The microphthalmia transcription factor (MITF), a basic-helix-loop-helix zipper factor, regulates distinct target genes in several cell types. We hypothesized that interaction with the Ets family factor PU.1, whose expression is limited to hematopoietic cells, might be necessary for activation of target genes like tartrate-resistant acid phosphatase (

The defective terminal differentiation of osteoclasts in mice homozygous for the mi allele of the microphthalmia transcription factor (MITF) gene implies that MITF plays a critical role in regulating gene expression during osteoclast ontogeny. To begin addressing the role of this transcription factor in the osteoclast, target genes need to be identified.

The activity of the TRACP promoter has been investigated as a model of gene regulation in osteoclasts. The murine TRACP gene promoter contains potential binding sites for a number of transcription factors in particular, candidate sites for the Ets factor PU.1 and for the microphthalmia transcription factor (MiTF). These are of relevance to osteoclast biology because the PU.1 knockout mouse has an osteopetrotic phenotype, and MiTF, when mutated in the mi/mi mouse, also results in osteopetrosis. The binding sites for both of these factors have been identified, and they have been determined to be functional in regulating TRACP expression. A novel assay system using the highly osteoclastogenic RAW/C4 subclone of the murine macrophage cell line RAW264.7 was used to perform gene expression experiments on macrophage and osteoclast cell backgrounds. We have shown that TRACP expression is a target for regulation by the macrophage/osteoclast transcription factor PU.1 and the osteoclast commitment factor MiTF and that these factors act synergistically in regulating this promoter. This directly links two controlling factors of osteoclast differentiation to the expression of an effector of cell function.

The microphthalmia transcription factor (MITF) regulates gene expression during differentiation of several distinct cell types, including osteoclasts. A structure/function analysis was performed to determine whether transcription activation domains were important for MITF action in osteoclasts. In addition to a previously characterized acidic activation necessary for melanocyte differentiation, the analysis defined a second potential activation domain located between amino acids 140 and 185. This second domain is required for MITF transactivation of two probable targets, the E-cadherin promoter and the tartrate-resistant acid phosphatase promoter, in transient transfection assays. An intact MITF gene rescued differentiation when introduced into osteoclasts derived from mi/mi mice using a retrovirus vector. In parallel experiments, an MITF gene lacking the acidic-activation domain rescued differentiation twofold less efficiently than wild type, and a gene lacking the region between amino acid residues 140 and 185 rescued differentiation tenfold less efficiently than wild type. The results indicate that the N-terminal region of MITF is necessary for activation of gene expression in osteoclasts and provides one mechanism by which this factor regulates distinct target genes in different cell types.