Townsend, Paul Andrew; (1997) The molecular basis of osteoclast adhesion. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The role of the integrin β1 and αV subunits in osteoclast functions was studied using an antisense oligodeoxynucleotide (ODN) approach. Novel integrin β subunits expressed by osteoclasts were also identified by homology cloning by PCR and characterised. An assay system to assess the effects of antisense ODNs to integrins was developed using ex vivo rabbit osteoclast preparations. Osteoclasts were settled onto either glass coated in serum, or different extracellular matrix proteins, or onto dentine to monitor adhesion and bone resorption. A fully phosphorothioated β1 ODN was designed that corresponds to a region of high β1-homology across species, but which was unrelated to other β subunits. The β1 ODN demonstrated a dose dependent reduction of both adhesion to glass and dentine and an inhibition of bone resorption. Changes in the ODN protection chemistry led to an increase in ODN efficacy. A number of αV ODNs were developed based on sequences around the ATG translation start sequence of the human vitronectin receptor. One ODN to αV, the S95 5543, was the most effective at inhibiting osteoclast adhesion and resorption. Antisense mechanisms of action were observed for both the β1 and αV ODNs, with reductions in the surface expression of their target subunits. However, in adhesion assays, both ODNs demonstrated some cross-ligand recognition. It was hypothesised that these effects may be accounted for by the expression a new integrin receptor. Therefore, the expression of novel β subunits was studied using homology cloning by PCR. Sequence comparisons revealed one 'tag', βoc, that shared a high nucleotide and peptide homology to β1. Molecular characterisation confirmed the βoc/β1 similarity, but a full length βoc clone was not isolated by cDNA library screening. A variety of detailed techniques were also utilised to aid in the characterisation of βoc, which provided preliminary data that their genomic organisation differed. All of the work presented in this thesis was performed by the author whilst a member of the Imperial Cancer Research Fund Haemopoiesis Research Group, Department of Medicine, University College London. Except where acknowledgement is made, the work is my own and has not been submitted for any other degree in this or any other university or institute of learning.

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