Beta1 integrin antisense oligodeoxynucleotides: utility in controlling osteoclast function.
The involvement of beta1 integrins in osteoclast function has been investigated by utilising an antisense oligodeoxynucleotide (ODN) approach. 18-mer antisense and control phosphorothioate ODNs were made to a conserved internal region of beta1 integrin sequence (nucleotide positions 1634-1651 of the human beta1 fibronectin receptor). These were tested on rabbit osteoclasts for anti-adhesive and resorptive effects mediated by alphaVbeta3 and alpha2beta1, the major integrins of osteoclasts. Antisense, but not control, beta1 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 were 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 alphaVbeta3 and alpha2beta1, 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 beta1 protein expression assessed by FACS and Western blot analysis; expression of other integrin subunits, alphaV and beta3, was unaffected. Similarly, the beta1 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 beta1 integrin(s).
Townsend
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PA
Ludwig Institute for Cancer Research, St. Mary's Hospital Medical School, Imperial College of Science, Technology and Medicine, London, UK. p.a.townsend@ic.ac.uk
Villanova
I
I
Teti
A
A
Horton
M A
MA
D000818:Animals; D001483:Base Sequence; D001798:Blood Proteins; D001854:Bone Marrow Cells / Q000187:drug effects; D001862:Bone Resorption / Q000188:drug therapy*; D002116:Calcitonin / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen; D003804:Dentin; D019281:Dimerization; D015536:Down-Regulation / Q000187:drug effects; D016326:Extracellular Matrix Proteins; D005340:Fibrinogen; D005353:Fibronectins; D005786:Gene Expression Regulation / Q000187:drug effects; D005898:Glass; D006801:Humans; D019012:Integrin beta1 / Q000235:genetics* / Q000502:physiology; D016023:Integrins / Q000502:physiology; D008545:Melanoma / Q000378:metabolism / Q000473:pathology; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010010:Osteoclasts / Q000187:drug effects*; D011817:Rabbits; D038981:Receptors, Collagen; D019030:Receptors, Vitronectin / Q000502:physiology; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014407:Tumor Cells, Cultured; D019096:Vitronectin
D001798:Blood Proteins; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D019012:Integrin beta1; D016023:Integrins; D016376:Oligonucleotides, Antisense; D038981:Receptors, Collagen; D019030:Receptors, Vitronectin; D019096:Vitronectin; C028815:salmon calcitonin; D005340:Fibrinogen; D002116:Calcitonin; D003094:Collagen