A COLLAGEN PEPTIDE MOTIF ACTIVATES TYROSINE KINASE-DEPENDENT CALCIUM SIGNALING PATHWAYS IN HUMAN OSTEOBLAST-LIKE CELLS

A collagen peptide motif (DGEA) which is a putative alpha(2) beta(1) i ntegrin binding site was examined for its ability to activate Ca2+ sig nalling pathways in the human osteoblast-like cell line SaOS-2. We sho w that these cells express both alpha(2) beta(1) integrin subunits (by immunocytochemistry) and that an anti-beta(1) monoclonal antibody (DF 5) mobilizes Ca2+ in these cells. DGEA elevated intracellular Ca2+ in fura-2-loaded cells, in a concentration-and sequence-dependent fashion , with an ED50 of 250 mu M. The tyrosine kinase inhibitor herbimycin A reduced the number of cells responding to DGEA and to transforming gr owth factor alpha. Thrombin also stimulated a rise in intracellular Ca 2+, but the number of cells responding was not reduced by herbimycin A . The DGEA response was dependent on extracellular Ca2+, but was not d ue to Ca2+ influx, since it was blocked by thapsigargin and not by lan thanum. Using three different anti-a, monoclonal antibodies, we were u nable to show that the DGEA-induced Ca2+ signal was mediated by the al pha(2) beta(1) integrin. In summary, the DGEA collagen motif does appe ar to activate receptor-mediated Ca2+ signalling events in SaOS-2 cell s, in a divalent cation-dependent manner, but we were unable to demons trate a role for alpha(2) beta(1) integrin in this response.