Localization of ADAM10 and notch receptors in bone

In Drosophila melanogaster, the role of the metallodisintegrin, Kuzbanian ( kuz), is thought to involve activation of the Drosophila Notch receptor tha t plays a role in cell-fate determination during neurogenesis and myoblast differentiation, To understand the possible function(s) of a-disintegrin an d metalloproteinase (ADAM10), the mammalian ortholog of kuz, in the skeleto n, we studied its expression as well as the messenger RNA (mRNA) encoding o ne candidate substrate, the mammalian Notch2 receptor in bone, bone cells, and cartilage. In sections of neonatal rat tibiae, ADAM10 is expressed in s pecific regions of articular cartilage and metaphyseal bone. Expression of ADAM10 in articular cartilage occurs predominantly in superficial chondrocy tes and becomes more sporadic with increasing distance from the articular s urface. In bone, ADAM10 is expressed by periosteal cells, osteoblasts, and osteocytes at locations of active hone formation, Osteoclasts did not expre ss ADAM10. Notch2 mRNA expression was not detectable in superficial chondro cytes. However it colocalized at all sites of ADAM10 expression in bone cel ls. In vitro, both primary human osteoblasts and osteoblast cell lines expr essed a single 4.5 kb and 7.5 kb transcript of ADAM10 and the Notch2 recept or homolog, respectively. Subcellular localization of the ADAM10 protein in MG-63 cells was determined using immunofluorescent techniques. These obser vations showed clearly that the ADAM10 protein was expressed in the trans-G olgi network and on the plasma membrane. Western blot analysis of fractiona ted cells showed that, in the plasma membrane fraction, the previously char acterized 58 kDa and 56 kDa isoforms were present, whereas, in the trans-Go lgi network, the ADAM10 protein was present in several additional bands, po ssibly indicative of further interdomain processing of the. ADAM10 protein. The metallodisintegrins (ADAMs) have several putative functions, including modulation of cell adhesion, membrane-associated proteolysis, and cell-cel l signaling. These observations suggest that, in bone but not cartilage, AD AM10 has catalytic activity within the trans-Golgi network and may play a r ole in the activation of Notch receptor homologs, This implicates ADAM10 in cell-fate determination of osteoblast progenitor cells, possibly during sk eletal development and normal bone remodeling. Plasmamembrane-associated AD AM10 may confer alternative functions. (C) 1999 by Elsevier Science Inc, Al l rights reserved.