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 signa...
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Published in | The Journal of biological chemistry Vol. 277; no. 13; pp. 11077 - 11083 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
United States
Elsevier Inc
29.03.2002
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Abstract | 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. |
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AbstractList | Receptor activator of NF- Kappa B 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 super(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 super(307) correlated with expression of the target gene tartrate-resistant acid phosphatase. MITF phosphorylation at Ser super(307) also correlated with persistent activation of p38 MAPK, and p38 MAPK could utilize MITF Ser super(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 super(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. 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. 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. |
Author | Mansky, Kim C. Sankar, Uma Han, Jiahuai Ostrowski, Michael C. |
Author_xml | – sequence: 1 givenname: Kim C. surname: Mansky fullname: Mansky, Kim C. organization: Department of Molecular Genetics and the Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210 – sequence: 2 givenname: Uma surname: Sankar fullname: Sankar, Uma organization: Department of Molecular Genetics and the Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210 – sequence: 3 givenname: Jiahuai surname: Han fullname: Han, Jiahuai organization: Department of Immunology, The Scripps Research Institute, La Jolla, California 92037 – sequence: 4 givenname: Michael C. surname: Ostrowski fullname: Ostrowski, Michael C. email: ostrowski.4@osu.edu organization: Department of Molecular Genetics and the Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210 |
BackLink | https://www.ncbi.nlm.nih.gov/pubmed/11792706$$D View this record in MEDLINE/PubMed |
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Snippet | Receptor activator of NF-κB ligand (RANKL) activates signaling pathways that regulate osteoclast differentiation, function, and survival. The microphthalmia... Receptor activator of NF-kappaB ligand (RANKL) activates signaling pathways that regulate osteoclast differentiation, function, and survival. The... Receptor activator of NF- Kappa B ligand (RANKL) activates signaling pathways that regulate osteoclast differentiation, function, and survival. The... |
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SubjectTerms | Amino Acid Sequence Carrier Proteins - metabolism Cell Differentiation Cell Line DNA-Binding Proteins - metabolism MAP Kinase Signaling System Membrane Glycoproteins - metabolism Microphthalmia-Associated Transcription Factor Mitf protein Mitogen-Activated Protein Kinases - metabolism MKK6 protein Molecular Sequence Data Osteoclasts - cytology Osteoclasts - enzymology Osteoclasts - metabolism p38 Mitogen-Activated Protein Kinases Phosphorylation rac1 GTP-Binding Protein - metabolism RANK Ligand RANKL protein Signal Transduction TRANCE protein Transcription Factors |
Title | Microphthalmia Transcription Factor Is a Target of the p38 MAPK Pathway in Response to Receptor Activator of NF-κB Ligand Signaling |
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