Molecular characterization and functional analysis of two V-ATPase subunits, Ac45 and d2, in osteoclasts

    Research output: ThesisDoctoral Thesis

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    Abstract

    [Truncated abstract] During bone-remodelling, proton secretion by osteoclasts generates an acidic microenvironment, which is critical for solubilisation of bone matrix and degradation of bone matrix. The coupling between vacuolar–type (H+)-ATPase (V-ATPase) and chloride ion channel (CLC-7), presented in the ruffled borders of osteoclasts, is responsible for the acidification. Multiple complexes of the V-ATPase consist of two distinct domains, a transmembrane V0 domain and a peripheral V1 domain. The aim of this study is to dissect the molecular mechanism regulating V-ATPase activity and its interaction during osteoclast development. The first part of this study focuses on the molecular identification and characterisation of the novel V-ATPase accessory subunit Ac45 in osteoclasts. The second part of the study aims to explore the transcription factors by which the d2 subunit is regulated during osteoclast formation. The accessory subunit Ac45 was identified in osteoclasts by using the technique of cDNA Subtractive Hybridisation. Examination of the Ac45 gene expression profiles during osteoclast formation by semi-quantitative PCR demonstrated that Ac45 was significantly up-regulated during osteoclastogenesis (~2 fold). Tissue distribution analysis of Ac45 showed that it was abundantly expressed in the brain, heart and kidney, however, with lower expressions in the muscle, spleen, thymus, liver and lung. Using pull-down immunoprecipitation, Ac45 was found to interact/associate with other V0 subunits including a3, c, and c". '...' Unexpectedly, during the EMSA analysis of NFAT binding sites, the presence of another unidentified transcription factor binding site was found adjacent to the N2 binding site (within the 29 oligonucleotide sequence). Following transcriptional factors mapping, it was predicated to be a putative myocyte enhancer factor 2 (MEF2) binding sequence. Further bioinformatic analysis mapped two putative MEF2 binding sites (E1 and E2) with the 1 kB V-ATPase d2 promoter. All four MEF2 isoforms were expressed during osteoclast formation, although in distinct patterns. Moreover, it is evident that throughout the osteoclast formation MEF2 consistently associated with the V-ATPase d2 promoter in ChIP and EMSA assays. Intriguingly, overexpression of MEF2 Wild type, but not its dominant negative form xvi (MEF2 R24L), together with caNFATc1, resulted in synergistic activation of the V-ATPase d2 promoter. However, such synergistic activation was significantly abrogated by either single or double mutation(s) of the MEF2 binding sites of the V-ATPase d2 promoter. Lastly, immunostaining analysis revealed, during RANKLtreated osteoclast differentiation, NFATc1 was greatly induced and translocated from the cytoplasm to the nucleus, where that co-localized with MEF2 in mature osteoclasts (day 5). In summary, the results presented in this thesis provided further insights into the composition of the osteoclast V-ATPase proton pump by: 1) identifying that the accessory subunit Ac45 is involved in the regulation and/or targeting the of V-ATPase complex and truncation of its targeting signal disrupts the proper interaction with other V0 subunits; and 2) revealing that NFATc1, MITF and MEF2 directly bind to the endogenous V-ATPase d2 promoter during osteoclastogenesis. Moreover, MEF2 and MITF cooperate with NFATc1 to synergistically transactivate the 1 kB V-ATPase d2 promoter. Further study is required to determine the precise role of Ac45 in V-ATPase complex as well as explore the molecular mechanism of d2-mediated osteoclast fusion.
    Original languageEnglish
    QualificationDoctor of Philosophy
    Publication statusUnpublished - 2008

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