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. 2017 Aug 7;127(9):3353–3366. doi: 10.1172/JCI87406

Figure 5. EREG/EGFR increases pain through a PI3K/AKT→mTOR→4E-BP1→eIF4F complex→MMP-9 signaling pathway.

Figure 5

(A) The signaling pathway investigated, with major proteins indicated in black and blocking drugs or mutants shown in red. (B) Treatment with wortmannin (5 μg, i.t.) blocks EREG‑induced increases in late-phase formalin-induced pain behavior (drug × drug: F1,23 = 4.7, P = 0.04). (C) Low doses of rapamycin (5 mg/kg) and CCI 779 (1 mg/kg) block EREG effects without affecting formalin-induced pain per se (rapamycin, drug × drug: F1,27 = 3.6, P = 0.04; CCI 779 drug × drug: F1,28 = 4.2, P = 0.03); higher doses (10 mg/kg) are analgesic (main effects: rapamycin, F1,28 = 22.9, P < 0.001; CCI 779, F1,28 = 30.2, P < 0.001). (D) No effect on EREG increases in formalin-induced pain behavior in SGK1/2 (Rps6kb1/Rps6kb2) double-null mutant mice (Rps6kb1/2–/–; main effect of drug: F1,18 = 25.8, P < 0.001). (E) Lack of EREG effects in 4E-BP1 (Eif4ebp1–/–) null mutant mice (genotype × drug: F1,33 = 7.1, P = 0.01). (F) Treatment with 4EGI-1 (25 μg, i.t.) blocks EREG effects (drug × drug: F1,20 = 7.6, P = 0.01). (G) Treatment with TIMP-1 (4 pmol, i.t.) blocks EREG effects (drug × drug: F1,30 = 5.6, P = 0.02). (H) Lack of EREG effects in MMP-9 null mutants (Mmp9–/–; genotype × drug: F1,20 = 16.1, P = 0.001). In all experiments, EREG was injected at 10 ng i.t. Bars in all graphs represent mean ± SEM for percentage of samples featuring licking/biting behavior; n = 6–8/drug/dose and n = 6–12/drug/genotype (dependent on breeding success). Two-way ANOVA for all panels followed by t test compared with EREG vehicle. *P < 0.05; **P < 0.01; ***P < 0.001, compared with wortmannin, rapamycin, CCI 779, 4EGI-1, or TIMP-1 vehicle, or +/+ genotype. #P < 0.05; ##P < 0.01; ###P < 0.001, compared with rapamycin/CCI 779 vehicle. †P < 0.05 compared with vehicle/vehicle group.