A role for αV integrin subunit in TGF-β-stimulated osteoclastogenesis

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Abstract

TGF-β increases bone resorption in vivo and greatly increases osteoclast formation stimulated by receptor activator of NF-κB ligand (RANKL) in vitro. TGF-β does not independently affect the differentiation state of RAW264.7 preosteoclasts, but increases cell attachment to vitronectin. This effect is mediated by increased expression of αV integrin subunit mRNA and protein. Concomitant with induction of osteoclast differentiation, RANKL causes relocation of αV to focal sites in the cell. This effect is potentiated by TGF-β. Integrin blockade disrupts both attachment to vitronectin and RANKL-induced osteoclast formation, but culture on vitronectin has little effect. Ectopic expression of αV stimulates multinucleation of RAW264.7 cells and increases the number of osteoclasts formed in the presence of RANKL. These data suggest that TGF-β potentiates RANKL-induced osteoclast formation, in part by increased expression of the αV integrin subunit, which may contribute to cell fusion.

Section snippets

Materials and methods

Cell culture and reagents. The macrophage-like cell line RAW264.7 was maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and incubated at 37 °C in 5% CO2. Depending on the experiments, treatments with 10 ng/ml human transforming growth factor-β1 (TGF-β; R&D Systems, MN) and/or 100 ng/ml recombinant mouse RANKL were used. Expression plasmids encoding cDNA inserts (Rc/CMVαV and pBABEpuro) of interest were transfected using the FuGENE 2 (Roche). After 24–48 h

Effect of TGF-β on RAW264.7 cells

As previously shown [9], TGF-β potently enhanced RANKL-induced osteoclast formation. No tartrate-resistant acid phosphatase positive (TRAP+) or multinucleated cells (MNCs) were formed in RAW264.7 cell cultures treated with TGF-β (Fig. 1A), while RANKL induced the formation of both TRAP+ MNCs and mononucleated cells. Treatment of RAW264.7 cells with both RANKL and TGF-β led to a 2.5-fold increase in TRAP+ mononucleated cells compared to RANKL treatment alone, and the increase in multinuclear

Discussion

These studies were designed to elucidate a possible mechanism for the role of TGF-β in RANKL-induced osteoclast formation [9]. Mice over-expressing TGF-β are osteoporotic [8], while mice over-expressing non-signalling TGF-β receptors are osteopetrotic [7]. These observations provided the rationale for our investigations. TGF-β alone affects RAW264.7 cells, but it alone does not act to promote osteoclast formation. Levels of RANK were not increased by TGF-β, nor did TGF-β alter the macrophage or

Acknowledgements

This work was supported by NHMRC RD Wright fellowship to DMT (Regkey No. 251752) and a University of Melbourne Early Career Researcher Grant.

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