MKK3/6-p38 MAPK Signaling Is Required for IL-1 and TNF- -Induced RANKL Expression in Bone Marrow Stromal Cells

Coupled bone turnover is directed by the expression of receptor-activated NFB ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG). Proinflammatory cytokines, such as interleukin-1 (IL-1 ) and tumor necrosis factor(TNF) induce RANKL expression in bone marrow stromal cells. Here, we report that IL-1 and TNF-induced RANKL requires p38 mitogen-activating protein kinase (MAPK) pathway activation for maximal expression. Real-time PCR was used to assess the p38 contribution toward IL-1 and TNF-induced RANKL mRNA expression. Steady-state RANKL RNA levels were increased approximately 17-fold by IL-1 treatment and subsequently reduced 70%–90% when p38 MAPK was inhibited with SB203580. RANKL mRNA stability data indicated that p38 MAPK did not alter the rate of mRNA decay in IL-1 -induced cells. Using a RANKL-luciferase cell line receptor containing a 120-kB segment of the 5 flanking region of the RANKL gene, reporter expression was stimulated 4–5-fold by IL-1 or TNFtreatment. IL-1 -induced RANKL reporter expression was completely blocked with specific p38 inhibitors as well as dominant negative mutant constructs of MAPK kinase-3 and -6. In addition, blocking p38 signaling in bone marrow stromal cells partially inhibited IL-1 and TNF-induced osteoclastogenesis in vitro. Results from these studies indicate that p38 MAPK is a major signaling pathway involved in IL-1 and TNF-induced RANKL expression in bone marrow stromal cells.