5-8).How osteoclasts communicate with and regulate the life history of other cells of the basic multicellular unit is a topic of intense debate. It is clear that osteoclast differentiation and activation are dependent on paracrine signals received from stromal cells in the multicellular unit (9). Within the past four years, chemical modulators of these processes have been identified, and recent evidence indicates that growth factors provide survival signals that result in bone cell proliferation and depression of the apoptotic process (5, 6, 10). In addition, it has been demonstrated that a number of pharmacological agents can induce osteoblast apoptosis in vitro (5, 10 -12). Surprisingly, however, little is known of events that promote osteoblast death in situ.Although the resorption process may generate agents that stimulate osteoblast proliferation, it is probable that products of the resorbing bone may also stimulate bone cell death. Recent work from this laboratory has clearly demonstrated that one of the ions present in the bone matrix, inorganic phosphate (P i ), induces apoptosis of cultured human osteoblasts and chondrocytes (1, 13). Since Ca 2ϩ as well as P i are released from the bone apatite lattice during the resorption process, the possibility exists that Ca 2ϩ may influence P i -mediated bone cell apoptosis. To test the hypothesis that this ion pair may trigger the death program, we examine the effect of P i and Ca 2ϩ on human osteoblast-like cells. We ask the questions, Can Ca 2ϩ modulate P i -induced cell death, and, if so, is death mediated by apoptosis? Using a cell culture system, we demonstrate that Ca 2ϩ accentuates the apoptogenic effect of P i . In addition, we provide evidence for the involvement of mitochondria and intracellular Ca 2ϩ in the apoptotic pathway activated by the ion pair.
MATERIALS AND METHODSCell Culture-Specimens of human bone were obtained during dental surgery performed at the Hospital of the University of Pennsylvania and during spinal surgery performed at the Children's Hospital of Philadelphia...