Fig 4. YAP interacts with the HDAC3-NCoR1 repressor complex to inhibit reparative macrophage phenotype.
(A) BMDMs were isolated from control and YAP/TAZ-dKO mice and stimulated with/without IL4/IL13 for 12 hours. qRT-PCR for reparative marker genes, Arg1, Egr2, Cd206, Ym1, Fizz1, and Fn1 using RNA isolated from untreated or IL4/IL13-treated control and YAP/TAZ-dKO BMDMs. n = 3 in each group. Gene expression results were normalized to gapdh, and results are represented as fold change. (B) Western blot analysis for Arg1 was performed using total lysates from untreated or IL4/IL13-treated control and YAP/TAZ-dKO BMDMs. β-actin is shown as a loading control. The relative protein expression was quantified. (C) BMDMs were isolated from control and YAP/TAZ-dKO mice and stimulated with/without IL4/IL13 for 15 and 30 minutes. The phosphorylated level of AKT was detected by WB analysis, and the relative protein expression was quantified. Vinculin is shown as a loading control. (D–F) WB analysis for YAP, pYAP (S127), and TAZ was performed using total lysates from RAW264.7 cells, wild-type PMs, and BMDMs treated with IL4/IL13 for 0 to 24 hours as indicated. β-actin, GAPDH, or vinculin are shown as a loading control. The relative protein expression was quantified. (G) Results of normalized luciferase reporter assays in HEK293T cells with Arg1-luciferase reporter in the presence of YAP or TAZ. (H) ChIP assay using chromatin from wild-type IL4/IL13 treated BMDMs with IgG or YAP antibody. (I–K) Results of normalized luciferase reporter assays in HEK293T cells with Arg1-luciferase reporter in the presence of YAP, TAZ, YAPS127A, and HDAC3 alone or as indicated combinations. (L) RAW264.7 cells were co-transfected with YAP-Myc and HDAC3-FLAG plasmids for 48 hours and then treated with IL4/IL13 for 12 hours. IP was performed using IgG control, anti-FLAG, and anti-Myc antibodies followed by WB for the Myc or FLAG tag. (M) Wild-type BMDMs were treated with IL4/IL13 for 12 hours, and IP was performed using IgG or an anti-YAP antibody followed by WB for YAP, HDAC3, and NCoR1. (N) BMDMs were isolated from control and YAP/TAZ-dKO mice and transfected with control, YAP, or HDAC3 siRNA for 72 hours, followed by IL4/IL13 stimulation for 16 hours. RNA was prepared for qRT-PCR analysis of Arg1. (O–Q) Results of normalized luciferase reporter assays in HEK293T cells with Arg1-luciferase reporter when YAP, HDAC3 alone, or their combinations were transfected in the presence or absence of vorinostat, scriptaid, or RGFP966. (R) Results of normalized luciferase reporter assays in HEK293T cells transfected with Arg1-luciferase reporter with or without YAP. After 24 hours, cells were infected with either HDAC3 or HDAC3H134A, H135A lentivirus and analyzed 48 hours after infection. Data from luciferase experiments are presented as mean ± SD. For numerical raw data, please see S1 Data. Arg1, Arginase-I; BMDMs, bone marrow–derived macrophages; Cd206, cluster of differentiation 206; Egr2, early growth response 2; Fizz1, found in inflammatory zone 1; Fn1, fibronectin 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDAC3, histone deacetylase 3; HEK293T, human embryonic kidney 293 T; IgG, immunoglobulin G; IL, interleukin; IP, immunoprecipitation; NCoR1, nuclear receptor corepressor 1; NS, non-significant; PMs, peritoneal macrophages; pYAP, phosphorylated YAP; qRT-PCR, real-time quantitative reverse transcription PCR; siRNA, short interfering RNA; WB, western blot; YAP, yes-associated protein; YAP/TAZ-dKO, YAP/TAZ double knockout.