Thapsigargin-induced grp78 expression is mediated by the increase of cytosolic free calcium in 9L rat brain tumor cells.

Exposure of 9L rat brain tumor cells to 300 nM thapsigargin (TG), a sarcoendoplasmic Ca(2+)-ATPases inhibitor, leads to an immediate suppression of general protein synthesis followed by an enhanced synthesis of the 78-kDa glucose-regulated protein, GRP78. Synthesis of GRP78 increases significantly and continues to rise after 4 h of treatment, and this process coincides with the accumulation of grp78 mRNA. TG-induced grp78 expression can be suppressed by the cytosolic free calcium ([Ca(2+)](c)) chelator dibromo-1, 2-bis(aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA) in a concentration-dependent manner. Induction of grp78 is completely abolished in the presence of 20 microM BAPTA under which the TG-induced increase of [Ca(2+)](c) is also completely prevented. By adding ethyleneglycol bis(beta-aminoethyl)ether-N,N,N',N' tetraacetic acid in the foregoing experiments, in a condition such that endoplasmic reticulum calcium ([Ca(2+)](ER)) is depleted and calcium influx from outside is prevented, TG-induced grp78 expression is also abolished. These data lead us to conclude that increase in [Ca(2+)](c), together with the depletion of [Ca(2+)](ER), are the major causes of TG-induced grp78 expression in 9L rat brain tumor cells. By using electrophoretic mobility shift assays (EMSA), we found that the nuclear extracts prepared from TG-treated cells exhibit an increase in binding activity toward the extended grp78 promoter as well as the individual cis-acting regulatory elements, CRE and CORE. Moreover, this increase in binding activity is also reduced by BAPTA. By competitory assays using the cis-acting regulatory elements as the competitors as well as the EMSA probes, we further show that all of the tested cis elements-CRE, CORE, and C1-are involved in the basal as well as in the TG-induced expression of grp78 and that the protein factor(s) that binds to the C1 region plays an important role in the formation and maintenance of the transcription complex.