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Microphthalmia Transcription Factor Is a Target of the p38 MAPK Pathway in Response to Receptor Activator of NF-κB Ligand Signaling

2002; Elsevier BV; Volume: 277; Issue: 13 Linguagem: Inglês

10.1074/jbc.m111696200

ISSN

1083-351X

Autores

Kim C. Mansky, Uma Sankar, Jiahuai Han, Michael C. Ostrowski,

Tópico(s)

NF-κB Signaling Pathways

Resumo

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-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. The osteoclast plays an important role in bone resorption in vertebrates during development and throughout life (1.Teitelbaum S.L. Science. 2000; 289: 1504-1508Crossref PubMed Scopus (3007) Google Scholar). This resorption is counterbalanced by new bone formation from osteoblasts. This process of coupling the actions of the bone-producing cells, the osteoblasts, and the bone-resorbing cells, the osteoclasts, is termed "bone remodeling," a process necessary for maintaining a constant bone mass throughout the lifetime of vertebrate organisms. Disruption of this process in humans results in diseases of bone, including osteoporosis and hypercalcemia of malignancy (2.Raisz L.G. Disorders of Bone and Mineral Metabolism. Raven Press, New York1992: 287-311Google Scholar,3.Mundy G.R. Raisz L.G. Cooper R.A. Schecter G.P. Salmon S.E. N. Engl. J. Med. 1974; 291: 1041-1046Crossref PubMed Scopus (510) Google Scholar).Osteoclasts differentiate from cells of the monocyte/macrophage lineage to become multinuclear, tartrate-resistant acid phosphatase (TRAP) 1The abbreviations used are: TRAPtartrate-resistant acid phosphataseRANKreceptor activator of NF-κBRANKLRANK ligandMAPKmitogen-activated protein kinaseMITFmicrophthalmia transcription factorCSFcolony-stimulating factorHAhemagglutininRIPAradioimmune precipitation assayMops4-morpholinepropanesulfonic acidGSTglutathione S-transferaseOCLosteoclast 1The abbreviations used are: TRAPtartrate-resistant acid phosphataseRANKreceptor activator of NF-κBRANKLRANK ligandMAPKmitogen-activated protein kinaseMITFmicrophthalmia transcription factorCSFcolony-stimulating factorHAhemagglutininRIPAradioimmune precipitation assayMops4-morpholinepropanesulfonic acidGSTglutathione S-transferaseOCLosteoclast-positive cells capable of resorbing bone (4.Roodman G.D. Exp. Hematol. 1999; 27: 1229-1241Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). Osteoclast differentiation is influenced by hormones and local factors produced by the osteoblasts and stromal cells (4.Roodman G.D. Exp. Hematol. 1999; 27: 1229-1241Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). One local factor expressed by osteoblasts is receptor activator of NF-κB ligand (RANKL) (5.Dougall W.C. Glaccum M. Charrier K. Rohrbach K. Brasel K. DeSmedt T. Daro E. Smith J. Tometsko M.E. Maliszewski C.R. Armstrong A. Shen V. Bain S. Cosman D. Anderson D. Morrissey P.J. Peschon J.J. Schuh J. Genes Dev. 1998; 13: 2412-2424Crossref Scopus (1188) Google Scholar, 6.Hsu, H., Lacey, D. L., Dunstan, C. R., Solovyev, I., Colombero, A., Timms, E., Tan, H.-L, Elliot, G., Kelley, M. J., Sarosi, I., Wang, L., Xia, X.-Z., Elliot, R., Chiu, L., Black, T., Scully, S., Caparelli, C., Morony, S., Shimamoto, G., Bass, M. B., and Boyle, W. J. @@[email protected]@Proc. Natl. Acad. Sci. U. S. [email protected]@/[email protected]@, 96, 3540–3545Google Scholar, 7.Lacey D.L. Timms E. Tan H.-L Kelley M.J. Dunstan C.R. Burgess T. Elliot R. Colombero A. Elliot G. Scully S. Hsu H. Sullivan J. Hawkins N. Davy E. Capparelli C. Eli A. Qian Y.-X. Kaufman S. Sarosi I. Shalhoub V. Senaldi G. Guo J. Delaney J. Boyle W.J Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4582) Google Scholar, 8.Kong Y.Y. Yoshida H. Sarosi I. Tan H.-L. Timms E. Capparelli C. Morony S. Oliveira S.A. Van G.I. Khoo W. Wakeham A. Dunstan C.R. Lacey D.L Mak T.W. Boyle W.J. Penninger J.M. Nature. 