AZM activates UPR activity through facilitating the dissociation of GRP78 from its interactors
The activation of ER stress occurred upon the dissociation of GRP78 from the luminal part of the ER integral membrane proteins: PERK, IRE1α and ATF6α (Yoo et al., 2013). Co-immunoprecipitation experiments showed that incubation of RA FLSs lysates with AZM decreased the GRP78 level in the PERK, IRE1α, and ATF6α immunoprecipitated fractions (Fig. 5A), thus indicating that AZM induced a dissociation of the GRP78, PERK, IRE1α and ATF6α complex and representing the initial event necessary to induce the observed ER stress.
Furthermore, SREBP-SCAP complex is like other key integral membrane proteins of the UPR associated with GRP78, and, the export of the complex requires its dissociation from GRP78 (Jin et al., 2000). GRP78 dissociates from SREBP allows its cleavage and nuclear translocation for its regulatory function (Katanasaka et al., 2010). Furthermore, attenuation of ER stress by overexpression of GRP78 blocked SREBP activation and decreased the expression of genes responsible for cholesterol and fatty acid biosynthesis (Kammoun et al., 2009). Here, an enhanced expression of m-SREBP-1c by AZM was seen in nuclear extract of RA FLSs, but not in cytoplasmic protein (Fig. 5B). Furthermore, the enhancement of m-SREBP-1c (mature SREBP-1c) nuclear translocation resulting from AZM treatment could be attenuated by overexpression of GRP78 in RA FLSs (Fig. 5B). These data supported that AZM inhibits the activity of GRP78, thereby provoking ER stress and activating SREBP-mediated cholesterol and lipid metabolism.