Fig 1.
YAP/TAZ expression is enhanced in pro-inflammatory macrophages.
(A and B) Western blot analysis for YAP, pYAP (S127), TAZ, and IL6 was performed using total lysates from wild-type BMDMs (A) and PMs (B) treated with LPS/IFNγ for 0 to 12 hours as indicated. β-actin or vinculin is shown as a loading control. The relative protein expression was quantified. (C–E) BMDMs were isolated from wild-type mice and transfected with control or YAP siRNA for 72 hours, followed by LPS/IFNγ or IL4/IL13 stimulation for 16 hours. Cell lysates were prepared for western blot and qRT-PCR analysis. (C) Western blot analysis for YAP, IL6, and Nos2 was performed using total lysates from wild-type BMDMs transfected with control or YAP siRNA. β-actin is shown as a loading control. The relative protein expression was quantified. (D) qRT-PCR for pro-inflammatory marker genes IL6, IL1β, IL12β, Nos2, and Fpr2 using RNA isolated from wild-type BMDMs transfected with control or YAP siRNA and stimulated with LPS/IFNγ. (E) qRT-PCR for reparative/anti-inflammatory marker genes Arg1, Egr2, Cd206, Ym1, and Fizz1 using RNA isolated from wild-type BMDMs transfected with control or YAP siRNA and stimulated with IL4/IL13. Data are shown as the mean ± SEM, n = 3 for each experimental group. Gene expression data were normalized with the reference gene Gapdh, and results are represented as fold change relative to the control treatment. 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; Fpr2, formyl peptide receptor 2; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; IFNγ, interferon gamma; IL, interleukin; LPS, lipopolysaccharide; Nos2; nitric oxide synthase 2; NS, non-significant; PMs, peritoneal macrophages; pYAP, phosphorylated YAP; qRT-PCR, real-time quantitative reverse transcription PCR; siRNA, short interfering RNA; TAZ, transcriptional coactivator with PDZ-binding motif; YAP, yes-associated protein.
Fig 2.
Macrophage-specific gene expression changes due to YAP/TAZ deletion.
(A) Venn analysis of RNA-seq data showing overlap between significantly differentially expressed genes (false discovery rate [FDR] ≤ 5%, absolute log2FC ≥1.0) between control and YAP/TAZ-dKO BMDMs with and without LPS/IFNγ treatment. (B) Heat map of 45 differentially expressed genes (out of a total of 86 genes) identified by Venn analysis of RNA-seq data. (C) Pathway enrichment analysis of the RNA-seq data from untreated or LPS/IFNγ treated control and YAP/TAZ-dKO BMDMs. (D) MA plots for selected pathways showing top differentially expressed pathway genes. MA plots for pro-inflammatory macrophage marker genes are also presented. (E) BMDMs were isolated from control and YAP/TAZ-dKO mice and stimulated with/without LPS/IFNγ for 12 hours. (A) qRT-PCR for pro-inflammatory marker genes, IL6, IL1β, IL12β, Nos2, TNFα, Ccl2, and Rantes using RNA isolated from untreated or LPS/IFNγ-treated control and YAP/TAZ-dKO BMDMs. n = 3 in each group. (F) NO production determined by nitrite (NO2−) levels in conditioned medium prepared from untreated or LPS/IFNγ-treated control and YAP/TAZ-dKO BMDMs. n = 3 in each group. For numerical raw data, please see S1 Data. BMDMs, bone marrow–derived macrophages; Ccl2, C–C motif chemokine ligand 2; FDR, false discovery rate; IFNγ, interferon gamma; IL, interleukin; LPS, lipopolysaccharide; NO, nitric oxide; Nos2, nitric oxide synthase 2; qRT-PCR, real-time quantitative reverse transcription PCR; RNA-seq, RNA sequencing; TAZ, transcriptional coactivator with PDZ-binding motif; TNFα, tumor necrosis factor alpha; UNT, untreated; YAP, yes-associated protein; YAP/TAZ-dKO, YAP/TAZ double knockout.
Fig 3.
YAP/TAZ deficiency impairs pro-inflammatory macrophage phenotype.
