Geldanamycin induction of grp78 requires activation of reactive oxygen species via ER stress responsive elements in 9L rat brain tumour cells

The molecular mechanism whereby anticancer agent geldanamycin (GA) impacts endoplasmic reticulum (ER) stress pathway is largely unknown. Here, we investigate the effect of GA on the expression of grp78 coding for ER stress protein and the mechanistic relationship of GA signalling to ER stress. GA induces the expression of mRNA and protein of grp78 by Northern blot analysis and metabolic labelling experiment in cultured rat brain tumour 9L cells. The induced grp78 expression is sensitive to antioxidant N-acetylcysteine (NAC) addition, indicating the involvement of reactive oxygen species (ROS) in GA-induced ER stress. Results from direct determination of oxidation status using dichlorodihydrofluorescein diacetate (H2DCFDA) showed that accumulation of ROS elicited GA was quenched by addition of NAC. Reporter genes harbouring deletions of transcription elements from grp78 promoter demonstrated that controlling elements of ERSE1, ERSE2 and CRE are required in GA treatment. The critical ROS-dependent elements in grp78 promoter can be confined within ER stress responsive element (ERSE) region, since reporter constructs loss of ERSE elements that lost the susceptibility to be modulated by NAC after GA treatment. Hence, ER stress elements correlate well with ROS-mediated elements in grp78 promoter. Reporter construct loss of ERSE element retains the susceptibility by NAC after GA treatment, indicating that CRE element might represent a ROS-independent, GA-inductive element. Conclusively, we show that ROS is required for GA to launch the transactivation of grp78, and a firm link was established between the ROS signalling pathway to specific promoter elements-ERSE1 and ERSE2 elements in ER stress marker gene grp78 promoter.