Activation of eIF2α-ATF4 by endoplasmic reticulum-mitochondria coupling stress enhances COX2 expression and MSC-based therapy for rheumatoid arthritis

Background Mesenchymal stem/stromal cell (MSCs) therapy represents a potential therapeutic tool to treat RA, but loss of secretory property post delivery restricted clinical application. It has been verified that endoplasmic reticulum stress (ERS)-MSCs exhibited better inhibition on rheumatoid arthritis (RA) T follicular helper cells (Tfh) via cyclooxygenase-2 (COX2)/prostaglandin E2 (PGE2) activation with unknown molecular mechanism, particulary the overall outcome of ERS-modified MSCs on RA. Methods To compare the therapeutic efficacy, thapsigargin (TG)-stimulated or unstimulated MSCs were transplantated into collagen-induced arthritis (CIA) mice. Joint inflammation was evaluated from general and histological aspects. Splenocytes were isolated and flow cytometry was performed to assess the proportion of T helper 1 (Th1), Th17 and Tfh subsets. During mechanism exploration, TRRUST and Cistrome Data Browser databases were used to analyze transcription factors related to COX2 regulation, as well as target genes regulated by activating transcription factor 4 (ATF4). Then western blot and qRT-PCR were employed to determine the level of ATF4 in ERS-MSCs. To verify the function of ATF4 in vivo , ATF4-overexpression MSCs were transplanted to CIA mice, joint inflammation, Th1, Th17 and Tfh subsets were analysed. To clear the molecular regulatory mechanism leading to ATF4 activation, protein levels of protein kinase RNA like endoplasmic reticulum kinase (PERK)/phosphorylated-PERK (p-PERK) and eukaryotic initiation factor 2α (eIF2α)/phosphorylated-eIF2α (p-eIF2α) were examined. Besides, ATF4 and eIF2α/p-eIF2α were checked after PERK blocking. Subsequently, mitochondrial stress was checked in ERS-MSCs. At last, blocking ERS and mitochondrial stress separately or simultaneously, ATF4 and eIF2α/p-eIF2α were checked again. Results Compared with MSCs, ERS-MSCs exhibited better therapeutic efficacy in CIA mice. Public databases and bioinformatics analysis confirmed the regulatory role of ATF4 on COX2 and experimental methods further confirmed ATF4-transfected MSCs diminished the joint inflammation of CIA mice. We also demonstrated that during ERS induction, PERK-mediated eIF2α phosphorylation contributes to elevated ATF4 expression. Besides, mitochondrial stress was also provoked in ERS-MSCs, coupling with ERS synergistically regulated ATF4. Conclusions ERS-MSCs exhibited better immunosuppresive ability than un-pretreated MSCs through COX2 overexpression, which was regulated by ATF4. Besides, ERS and mitochondrial stress co-regulate ATF4 expression. This study established a new role of ATF4 in promoting secretory properties of MSC and provided a promising MSC-based therapeutic strategy for RA treatment.