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

Expression of thecytokine, receptor activator of NF-κB 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 adenylatecyclase stimulator forskolin, and calcitriol, alone and in combination, on endogenous RANKL protein expression in UMR-106 rat osteoblasticosteosarcoma eel Is by Western blotting and enzyme immunoassay (EIA). PTH and forskolin dose dependently increased a∼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 ∼52 kDa band, but markedly increased the expression of a ∼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 ∼52 and ∼32 kDa RANKL products appear to interact differently with OPG, which could affect responses to the agents in target cells.