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-kappaB 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.
Highlights
When colony-stimulating factor-1 (CSF-1) was withdrawn from bone marrow-derived macrophage cultures for 12 h, indirect immunofluorescence demonstrated that MITF was found predominantly in the cytoplasm in 80%
Osteoclast differentiation is regulated by a group of ubiquitous transcription factors instead of by cell type-specific transcription factors
These factors, including MITF, PU.1, NFATc1, NF-B, and c-Fos, individually are expressed in many different cell types and cell lineages but collectively orchestrate a program of gene expression that leads to osteoclast differentiation [2]
Summary
Antibodies—Antibodies against MITF and phospho-S307MITF were described previously [11], anti-phospho-S73 antibody was kindly provided by David E. 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. Osteoclast precursors were plated at a density of 3 ϫ 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. For ReChIP assays, pre-cleared soluble chromatin from 6 ϫ 106 cells harvested from two 100-mm culture dishes were immunoprecipitated with PU. antibody. 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.
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