Abstract
ABSTRACTExposure of 9L rat brain tumor cells to 300 nM thapsigargin (TG), a sarco-endoplasmic Ca2+-ATPases inhibitor, leads to immediately suppression of protein synthesis followed by an enhanced synthesis of the 78 kDa glucose-regulated protein (GRP78). Translation of GRP78 increases significantly and continues to rise after 4 h of treatment and this process coincides to the accumulation of it mRNA. TG-induced grp78 expression can be suppressed by dibromo-1,2-bis(aminophenoxy)ethane N, N, N’, N’-tetraacetic acid (BAPTA) in a concentration-dependent manner and is completely abolished in the presence of 20 □M BAPTA under which the TG-induced rise of cytoplasmic free calcium ([Ca2+]c) is also prevented. By adding ethyleneglycol bis(b-aminoethyl)ether-N, N, N’, N’ tetra acetic acid (EGTA) in the above experiments, in a condition 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, rather depletion of [Ca2+]ER, is the major cause 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 and three separate cis-acting regulatory elements CRE, CORE, and C1. 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 all of the aforementioned cis elements are involved in the basal as well as in the TG-induced expression of grp78 and that the C1 region plays the most