Journal: PLoS ONE

Article Title: TGF-β-Activated Kinase 1 (TAK1) Signaling Regulates TGF-β-Induced WNT-5A Expression in Airway Smooth Muscle Cells via Sp1 and β-Catenin

doi: 10.1371/journal.pone.0094801

Figure Lengend Snippet: (A, E) Airway smooth muscle cells were either left unstimulated (vehicle basal) or stimulated with TGF-β (2 ng/ml) in the presence or absence of LL-Z1640-2 (0.1 µM, 0.5 µM, 1.0 µM) for 24 hours. Expression of WNT-5A mRNA(A) and collagen IαI and fibronectin mRNA (E) was determined by qRT-PCR, corrected for 18S rRNA and expressed relative to vehicle basal. Data represent mean ± SEM of 4-5 independent experiments. **p<0.01, ***p<0.001 compared to vehicle basal, # p<0.05, ## p<0.01, ### p<0.001 compared to TGF-β-stimulated cells; 2-way ANOVA followed by Bonferroni multiple comparisons test. (B) Airway smooth muscle cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of LL-Z1640-2 (0.5 µM) for 48 hours. Western analysis was performed on whole cells extracts for WNT-5A protein. Expression of GAPDH was analyzed as loading control. (C–D, F) Airway smooth muscle cells were transfected with TAK1-specific siRNA or a non-targeting siRNA as control. Subsequently, cells were stimulated with TGF-β (2 ng/ml) for 24 hours and analyzed for the expression of TAK1 mRNA (C), WNT-5A mRNA (D) and collagen IαI and fibronectin mRNA (F) by qRT-PCR and expressed relative to non-targeting siRNA-transfected, untreated control. Data represent mean ± SEM of 4 independent experiments. *p<0.05, **p<0.01, ***p<0.001 compared to non-targeting siRNA-transfected untreated control, #p<0.05, ## p<0.01 compared to non-targeting siRNA-transfected, TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test.

Article Snippet: Recombinant human TGF-β 1 and rat anti-WNT-5A antibody were from R&D systems (Abingdon, UK). siRNAs specific for human TAK1, human CUTL1, human TCF4 and human ETS1, rabbit anti-Sp1 (PEP2) X TransCruz, mouse anti-GAPDH, mouse anti-β-actin, horseradish peroxidase (HRP)-conjugated chicken anti-rat antibody and Protein A-agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Transfection

Journal: PLoS ONE

Article Title: TGF-β-Activated Kinase 1 (TAK1) Signaling Regulates TGF-β-Induced WNT-5A Expression in Airway Smooth Muscle Cells via Sp1 and β-Catenin

doi: 10.1371/journal.pone.0094801

Figure Lengend Snippet: (A) TAK1 activates p38 and JNK. Airway smooth muscle cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of LL-Z1640-2 (0.5 µM) for 30 and 60 minutes. Whole cells extracts were immunoblotted for phospho-p38 and phospho-JNK using specific antibodies. Equal protein loading was verified by the analysis of β-actin. (B–D) p38 and JNK involvement in WNT-5A expression. Airway smooth muscle cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of SB203580 (10 µM) or SP600125 (10 µM) or combination of both SB203580 and SP600125 (10 µM each) for 24 hours. RNA was isolated and WNT-5A mRNA expression was determined by qRT-PCR, corrected for 18S rRNA and expressed relative to vehicle basal. Data represent mean ± SEM of 4–6 independent experiments. **p<0.01, ***p<0.001 compared to vehicle basal, ### p<0.001 compared to TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test.

Article Snippet: Recombinant human TGF-β 1 and rat anti-WNT-5A antibody were from R&D systems (Abingdon, UK). siRNAs specific for human TAK1, human CUTL1, human TCF4 and human ETS1, rabbit anti-Sp1 (PEP2) X TransCruz, mouse anti-GAPDH, mouse anti-β-actin, horseradish peroxidase (HRP)-conjugated chicken anti-rat antibody and Protein A-agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Isolation, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: TGF-β-Activated Kinase 1 (TAK1) Signaling Regulates TGF-β-Induced WNT-5A Expression in Airway Smooth Muscle Cells via Sp1 and β-Catenin

