M-CSF induces the stable interaction of cFms with αVβ3 integrin in osteoclasts

The macrophage colony stimulating factor receptor (cFms) and αVβ3 integrin are both abundantly expressed and play critical roles in the differentiation, survival and migration of osteoclasts. We have previously demonstrated that cross-talk between cFms- and αVβ3-mediated signaling pathways regulated the cytoskeletal organization required for osteoclast migration. To investigate the nature of interaction between the two receptors, we sequentially used anion-exchange chromatography and immunoprecipitation to purify αVβ3-associated protein complexes. We have demonstrated that cFms stably associated with αVβ3 in osteoclasts during adhesion, and that the association was induced by macrophage colony stimulating factor (M-CSF) stimulation. However, the kinetics of association of αVβ3 and cFms did not correlate with the kinetics of tyrosine phosphorylation of cFms. Instead, maximally observed αVβ3/cFms association was after the peak of cFms tyrosine phosphorylation and correlated inversely with the total amount of cFms remaining. Furthermore, the complex containing cFms and αVβ3 also contained a number of other signaling molecules including Pyk2, p130Cas and c-Cbl, known downstream regulators of the integrin-mediated signaling pathways in osteoclasts. In the presence of M-CSF, co-localization of αVβ3 integrin and cFms was identified in the podosomal actin ring of the osteoclast during adhesion on glass. Interestingly, co-localization of both receptors was not found in the sealing zone, but in punctate structures associated with adhesion- or transcytosis-like structures in osteoclasts on bone. Taken together, we suggest that the association of αVβ3 and cFms could be the result of signaling following tyrosine phosphorylation of cFms. The recruitment of cFms to αVβ3 integrin may be an integral part of a larger signaling complex via which both of adhesion- and growth factor receptors coordinately regulate osteoclast adhesion, motility and membrane trafficking.