Tumor Necrosis Factor-alpha utilizes MAPK/NFκB pathways to induce cholesterol-25 hydroxylase for amplifying pro-inflammatory response via 25-hydroxycholesterol-integrin-FAK pathway
Fig 6
MAPK and NFκB pathways are required for TNF-mediated C25H expression.
(A) MEFs treated with TNF (10ng/ml) for 4h in the absence or presence of NFκB inhibitor Bay-11 (10μM) were analyzed for C25H mRNA expression by RT-qPCR (n = 5–6; two independent experiments). (B) MEFs treated with TNF (10ng/ml) for 4h in the absence or presence of p38 inhibitor SB203580 (10μM) were analyzed for C25H mRNA expression by RT-qPCR (n = 5–6; two independent experiments). (C) MEFs treated with TNF (10ng/ml) for 4h in the absence or presence of JNK inhibitor SP600125 (25μM) were analyzed for C25H mRNA expression by RT-qPCR (n = 5–6; two independent experiments). (D) MEFs treated with TNF (10ng/ml) for 4h in the absence or presence of ERK inhibitor PD98059 (25μM) were analyzed for C25H mRNA expression by RT-qPCR (n = 5–6; two independent experiments). (E) MEFs treated with TNF (10ng/ml) for 8h in the absence or presence of NFκB inhibitor Bay-11 (10μM) were analyzed for C25H protein expression by western blotting. (F) Densitometry analysis of C25H protein levels relative to actin (C25H/actin) in MEFs treated with TNF for 8h in the absence or presence of NFκB inhibitor Bay-11 (10μM). C25H/actin ratio levels were normalized to vehicle treated cells. G) MEFs treated with TNF (10ng/ml) for 8h in the absence or presence of p38 inhibitor SB203580 (10μM) were analyzed for C25H protein expression by western blotting. (H) Densitometry analysis of C25H protein levels relative to actin (C25H/actin) in MEFs treated with TNF for 8h in the absence or presence of p38 inhibitor SB203580 (10μM). C25H/actin ratio levels were normalized to vehicle treated cells. RT-qPCR data are shown as Mean ± SEM by using one-way ANOVA multiple comparison test. *p ≤ 0.05. ns; not significant. Immunoblot images are representative from three independent experiments. The densitometric values represent the mean ± SEM from three independent experiments. *p < 0.05 determined by Student’s t-test.