GRP78 is required for the anti-arthritis effects of AZM
We deleted GRP78 gene using the CRISPR-Cas9 technique and this technique produced nearly complete deletion of GRP78 (Fig. 6A). Although GRP78 deficiency resulted in the increase of PERK and elF2α phosphorylation, IRE1α, ATF4 and CHOP expression, as well as SREBP nuclear translocation in the absence of AZM, no further changes were observed when GRP78-/- were challenged with AZM (Fig. 6B). In addition, re-establishing expression of GRP78 by transfecting GRP78 knockout cells with a GRP78 expression plasmid reinstated AZM’s UPR and SREBP activation (Fig. 6B). However, transfection of GRP78 knockout cells with an expression plasmid encoding a GRP78 point mutant Asp-178-Ala (GRP78 D178A), which inactivates GRP78 enzymatic activity and fails to hydrolyze ATP, could not rescue AZM’s activity (Fig. 6B). Taken together, these findings indicate the dependence of AZM’s UPR activation on GRP78 and GRP78-mediated ATPase activity.
To further ascertain that the anti-arthritis effect of AZM is mediated by GRP78, GRP78 was inhibited by siRNA in RA FLSs (Fig. 6C). The downregulation of GRP78 reduced the production of inflammatory factors (Fig. 6D) and genes involved in cholesterol and lipid metabolism process (online Supplementary Fig. 4), but induced apoptosis (Fig. 6E), which was the same as the effect of AZM. However, coculture with AZM had no synergistic effect on inflammatory factors, genes of cholesterol and lipid metabolism, and cell apoptosis with GRP78 knockdown (Fig. 6D, 6E, and online Supplementary Fig. 4). Taken together, our results showed that AZM directly bound to GRP78 and exerted anti-inflammatory effects in RA.