Reduction of GRP78 increases expression of ATF4, GRP94, GADD153 in mIMCD3 cells.

Abstract

We previously demonstrated that mRNA and protein expression of the 78kDa glucose‐regulated protein (GRP78) increased in the renal IM of 7 day dDAVP infused AQP1 null mice. GRP78 is an ER chaperone protein with an important role in protein folding, Ca2+ binding, and ER homeostasis. In order to investigate the role of GRP78 in osmotic tolerance, GRP78 siRNA was transfected into mIMCD3 cells to knockdown GRP78 expression. mIMCD3 cells were harvested 24hrs and 48hrs after transfection for mRNA and protein analysis respectively. We found, however, that siRNA knockdown of GRP78 resulted in increased expression of several genes involved in UPR signaling pathways. Quantitative real time PCR showed an increase in the expression of 94kDa glucose‐regulated protein (GRP94), activating transcription factor 4 (ATF4), and growth arrest and DNA damaging protein (GADD153); 3.36 ± 0.03, 1.43 ± 0.1, and 3.51 ± 0.03 fold change over control, respectively, p<0.05. Immunoblotting confirmed an increase in protein expression of GRP94 and GADD153 after GRP78 knockdown. Preliminary trypan blue staining data suggests that despite the observed GADD153 upregulation, no significant cell death occurs 48 hours after GRP78 siRNA transfection. To investigate the possible role of Ca2+ in the activation of UPR pathways in mIMCD3 cells Fura‐PE3AM will be used to monitor intracellular Ca+2 flux in GRP78 siRNA transfected mIMCD3 cells.