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• W2018887276 abstract "Transcription factors NFATc1, PU.1, and MITF collaborate to regulate specific genes in response to colony-stimulating factor-1 (CSF-1) and receptor activator of NF-κB ligand (RANKL) signaling during osteoclast differentiation. However, molecular details concerning timing and mechanism of specific events remain ill-defined. In bone marrow-derived precursors, CSF-1 alone promoted assembly of MITF-PU.1 complexes at osteoclast target gene promoters like cathepsin K and acid 5 phosphatase without increasing gene expression. The combination of RANKL and CSF-1 concurrently increased the levels of MAPK-phosphorylated forms of MITF, p38 MAPK, and SWI/SNF chromatin-remodeling complexes bound to these target promoters and markedly increased expression of the genes. NFATc1 was subsequently recruited to complexes at the promoters during terminal stages of osteoclast differentiation. Genetic analysis of Mitf and Pu.1 in mouse models supported the critical interaction of these genes in osteoclast differentiation. The results define MITF and PU.1 as nuclear effectors that integrate CSF-1/RANKL signals during osteoclast differentiation to initiate expression of target genes, whereas a complex that includes NFATc1 may act to maintain target gene expression in differentiated cells. Transcription factors NFATc1, PU.1, and MITF collaborate to regulate specific genes in response to colony-stimulating factor-1 (CSF-1) and receptor activator of NF-κB ligand (RANKL) signaling during osteoclast differentiation. However, molecular details concerning timing and mechanism of specific events remain ill-defined. In bone marrow-derived precursors, CSF-1 alone promoted assembly of MITF-PU.1 complexes at osteoclast target gene promoters like cathepsin K and acid 5 phosphatase without increasing gene expression. The combination of RANKL and CSF-1 concurrently increased the levels of MAPK-phosphorylated forms of MITF, p38 MAPK, and SWI/SNF chromatin-remodeling complexes bound to these target promoters and markedly increased expression of the genes. NFATc1 was subsequently recruited to complexes at the promoters during terminal stages of osteoclast differentiation. Genetic analysis of Mitf and Pu.1 in mouse models supported the critical interaction of these genes in osteoclast differentiation. The results define MITF and PU.1 as nuclear effectors that integrate CSF-1/RANKL signals during osteoclast differentiation to initiate expression of target genes, whereas a complex that includes NFATc1 may act to maintain target gene expression in differentiated cells. Bone is a dynamic tissue that maintains both physical integrity and calcium homeostasis in vertebrate species. Consequently, bone is modeled and continuously remodeled beginning in embryogenesis and throughout the lifetime of mammals and other animals. Bone integrity and function are maintained by an exquisite balance between the two major cell types involved in the remodeling process, the osteoblast and the osteoclast (1Manolagas S.C. Endocr. Rev. 2000; 21: 115-137Crossref PubMed Scopus (1978) Google Scholar, 2Boyle W.J. Simonet W.S. Lacey D.L. Nature. 2003; 423: 337-342Crossref PubMed Scopus (4847) Google Scholar, 3Teitelbaum S.L. Ross F.P. Nat. Rev. Genet. 2003; 4: 638-649Crossref PubMed Scopus (1296) Google Scholar). Osteoblasts, originating from mesenchymal progenitor cells, are responsible for the mineralization of bone matrix. Osteoclasts are of hematopoietic origin and are highly specialized multinuclear cells capable of resorbing bone. It is now well accepted that many human disorders of bone, including common human disorders like osteoporosis, Paget's disease, rheumatoid arthritis, and cancer bone metastases, reflect an imbalance in the differentiation and function of these two cell types (1Manolagas S.C. Endocr. Rev. 2000; 21: 115-137Crossref PubMed Scopus (1978) Google Scholar, 4Rodan G.A. Martin T.J. Science. 2000; 289: 1508-1514Crossref PubMed Scopus (1463) Google Scholar. Two essential cytokines, macrophage colony-stimulating factor-1 (CSF-1) 2The abbreviations used are: CSF-1, colony-stimulating factor-1; RANKL, receptor activator of NF-κB ligand; MITF, microphthalmia-associated transcription factor; TFE, transcription factor E; MAPK, mitogen-activated protein kinase; OCL, osteoclast-like cell; ChIP, chromatin immunoprecipitation; qPCR, quantitative real-time PCR; GST, glutathione S-transferase; TRAP, tartrate-resistant acid phosphatase; Acp5, acid phosphatase 5; Ctsk, cathepsin K; Erk, extracellular signal-regulated kinase; pol II, polymerase II; ReChIP, sequential chromatin immunoprecipitation.2The abbreviations used are: CSF-1, colony-stimulating factor-1; RANKL, receptor activator of NF-κB ligand; MITF, microphthalmia-associated transcription factor; TFE, transcription factor E; MAPK, mitogen-activated protein kinase; OCL, osteoclast-like cell; ChIP, chromatin immunoprecipitation; qPCR, quantitative real-time PCR; GST, glutathione S-transferase; TRAP, tartrate-resistant acid phosphatase; Acp5, acid phosphatase 5; Ctsk, cathepsin K; Erk, extracellular signal-regulated kinase; pol II, polymerase II; ReChIP, sequential chromatin immunoprecipitation. and receptor activator of NF-κB ligand (RANKL), are produced by osteoblasts and are necessary and sufficient for osteoclast differentiation (5Yoshida H. Hayashi S. Kunisada T. Ogawa M. Nishikawa S. Okamura H. Sudo T. Shultz L.D. Nishikawa S. Nature. 1990; 345: 442-444Crossref PubMed Scopus (1514) Google Scholar, 6Wiktor-Jedrzejczak W. Bartocci A. Ferrante Jr., A.W. Ahmed-Ansari A. Sell K.W. Pollard J.W. Stanley E.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4828-4832Crossref PubMed Scopus (877) Google Scholar, 7Kong Y.Y. Yoshida H. Sarosi I. Tan H.L. Timms E. Capparelli C. Morony S. Oliveira-dos-Santos A.J. Van G. Itie A. 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 (2844) Google Scholar, 8Lacey D.L. Timms E. Tan H.L. Kelley M.J. Dunstan C.R. Burgess T. Elliott R. Colombero A. Elliott 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 (4594) Google Scholar). Signaling pathways engaged by these cytokines can activate a number of transcription factors required for osteoclast differentiation, including NF-κB, c-Fos, NFATc1, PU.1, and MITF (2Boyle W.J. Simonet W.S. Lacey D.L. Nature. 2003; 423: 337-342Crossref PubMed Scopus (4847) Google Scholar, 9Wong B.R. Josien R. Lee S.Y. Vologodskaia M. Steinman R.M. Choi Y. J. Biol. Chem. 1998; 273: 28355-28359Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 10Matsuo K. Owens J.M. Tonko M. Elliott C. Chambers T.J. Wagner E.F. Nat. Genet. 2000; 24: 184-187Crossref PubMed Scopus (268) Google Scholar, 11Mansky K.C. Sankar U. Han J. Ostrowski M.C. J. Biol. Chem. 2002; 277: 11077-11083Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 12Weilbaecher 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 (133) Google Scholar, 13Takayanagi H. Kim S. Koga T. Nishina H. Isshiki M. Yoshida H. Saiura A. Isobe M. Yokochi T. Inoue J. Wagner E.F. Mak T.W. Kodama T. Taniguchi T. Dev. Cell. 2002; 3: 889-901Abstract Full Text Full Text PDF PubMed Scopus (1964) Google Scholar). Of these, MITF (microphthalmia-associated transcription factor) is a basic helix-loop-helix leucine zipper protein closely related to the transcription factor E (TFE) family, composed of TFE3, TFEB, and TFEC gene products (14Hodgkinson C.A. Moore K.J. Nakayama A. Steingrimsson E. Copel- and N.G. Jenkins N.A. Arnheiter H. Cell. 1993; 74: 395-404Abstract Full Text PDF PubMed Scopus (942) Google Scholar, 15Hemesath 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 (546) Google Scholar). MITF has been implicated in the survival and differentiation of developmentally unrelated cell types, including melanocytes and osteoclasts (15Hemesath 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 (546) Google Scholar, 16Hershey C.L. Fisher D.E. Bone. 2004; 34: 689-696Crossref PubMed Scopus (98) Google Scholar). Strong evidence for a critical role of MITF and TFE3 in terminal osteoclast differentiation is provided by the severe osteopetrotic and osteoclast phenotype in mice homozygous for Mitf- (mi) or Mitf-or alleles, or double mutants homozygous for the Mitf-vga hypomorphic allele and a Tfe3 null allele (16Hershey C.L. Fisher D.E. Bone. 2004; 34: 689-696Crossref PubMed Scopus (98) Google Scholar, 17Steingrimsson E. Moore K.J. Lamoreux M.L. Ferre-D'Amare A.R. Burley S.K. Zimring D.C. Skow L.C. Hodgkinson C.A. Arnheiter H. Copeland N.G. Jenkins N.A. Nat. Genet. 