Microphthalmia transcription factor is a target of the p38 MAPK pathway in response to receptor activator of NF-kappa B ligand signaling.
Receptor activator of NF-kappaB 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 Ser(307), 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 Ser(307) correlated with expression of the target gene tartrate-resistant acid phosphatase. MITF phosphorylation at Ser(307) also correlated with persistent activation of p38 MAPK, and p38 MAPK could utilize MITF Ser(307) 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 Ser(307). Dominant negative p38 alpha and beta 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.
Mansky
Kim C
KC
Department of Molecular Genetics and the Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210, USA.
Sankar
Uma
U
Han
Jiahuai
J
Ostrowski
Michael C
MC
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D002454:Cell Differentiation; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051739:Microphthalmia-Associated Transcription Factor; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010010:Osteoclasts / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D010766:Phosphorylation; D053245:RANK Ligand; D015398:Signal Transduction; D014157:Transcription Factors; D048051:p38 Mitogen-Activated Protein Kinases; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D008562:Membrane Glycoproteins; D051739:Microphthalmia-Associated Transcription Factor; D053245:RANK Ligand; D014157:Transcription Factors; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020830:rac1 GTP-Binding Protein