Evidence for multiple bone resorption-stimulating factors produced by normal human keratinocytes in culture

Conditioned medium from cultured normal human foreskin keratinocytes enhanced the release of calcium from neonatal mouse calvaria in organ culture. Unfractionated keratinocyte-conditioned medium (KCM) stimulated bone resorption in a dose-dependent manner, but it did not increase the concentration of prostaglandin E₂ (PGE₂) in the bone culture medium until a maximal dose of KCM for resorption was used. Furthermore, inhibitors of PGE₂ synthesis, indomethacin, ibuprofen, and piroxicam, did not inhibit KCM-induced calcium release. High concentrations of KCM increased cAMP production by calvaria in the presence of isobutylmethylxanthine, but the increase was small compared with that produced by a dose of bovine PTH that caused a similar level of bone resorption. The bone resorption-stimulating activity of KCM was not lost after incubation at 56 C for 60 min, but it was lost after heating at 100 C for 10 min. Fractionation of KCM by gel filtration chromatography revealed two distinct peaks of bone resorption-stimulating activity. One peak, KCM(I), caused a significant increase in bone resorption at 2 μg protein/ml. KCM(I) did not increase medium PGE₂, and inhibition of PGE₂ synthesis in bone had no effect on KCM(I)-induced bone resorption. KCM(I) failed to increase cAMP production by human osteosarcoma SaOS-2 cells. Another peak, KCM(II), caused a dose-dependent increase in bone resorption, and a significant increase in medium calcium was noted at a 20-fold lower concentration (0.1 μg protein/ml) than with KCM(I). In contrast to KCM(I), the increase in bone resorption stimulated by KCM(II) was accompanied by a parallel increase in the production of PGE₂, and inhibition of PGE₂ synthesis completely inhibited the bone resorption-stimulating activity of KCM(II). KCM(II) also caused an increase in cAMP production by SaOS-2 cells. We conclude that KCM contains at least two distinct bone resorption-stimulating factors, one of which acts via a PG-mediated mechanism and the other by a PG-independent pathway.