Abstract
Exposure of 9L rat brain tumor cells to 100 mM ethanol for 36 h leads to a specific induction of the 78 kDa glucose-regulated protein (GRP78). By exploiting protein kinase inhibitors, we further analyzed the possible participation of specific protein kinases in the above processes. It was found that induction of GRP78 in cells treated with ethanol is diminished by genistein and SB203580, which inhibit protein tyrosine kinases (PTKs) and p38 mitogen-activated protein kinase (p38MAPK), respectively. Subsequent electrophoretic mobility shift assay (EMSA) using probes encompassing CRE-like, CORE, and CCAAT box (C1) which were derived from the promoter regions of the grp78 gene showed that nuclear factors prepared from untreated cells were able to form complexes with all three cis-regulatory elements. These observations indicated that the three elements might confer the basal expression of grp78. Interestingly, while these probes were reacted with nuclear extracts prepared from ethanol-treated cells, the formed complexes were disappeared, indicated that these element-bound transcription factors might exert inhibitory effects on ethanol-induced GRP78 synthesis. By competitory assays using the cis-acting regulatory elements as the competitors as well as the EMSA probes, we further showed all the formed complexes are specific to used probes.