1994; 8: 256-263Crossref PubMed Scopus (441) Google Scholar, 18Steingrimsson E. Tessarollo L. Pathak B. Hou L. Arnheiter H. Copel- and N.G. Jenkins N.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4477-4482Crossref PubMed Scopus (171) Google Scholar). How MITF selectively affects gene expression and differentiation of developmentally unrelated cell types is a question of general biological interest. In osteoclasts MITF has been shown to regulate three definitive osteoclast target genes, tartrate-resistant acid phosphatase/acid phosphatase 5 (TRAP/Acp5) (19Luchin 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 (108) Google Scholar), cathepsin K (Ctsk) (20Motyckova G. Weilbaecher K.N. Horstmann M. Rieman D.J. Fisher D.Z. Fisher D.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5798-5803Crossref PubMed Scopus (192) Google Scholar), and Oscar (21So H. Rho J. Jeong D. Park R. Fisher D.E. Ostrowski M.C. Choi Y. Kim N. J. Biol. Chem. 2003; 278: 24209-24216Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Interactions between MITF and the Erythroblastosis virus E26 oncogene homolog (ETS) family transcription factor PU.1 at least partly account for the ability to regulate these target genes in osteoclasts (21So H. Rho J. Jeong D. Park R. Fisher D.E. Ostrowski M.C. Choi Y. Kim N. J. Biol. Chem. 2003; 278: 24209-24216Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 22Luchin A. Suchting S. Merson T. Rosol T.J. Hume D.A. Cassady A.I. Ostrowski M.C. J. Biol. Chem. 2001; 276: 36703-36710Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). However, MITF and PU.1 are expressed in macrophages and osteoclasts, as well as the mononuclear precursors for both of these cell types, posing an additional question about how correct gene regulation patterns are maintained in different cell types. In recent work, we demonstrated that subcellular localization of MITF was affected by signaling-dependent interactions with 14-3-3 proteins, providing one mechanism that regulates MITF activity in myeloid precursors cells (23Bronisz A. Sharma S.M. Hu R. Godlewski J. Tzivion G. Mansky K.C. Ostrowski M.C. Mol. Biol. Cell. 2006; 17: 3897-3906Crossref PubMed Scopus (59) Google Scholar). In addition, MITF, PU.1, and NFATc1 can act in combination to synergistically activate reporter genes, and both PU.1 and NFATc1 have been localized to the Ctsk promoter by the chromatin immunoprecipitation technique (24Matsumoto M. Kogawa M. Wada S. Takayanagi H. Tsujimoto M. Katayama S. Hisatake K. Nogi Y. J. Biol. Chem. 2004; 279: 45969-45979Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). These data suggest that NFATc1 may be the key factor whose expression and activation distinguish between genes regulated by MITF and PU.1 in macrophages versus osteoclasts (21So H. Rho J. Jeong D. Park R. Fisher D.E. Ostrowski M.C. Choi Y. Kim N. J. Biol. Chem. 2003; 278: 24209-24216Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 24Matsumoto M. Kogawa M. Wada S. Takayanagi H. Tsujimoto M. Katayama S. Hisatake K. Nogi Y. J. Biol. Chem. 2004; 279: 45969-45979Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 25Kim K. Kim J.H. Lee J. Jin H.M. Lee S.H. Fisher D.E. Kook H. Kim K.K. Choi Y. Kim N. J. Biol. Chem. 2005; 280: 35209-35216Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). In the present work, CSF-1 and RANKL are demonstrated to regulate the nuclear activity of MITF and PU.1, resulting in increased occupancy of osteoclast target promoters by the phosphorylated, activated form of MITF, and in the co-recruitment of the SWI/SNF chromatin-remodeling complex and activated p38 mitogen-activated protein kinase (MAPK). Following these events, NFATc1 is recruited to target promoters. These results indicate that signaling-dependent regulation of MITF and PU.1 is critical in the initiation of transcription of