1999; 397: 315-323Crossref PubMed Scopus (2834) Google Scholar, 9.Li J. Sarosi I. Yan X-Q. Morony S. Capparelli C. Tan H.-L. McCabe S. Elliot R. Scully S. Van G. Kaufman S. Juan S.-C. Sun Y. Tarpley J. Martin L. Christensen K. McCabe J. Kostenuik P. Hsu H. Fletcher F. Dunstan C.R. Lacey D.L. Boyle W.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 97: 1566-1571Crossref Scopus (936) Google Scholar, 10.Yasuda H. Shima N. Nakagawa N. Yamaguchi K. Kinosaki M. Mochizuki S.-I. Tomoyasu A. Yano K. Goto M. Murakami A. Tsuda E. Morinaga T. Higashio K. Udagawa N Takahashi N. Suda T. Proc. Natl. Acad. Sci. U. S. A. 2000; 95: 3597-3602Crossref Scopus (3526) Google Scholar). RANKL is a member of the tumor necrosis factor superfamily that is important for maturation of T- and B-cells as well as osteoclasts (7.Lacey D.L. Timms E. Tan H.-L Kelley M.J. Dunstan C.R. Burgess T. Elliot R. Colombero A. Elliot G. Scully S. Hsu H. Sullivan J. Hawkins N. Davy E. Capparelli C. Eli A. Qian Y.-X. Kaufman S. Sarosi I. Shalhoub V. Senaldi G. Guo J. Delaney J. Boyle W.J Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4582) Google Scholar). Mice containing a targeted deletion in either the RANK receptor or RANKL lack differentiated osteoclasts and develop severe osteopetrosis (7.Lacey D.L. Timms E. Tan H.-L Kelley M.J. Dunstan C.R. Burgess T. Elliot R. Colombero A. Elliot G. Scully S. Hsu H. Sullivan J. Hawkins N. Davy E. Capparelli C. Eli A. Qian Y.-X. Kaufman S. Sarosi I. Shalhoub V. Senaldi G. Guo J. Delaney J. Boyle W.J Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4582) Google Scholar, 8.Kong Y.Y. Yoshida H. Sarosi I. Tan H.-L. Timms E. Capparelli C. Morony S. Oliveira S.A. Van G.I. Khoo W. Wakeham A. Dunstan C.R. Lacey D.L Mak T.W. Boyle W.J. Penninger J.M. Nature. 1999; 397: 315-323Crossref PubMed Scopus (2834) Google Scholar, 9.Li J. Sarosi I. Yan X-Q. Morony S. Capparelli C. Tan H.-L. McCabe S. Elliot R. Scully S. Van G. Kaufman S. Juan S.-C. Sun Y. Tarpley J. Martin L. Christensen K. McCabe J. Kostenuik P. Hsu H. Fletcher F. Dunstan C.R. Lacey D.L. Boyle W.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 97: 1566-1571Crossref Scopus (936) Google Scholar).RANKL/RANK signal through tumor necrosis factor receptor-associated factors to activate multiple signaling pathways thought to be important for osteoclast differentiation and function, including NF-κB, mitogen-activated protein kinase (MAPK) pathways, Src kinase, and phosphatidylinositol 3-kinase pathways (11.Wong B.R. Besser D. Kim N. Arron J. Vologodskaia M. Hanafusa H. Choi Y. Mol. Cell. 1999; 4: 1041-1049Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar, 12.Wong B.R. Josien R. Lee S.Y. Yologodskaia M. Steinman R.M. Choi Y. J. Biol. Chem. 1998; 273: 28355-28359Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). Recently, it has been shown that inhibition of the p38 signaling pathway in bone marrow-derived osteoclast precursor cells by treatment with the drug SB203580 inhibited the formation of multinuclear, functional osteoclasts that expressed TRAP in response to RANKL treatment (13.Matsumoto M. Sudo T. Osada H. Tsujimoto M. J. Biol. Chem. 2000; 275: 31155-31161Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar). In contrast, the drug PD98059, a specific inhibitor of the MAPK p42/44 pathway, had no effect on the differentiation of bone marrow cells. These results indicate that the p38 MAPK signaling pathway is involved in RANKL-induced differentiation of bone marrow-derived precursor cells (13.Matsumoto M. Sudo T. Osada H. Tsujimoto M. J. Biol. Chem. 2000; 275: 31155-31161Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar).The microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix leucine zipper protein (14.Hodgkinson C.A. Moore K.J. Nakayama A. Steingrimsson E. Copeland N.G. Jenkins N.A. Arnheiter H. Cell. 1993; 74: 395-404Abstract Full Text PDF PubMed Scopus (940) Google Scholar, 15.Hughes M.J. Lingrel J.B. Krakowsky J.M. Anderson K.P. J. Biol. Chem. 1993; 268: 20687-20690Abstract Full Text PDF PubMed Google Scholar) closely related to the transcription factors TFE3, TFEB, and TFEC (16.Beckman H. Su L.K. Kadesch T. Genes Dev. 1990; 4: 167-179Crossref PubMed Scopus (352) Google Scholar, 17.Carr C.S. Sharp P.A. Mol. Cell. Biol. 1990; 10: 4384-4388Crossref PubMed Scopus (189) Google Scholar, 18.Zhao G.