(A) BMDMs were isolated from control and YAP/TAZ-dKO mice and stimulated with/without LPS/IFNγ for 12 hours. Western blot analysis for IL6, IL1β, and Nos2 was performed using total lysates from untreated or LPS/IFNγ-treated control and YAP/TAZ-dKO BMDMs. β-actin is shown as a loading control. The relative protein expression was quantified. (B) BMDMs were isolated from control and YAP/TAZ-dKO mice and stimulated with/without LPS/IFNγ for 15 and 30 minutes. The phosphorylated level of JNK, p38, Erk1/2, IKKɑβ, and Tak1 was detected by western blot analysis, and the relative expression was quantified. β-actin is shown as a loading control. (C) Reported TBSs. (D) Predicted TBSs in IL6 promoter. IL6-promoter fragments I (Pro-I) and III (Pro-III) harbor 3 TBSs, while fragment II (Pro-II) harbors 4 TBSs. (E) Results of normalized luciferase reporter assays in HEK293T cells with full-length IL6-luciferase reporter (approximately 2Kb promoter) in the presence of YAP or TAZ. (F) ChIP assay using chromatin from wild-type untreated or LPS/IFNγ treated BMDMs with IgG, YAP, and TAZ antibody. (G) IL6-promoter fragments (Pro-I, Pro-II, and Pro-III) containing wild-type TBSs driving luciferase were compared to mutant fragments with mutated TBSs. (H) IL6 and Tead-luciferase reporters were transfected in HEK293T cells with/without YAP or TAZ in the presence or absence of verteporfin (5 μM). Data from luciferase experiments are presented as mean ± SD. For numerical raw data, please see S1 Data. BMDMs, bone marrow–derived macrophages; ChIP, chromatin immunoprecipitation; Erk1/2, extracellular signal-regulated kinase 1/2; HEK293T, human embryonic kidney 293 T; IFNγ, interferon gamma; IgG, immunoglobulin G; IKKɑβ, inhibitor of nuclear factor kappa-ɑβ-kinase; IL, interleukin; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; Nos2, nitric oxide synthase 2; SD, standard deviation; Tak1, transforming growth factor beta-activated kinase 1; TAZ, transcriptional coactivator with PDZ-binding motif; TBSs, Tead-binding sequences; Tead, Transcriptional enhanced associate domain; VP, verteporfin; YAP, yes-associated protein; YAP/TAZ-dKO, YAP/TAZ double knockout.
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.
Fig 5.
Improved recovery of cardiac function in YAP/TAZ-deficient mice post-MI.
(A–E) Echocardiographic measurements of ventricular functional parameters at baseline and 4-week post-MI from control and YAP/TAZ-dKO mice: (A) LVEF; (B) FS; (C) LVESV; (D) FAC; (E) Representative echocardiographic M-mode images of LV. Data are shown as mean ± SEM (n = 4 to 7 per group). (F–H) Flow cytometry analysis of pro-inflammatory (iNOS+/F4/80+) and reparative (CD206+/F4/80+) macrophages from the 2 and 6 days post-MI hearts: (F) percentage of pro-inflammatory (F4/80+iNOS+) and reparative (F4/80+CD206+) macrophages. Underlying raw data can be found in S1 Data and S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20, S21 and S22 FCS files. (G) qRT-PCR for pro-inflammatory cytokines IL6, IL1β, and Nos2 in isolated iNOS+/F4/80+ macrophages from the 2 days post-MI hearts. (H) qRT-PCR for anti-inflammatory markers Arg1, Ym1, and Fizz1 in isolated CD206+/F4/80+ macrophages from the 6 days post-MI hearts. (I) qRT-PCR for pro-inflammatory marker genes, IL6, IL1β, TNFα, Nos2, Ccl2, Ccr7, and Rantes using RNA isolated from control and YAP/TAZ-dKO hearts (LV) at 3 days post-MI. n = 4 in each group. (J) qRT-PCR for anti-inflammatory marker genes, Ym1, Egr2, Arg1, Fizz1, Cd206, IL10, and TGFβ using RNA isolated from control and YAP/TAZ-dKO hearts (LV) at 7 days post-MI. n = 3 to 4 in each group. Gene expression results were normalized to Gapdh, and results are represented as fold change. For numerical raw data, please see S1 Data. Arg1, Arginase-I; Ccl2, C–C motif chemokine ligand 2; Ccr7, C-C chemokine receptor type 7; Cd206, cluster of differentiation 206; Egr2, early growth response 2; FAC, fractional area change; Fizz1, found in inflammatory zone 1; FS, fractional shortening; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin; LVEF, left ventricular ejection fraction; LVESV, left ventricular end-systolic volume; MI, myocardial infarction; Nos2, nitric oxide synthase 2; NS, non-significant; qRT-PCR, real-time quantitative reverse transcription PCR; TAZ, transcriptional coactivator with PDZ-binding motif; TGFβ, transforming growth factor beta; TNFα, tumor necrosis factor alpha; YAP, yes-associated protein; YAP/TAZ-dKO, YAP/TAZ double knockout.
Fig 6.
Improved post-MI ventricular remodeling and angiogenesis in YAP/TAZ-deficient hearts.
