Conditioned medium from ras oncogene-transformed NIH 3T3 cells induces bone resorption in vitro.
Tumor-associated hypercalcemia is due, in part, to enhanced osteoclastic bone resorption induced by soluble factors elaborated from malignant cells. ras transformation of NIH 3T3 cells results in a 50-fold induction of cathepsin L mRNA and secretion of the corresponding protein. Since cathepsin L is an acid proteinase we asked whether conditioned medium from these cells would directly increase calcium release from bone in vitro. We tested conditioned medium obtained after 72 h culture of NIH 3T3 ras-transformed cells (DT) or nontransformed NIH 3T3 cells (3T3) and identical medium not exposed to cells (Ctl). Incubation of either live or dead neonatal mouse calvaria for 48 h in DT-conditioned medium increased calcium release compared to bones incubated with 3T3 medium. In both states the increased calcium release with DT medium was blocked by 0.25 mM E-64, a general cysteine proteinase inhibitor, and 1 microM Z-Phe-Ala-CH2F, a specific inhibitor of cathepsin L activity. Thus, conditioned medium from ras-transformed cells enhances calcium release in both live and dead bone. Since cathepsin L is the major protein secreted by these cells and the effect of DT-conditioned medium is blocked by a specific inhibitor of cathepsin L, these studies suggest that this acid proteinase acts directly on bone mineral to enhance net calcium release.
Krieger
N S
NS
Nephrology Program, Pritzker School of Medicine, University of Chicago 60637.
Sukhatme
V P
VP
Bushinsky
D A
DA
D000818:Animals; D015152:Blotting, Northern; D001862:Bone Resorption / Q000139:chemically induced*; D002118:Calcium / Q000378:metabolism; D056668:Cathepsin L; D002403:Cathepsins / Q000096:biosynthesis; D002461:Cell Line, Transformed; D003470:Culture Media / Q000494:pharmacology*; D003546:Cysteine Endopeptidases / Q000096:biosynthesis; D010450:Endopeptidases; D011905:Genes, ras / Q000502:physiology*; D051379:Mice; D009924:Organ Culture Techniques; D012886:Skull / Q000187:drug effects
D003470:Culture Media; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534278:Ctsl protein, mouse; D002118:Calcium