Transforming growth factor-beta stimulates bone resorption in neonatal mouse calvariae by a prostaglandin-unrelated but cell proliferation-dependent pathway.

The relationships between bone resorption, prostanoid formation, and cell proliferation in cultured neonatal mouse calvariae stimulated with transforming growth factor-beta (TGF-beta) have been examined. Bone resorption was assessed by analyzing the mobilization of minerals (45Ca, Ca2+., Pi) and the release of 3H from bones prelabeled with [3H]proline. Prostanoid formation was determined by analyzing the amounts of prostaglandin E2 (PGE2) and 6-keto-prostaglandin F1 alpha (the stable breakdown product of PGI2) in culture media. Purified porcine TGF-beta 1 and recombinant human TGF-beta 2 stimulated the release of 45Ca and the formation of prostanoids. The effects were time and dose dependent. The concentrations of TGF-beta 1 and TGF-beta 2 causing half maximal stimulation of 45Ca release were 1 and 0.1 ng/ml, respectively. TGF-beta 1 also enhanced the release of 3H from [3H]proline labeled bones and the mobilization of Ca2+ and Pi from unlabeled bones, as well as the release of lysosomal enzymes (beta-N-acetylglucosaminidase). The degree of stimulation of mineral mobilization and matrix degradation was less than that obtained in bones stimulated with parathyroid hormone or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). TGF-beta 1-induced stimulation of 45Ca release was inhibited by calcitonin, acetazolamide, and the biphosphonate AHPrBP, three different osteoclast inhibitors. In contrast to the escape from calcitonin-induced inhibition seen in parathyroid hormone (PTH)-stimulated bones, the inhibitory effect of calcitonin in TGF-beta 1-stimulated bones persisted in long-term cultures (144 h). The stimulatory effect of TGF-beta 1 was inhibited by anti-TGF-beta 1 and by gamma-interferon (1000 U/ml). Indomethacin (1 microM), flurbiprofen (1 microM), and meclofenamic acid (1 microM) completely abolished the stimulatory effect of TGF-beta 1 on PGE2 and 6-keto-PGF 1 alpha formation without affecting TGF-beta 1-induced stimulation of 45Ca release. Similarly, the stimulatory effect of TGF-beta 2 on 45Ca release was unaffected by indomethacin. In bones in which prostaglandin formation was abolished by indomethacin, a 45Ca release response to TGF-beta 1 was obtained at 12 h. The mitotic inhibitor hydroxyurea inhibited TGF-beta 1 but not PTH-induced 45Ca release. These data demonstrate that TGF-beta 1 and TGF-beta 2 have the capacity to stimulate bone resorption and prostanoid formation in neonatal mouse calvariae, but that the effect of TGF-beta on bone resorption is unrelated to prostanoid formation. In addition, it is shown that bone resorption stimulated by TGF-beta is dependent on cell replication.