title: Microphthalmia transcription factor regulates the expression of the novel osteoclast factor GPNMB.
abstract: Microphthalmia transcription factor (MITF) regulates bone homeostasis by inducing expression of critical genes associated with osteoclast function. Gpnmb is a macrophage-enriched gene that has also been shown to be expressed in osteoblasts. Here, we have shown gpnmb to be highly induced in maturing murine osteoclasts. Microarray expression profile analysis identified gpnmb as a potential target of MITF in RAW264.7 cells, subclone C4 (RAW/C4), that overexpress this transcription factor. Electrophoretic mobility shift assays identified a MITF-binding site (M-box) in the gpnmb promoter that is conserved in different mammalian species. Anti-MITF antibody supershifted the DNA-MITF complex for the promoter site while MITF binding was abolished by mutation of this site. The gpnmb promoter was transactivated by co-expression of MITF in reporter gene assays while mutation of the gpnmb M-box prevented MITF transactivation. The induction of gpnmb expression during osteoclastogenesis was shown to exhibit similar kinetics to the known MITF targets, acp5 and clcn7. GPNMB expressed in RAW/C4 cells exhibited distinct subcellular distribution at different stages of osteoclast differentiation. At days 5 and 7, GPNMB protein co-localised with the osteoclast/macrophage lysosomal/endocytic marker MAC-3/LAMP-2, suggesting that GPNMB resides in the endocytic pathway of mature macrophages and is possibly targeted to the plasma membrane of bone-resorbing osteoclasts. The inclusion of gpnmb in the MITF regulon suggests a role for GPNMB in mature osteoclast function.
authors.affiliation: Institute for Molecular Biosciences, Co-operative Research Centre for Chronic Inflammatory Diseases, The University of Queensland, St. Lucia, QLD 4072, Australia.
authors.forename: Vera M
authors.initials: VM
authors.lastname: Ripoll
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Meadows
authors.forename: Liza-Jane
authors.initials: LJ
authors.lastname: Raggatt
authors.forename: Ming K
authors.initials: MK
authors.lastname: Chang
authors.forename: Allison R
authors.initials: AR
authors.lastname: Pettit
authors.forename: Alan I
authors.initials: AI
authors.lastname: Cassady
authors.forename: David A
authors.initials: DA
authors.lastname: Hume
mesh_terms: D000135:Acid Phosphatase / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D018118:Chloride Channels / Q000235:genetics; D017124:Conserved Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004705:Endocytosis; D005136:Eye Proteins / Q000235:genetics*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D008264:Macrophages / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D051739:Microphthalmia-Associated Transcription Factor / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D053245:RANK Ligand / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D018085:Regulon; D012689:Sequence Homology, Nucleic Acid; D000071681:Tartrate-Resistant Acid Phosphatase; D015533:Transcriptional Activation
chemical_list: D018118:Chloride Channels; C490803:Clcn7 protein, mouse; D017931:DNA Primers; D005136:Eye Proteins; C445671:Gpnmb protein, mouse; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D051739:Microphthalmia-Associated Transcription Factor; C497235:Mitf protein, mouse; D053245:RANK Ligand; D011994:Recombinant Proteins; C505688:TNFSF11 protein, human; D004247:DNA; C000605510:ACP5 protein, human; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase