Biochemical and Biophysical Research Communications
A role for αV integrin subunit in TGF-β-stimulated osteoclastogenesis
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.
References (28)
Transforming growth factor-β
Pathogenesis of osteoporosis and challenges for drug delivery
Adv. Drug Deliv. Rev.
(2000)- et al.
Osteoprotegerin: a novel secreted protein involved in the regulation of bone density
Cell
(1997) - et al.
TGF-β enhances osteoclast differentiation in hematopoietic cell cultures stimulated with RANKL and M-CSF
Biochem. Biophys. Res. Commun.
(1999) - et al.
The retinoblastoma protein acts as a transcriptional coactivator required for osteogenic differentiation
Mol. Cell
(2001) - et al.
Gene expression during the differentiation of myogenic cells of the L6 line
Biochimie
(1976) - et al.
An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion
Cell
(1975) - et al.
An adherent condition is required for formation of multinuclear osteoclasts in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor kappa B ligand
Blood
(2000) The alpha v beta 3 integrin “vitronectin receptor”
Int. J. Biochem. Cell Biol.
(1997)- et al.
Influence of stereochemistry of the sequence Arg-Gly-Asp-Xaa on binding specificity in cell adhesion
J. Biol. Chem.
(1987)
Cell adhesion is a prerequisite for osteoclast survival
Biochem. Biophys. Res. Commun.
Chemotactic response of osteoblast like cells to transforming growth factor β
J. Bone Miner. Res.
Role of transforming growth factor-β in bone remodeling
Clin. Orthop.
Effects of transforming growth factor beta 1 on the regulation of osteoclastic development and function
J. Bone Miner. Res.
Cited by (13)
Current insights into the role of transforming growth factor-β in bone resorption
2005, Molecular and Cellular EndocrinologyMolecular profiling of giant cell tumor of bone and the osteoclastic localization of ligand for receptor activator of nuclear factor κB
2005, American Journal of PathologyCitation Excerpt :As a final assay for RANKL as a secreted or membrane-bound signal mediating the recruitment of monocytic cells by stromal cells, we used RAW264.7 cells as a bioassay for RANKL production. RAW264.7 cells form TRAP-positive mono- and multinucleated osteoclast-like cells in the presence of recombinant human RANKL (in our hands, between 10 and 50 TRAP-positive cells/high-power field20). However, in repeated experiments, neither GCT stromal cell supernatants nor co-culture with GCT-derived stromal cells resulted in the formation of TRAP-positive cells (data not shown).
Effects of TGF-β1 on the migration and morphology of RAW264.7 cells in vitro
2019, Molecular Medicine ReportsEnhancement of RANKL-induced MITF-E expression and osteoclastogenesis by TGF-β
2014, Cell Biochemistry and FunctionPathophysiology and pharmacological targeting of tumor-induced bone disease: Current status and emerging therapeutic interventions
2011, Current Medicinal Chemistry