-Q. Zhao Q. Zhou X. Mattei M.-G. De Crombrugghe B. Mol. Cell. Biol. 1993; 13: 4505-4512Crossref PubMed Scopus (106) Google Scholar). MITF regulates osteoclast target genes like TRAP, cathepsin K, and E-cadherin by binding to a 7-base pair conserved sequence TCANGTG found in the promoter regions of these genes (19.Luchin A. Purdom G. Murphy K. Clark M.-Y. Angel N. Cassady A.I. Hume D.A. Ostrowski M.C. J. Bone Miner. Res. 2000; 15: 451-460Crossref PubMed Scopus (107) Google Scholar, 20.Mansky K.C. Marfatia K. Purdom G. Luchin A. Hume D.A. Ostrowski M.C. J. Leukocyte Biol. 2002; 71: 295-303PubMed Google Scholar, 21.Motyckova G. Weilbaecher K.N. Horstmann M.A. Rieman D.J. Fisher D.Z. Fisher D.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5798-5803Crossref PubMed Scopus (190) Google Scholar). In situhybridization experiments confirmed that MITF is expressed in osteoclasts beginning at the earliest stages of endochondral ossification of long bones (19.Luchin A. Purdom G. Murphy K. Clark M.-Y. Angel N. Cassady A.I. Hume D.A. Ostrowski M.C. J. Bone Miner. Res. 2000; 15: 451-460Crossref PubMed Scopus (107) Google Scholar). A mutant allele of the MITF gene, mi, encodes a protein product lacking one of four arginines in the basic region of the MITF protein critical for binding to target genes (14.Hodgkinson C.A. Moore K.J. Nakayama A. Steingrimsson E. Copeland N.G. Jenkins N.A. Arnheiter H. Cell. 1993; 74: 395-404Abstract Full Text PDF PubMed Scopus (940) Google Scholar, 22.Hemesath T.J. Steingrimsson E. McGill G. Hansen M.J. Vaught J. Hodgkinson C.A. Arnheiter H. Copeland N.G. Jenkins N.A. Fisher D.E. Genes Dev. 1994; 8: 2770-2780Crossref PubMed Scopus (545) Google Scholar). Osteoclast-like precursor cells derived from mice homozygous for the mi mutation are incapable of fusing to form multinuclear cells, lack a ruffled border, express low levels of TRAP and cathepsin K, and cannot efficiently resorb bone (19.Luchin A. Purdom G. Murphy K. Clark M.-Y. Angel N. Cassady A.I. Hume D.A. Ostrowski M.C. J. Bone Miner. Res. 2000; 15: 451-460Crossref PubMed Scopus (107) Google Scholar, 21.Motyckova G. Weilbaecher K.N. Horstmann M.A. Rieman D.J. Fisher D.Z. Fisher D.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5798-5803Crossref PubMed Scopus (190) Google Scholar, 23.Thesingh C.W. Scherft J.P. Bone. 1985; 6: 43-52Crossref PubMed Scopus (75) Google Scholar,24.Holtrop M.E. Cox K.A. Elion G. Simmons H.A. Raisz L.G. Metab. Bone Dis. Relat. Res. 1981; 3: 123-129Abstract Full Text PDF PubMed Scopus (42) Google Scholar). Based on the similarity of the phenotype of the bone marrow-derived precursor cells blocked for p38 MAPK activity and the mi/mi osteoclasts-like cells, we hypothesized that MITF may be a target of the p38 MAPK in osteoclasts.In this study, we investigate whether MITF is a potential target of the p38 MAPK in osteoclasts. We show that MITF was persistently phosphorylated on a conserved serine 307 in primary osteoclast-like cells in response to RANKL signaling and that phosphorylation of serine 307 correlated with expression of the target gene, TRAP. Further, p38 MAPK could phosphorylate serine 307 in vitro, and phosphorylation of MITF at serine 307 increased the ability of the factor to stimulate the TRAP promoter activity in transient transfection assays. We conclude that MITF may be a direct target of a RANKL/p38 signaling pathway that is