An analysis of Ca2+ release by DGEA: mobilization of two functionally distinct internal stores in Saos-2 cells.
Osteoblasts can be activated by their collagen matrix and in particular the DGEA peptide motif. We have reported that DGEA is able to activate Ca2+ 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 Ca2+ stores, since the DGEA action is not blocked by La3+ but is lost when Ca2+ stores are depleted with 2 microM and blocked by 10 microM ryanodine. The activated stores also differ functionally from those activated by thrombin, as blockade with U-73122 obstructs only thrombin-activated Ca2+ 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 Ca2+ 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.
Department of Orthodontics and Paediatric Dentistry, United Medical and Dental Schools, London SE1 9RT, United Kingdom.
T J
TJ
McCann
G
G
Terranova
J W
JW
Keyte
S S
SS
Papaioannou
W T
WT
Mason
M C
MC
Meikle
F
F
McDonald
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D002461:Cell Line, Transformed; D016895:Culture Media, Serum-Free; D004963:Estrenes / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D007811:Lanthanum / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology*; D010006:Osteoblasts; D012516:Osteosarcoma; D011760:Pyrrolidinones / Q000494:pharmacology; D012433:Ryanodine / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
D016895:Culture Media, Serum-Free; D004963:Estrenes; D009842:Oligopeptides; D011760:Pyrrolidinones; C113781:aspartyl-glycyl-glutamyl-alanine; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D012433:Ryanodine; D007811:Lanthanum; D010738:Type C Phospholipases; D013917:Thrombin; D002118:Calcium