The Influence Of EDA~+FN-induced ERS On Formation Of Foam Cell In AS

EDA+FN is an extra domain A (EDA) of fibronectin (FN) mRNA, which arises from alternatively spliced transcripts. Recent studies have shown that EDA plays an important role in atherosclerosis (AS). Compared with single ApoE-/- mutant mice, double ApoE" and EDA" mutant mice showed reduced atherosclerosis after an atherogenic-diet treatment. EDA-/- ApoE-/- macrophages accumulated less intracellular lipid than ApoE-/-control cells. However, the underlying mechanism of EDA+FN in the the development and progression of AS has been poorly understood.To explore the possible role of EDA+FN in AS, we hypothesize that EDA+FN influence the formation of foamy macrophages by inducing endoplasmic reticulum stress (ERS). First of all, stable macrophage cell lines with tetracycline-regulated EDA+FN or EDA-FN expression were generated, providing the possibility for further studying the function of EDA segment in the cellular level. Two recombinant plasmids, pRevTRE/EDA+FN and pRevTRE/EDA-FN, were constructed using genetic engineering techniques. pRevTet-on and pGag/Pol,pVSV-G were co-transfected into 293T using calcium phosphate precipitation. Supernatants were collected, concentrated, and infected into mouse microphage cell line, Raw264.7. Drug-resistant cell clones were obtained through G418 selection, and named MCTet-on. pRevTRE/EDA+FN and pRevTRE/EDA-FN containing the target gene were transformed into MCTet-on using the same approach. Hygromycin-resistant cell monoclines were collected by limiting dilution, and termed MCTeton/EDA+FN and MCTeton/EDA-FN, respectively. RT-PCR and immunoblotting were then applied to identify the cell line. Results demonstrated that MCTeton/EDA+FN and MCTeton/EDA-FN expressed EDA+FN and EDA-FN after Dox induction, respectively, suggesting that we successfully generate stable macrophage cell lines with tetracycline-regulated EDA+FN or EDA-FN expression. To understand the relationship of EDA+FN to ERS, MCTeton/EDA+FN and MCTeton/EDA-FN were collected after 0,24,48 and 72hrs of Dox induction. Expression of ERS markers, GRP78/BiP and CHOP, were detected using the immnunoblotting. The results displayed that GRP78/BiP expression reached the peak at 48 hrs (P＜0.01, n=3), and decreased at 72hrs, while CHOP expression begin to appear at 72 hrs. However, expression of GRP78/BiP and CHOP in MCTeton /EDA-FN control cells had no significant changes. This indicated that overexpression of EDA+FN induces ER stress after DOX induction and activates UPR. We also applied real-time PCR to examine sterol regulatory element binding protein (SREBPs) mRNA at 0,24,48 and 72 hrs after Dox induction. SREBP-1 mRNA expression slightly changed after induction, but SREBP-2 obviously increased over the induction time. At 48hrs, it increased by 3.4 times (P＜0.01, n=5), indicating that upregulation of SRERP-2 may be induced by ERS. Oil red O staining, uptake of LDL and total cholesterol/triglyceride assays were used to examine the formation of foamy macrophages, uptake and synthesis of the lipid in the MCTeton/EDA+FN at 0 and 48hrs of induction. The results showed that macrophages overexpressing EDA+FN enhanced lipid uptake and the amount of cholesterol which is 1.86 fold higher than the control group (P＜0.05, n=3), but not the amount of triglyceride. Treatment with an ERS/UPR inhibitor, PBA, inhibited upregulation of ER chaperone, GRP78/BiP, prevented the lipid uptake by MCTeton/EDA+FN for, and decreased the amount of cholesterol. Overexpression of SREBP-2 induced by EDA+FN also decreased to 2.2 times at 48hrs of induction after treating with PBA. These findings suggest that EDA+FN-induced ERS/UPR may affect Lipid Metabolism by activating SREBP-2, thereby promoting the formation of foamy macrophages.In conclusion, we successfully generate stable macrophage cell lines with tetracycline-regulated EDA+FN or EDA-FN expression. We reported at the first time that overexpression of EDA+FN may induce ERS, initially confirming that EDA+FN-induced ERS/UPR may increase lipid uptake and synthesis by activating SREBP-2, and then promote the formation of macrophage cells. These data provide the experimental evidence for further explaining the mechanism of EDA+FN in the AS.