necessary for osteoclast differentiation and function.EXPERIMENTAL PROCEDURESCulture and Analysis of OCLsHematopoietic precursors were obtained from the bone marrow of wild type mice. OCLs were grown in the presence of 50 ng/ml colony-stimulating factor-1 (CSF-1) for 3 days. CSF-1 was lowered to 5 ng/ml, and then RANKL was added at 100 ng/ml for the indicated times.SB203580 (Calbiochem) was used at the indicated concentration, 10 μm, and was applied to the cells 30 min before stimulation with RANKL.DNA Constructs and TransfectionsFor all experiments presented here, the melanocyte form of the MITF cDNA was used (14.Hodgkinson C.A. Moore K.J. Nakayama A. Steingrimsson E. Copeland N.G. Jenkins N.A. Arnheiter H. Cell. 1993; 74: 395-404Abstract Full Text PDF PubMed Scopus (940) Google Scholar,25.Steingrimsson E. Moore K.J. Lamoreux M.L. Rerre-D'Amare A.R. Burley S.K. Zimring D.C.S. Skow L.C. Hodgkinson C.A. Arnheiter H. Copeland N.G. Jenkins N.A. Nat. Genet. 1994; 8: 256-263Crossref PubMed Scopus (440) Google Scholar). The MITF phosphorylation mutant, termed S307A/P308A, was made by replacing conserved serine and proline with alanine residues. The point mutant was verified by sequencing. The full-length form or point mutants were cloned into a vector providing the influenza hemagglutinin tag (HA) (Roche Molecular Biochemicals). Wild type TRAP promoter and the TRAP promoter containing the E box mutation were previously described (19.Luchin A. Purdom G. Murphy K. Clark M.-Y. Angel N. Cassady A.I. Hume D.A. Ostrowski M.C. J. Bone Miner. Res. 2000; 15: 451-460Crossref PubMed Scopus (107) Google Scholar).Bacterial expression constructs of His-tagged MKK6b(E) and p38α (26.Jiang Y. Gram H. Zhao M. New L. Gu J. Feng L. Padova F.D. Ulevitch R.J. Han J. J. Biol. Chem. 1997; 272: 30122-30128Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar), dominant negative expression plasmids for p38 α, β, γ, and δ isoforms (27.Zhao M. New L. Kravchenko V.V. Kato Y. Gram H. Padova F.D. Olson E.N. Ulevitch R.J. Han J. Mol. Cell. Biol. 1999; 19: 21-30Crossref PubMed Scopus (376) Google Scholar), and MKK6(E) expression plasmid were all previously described (28.Han J. Lee J.-D. Jiang Y. Li Z. Feng L. Ulevitch R.J. J. Biol. Chem. 1996; 271: 2886-2891Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar).DNA transfections of RAW 264 cells have been previously described (19.Luchin A. Purdom G. Murphy K. Clark M.-Y. Angel N. Cassady A.I. Hume D.A. Ostrowski M.C. J. Bone Miner. Res. 2000; 15: 451-460Crossref PubMed Scopus (107) Google Scholar).Construction of Cell LinesRAW 264.7 cells were transfected with Superfect (Qiagen) following the manufacturer's directions with either the plasmid expressing HA-tagged S73AMITF or HA-tagged S73A/S307A/P308AMITF, and cells were selected using 100 μg/ml Geneticin (Invitrogen). Individual clones were picked and screened for expression of HA-MITF.Preparation of Recombinant ProteinsRecombinant p38 isoforms and MKK6(E) were prepared by a previously described method (27.Zhao M. New L. Kravchenko V.V. Kato Y. Gram H. Padova F.D. Olson E.N. Ulevitch R.J. Han J. Mol. Cell. Biol. 1999; 19: 21-30Crossref PubMed Scopus (376) Google Scholar). MITF corresponding to amino acids 297–377 (nucleotides 1018–1263) was cloned into pGEX2t (Pharmacia) and was purified to 95% purity by glutathione Sepharose affinity chromatography. Versions of the protein with either serine or alanine at amino acid position 307 were produced and purified.RANKL Bacterial Expression and PurificationRANKL corresponding to amino acids 158–316 (nucleotides 472–978) was placed into vector pET 32b (Novagene), purified by native lysis to 95% purity by nickel-Sepharose (Qiagen) affinity chromatography and eluted in imidazole. The concentration of soluble RANKL was optimized for induction of p38 phosphorylation and formation of TRAP-positive osteoclasts with