target genes during osteoclast differentiation. Antibodies—Antibodies against MITF and phospho-S307-MITF were described previously (11Mansky K.C. Sankar U. Han J. Ostrowski M.C. J. Biol. Chem. 2002; 277: 11077-11083Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), anti-phospho-S73 antibody was kindly provided by David E. Fisher (Boston, MA). Anti-PU.1 antibody was raised in rabbits against the peptide DLVTYDSELYQRPMHDYC representing amino acids 18-35 of mouse PU.1 and affinity-purified using a column containing the target peptide (BIOSOURCE, Hopkinton, MA). Anti-NFATc1 antibody and RNA polymerase antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against phospho-p38 MAPK and histone H3 were purchased from Upstate Cell Signaling (Charlottesville, VA). Anti-BRG1, anti-BAF57, and anti-BAF155 antibodies are described elsewhere (26Wang L. Baiocchi R.A. Pal S. Mosialos G. Caligiuri M. Sif S. Mol. Cell Biol. 2005; 25: 7953-7965Crossref PubMed Scopus (64) Google Scholar). Monoclonal RNA polymerase II H5 antibody, which recognizes phosphorylated serine 2 in the C-terminal repeat domain, was from Covance (Covance Research Products, Berkley, CA). Culture and Analysis of Osteoclast-like Cells—Detailed procedures for OCL differentiation have been described previously (27Mansky K.C. Marfatia K. Purdom G.H. Luchin A. Hume D.A. Ostrowski M.C. J. Leukoc. Biol. 2002; 71: 295-303PubMed Google Scholar, 28Sankar U. Patel K. Rosol T.J. Ostrowski M.C. J. Bone Miner. Res. 2004; 19: 1339-1348Crossref PubMed Scopus (48) Google Scholar). Briefly, hematopoietic precursors were obtained from spleens of wild type and mutant mice. OCLs were grown in Dulbecco's modified Eagle's medium containing 50 ng/ml CSF-1 for 3 days on non-tissue culture plastic dishes; at this point, the non-adherent cell fraction was isolated for further studies. For CSF-1 starvation, non-adherent cells were washed twice, plated on tissue culture dishes, and maintained in medium without CSF-1 for 12 h. For differentiation, non-adherent cells were transferred to tissue culture dishes and treated with 50 ng/ml CSF-1 and 100 ng/ml RANKL for different times indicated in the figures. Chromatin Immunoprecipitation, Sequential Chromatin Immunoprecipitation (ReChIP), and qPCR—Chromatin immunoprecipitation (ChIP) assays were performed as described by Luo et al. (29Luo R.X. Postigo A.A. Dean D.C. Cell. 1998; 92: 463-473Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar). Briefly, osteoclast precursors were plated at a density of 3 × 106 cells per 10-cm dish and treated with cytokines for various times as indicated in the figure legends, and cells were cross-linked with 1% final concentration of formaldehyde at 37 °C for 10 min before harvest. Soluble chromatin was prepared following sonication with a Branson 250 digital sonifier (Branson Ultrasonics, Danbury, CT) to an average DNA length of 200-1000 bp. ∼5 × 105 cell equivalent (one-sixth) of the sheared soluble chromatin was pre-cleared with tRNA-blocked Protein G-agarose, and 10% of the pre-cleared chromatin was set aside as input control. Immunoprecipitation was carried out with 5 μg of antibodies as indicated in the figures overnight at 4 °C. Immune complexes were pulled down using Protein G-agarose, washed, and eluted twice with 250 μl of elution buffer (0.1 m NaHCO3, 1% SDS), and cross-linking was reversed in 200 mm NaCl at 65 °C overnight with 20 μg of RNase A (Sigma). DNA was purified following proteinase K treatment (Invitrogen) with the Qiagen PCR purification kit using the manufacturer's instructions. Samples were analyzed by real-time PCR either by SYBR Green super mix (Bio-Rad) for Ctsk promoter or by the Roche universal probe library (Roche Diagnostics, Indianapolis, IN) probe using the Faststart TaqMan master kit (Roche Diagnostics) for Acp5 promoter. The threshold for the promoter being studied was adjusted by that of input values and represented as relative abundance. All qPCR reactions were analyzed by melt curve analysis and agarose gels to confirm the presence of a single specific band. For ReChIP assays, pre-cleared soluble chromatin from 6 × 106 cells