The Expression of Clcn7 and Ostm1 in Osteoclasts Is Coregulated by Microphthalmia Transcription Factor

Abstract

Microphthalmia transcription factor (MITF) regulates osteoclast function by controling the expression of genes, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K in response to receptor activator of nuclear factor-B ligand (RANKL)-induced signaling. To identify novel MITF target genes, we have overexpressed MITF in the murine macrophage cell line RAW264.7 subclone 4 (RAW/C4) and examined the gene expression profile after sRANKL-stimulated osteoclastogenesis. Microarray analysis identified a set of genes superinduced by MITF overexpression, including Clcn7 (chloride channel 7) and Ostm1 (osteopetrosis-associated transmembrane protein 1). Using electrophoretic mobility shift assays, we identified two MITF-binding sites (M-boxes) in the Clcn7 promoter and a single M-box in the Ostm1 promoter. An anti-MITF antibody supershifted DNA-protein complexes for promoter sites in both genes, whereas MITF binding was abolished by mutation of these sites. The Clcn7 promoter was transactivated by coexpression of MITF in reporter gene assays. Mutation of one Clcn7 M-box prevented MITF transactivation, but mutation of the second MITF-binding site only reduced basal activity. Chromatin immunoprecipitation assays confirmed that the two Clcn7 MITF binding and responsive regions in vitro bind MITF in genomic DNA. The expression of Clcn7 is repressed in the dominant negative mutant Mitf mouse, mi/mi, indicating that the dysregulated bone resorption seen in these mice can be attributed in part to transcriptional repression of Clcn7. MITF regulation of the TRAP, cathepsin K, Clcn7, and Ostm1 genes, which are critical for osteoclast resorption, suggests that the role of MITF is more significant than previously perceived and that MITF may be a master regulator of osteoclast function and bone resorption.Bone-resorbing osteoclasts are multinucleated cells that are derived from the hematopoietic myeloid/monocyte lineage. Normal bone remodeling is controlled by the close interactions between osteoclasts and osteoblasts, the primary bone-synthesizing cells. The balance between the activities of these two cell types is controlled by tight transcriptional regulation of both osteoclast-specific and osteoblast-specific genes along with signaling mechanisms that couple the actions of these cells. Defects in the transcriptional control of osteoclasts can result in a failure of either cell differentiation or function. The resulting loss of bone resorption results in the development of osteopetrosis, a condition characterized by dense brittle bones and the lack of a marrow cavity. The use of transgenic gene knock-out mice has elucidated the role of several transcription factors in regulating osteoclast differentiation and function. Null mutations in PU.1 (1), c-Fos (2), and NF-B p50/p52 (3-5) all result in osteopetrosis, thereby indicating that these transcription factors play a critical role in regulating osteoclast biology.Microphthalmia transcription factor (MITF) 2 has been shown to be a regulator of osteoclast function by activ...

Molecular Stress-inducing Compounds Increase Osteoclast Formation in a Heat Shock Factor 1 Protein-dependent Manner

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miRNA-based mechanism for the commitment of multipotent progenitors to a single cellular fate

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