each preparation.Immunological Reagents and AnalysisThe peptide PSTGLSpSPDLVN (where pS represents phosphoserine), corresponding to amino acids 301–312 of MITF was synthesized (QCB/BIOSOURCE, Hopkinton, MA). The position of the phosphate at amino acid 307 was confirmed by NMR. The phosphopeptide was used to immunize two New Zealand White rabbits. Collected serum was pooled and passed over a column to which the nonphosphopeptide was coupled, and material that did not bind to this column was collected and passed over a phosphopeptide affinity column. Bound material was eluted from this second column with glycine buffer, dialyzed against phosphate-buffered saline, and stored at −70 °C. Polyclonal phosphorylation-independent MITF antibody has been described previously (20.Mansky K.C. Marfatia K. Purdom G. Luchin A. Hume D.A. Ostrowski M.C. J. Leukocyte Biol. 2002; 71: 295-303PubMed Google Scholar). p38 α- and β-specific antibodies were previously described (26.Jiang Y. Gram H. Zhao M. New L. Gu J. Feng L. Padova F.D. Ulevitch R.J. Han J. J. Biol. Chem. 1997; 272: 30122-30128Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). Antibodies that recognized p38 MAPK were obtained from New England Biolabs Cell Signaling.Western blotting was performed with nitrocellulose membranes and the ECL detection system (Kirkegaard and Perry Laboratories). For Western blots using the antibodies that recognize total or phosphorylated p38, the manufacturer's protocol was followed. For the phosphospecific MITF antibody, the blot was blocked in TBS (50 mm Tris, pH 7.5, 150 mm NaCl), 0.1% Tween 20, 5% powdered milk for 2 h at room temperature. The blot was washed three times in TBS, 0.1% Tween 20. The phosphospecific MITF antibody was added to fresh blocking buffer and incubated for 2 h at room temperature. The blot was washed three times in TBS, 0.1% Tween 20. The secondary antibody was incubated in TBS, 0.1% Tween 20 for 1 h at room temperature. The blot was washed three times in TBS, 0.1% Tween 20 and developed.For [35S]methionine labeling experiments, cells were starved in Dulbecco's modified Eagle's medium lacking methionine. 90 μCi of [35S]methionine (ICN) was added to the cells, and the cells were labeled for 3 h. Cells were lysed in RIPA (50 mm Tris-HCl, pH 7.6, 125 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate) that included 10 μg/ml antipain, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mm sodium vanadate. Cells were incubated in RIPA at 4 °C for 20 min and then centrifuged at 25,000 rpm for 20 min. Protein G beads (Amersham Biosciences) were added to the lysate, and the antibody (HA from Babco) was added. The immunoprecipitates were incubated overnight at 4 °C. The immunoprecipitates were washed three times in RIPA and run on an 8% SDS-PAGE.Kinase AssayFull activation of recombinant p38 αin vitro was achieved by incubation with recombinant MKK6(E) at a 5:1 molar ratio at 37 °C for 15 min in the presence of ATP as previously described (27.Zhao M. New L. Kravchenko V.V. Kato Y. Gram H. Padova F.D. Olson E.N. Ulevitch R.J. Han J. Mol. Cell. Biol. 1999; 19: 21-30Crossref PubMed Scopus (376) Google Scholar). In vitro kinase assays were carried out at 37 °C for 15 min, using 0.2 μg of recombinant kinase, 5 μg of GST-MITF, 250 μm ATP, and 12 μCi of [γ-32P]ATP in 20 μl of kinase reaction buffer. Reactions were terminated by the addition of SDS sample buffer. Reaction products were resolved on a 10% SDS-PAGE. Phosphorylated proteins were visualized by autoradiography.p38 MAPK activities were measured in an immune complex kinase assay. RAW264.7 cells were stimulated with RANKL at 100 ng/ml for 15 min. Cells were lysed in RIPA. Cells were incubated in RIPA at 4 °C for 20 min and then centrifuged at 25,000 rpm for 20 min. The immunoprecipitates were incubated with anti-p38 antibody (Cell Signaling Technology) overnight at 