EtOH + HFD-fed DEN-challenged Il-17raΔHep mice exhibit a defect in caspase 2-dependent activation of SREBP1/2.
(A) Expression of cleaved (C) or full-length (F) SREBP1, SREBP2, caspase-2, S1P, and TNFR1 were analyzed by Western blotting in livers from patients with no liver injury (normal), NASH, or ALD. (B) Expression of cleaved (C) or full-length (F) SREBP1, SREBP2, caspase-2, TNFR1, and TACE in livers of EtOH + HFD-fed Il-17raF/F mice and IL-17RAΔHep littermates; representative images of ≥3 independent experiments are shown. (C) Expression of full-length (F) TNFR1 was analyzed by Western blot of livers of EtOH + HFD-fed Il-17raF/F mice, Il-17raΔMΦ, and Il-17raΔHSC littermates; representative images of ≥3 independent experiments are shown. (D) Expression of SREBP2, caspase-2, TNFR1 (Ab1: Santa Cruz, upper panel; Ab2, Abcam, lower panel), and TACE was analyzed in livers of EtOH + HFD-fed Il-17raF/F mice and Il-17raΔHep mice using immunohistochemistry (×60, ×4, and ×10 objectives); positive area was calculated as a percent. (E) Primary WT and IL-17RA-deficient steatotic hepatocytes from EtOH + HFD-fed Il-17raF/F mice and Il-17raΔHep mice were cultured ±Marimastat (10 μM), GW280264X (2 μM), or ±brefeldin A (BFA, 1 or 5 μg/ml) for 6 h. Expression of full-length (F) or cleaved (C) TNFR1 and ARTS-1 was analyzed using Western blotting of cell lysates and supernatants from WT mice and Il-17raΔHep. BFA inhibited TNFR1 exocytosis in WT and IL-17RA-deficient hepatocytes in a dose-dependent manner. Statistical analysis was performed using 2-tailed Student’s t test. Data are presented mean ± SD. ***p <0.001. ALD, alcoholic liver disease; ARTS-1, aminopeptidase regulator of TNFR1 shedding; DEN, diethylnitrosamine; EtOH, ethanol; HFD, high-fat diet; HSP90, heat shock protein 90; NASH, non-alcoholic steatohepatitis; SREBP, sterol regulatory element-binding protein; TACE, tumor necrosis factor-α-converting enzyme; TNFR1, tumor necrosis factor receptor 1; WT, wild-type.