Abnormal localisation and hyperclustering of alpha(v)beta(3) integrins andassociated proteins in Src-deficient or tyrphostin A9-treated osteoclasts

The non-receptor tyrosine kinase Src was shown to be essential for osteocla st function in vivo. We have previously reported that engagement of alpha ( v)beta (3) integrin in osteoclasts induces tyrosine phosphorylation and act ivation of the adhesion kinase PYK2 and the adaptor protein p130(Cas) in a Src-dependent manner. The objective of this study was to analyse the role o f c-Src in the alpha (v)beta (3) integrin-dependent recruitment of signalli ng and cytoskeletal molecules in osteoclasts during bone resorption, Using prefusion osteoclasts (pOCs) obtained from cocultures of osteoblasts and sp leen cells isolated from Src(-/-) mice or their normal littermates, we foun d: (1) similar expression levels and ligand binding affinities of alpha (v) beta (3) integrins in Src(-/-) and Src(+/?) pOCs, (2) reduced adhesion and spreading of Src(-/-) pOCs, (3) defective organisation of the microfilament proteins, F-actin, uinculin and paxillin, and of PYK2 and p130(Cas) in the sealing zone of Src(-/-) OCLS, and (4) hyperclustering of alpha (v)beta (3 ) integrins together with microfilament and signalling proteins in the basa l membrane of Src-deficient OCLs, In normal OCLs, the tyrosine kinase inhib itor tyrphostin A9 inhibits actin ring formation, bone resorption and tyros ine phosphorylation of several proteins, including c-Src, Furthermore, tyrp hostin A9 induced similar hyperclustering of alpha (v)beta (3) integrins in osteoclasts as observed in Src(-/-) OCLs, Taken together, these findings s uggest that normal localisation of alpha (v)beta (3) and recruitment of its downstream effecters to the appropriate compartments of the osteoclast dur ing resorption depend on Src kinase activity.