Studies on annexins in primary cultures of human osteoblasts and in the human osteosarcoma cell line MG-63.

Studies on annexins in primary cultures of human osteoblasts and in the human osteosarcoma cell line MG-63 JAVAD MOHITI*, JOHN H. WALKER*AND ALISON M.CASWF.LL0. *Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT UK. OHealth Sciences Group, Leeds Metropolitan University, Leeds. Calcium plays a central role in the regulation of many physiological processes. Calmodulin is a major calcium binding protein that regulates cell function. Over the past few years another major class of calcium-binding protein has emerged which binds calcium through an alternative calcium-binding site. These proteins are called “ annexins”. Various physiological roles have been suggested for annexins including phospholipase A, inhibition, membrane fusion, anti-coagulation, cell differentiation and interaction with cytoskeletal proteins ( 1,2). These proteins could also play a role in the regulation of calcium in osteoblasts and in the mineralization of bone through their ability to bind to calcium and phospholipids (3). I t has been reported that annexins I , II and V bind to actin and annexin V binds to type 1,11 and X collagen (4). Hence annexins could contribute to a variety of processes required for mineralization including the regulation of calcium transport into human osteoblasts and interactions between macromolecules in the extracellular matrix. We have attempted to identify the annexins present in primary cultures of human osteoblasts and in the osteosarcoma cell line MG-63 (S), and to quantitate annexin V. We have also studied the presence of annexin V in the extracellular medium and the effect of cell culture conditions on the expression of annexin V in MG63 cells. The cells were grown in 90 mm tissue culture dishes with Dulbecco’s modified minimum essential medium containing IO%(v/v) fetal calf serum and then proteins were extracted with 2% SDS. Proteins were analysed by SDS-PAGE (6) on 10% polyacrylamide gels. Separated proteins were transferred from the gel onto nitrocellulose membranes according to Burnette (7) using an electroblotting apparatus operated at constant current W m A for 3 h or IOOmA overnight. lmmunoprobing was carried out by exposing nitrocellulose paper to antibodies against each annexin and then incubating with peroxidase-conjugated second antibody. baminobenzidine (DAB) and ECL methods were used to detect immobilized peroxidase. Finally, we used densitometry to quantitate the amount of annexin V in primary cultures of humam osteoblasts and in M G 4 3 cells.