Sphingosine kinase mediates cyclic AMP suppression of apoptosis in rat periosteal cells.

Prostaglandin E stimulates bone formation in humans and animals, and increases intracellular cAMP in osteoblastic cells. We found that cAMP inhibits apoptosis in osteoblastic cells, and examined the mechanism of this effect. We report that the cAMP elevating agent, forskolin, increases cell number in the rat periosteal cell line (RP-11), by suppressing apoptosis in a cell type-specific manner. In RP-11, forskolin transiently up-regulates extracellular signal-regulated kinase activity, a known suppressor of apoptosis. PD98059, a selective inhibitor of the extracellular signal-regulated kinase pathway, only partially reverses the antiapoptotic effect of forskolin, which suggests an additional mechanism for cAMP action. We found that forskolin stimulates cytosolic sphingosine kinase (SPK) activity in these cells; in two other osteoblastic cell lines, however, forskolin does not suppress apoptosis. In contrast to the partial opposing effect of PD98059 to forskolin action, N, N-dimethylsphingosine, a specific inhibitor of SPK, completely reverses the antiapoptotic effect of forskolin, and has no effect on apoptosis in the absence of forskolin. These findings show for the first time that cAMP activates SPK in a cell-type-specific manner, and suggest that cAMP suppression of apoptosis in RP-11 periosteal cells is mediated by its stimulation of SPK.

Stimulation of mouse osteopontin promoter by v-Src is mediated by a CCAAT box-binding factor.

Osteopontin is an arginine-glycine-aspartic acid-containing cell adhesion protein, which is frequently expressed in transformed cells and is thought to play a role in tumorigenesis. v-Src is a transforming viral oncogene product encoded by Rous sarcoma virus (RSV). We report that v-Src expression in HT1080 fibrosarcoma cells significantly stimulates mouse osteopontin promoter activity. We also determined the v-Src response element in the osteopontin promoter as an inverted CCAAT box located at -53 to -49 from the transcription start site. Mutations of the CCAAT box disrupts protein-DNA interaction and diminishes both v-Src stimulation and basal promoter activity. A CCAAT box-containing fragment corresponding to -155 to -122 of RSV long terminal repeat competed with the -72 to -38 fragment of mouse osteopontin promoter for specific protein binding in the gel shift assay. A polyclonal antibody against CBF, a CCAAT box-binding factor, supershifted in gel shift assays the protein-DNA complex formed by nuclear extract of HT1080 with either the RSV CCAAT box fragment or with the osteopontin -72 to -38 fragment. Moreover, both osteopontin mRNA levels and enhancer activity of CCAAT box-containing -72 to -38 fragment were significantly elevated in v-src-transformed NIH 3T3 cells relative to parental cells. These findings suggest that the elevated osteopontin expression in transformed cells could be due, at least in part, to v-Src stimulation of the osteopontin promoter and that this effect is mediated by a CBF-like factor.

The role of prostaglandins in bone formation.

Prostaglandins of the E series have been shown to be effective inducers of bone formation in vivo. In this study, the effects of PGE2 were evaluated in vivo using subcutaneous administration (3 mg/kg/d for 25 days) to ovariectomized rats or local application in the marrow cavity of tibiae of rats using biodegradable implants (0.13, 1.4 and 32 microg released over 8 days). Systemic treatment of rats with PGE2 stimulated cancellous bone formation in the metaphysis of the proximal tibiae as well as endocortical bone formation and de novo trabecular bone formation in the marrow cavity. Local delivery of PGE2 increased cancellous bone volume in the secondary spongiosa and cortical thickness (at 32 microg). Comparisons of prostanoid effects in vitro, in a bone-derived cell line, showed that PGF2alpha was a better stimulator of DNA synthesis than PGE2. PGF2alpha increased the steady state levels of IGF-I receptor mRNA while PGE2 increased IGF-I expression. Although the mechanism of bone formation by PGE2 is not known at this time, it is clear that PGE2 has powerful local anabolic effects on bone formation in vivo possibly by mediating responses to signals such as changes in mechanical force.