Biochemical and Biophysical Research Communications
Regulation of tissue transglutaminase by prolonged increase of intracellular Ca2+, but not by initial peak of transient Ca2+ increase☆
Section snippets
Materials and methods
Chemicals and reagents. Fetal bovine serum, penicillin/streptomycin solution, and RPMI 1640 medium were obtained from Gibco-BRL (Gaithersburg, MD). 5-(Biotinamido)pentylamine and FITC-conjugated streptavidin were purchased from Pierce (Pockford, IL). AA, LPA, and FITC-conjugated anti-mouse IgG were obtained from Sigma (St. Louis, MO), and maitotoxin was obtained from Wako Chemicals (Japan). Monoclonal antibody against tTGase was from NeoMarkers (Fermont, CA).
Cell culture. AGS cells obtained
Activation of in situ tTGase by AA or MTX in AGS cells
Since there is no information on the existence of tTGase in human gastric adenocarcinoma cells, initially, we identified tTGase by Western blotting and immunostaining using a monoclonal anti-tTGase in the AGS cells (Fig. 1A). tTGase was identified as approximately a 77 kDa protein by Western blotting in the AGS cells, and the expression level of tTGase was much higher in the AGS cells than in rat gastric mucosal cells or NIH 3T3 fibroblasts. The AGS cells were stained with a monoclonal
Discussion
In this report, we presented evidences to support the new regulation mechanism of tTGase by the prolonged increase of intracellular Ca2+ in the human gastric adenocarcinoma cells. AA and MTX activated tTGase in a dose- and time-dependent manner, and the tTGase activation by MTX was suppressed by transfection with tTGase siRNA. AA induced a rapid initial increase of intracellular Ca2+ followed by a late prolonged Ca2+ increase, and the removal of extracellular Ca2+ with EGTA blocked the
Acknowledgment
This work was supported in part by the grant from Vascular System Research Center of KOSEF (1999-2-20700-004-5).
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Abbreviations: AA, arachidonic acid; AGS, human gastric adenocarcinoma; LPA, lysophosphatidic acid; MTX, maitotoxin; ROS, reactive oxygen species; tTGase, tissue transglutaminase.