Microphthalmia transcription factor regulates the expression of the novel osteoclast factor GPNMB.

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.

Reduction of the in vitro pro-inflammatory response by macrophages to poly(3-hydroxybutyrate-co-3-hydroxyvalerate).

This study evaluates the pro-inflammatory response to the thermoplastic biopolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) through the analysis of cellular responses in vitro. The murine macrophage RAW264.7 cell line was cultured on solvent cast PHBV films, which was found to induce pro-inflammatory activity that required direct contact between the material and the macrophages. The identity of the pro-inflammatory stimulus was determined by culturing bone marrow-derived macrophages from bacterial lipopolysaccharide (LPS) hyporesponsive C3H/HeJ mice and CpG non-responsive TLR9-/- mice on PHBV. The lack of a pro-inflammatory response by the C3H/HeJ cells indicates that the pro-inflammatory agent present within PHBV is predominately LPS while the TLR9-/- macrophages confirmed that CpG-containing bacterial DNA is unlikely to contribute to the activity. A series of purification procedures was evaluated and one procedure was developed that utilized hydrogen peroxide treatment in solution. The optimized purification was found to substantially reduce the pro-inflammatory response to PHBV without adversely affecting either the molecular structure or molecular weight of the material thereby rendering it more amenable for use as a biomaterial in vivo.

Reducing the radiation sterilization dose improves mechanical and biological quality while retaining sterility assurance levels of bone allografts.

BACKGROUND: Bone allografts carry a risk of infection, so terminal sterilization by gamma irradiation at 25kGy is recommended; but is deleterious to bone quality. Contemporary bone banking significantly reduces initial allograft bioburden, questioning the need to sterilize at 25kGy. METHODS: We inoculated allograft bone with Staphylococcus epidermidis and Bacillus pumilus, then exposed them to gamma irradiation at 0, 5, 10, 15, 20 and 25kGy. Mechanical and biological properties of allografts were also assessed. Our aim was to determine an optimal dose that achieves sterility assurance while minimizing deleterious effects on allograft tissue. RESULTS: 20-25kGy eliminated both organisms at concentrations from 10(1) to 10(3)CFU, while 10-15kGy sterilized bone samples to a bioburden concentration of 10(2)CFU. Irradiation did not generate pro-inflammatory bone surfaces, as evidenced by macrophage activation, nor did it affect attachment or proliferation of osteoblasts. At doses ≥10kGy, the toughness of cortical bone was reduced (P<0.05), and attachment and fusion of osteoclasts onto irradiated bone declined at 20 and 25kGy (P<0.05). There was no change in collagen cross-links, but a significant dose-response increase in denatured collagen (P<0.05). CONCLUSIONS: Our mechanical and cell biological data converge on 15kGy as a threshold for radiation sterilization of bone allografts. Between 5 and 15kGy, bone banks can undertake validation that provides allografts with an acceptable sterility assurance level, improving their strength and biocompatibility significantly. CLINICAL RELEVANCE: The application of radiation sterilization doses between 5 and 15kGy will improve bone allograft mechanical performance and promote integration, while retaining sterility assurance levels. Improved quality of allograft bone will promote superior clinical outcomes.

The expression of Clcn7 and Ostm1 in osteoclasts is coregulated by microphthalmia transcription factor.

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-kappaB 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.