(A) Masson’s trichrome staining on control and YAP/TAZ-dKO hearts at 28 days post-MI. (B) Quantification of Masson’s trichrome stained fibrotic area in control and YAP/TAZ-dKO hearts at 28 days post-MI (n = 6 to 7 per group). Fibrotic area was normalized to the remaining heart for each heart section. (C) Immunostaining showing the expression of collagen type I in infarct zone on control and YAP/TAZ-dKO heart sections at 28 days post-MI. DAPI was used to stain nuclei. Scale bar 100 μM. (D) Quantification of collagen type I positive area in control and YAP/TAZ-dKO heart sections. n = 3 per group. (E) qRT-PCR for profibrotic genes involved in collagen biosynthesis using RNA isolated from control and YAP/TAZ-dKO hearts (LV) at 7 and 28 days post-MI. n = 3 to 4 in each group. (F) qRT-PCR for angiogenic factors Vegf and Vcam1 using RNA isolated from control and YAP/TAZ-dKO hearts (LV) at 7 and 28 days post-MI. n = 3 to 4 in each group. (G) Immunohistochemistry and quantification for Emcn on control and YAP/TAZ-dKO heart sections. For numerical raw data, please see S1 Data. Scale bar 100μM. Acta2, smooth muscle actin alpha 2; Col1ɑ1, collagen type I ɑ 1; Col1ɑ2, collagen type I ɑ 2; Emcn, endomucin; Epi, epicardium; Lox, lysyl oxidase; LV, left ventricular; MI, myocardial infarction; Myo, myocardium; NS, non-significant; p4ha1, prolyl 4-hydroxylase subunit alpha-1; plod2, procollagen-lysine,2-oxoglutarate 5-dioxygenase 2; Plod2b, procollagen-lysine,2-oxoglutarate 5-dioxygenase 2b; qRT-PCR, real-time quantitative reverse transcription PCR; Tagln, transgelin; TAZ, transcriptional coactivator with PDZ-binding motif; Timp1, tissue inhibitor matrix metalloproteinase 1; Vcam1, vascular cell adhesion molecule 1; Vegf, vascular endothelial growth factor; YAP, yes-associated protein; YAP/TAZ-dKO, YAP/TAZ double knockout.
Fig 7.
YAP activation enhances pro-inflammatory but inhibits reparative macrophage phenotype.
(A) Generation of transgenic mice expressing YAP5SA, a constitutively active form of YAP in macrophages by crossing Rosa26YAP5SA mice with LysMCre mice. (B) Western blot analysis for YAP was performed using total lysates from control and LysMCre;Rosa26YAP5SA BMDMs. β-actin is shown as a loading control. (C) Double immunofluorescence for β-galactosidase (red) and CD68 (green) was performed on control and LysMCre;Rosa26YAP5SA BMDMs. Nuclei were visualized by DAPI staining (blue). Scale bar 50 μM. (D–F) BMDMs were isolated from control and LysMCre;Rosa26YAP5SA mice and stimulated with/without LPS/IFNγ or IL4/IL13 for 24 hours. (D) qRT-PCR for pro-inflammatory marker genes, IL6, IL1β, IL12β, Nos2, TNFα, Ccl2, Rantes, and Ccr7 using RNA isolated from untreated or LPS/IFNγ-treated control and LysMCre;Rosa26YAP5SA BMDMs. n = 3 in each group. (E) NO production determined by nitrite (NO2−) levels in conditioned medium prepared from untreated or LPS/IFNγ-treated control and LysMCre;Rosa26YAP5SA BMDMs. n = 3 in each group. (F) qRT-PCR for reparative marker genes, Arg1, Ym1, Cd206, and Fizz1 using RNA isolated from untreated or IL4/IL13-treated control and LysMCre;Rosa26YAP5SA BMDMs. n = 3 in each group. Gene expression results were normalized to gapdh, and results are represented as fold change. (G) Masson’s trichrome staining on control and LysMCre;Rosa26YAP5SA hearts at 28 days post-MI. Fibrotic area was normalized to the remaining heart for each heart section. (H) Quantification of Masson’s trichrome stained fibrotic area in control and LysMCre;Rosa26YAP5SA hearts at 28 days post-MI (n = 4 per group). (I) Immunostaining showing the expression collagen type I in infarct zone on control and LysMCre;Rosa26YAP5SA heart sections at 28 days post-MI. DAPI was used to stain nuclei. Scale bar 100 μM. (J) Quantification of collagen type I positive area in control and LysMCre;Rosa26YAP5SA heart sections. n = 3 per group. For numerical raw data, please see S1 Data. Arg1, Arginase-I; BMDMs, bone marrow–derived macrophages; Ccl2, C–C motif chemokine ligand 2; Cd206, cluster of differentiation 206; Fizz1, found in inflammatory zone 1; IFNγ, interferon gamma; IL, interleukin; IRES, internal ribosome entry site; LPS, lipopolysaccharide; NO, nitric oxide; Nos2, nitric oxide synthase 2; NS, non-significant; qRT-PCR, real-time quantitative reverse transcription PCR; TNFα, tumor necrosis factor alpha; YAP, yes-associated protein.