THE PARATHYROID HORMONE-RELATED PROTEIN STIMULATES HUMAN OSTEOBLAST-LIKE CELLS TO SECRETE A 9000 DALTON BONE-RESORBING PROTEIN

The mechanism by which parathyroid hormone-related protein (PTH-RP) stimulates bone resorption is not known. Like certain other resorbing agents it may act to release bone-resorbing cytokines from the osteoblast. To examine this hypothesis, we used serum-free conditioned media (CM) from SAOS II cells incubated with 10-8M h(l-7A) PTH-RP for 48 h. Treated CM contained substantially more bone-resorbing activity (BRA) in the fetal-rat long-bone assay than CM from untreated cells (2.17+0.21 vs 1.38+0.16 fold stimulation over basal [f]; p<0.05)). After centrifugation and dialysis, 1 liter of treated CM contained a total BRA of 7102 ngeq b(l-34) PTH with a specific activity (SA) of 447 ngeq b(l-34) PTH/mg protein. Treated CM did not stimulate the ROS assay and the cytokines PGE2, TGF-α, EGF, GM-CSF and IL-1 were present in low concentrations. The BRA was heat sensitive. Ultrafiltration revealed that 97% of the BRA was in a 3-30 kD fraction. Further purification was achieved by sequential reverse phase HPLC and size exclusion-HPLC (SE-HPLC). A single fraction containing BRA from SE-HPLC was purified 277-fold to a SA of 123, 810 ngeq b(l-34) PTH/mg protein and had an apparent MW of 9 kD. SDS-PAGE revealed 4 bands in this SE-HPLC fraction with 1 band at 9 kD unique to that fraction. PTH-RP may cause bone resorption in part by stimulating the release of a 9 kD protein from osteoblasts which is responsible for activating osteoclasts. © 1990 by The Endocrine Society.