beta 1 Integrin antisense oligodeoxynucleotides: utility in controlling osteoclast function

The involvement of beta 1 integrins in osteoclast function has been investigated by utilising an antisense oligodeoxynucleotide (ODN) approach. 18-mer antisense and control phosphorothioate ODNs mere made to a conserved internal region of beta 1 integrin sequence (nucleotide positions 1634-1651 of the human beta 1 fibronectin receptor), These were tested on rabbit osteoclasts for anti-adhesive and resorptive effects mediated by alpha V beta 3 and alpha 2 beta 1, the major integrins of osteoclasts. Antisense, but not control, beta 1 ODNs inhibited osteoclast adhesion to collagen-coated glass (by up to 70 %), but not to glass coated with vitronectin, fibronectin or fibrinogen, Adhesion to dentine and subsequent resorption mere also inhibited (up to 60 %) in a sequence-specific manner. The mechanism of action was verified using both a melanoma cell line, DX3, which expresses multiple integrins at high level including alpha V beta 3 and alpha 2 beta 1, and in a rabbit osteoclast marrow culture (BMC) system. Exposure of DX3 cells to antisense ODN for up to 48 hours reduced adhesion to FCS- and collagen-coated glass, and concomitantly inhibited beta 1 protein expression assessed by FACS and Western blot analysis; expression of other integrin subunits, alpha V and beta 3, was unaffected. Similarly the beta 1 protein levels in the BMC were reduced by >75 % without any effect on actin expression, These data reveal the utility of antisense ODNs in exploring osteoclast biology and further define the functional role of osteoclastic beta 1 integrin(s).