4 °C. The immunoprecipitates were collected and washed three times in RIPA and two times with kinase buffer (20 mm Mops, pH 7.5, 25 mm β-glycerol phosphate, 10 mm MgCl2, 1 mmdithiothreitol, 1 mm sodium vanadate). Immunoprecipitates of anti-p38 were mixed with 0.5 μg of GST-MITF, 150 μmATP, and 12 μCi of [γ-32P]ATP in 50 μl of kinase buffer. The reactions were further incubated at 30 °C for 30 min. Reactions were terminated by the addition of SDS sample buffer. Reaction products were resolved on a 10% SDS-PAGE. Phosphorylated proteins were visualized by autoradiography.RESULTSProduction and Characterization of Antibodies Specific for MITF Phosphoserine 307Osteoclasts that are grown in the presence of CSF-1 and RANK ligand but have been blocked for the activation of the p38 MAPK by the specific inhibitor SB203580 remain mononuclear and stain weakly for the osteoclast marker TRAP (13.Matsumoto M. Sudo T. Osada H. Tsujimoto M. J. Biol. Chem. 2000; 275: 31155-31161Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar), a similar phenotype to the osteoclasts that are cultured from the mice homozygous for the mi mutation (23.Thesingh C.W. Scherft J.P. Bone. 1985; 6: 43-52Crossref PubMed Scopus (75) Google Scholar, 24.Holtrop M.E. Cox K.A. Elion G. Simmons H.A. Raisz L.G. Metab. Bone Dis. Relat. Res. 1981; 3: 123-129Abstract Full Text PDF PubMed Scopus (42) Google Scholar). This led us to test the hypothesis that MITF might be a target of the p38 MAPK pathway during osteoclast differentiation. We analyzed the amino acid sequence of MITF and discovered a potential p38 phosphorylation site at serine 307 that is conserved among different species including human, mouse, and chicken (Fig. 1A). However, the serine at amino acid position 307 is not conserved among the other related transcription factors, TFEC, TFE3, and TFEB (29.Rehli M. Elzen N.D. Cassady A.I. Ostrowski M.C. Hume D.A. Genomics. 1999; 56: 111-120Crossref PubMed Scopus (74) Google Scholar).To begin to investigate if MITF was phosphorylated at serine 307 in osteoclasts, an antibody that was specific for the phosphorylated serine at residue 307 was developed (see "Experimental Procedures"). For this purpose, the peptide PSTGLSpSPDLVN (corresponding to amino acids 301–312 of MITF) was synthesized and used to produce polyclonal rabbit serum. Following affinity purification, the specificity of the antibody for detecting phosphoserine 307 MITF in Western blotting experiments was tested with recombinant GST-MITF corresponding to amino acids 297–377. For these experiments, the GST-MITF region was incubated in vitro with activated p38 α and ATP (Fig. 1B). These experiments showed that only GST-MITF that had been incubated with the p38 preparation was recognized by the antibody (Fig. 1B, lane 1 versus lane 2). Recombinant protein containing alanine substituted for serine at amino acid position 307 was not recognized by the phosphospecific antibody in either the presence or absence of activated p38 MAPK. (Fig. 1B, compare lanes 3 and 4 with lane 1). When the blot in Fig. 1B was stripped and reprobed with an antibody specific for the GST moiety, we were able to detect the recombinant protein in all four lanes (Fig. 1B, bottom panel).To further characterize the phospho-Ser307-specific antibody, RAW 264.7 cell lines that stably expressed either HA-tagged MITF or S307A/P308A HA-tagged MITF were created. The cells were stimulated with RANKL for various times, and cell extracts were prepared and immunoprecipitated with the antibody recognizing the HA epitope. The phosphorylation status of MITF Ser307 was determined using the phosphospecific MITF antibody (Fig. 1C, top panel). This analysis demonstrated that Ser307 was phosphorylated following 15, 30, or 60 min with RANKL stimulation but that the antibody did not react with the S307A/P308A MITF protein. The bottom panel of Fig. 1C