An analysis of Ca2+ release by DGEA: mobilization of two functionally distinct internal stores in Saos-2 cells

1 Department of Orthodontics and Paediatric Dentistry, United Medical and Dental Schools, London SE1 9RT; 2 Department of Neurobiology, The Babraham Institute, Babraham, Cambridge CB2 4AT; and 3 Biopolymer Synthesis and Analysis Unit, School of Biomedical Science, Queens Medical Centre, Nottingham NG7 2UH, United Kingdom Osteoblasts can be activated by their collagen matrix and in particular the DGEA peptide motif. We have reported that DGEA is able to activate Ca 2+ signaling pathways in the human osteoblast-like cell line, Saos-2, by a tyrosine kinase-dependent pathway (T. J. McCann, W. T. Mason, M. C. Meikle, and F. McDonald. Matrix Biol. 16: 271-280, 1997). In the present study, we show that this activity is due to coupling of the signal to intracellular Ca 2+ stores, since the DGEA action is not blocked by La 3+ but is lost when Ca 2+ stores are depleted with 2 µM and blocked by 10 µM ryanodine. The activated stores also differ functionally from those activated by thrombin, as blockade with U-73122 obstructs only thrombin-activated Ca 2+ release. We have shown that the DGEA activity was not due to its high-charge density, since the two acidic residues can be substituted with their uncharged homologues (asparagine and glutamine) without significant loss of activity. This was in turn measured by an adhesion assay that also demonstrated this level of specificity. Furthermore, by constructing DGEA bound to FITC, we have shown that DGEA binding was dependent on divalent cations. We have also demonstrated that an intact actin cytoskeleton is not required for Ca 2+ activation by inhibiting actin polymerization with the addition of cytochalasin B. These data strengthen the argument that collagen has a significant role in regulating osteoblast function via this peptide motif. calcium signaling; type I collagen