OTX015 was administrated by oral gavage twice daily at 25 mg/kg (35) starting at week 3 (day 21) after bleomycin injury till week 6 (day 42)

OTX015 was administrated by oral gavage twice daily at 25 mg/kg (35) starting at week 3 (day 21) after bleomycin injury till week 6 (day 42). siRNA compared with NT control, by 2-tailed test. (C) RNA from NT and Brd4 siRNACtreated cells was analyzed for Nox4 mRNA by real-time PCR. * 0.05, compared with the NT control of the same cell line, by 2-tailed test. (D) BET inhibitors, BET-762 (0.5 M), JQ1 (1 M), and OTX015 (0.5 M) were added to primary IPF lung fibroblasts at 70% confluence for 48 hours, and RNA was collected and Nox4 mRNA expression analyzed by real-time PCR. Triangles, squares, or circles indicate 3 different IPF individuals from whom primary cells were derived. Expressed values represent mean SD; = 3 experimental replicates of each cell line. * 0.05, treated group vs. control (vehicle) group, by 2-tailed test. Brd4 inhibition blocks TGF-1Cinduced Nox4 expression. TGF-1 is usually a cardinal profibrotic cytokine (8) that induces Nox4 expression in fibroblasts (5); we further examined the effects of BET inhibitors on TGF-1Cinduced Nox4 expression. Normal human lung fibroblasts (IMR90) were transfected with Brd4 siRNA or NT siRNA control, followed by treatment with TGF-1 (2 ng/mL) for 48 hours. Fibroblasts transfected with Brd4 siRNA failed to upregulate Nox4 expression (Physique 2, ACC). We then examined the effect of BET inhibitors on Nox4 expression in response to TGF-1. IMR90 fibroblasts were pretreated with BET inhibitors for 2 hours before TGF-1 (2 ng/mL) treatment for 48 hours; the upregulation of Nox4 mRNA was suppressed by all 3 Brd4 inhibitors, although OTX015 was the most potent with 95% inhibition at 0.5 M (Figure 2D; corresponding changes at the protein levels were also observed, Supplemental Physique 2B). In subsequent experiments, we focused on the effects of OTX015 for both in vitro and in vivo studies. The effects of OTX015 on Nox4 expression were confirmed at the protein level (Physique 2, E and F) and at the level of enzymatic activity, as assessed by extracellular H2O2 release (Physique 2G). Although Nox4 has been reported to promote myofibroblast differentiation and profibrotic responses, it is not known whether putative antifibrotic effects of BET inhibition can be fully accounted for by Nox4 inhibition. We tested the effect of OTX015 treatment on Nox4-silenced cells and observed a small, but appreciable, additive inhibitory effect on TGF-1Cinduced expression of -SMA and collagen. The effect was shown in Supplemental Physique 3. Together, our data indicate that Brd4 inhibition, either by siRNA-mediated gene silencing or by pharmacologic BET inhibitors, downregulates not only the constitutive but also TGF-1Cinducible Nox4 Tap1 expression/activity in lung fibroblasts. Open in a separate window Physique 2 Brd4 inhibition blocks TGF-1Cinduced Nox4 gene upregulation in human lung fibroblasts.(ACC) Normal human lung fibroblasts (IMR90) were transfected with siRNA Brd4 or NT and then treated with vehicle or TGF-1 (2 ng/mL) for 48 hours. (A) The whole cell lysate were collected to examine Brd4 expression by Western blots. (B) Densitometry of Brd4-associated signals detected (ratio to -actin) in A. * 0.05, EP1013 Brd4 siRNACtransfected cells compared with NT control of the same cell line, by 2-tailed test. (C) Treated as in A, cells were analyzed for Nox4 mRNA by real-time PCR (mean SD; = 3 in each group). * 0.05, each group compared with vehicle only; # 0.05, TGF-1Ctreated siRNA Brd4 vs. NT cells, by 2-tailed EP1013 test. (D) IMR90 fibroblasts were incubated overnight with 1% fetal bovine serum at 70% confluence and then treated with vehicle or various Brd4 inhibitors with the same concentration as in Physique 1 for 2 hours before stimulation with TGF-1 (2 ng/mL) EP1013 for 48 hours. Cells were analyzed for Nox4 mRNA by real-time PCR (mean SD; = 3 in each group). * 0.05, each group compared with TGF-1 with vehicle only (Vehl/TGF-1), by 2-tailed test. (E) IMR90 fibroblasts were pretreated with or without OTX015 for 2 hours and then with or without TGF-1 for 48 hours. Cells were collected and subjected to SDS-PAGE and Western blot analysis for Nox4 and -actin (loading control). (F) The densitometry of Nox4-associated signals detected (ratio to -actin) in E. * 0.05, OTX015 pretreated cells with TGF-1 compared with TGF-1, by EP1013 2-tailed test. (G) IMR90 fibroblasts stimulated with/without EP1013 TGF-1 (2 ng/mL for 24 hours) in the presence/absence of OTX015 (0.5.