harvested from two 100-mm culture dishes were immunoprecipitated with PU.1 antibody. After washing, the PU.1-immune complex was disrupted with 10 mm dithiothreitol at 37 °C for 30 min with shaking and diluted 50-fold with the ChIP buffer. This eluted immune complex was divided and immunoprecipitated with the second specific antibodies as indicated in the figures, and the complexes were analyzed as above. Immunoprecipitation, Immunofluorescence, Western Blotting, and GST Pulldown Assays—Procedures for immunoprecipitation, Western blotting, immunofluorescence, and GST pulldown assays have all been recently described (23Bronisz A. Sharma S.M. Hu R. Godlewski J. Tzivion G. Mansky K.C. Ostrowski M.C. Mol. Biol. Cell. 2006; 17: 3897-3906Crossref PubMed Scopus (59) Google Scholar). Analysis of RNA Expression—RNA was extracted using TRIzol (Invitrogen). Residual contaminating genomic DNA was removed using 2 units of DNase 1 (Roche Applied Science) for 20 min at room temperature, and RNA was purified with the RNeasy kit (Qiagen). 2 μg of purified total RNA was reverse transcribed by Superscript III reverse transcriptase (Invitrogen) with random hexamer primers. Primers used for real-time PCR were picked by Oligo v4.0 software, and sequences used are available upon request. The real-time PCR was conducted using SYBR Green super mix (Bio-Rad) in an iCycler real-time detection system (Bio-Rad). The PCR threshold was determined by using the iCycler PCR baseline-subtracted curve fit method. The threshold for the gene being studied was adjusted by that of a reference gene (ribosomal protein L4). All reactions were examined by agarose gels to confirm the presence of a single specific band. Mouse Mutants and Bone Histomorphometry—All mice were maintained on a C57B/6J background. Wild-type mice were used to make bone marrow precursors for the in vitro experiments. To generate mice for the genetic experiments mice heterozygous for both the Pu.1 null allele (22Luchin A. Suchting S. Merson T. Rosol T.J. Hume D.A. Cassady A.I. Ostrowski M.C. J. Biol. Chem. 2001; 276: 36703-36710Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) and the Mitfvga allele were crossed to mice heterozygous for both the Mitfmi and Mitfvga alleles. Mitfmi/vga mice have a white coat color (18Steingrimsson E. Tessarollo L. Pathak B. Hou L. Arnheiter H. Copel- and N.G. Jenkins N.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4477-4482Crossref PubMed Scopus (171) Google Scholar) but were also identified by genotyping for both alleles. The Pu.1 and mi alleles were genotyped as previously described (22Luchin A. Suchting S. Merson T. Rosol T.J. Hume D.A. Cassady A.I. Ostrowski M.C. J. Biol. Chem. 2001; 276: 36703-36710Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The vga allele was genotyped by PCR with primers that recognized the lacZ transgene inserted into the Mitf locus. Use and care of mice in this study was approved by The Ohio State University Institutional Animal Care and Use Committee. Procedures for radiography and histomorphometry have been previously described (28Sankar U. Patel K. Rosol T.J. Ostrowski M.C. J. Bone Miner. Res. 2004; 19: 1339-1348Crossref PubMed Scopus (48) Google Scholar). For radiography, skeletons were fixed in 4% paraformaldehyde and analyzed with Faxitron (Hewlett-Packard model 43855A) for 2 min at 45 kV using X-OMAT V film (Eastman Kodak, Rochester, NY). Fixed femurs infiltrated with glycol methacrylate solution (Polysciences Inc.) at 4 °C were embedded using the JB-4 embedding kit (Polysciences Inc). Serial sagittal sections, 6-μm thick, were made and stained with hematoxylin & eosin and tartrate-resistant acid phosphatase (TRAP) to visualize osteoclasts. Images of stained sections were acquired using a Spot 2 digital charge-coupled device camera on a Nikon E-1000 microscope, and ImagePro Plus was used to quantify the data. For all histomorphometric measures, four different sections from four different mice were quantified, and the values were averaged. CSF-1 Promotes Recruitment of MITF-PU.1 Complexes to Cathepsin K and Acid Phosphatase Promoters—We recently demonstrated