represents the blot in the top panel reprobed with an antibody that recognizes the HA tag to show approximately equal loading of the HA-containing immunoprecipitates.MITF Is Rapidly and Persistently Phosphorylated in Osteoclasts after RANKL StimulationThe anti-phospho-MITF antibody was used to determine the phosphorylation status of endogenous MITF in primary osteoclast precursors after stimulation by RANKL. Bone marrow-derived osteoclast precursors were obtained from wild type mice and cultured in the presence of CSF-1 for 3 days. At this time, RANKL was added to the bone marrow cell cultures, and nuclear extracts were prepared and analyzed by Western blotting (Fig. 2). Using the anti-phospho-Ser307 antibody, MITF phosphorylation could be detected following 30 min of RANKL stimulation and persisted following 24 h of continuous RANKL treatment (Fig. 2A, upper panel). When the same blot was reprobed with a nondiscriminating MITF antibody, approximately equal levels of MITF protein are seen in all lanes (Fig. 2A, lower panel). MITF appeared as a doublet with electrophoretic mobility of around 55 and 57 kDa in these experiments, and both bands are detected by the anti-phospho-Ser307antibody (Fig. 2A). Previous work in melanocytes has also indicated that MITF is resolved as a doublet in extracts prepared from this cell type and that the upper band is due to c-Kit-mediated phosphorylation of conserved serine residue 73 (30). Ser73is also phosphorylated in osteoclasts in response to CSF-1, accounting for the doublet band pattern in this cell line as well (31.Weilbaecher K.N. Motyckova G. Huber W.E. Takemoto C.M. Hemesath T.J. Xu Y. Hershey C.L. Dowland N.R. Wells A.G. Fisher D.E. Mol. Cell. 2001; 8: 749-758Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). As shown below (see Fig. 5) mutation of Ser73 to alanine results in a loss of the slower migrating band (see Fig. 5).Figure 2RANKL stimulates phosphorylation of MITF at serine 307. A, phosphospecific MITF antibody that recognizes phosphorylated MITF after RANKL stimulation of osteoclasts. The top panel is a Western blot of nuclear extracts from osteoclasts run on an 8% SDS-PAGE and probed using phosphospecific MITF antibody. The numbers abovethe lanes indicate the hours after RANKL stimulation that the extracts were collected. The bottom panel is the same blot in the top panel reprobed with an antibody that recognizes all forms of MITF. B, real time quantitative RT-PCR (Taqman assay) was performed using RNA from bone marrow cultures treated for the same time with RANKL as indicated in A. Both TRAP and glyceraldehyde-3-phosphate dehydrogenase RNA were quantitated. All values in the chart are expressed as the ratio of TRAP to glyceraldehyde-3-phosphate dehydrogenase expression and then further normalized to the CSF-1-only control. C, p38 MAPK is phosphorylated in response to RANKL stimulation in osteoclasts. The top panel is a Western blot of whole cell extracts from osteoclasts, run on a 10% SDS-PAGE and probed using phosphospecific p38 antibody. The numbers above the lanes indicate the hours after RANKL stimulation that the extracts were collected. The bottom panel is the same blot in the top panel reprobed with an antibody that recognizes all forms of p38 MAPK.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5MITF Ser307 is necessary for collaboration with Rac1 or MKK6. A, activity of point mutants measured in transient transfection assays in RAW 264.7 cells. TRAP luciferase reporter construct (2.5 μg) was co-transfected with 0.5 μg of expression vector for either wild type MITF, MITF containing the indicated point mutant, or 0.4 μg of Rac1 15L or MKK6(E) alone or together. Activity is expressed as relative luciferase activity. The averages of three independent experiments performed in duplicate are show

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