ADAM gene expression and regulation during human osteoclast formation.
34 - 46.
In this study, we identified the expression and the regulation of ADAM members (a disintegrin and metalloprotease) at both gene and protein levels during human osteoclast differentiation and activity. Human peripheral blood monocytes (HPBMC) treated with M-CSF and RANKL were used as an in vitro fusion model. In parallel, we used human osteoclastoma (OCL) tumor as a source of mature osteoclasts, and human osteoblastic cells as a control representing nonfusing and non-resorbing bone cells. RT-PCR using ADAM-specific primers enabled us to identify the expression of ADAM 8, 9, 10, 15, 17, and 28 in both osteoclasts and osteoblasts. Using primers specific for each ADAM 12 isoform (L and S), we observed a strong signal for both forms (ADAM 12L and ADAM 12S) in osteoblastic cells, while only ADAM 12S was detectable in HPBMC-derived osteoclasts and osteoclastoma. Gene regulation was studied using real-time PCR analysis performed during HPBMC differentiation; this showed a progressive increase of ADAM 12 mRNA level from day 1 to 8 of the culture, while at around day 9, ADAM 12 mRNA level decreased 2-fold. We also showed that ADAM 8, ADAM 17, and ADAM 28 decreased according to the stage of HPBMC differentiation or fusion. ADAM 10 was unaltered during cell fusion. However, confocal immunolocalization showed that ADAM 10 protein re-localized from the nuclei and cytoplasm to the plasma membrane during culture and to the ruffled border in resorbing cells. The same re-localization process was observed using an ADAM 12S-specific antibody during HPBMC differentiation. Between days 12 and 14, ADAM 12 co-localized with the F-actin ring, and at day 15, a strong signal was also present in ruffled border or sealing zone area of osteoclasts. Our results describe the expression and regulation of various ADAMs in human bone cells and the selective expression of ADAM 12L in osteoblasts. Our gene regulation and protein localization studies suggest a function for ADAM 10 and ADAM 12S in the formation of osteoclasts from HPBMC and resorption activity.
|Title:||ADAM gene expression and regulation during human osteoclast formation.|
|Keywords:||Carrier Proteins, Cell Differentiation, Cell Fusion, Cell Line, Tumor, Disintegrins, Gene Expression Regulation, Humans, In Vitro Techniques, Macrophage Colony-Stimulating Factor, Membrane Glycoproteins, Metalloendopeptidases, Monocytes, Osteoblasts, Osteoclasts, RANK Ligand, RNA, Messenger, Receptor Activator of Nuclear Factor-kappa B|
|UCL classification:||UCL > School of BEAMS > Faculty of Maths and Physical Sciences > London Centre for Nanotechnology|
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