No abstract

Several clonal cell lines from a transplantable rat osteosarcoma, selected on the basis of parathyroid hormone (PTH)-sensitive adenylate cyclase, were established in culture. Bovine PTH-(1-84) (0.1 microM) stimulation of adenylate cyclase varied among clones from 8-fold to none. The level of PTH response was a stable property of each clonal line that was retained through numerous passages in vitro (nearly 3 yr in the oldest clone). Highly PTH-responsive lines had a cuboidal-eliptoid morphology and differed from the nonresponsive lines, which had a more fibroblastic appearance. PTH responsiveness correlated with several properties, presumably associated with the osteoblastic phenotype: elevated alkaline phosphatase activity, synthesis of the gamma-carboxyglutamic acid-containing bone protein, and production of mineralized tumors in host rats. PTH (1.0 nM; 24 h) reduced the alkaline phosphatase activity by 40% when tested in a responsive clone. The acid phosphatase activity of the various cell lines was uniformly low. These osteosarcoma-derived cell lines which are stable in vitro thus seem to reflect the phenotypic heterogeneity observed in the tumor in situ. They could be useful in studies of phenotypic expression, PTH action, and a possible relationship between the two.

PGE, and PGE2 are potent stimulators of bone formation. Osteogenesis is strongly dependent on angiogenesis. Vascular endothelial growth factor (VEGF), a secreted endothelial cellspecific mitogen, has been implicated in physiological and pathological angiogenesis. The aim of this study was to examine the possible role of VEGF in PG stimulation of bone formation. We found that in rat calvaria-derived osteoblast-enriched cells and in the osteoblastic RCT-3 cell line PGE2 and El increased VEGF mRNA and protein levels. The increased expression of VEGF mRNA produced by PGE2 was rapid (maximal at 1 h), transient (declined by 3 h), potentiated by cycloheximide, and abolished by actinomycin D. PGE2 had no effect on VEGF mRNA stability, suggesting transcriptional regulation ofVEGF expression by PGE2. Rp-cAMP, a cAMP antagonist, suppressed VEGF mRNA induced by PGE2, indicating cAMP mediation. The upregulation of VEGF expression by PGE2 in the preosteoblastic RCT-1 cells was potentiated by treatment with retinoic acid, which induces the differentiation of these cells. The upregulation of VEGF mRNA by PGE2 was inhibited by dexamethasone treatment. In addition, Northern blot analysis showed that VEGF mRNA is expressed in adult rat tibia. In summary, we documented, for the first time, the expression of VEGF in osteoblasts and in bone tissue. Stimulation of VEGF expression by PGs and its suppression by glucocorticoids, which, respectively, stimulate and suppress bone formation, strongly implicate the involvement of VEGF in bone metabolism. (J. Clin. Invest. 1994.93:2490-2496

Integrins are heterodimeric adhesion receptors that mediate cell-matrix interaction. Osteoclast exhibits high expression of the alpha(v)beta(3) integrin, which binds to a variety of extracellular matrix proteins including vitronectin, osteopontin, and bone sialoprotein. Arg-Gly-Asp (RGD)-containing peptides, RGD-mimetics, and blocking antibodies to alpha(v)beta(3) integrin were shown to inhibit bone resorption in vitro and in vivo, suggesting that this integrin may play an important role in regulating osteoclast function. Several lines of evidence have demonstrated that a number of signaling molecules are involved in the alpha(v)beta(3) integrin-dependent signaling pathway, including c-Src, Pyk2, c-Cbl, and p130(Cas). In this article, we review the history of "alpha(v)beta(3) integrin and osteoclasts" and discuss the involvement of alpha(v)beta(3) integrins in osteoclast function at tissue, cellular, and molecular levels. A better understanding of the role of alpha(v)beta(3) integrin in osteoclastic bone resorption would provide opportunities for developing new therapeutics to treat human bone diseases, including rheumatoid arthritis, osteoporosis, and periodontal disease.

Prostaglandin (PG) E(2) is a potent inducer of cortical and trabecular bone formation in humans and animals. Although the bone anabolic action of PGE(2) is well documented, the cellular and molecular mechanisms that mediate this effect remain unclear. This study was undertaken to examine the effect of pharmacological inactivation of the prostanoid receptor EP(4), one of the PGE(2) receptors, on PGE(2)-induced bone formation in vivo. We first determined the ability of EP(4)A, an EP(4)-selective ligand, to act as an antagonist. PGE(2) increases intracellular cAMP and suppresses apoptosis in the RP-1 periosteal cell line. Both effects were reversed by EP(4)A, suggesting that EP(4)A acts as an EP(4) antagonist in the cells at concentrations consistent with its in vitro binding to EP(4). We then examined the effect of EP(4) on bone formation induced by PGE(2) in young rats. Five- to 6-week-old rats were treated with PGE(2) (6 mg/kg/day) in the presence or absence of EP(4)A (10 mg/kg/day) for 12 days. We found that treatment with EP(4)A suppresses the increase in trabecular bone volume induced by PGE(2). This effect is accompanied by a suppression of bone formation indices: serum osteocalcin, extent of labeled surface, and extent of trabecular number, suggesting that the reduction in bone volume is due most likely to decreased bone formation. The pharmacological evidence presented here provides strong support for the hypothesis that the bone anabolic effect of PGE(2) in rats is mediated by the EP(4) receptor.