Microphthalmia transcription factor is a target of the p38 MAPK pathway in response to receptor activator of NF-κB ligand signaling

Receptor activator of NF-κB ligand (RANKL) activates signaling pathways that regulate osteoclast differentiation, function, and survival. The microphthalmia transcription factor (MITF) is required for terminal differentiation of osteoclasts. To determine whether MITF could be a target of RANKL signaling, a phosphospecific MITF antibody directed against conserved residue Ser³⁰⁷, a potential mitogen-activated protein kinase (MAPK) site, was produced. Using this antibody, we could demonstrate that MITF was rapidly and persistently phosphorylated upon stimulation of primary osteoclasts with RANKL and that phosphorylation of Ser³⁰⁷ correlated with expression of the target gene tartrate-resistant acid phosphatase. MITF phosphorylation at Ser³⁰⁷ also correlated with persistent activation of p38 MAPK, and p38 MAPK could utilize MITF Ser³⁰⁷ as a substrate in vitro. The phosphorylation of MITF and activation of target gene expression in osteoclasts were blocked by p38 MAPK inhibitor SB203580. In transient transfections, a constitutively active Rac1 or MKK6 gene could collaborate with MITF to activate the tartrate-resistant acid phosphatase gene promoter dependent on Ser³⁰⁷. Dominant negative p38 α and β could inhibit the collaboration between upstream signaling components and MITF in the transient assays. These results indicate that MITF is a target for the RANKL signaling pathway in osteoclasts and that phosphorylation of MITF leads to an increase in osteoclast-specific gene expression.