The parathyroid hormone-related protein stimulates human osteoblast-like cells to secrete a 9,000 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(-8) M h(1-74) 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 less than 0.05]. After centrifugation and dialysis, 1 liter of treated CM contained a total BRA of 7102 ngeq b(1-34) PTH with a specific activity (SA) of 447 ngeq b(1-34) PTH/mg protein. Treated CM did not stimulate the ROS assay and the cytokines PGE2, TGF-alpha, 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(1-34) 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.
Morris
C A
CA
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
Mitnick
M E
ME
Weir
E C
EC
Horowitz
M
M
Kreider
B L
BL
Insogna
K L
KL
D001685:Biological Factors / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D016207:Cytokines; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008970:Molecular Weight; D010006:Osteoblasts / Q000378:metabolism*; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins / Q000494:pharmacology*
D001685:Biological Factors; D003470:Culture Media; D016207:Cytokines; C470121:PTHLH protein, human; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; C025940:bone resorption factor