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 Ca2+‐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 ([Ca2+]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 μM BAPTA under which the TG‐induced increase of [Ca2+]c is also completely prevented. By adding ethyleneglycol bis(β‐aminoethyl)ether‐N,N,N′,N′ tetraacetic acid in the foregoing experiments, in a condition such that endoplasmic reticulum calcium ([Ca2+]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 [Ca2+]c, together with the depletion of [Ca2+]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. J. Cell. Biochem. 78:404–416, 2000. © 2000 Wiley‐Liss, Inc.