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.
Chen
L Y
LY
Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
Chiang
A S
AS
Hung
J J
JJ
Hung
H I
HI
Lai
Y K
YK
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D001932:Brain Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel; D000091342:Endoplasmic Reticulum Chaperone BiP; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D018316:Gliosarcoma / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D015398:Signal Transduction; D019284:Thapsigargin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000378:metabolism
D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D004791:Enzyme Inhibitors; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D012333:RNA, Messenger; D004533:Egtazic Acid; D019284:Thapsigargin; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium