Transforming growth factor‐β 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‐β (TGF‐β) have been examined. Bone resorption was assessed by analyzing the mobilization of minerals ( 45 Ca, Ca 2+ , P i ) and the release of 3 H from bones prelabeled with [ 3 H] proline. Prostanoid formation was determined by analyzing the amounts of prostaglandin E 2 (PGE 2 ) and 6‐keto‐prostaglandin F 1α (the stable breakdown product of PGI 2 ) in culture media. Purified porcine TGF‐β 1 and recombinant human TGF‐β 2 stimulated the release of 45 Ca and the formation of prostanoids. The effects were time and dose dependent. The concentrations of TGF‐β 1 and TGF‐β 2 causing half maximal stimulation of 45 Ca release were 1 and 0.1 ng/ml, respectively. TGF‐β 1 also enhanced the release of 3 H from [ 3 H]proline labeled bones and the mobilization of Ca 2+ and P i from unlabeled bones, as well as the release of lysosomal enzymes (β‐N‐acetylglu‐cosaminidase). 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 D 3 (1,25(OH) 2 D 3 ). TGF‐β 1 ‐induced stimulation of 45 Ca release was inhibited by calcitonin, acetazolamide, and the bisphosphonate 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‐β 1 ‐stimulated bones persisted in long‐term cultures (144 h). The stimulatory effect of TGF‐β 1 was inhibited by anti‐TGF‐β 1 and by γ‐interferon (1000 U/ml). Indometacin (1 μM), flurbiprofen (1 μM), and meclofenamic acid (1 μM) completely abolished the stimulatory effect of TGF‐β 1 on PGE 2 and 6‐keto‐PGF 1α formation without affecting TGF‐β 1 ‐induced stimulation of 45 Ca release. Similarly, the stimulatory effect of TGF‐β 2 on 45 Ca release was unaffected by indomethacin. In bones in which prostaglandin formation was abolished by indomethacin, a 45 Ca release response to TGF‐β 1 was obtained at 12 h. The mitotic inhibitor hydroxyurea inhibited TGF‐β 1 but not PTH‐induced 45 Ca release. These data demonstrate that TGF‐β 1 and TGF‐β 2 have the capacity to stimulate bone resorption and prostanoid formation in neonatal mouse calvariae, but that the effect of TGF‐β on bone resorption is unrelated to prostanoid formation. In addition, it is shown that bone resorption stimulated by TGF‐β is dependent on cell replication. (J Bone Miner Res 1996;11:1628‐1639)