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.