that the combined action of CSF-1 and RANKL regulated the subcellular localization of MITF (23Bronisz A. Sharma S.M. Hu R. Godlewski J. Tzivion G. Mansky K.C. Ostrowski M.C. Mol. Biol. Cell. 2006; 17: 3897-3906Crossref PubMed Scopus (59) Google Scholar). To extend these observations and dissect the effect of CSF-1 alone on MITF function, the subcellular localization of MITF and its association with target promoters was analyzed as a function of CSF-1 stimulation. When CSF-1 was withdrawn from bone marrow-derived macrophage cultures for 12 h, indirect immunofluorescence demonstrated that MITF was found predominantly in the cytoplasm in 80% of cells studied (Fig. 1A and supplemental Fig. S1A). Addition of CSF-1 to these cytokine-deprived cells for 12 h resulted in the partial nuclear localization of MITF, with >80% of cells analyzed having both nuclear and cytoplasmic staining for MITF (Fig. 1A and supplementary Fig. 1A). In contrast, CSF-1 withdrawal and restimulation didn't significantly affect the nuclear localization of PU.1 (Fig. 1A). The affect of CSF-1 on MITF and PU.1 nuclear function was studied using the technique of chromatin immunoprecipitation (ChIP). Fig. 1B represents the regions of Ctsk and Acp5 genes that were analyzed following the ChIP technique, regions previously demonstrated to contain binding sites for these transcription factors (19Luchin 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 (108) Google Scholar, 20Motyckova G. Weilbaecher K.N. Horstmann M. Rieman D.J. Fisher D.Z. Fisher D.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5798-5803Crossref PubMed Scopus (192) Google Scholar) (see supplemental Fig. S2A for sequences of the promoter regions containing binding sites for these factors). Upon withdrawal of CSF-1, as expected from previous results, MITF was not found at Ctsk and Acp5 promoters (Fig. 1C). In addition, PU.1, despite the nuclear localization of this factor, could not be detected at either of these promoters (Fig. 1C). After addition of CSF-1, MITF and PU.1 were enriched at both promoters (Fig. 1C), indicating that MITF plays a crucial role in the initial recruitment of PU.1 to these promoters. Quantitative real-time PCR (qPCR) was used to confirm the results (supplemental Fig. S1B). In addition to the 5′-region of the Ctsk and Acp5 genes, 3′-regions corresponding to internal exons/introns (also depicted in Fig. 1B) were used as negative controls. MITF and PU.1 could not be detected at this portion of the Ctsk or Acp5 genes (Fig. 1C). Additional negative controls performed were a reaction in which preimmune IgG (no antibody) was included, and analysis of the 5′-regulatory region of the Hprt gene, a gene that is not regulated by MITF or PU.1 (Fig. 1C). Histone H3 was routinely used as a positive control for all ChIP experiments, and signals were observed for specific antibodies with all primer sets used (Fig. 1C). The same set of negative controls was used for all subsequent ChIP experiments presented for both Ctsk and Acp5 genes (see supplemental Figs. S2-S5 for data). Although CSF-1 promoted nuclear localization of MITF and recruitment to osteoclast target promoters, CSF-1 alone was unable to promote differentiation of osteoclast-like cells (OCLs) in vitro (Fig. 2A) or to activate expression of the Ctsk and Acp5 genes (Fig. 2B). In contrast, the combination of CSF-1 and RANKL induced visible OCL formation after 3 days of treatment and resulted in robust expression of Ctsk and Acp5 genes. After 5 days of cytokine treatment, OCL differentiation was complete, and expression of target genes was at maximal levels (Fig. 2, A and B). ChIP assays were used over this time course of differentiation to determine if MITF-PU.1 complexes were further enriched at target promoters by the action of both cytokines versus CSF-1 alone (Fig. 2C; supplemental Fig. S2B for controls). The combination of CSF-1 and RANKL together for either 0.5 or 3 days resulted in only a marginal increase of <2-fold in occupancy of these two promoters by MITF or PU.1, a change that was not significantly different from the CSF-1 treatment (Fig. 2C, second and third