Abstract
Geldanamycin (GA), a benzoquinone ansamycin, is an inhibitor of heat shock protein 90 (HSP90)/ glucose-regulated protein 94 (GRP94) and has been implicated as a potent anti-cancer drug. In our previous study, we found that GA with sublethal dose provoked the ER stress in 9L rat brain tumor (RBT) cells and induced glucose-regulated proteins under unfold protein response (UPR) at transcriptional level. The promoter of grp genes contain multiple copies of the ER stress response element (ERSE), with a consensus of CCAAT(N9)CCACG, which is critical and necessary for transcription induction. Herein, we showed that GRP94, an ER resident chaperone, was induced under GA treatment in 9L rat brain tumor (RBT) cells and the mRNA level of grp94 was peaked at 8 h for about 9 fold. We analyzed the promoter sequence of grp94 according to rat genome resource and designed reporter vectors containing progressive-deleted promoters of grp94. In reporter gene assays, ERSE4 and CRE-BP1/c-Jun played the major roles in GA-induced grp94 expression. Moreover, with mutagenesis clones we further confirmed this result although other ERSEs still have involved in. By inhibitors screening, pretreatment of KT5720, BIM I, or Gö6983 partially decreased GA-induced GRP94 expression, suggesting the involvement of PKA and PKC. Pretreatment of AEBSF blocked proteolysis of ATF6 abolished GA-induced GRP94 expression. Taken together, we found that under GA induced ER stress in 9L cells, activation of GRP94 were mainly through ERSEs by the transcription factor ATF6.