Receptor activator of NF-kappaB ligand protein expression in UMR-106 cells is differentially regulated by parathyroid hormone and calcitriol

Expression of the cytokine, receptor activator of NF-kappaB ligand (RANKL), is stimulated by both parathyroid hormone (PTH) and calcitriol in osteoblasts. Most studies have examined the effects on RANKL mRNA, and less information is available on the protein products. We have determined the effects of PTH, the adenylate cyclase stimulator forskolin, and calcitriol, alone and in combination, on endogenous RANKL protein expression in UMR-106 rat osteoblastic osteosarcoma cells by Western blotting and enzyme immunoassay (EIA). PTH and forskolin dose dependently increased a approximately 52 kDa band in whole cell lysates that was detected by both C- and N-terminal directed RANKL antibodies. Calcitriol treatment produced little or no expression of this approximately 52 kDa band, but markedly increased the expression of a approximately 32 kDa band that was only detected with an antibody directed to the N-terminus of RANKL. An EIA based on RANKL binding to OPG detected a large increase in RANKL expression following calcitriol treatment, and much smaller increases with PTH or forskolin. The combination of PTH and calcitriol or forskolin and calcitriol elicited effects similar to those of PTH and forskolin alone, as detected by both Western blotting and EIA. In contrast to the effects on protein, all agents increased RANKL mRNA expression, with the greatest effects seen with the co-treatments. The results indicate that PTH, likely through effects on cyclic AMP, has a different effect on RANKL processing than calcitriol. The approximately 52 and approximately 32 kDa RANKL products appear to interact differently with OPG, which could affect responses to the agents in target cells.