bar graphs in each panel). After addition of both cytokines for 5 days, when OCL differentiation is completed, occupancy of Ctsk and Acp5 promoters by both MITF and PU.1 were maximal but only 2- to 3.5-fold higher than the CSF-1-only treatment (Fig. 2C, fourth bar graph). These data indicate that there are only small changes in the occupancy of these target promoters by MITF and PU.1 over the time course of OCL differentiation, which correlate well with the robust increases in gene expression observed after 3 days of CSF-1/RANKL treatment. Phosphorylated, Activated Forms of MITF and p38 MAPK Are Present at Target Promoters following CSF-1/RANKL Stimulation—MITF is targeted by both Erk and p38 MAPK pathways in a CSF-1/RANKL-dependent fashion, at residues Ser-73 and Ser-307, respectively, and both phosphorylation events increase MITF activity (11Mansky K.C. Sankar U. Han J. Ostrowski M.C. J. Biol. Chem. 2002; 277: 11077-11083Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 12Weilbaecher 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 (133) Google Scholar). Using phospho-specific MITF antibodies available for each of these residues, we analyzed whether chromatin-associated MITF located at the Ctsk promoter was phosphorylated at these positions (Fig. 3, A and B; supplemental Fig. S3A for controls). In bone marrow-derived cells that were grown with CSF-1 alone an enrichment of pSer73-MITF at the Ctsk promoter was observed, but pSer307-MITF was not detected. CSF-1 and RANKL treatment for 0.5 days had no additional effect on the level of pSer73-MITF at the Ctsk promoter, but the abundance of pSer307-MITF was substantially increased (Fig. 3B). After 3 days of combined CSF-1/RANKL stimulation, the levels of both pSer73 and pSer307 forms of MITF at the Ctsk promoter were maximal. The pSer73 form of MITF increased ∼10-fold at this time, whereas the pSer307 form increased an additional 3-fold above levels detected following 0.5 day of treatment. After 5 days of cytokine treatment, the chromatin-associated levels of both phosphorylated forms of MITF remained elevated and not significantly different from the levels seen at the 3-day time point. Whether the kinases responsible for phosphorylation of these sites were also enriched at the Ctsk promoter was examined (Fig. 3C and supplemental Fig. S3B for controls). This analysis demonstrated that the activated, phosphorylated form of p38 MAPK was not detected in cells treated with CSF-1 alone, but chromatin-associated phospho-p38 MAPK could be observed in cells treated with both CSF-1 and RANKL (Fig. 3C, gray bar graphs). The levels of phospho-p38 were also maximal after 3-5 days of treatment, increasing ∼3-fold over the level seen at 0.5 day of cytokine treatment. In contrast, Erk1 and -2 were not detected at the Ctsk promoter under the same conditions (Fig. 3C, black bar graphs). To more directly address whether phosphorylation of MITF and recruitment of p38 MAPK correlated with transcriptional activation of the target genes, ChIP was used to determine occupancy of the target promoters by RNA polymerase II (pol II) (Fig. 3D and supplemental Fig. S3B). An antibody that recognizes total RNA pol II, as well as one that recognizes pol II phosphorylated on the C-terminal tail, were used. The experiments demonstrated that levels of RNA pol II at the promoters correlate well with osteoclast differentiation, target gene expression, and the presence of phosphorylated MITF phospho-p38 MAPK than with the presence of the MITF-PU.1 complex alone. Enrichment of NFATc1-associated Ctsk and Acp5 Promoters Is Observed in Activated Osteoclasts—NFATc1 plays a central role in osteoclast differentiation and has been shown to collaborate with MITF and PU.1 in activation of Ctsk and Acp5 promoters (13Takayanagi H. Kim S. Koga T. Nishina H. Isshiki M. Yoshida H. Saiura A. Isobe M. Yokochi T. Inoue J. Wagner E.F. Mak T.W. Kodama T. Taniguchi T. Dev. Cell. 2002; 3: 889-901Abstract Full Text Full Text PDF PubMed Scopus (1964) Google Scholar, 24Matsumoto M. Kogawa M. Wada S. Takayanagi H. T" @default.
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