doi: 10.1371/journal.pone.0094801

Figure Lengend Snippet: (A) De novo protein synthesis is required for TGF-β-induced WNT-5A expression. Airway smooth muscle cells were either left unstimulated (vehicle basal) or stimulated with TGF-β (2 ng/ml) in the presence or absence of the protein synthesis inhibitor cycloheximide (5 µg/ml) for 24 hours. WNT-5A mRNA induction was evaluated by qRT-PCR. Data represent mean ± SEM of 4 independent experiments. **p<0.01, ***p<0.001 compared to vehicle basal, ## p<0.01 compared to TGF-β-stimulated cells; 2-tailed Student's t test for paired observations. (B-D) β-Catenin silencing reduces TGF-β-induced WNT-5A expression. Airway smooth muscle cells were transfected with β-catenin-specific siRNA or a non-targeting siRNA as control. Subsequently, cells were stimulated with TGF-β (2 ng/ml) for 24 hours (mRNA; B,C) or 48 hours (protein; D). (B,C) Expression of β-catenin mRNA (B) and WNT-5A mRNA (C) was determined by qRT-PCR and expressed relative to non-targeting siRNA transfected, untreated control. Data represent mean ± SEM of 5 independent experiments. *p<0.05, **p<0.01 compared to non-targeting siRNA-transfected, untreated control, # p<0.05, ### p<0.001 compared to non-targeting siRNA-transfected, TGF-β-stimulated cells; 2-tailed Student's t test for paired observations. (D) Western blot analysis was performed to analyze WNT-5A and β-catenin protein expression in whole cell extracts. Equal protein loading was verified by the analysis of GAPDH. (E) Forced increase in β-catenin abundance elevates WNT-5A protein level. Cells were transfected with S33Y-β-catenin mutant or a GFP expression vector as control. Subsequently, cells were either left untreated or stimulated with TGF-β (2 ng/ml) for 48 hours. Western blot analysis was performed to determine the abundance of WNT-5A and total β-catenin at protein level. GAPDH expression assessed as loading control. (F) Canonical WNT ligand stimulation increases WNT-5A gene expression. Cells were stimulated with L-cells-derived WNT-3A conditioned medium or control conditioned medium for 24 hours. Expression of WNT-5A mRNA was evaluated by qRT-PCR and expressed relative to control conditioned medium. Data represent mean ± SEM of 5 independent experiments. **p<0.01 compared to control conditioned medium; 2-tailed Student's t test for paired observations.

Article Snippet: Recombinant human TGF-β 1 and rat anti-WNT-5A antibody were from R&D systems (Abingdon, UK). siRNAs specific for human TAK1, human CUTL1, human TCF4 and human ETS1, rabbit anti-Sp1 (PEP2) X TransCruz, mouse anti-GAPDH, mouse anti-β-actin, horseradish peroxidase (HRP)-conjugated chicken anti-rat antibody and Protein A-agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Western Blot, Mutagenesis, Plasmid Preparation, Gene Expression, Derivative Assay

Journal: PLoS ONE

Article Title: TGF-β-Activated Kinase 1 (TAK1) Signaling Regulates TGF-β-Induced WNT-5A Expression in Airway Smooth Muscle Cells via Sp1 and β-Catenin

doi: 10.1371/journal.pone.0094801

Figure Lengend Snippet: (A) In silico analysis of WNT-5A promoter. Schematic representation of WNT-5A promoter A indicating the transcription factor binding sites as predicted by PROMO version 3. Only selective transcription factors are depicted here. The schematic is not to scale. TSS: Transcriptional Start Site. (B–G) Silencing of various transcription factors and WNT-5A gene expression. Airway smooth muscle cells were transfected with a non-targeting siRNA as control or with CUTL1-specific (B, C), TCF4-specific (D, E) or ETS1-specific (F, G) siRNA. Subsequently, cells were stimulated with TGF-β (2 ng/ml) for 24 hours and analyzed for the expression of genes as indicated in panels by qRT-PCR, corrected for 18S rRNA and expressed relative to non-targeting siRNA transfected, untreated control. Data represent mean ± SEM of 3-5 independent experiments. *p<0.05, **p<0.01, ***p<0.001 compared to non-targeting transfected, untreated control; 1-way ANOVA followed by Newman-Keuls multiple comparisons test.

Article Snippet: Recombinant human TGF-β 1 and rat anti-WNT-5A antibody were from R&D systems (Abingdon, UK). siRNAs specific for human TAK1, human CUTL1, human TCF4 and human ETS1, rabbit anti-Sp1 (PEP2) X TransCruz, mouse anti-GAPDH, mouse anti-β-actin, horseradish peroxidase (HRP)-conjugated chicken anti-rat antibody and Protein A-agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: In Silico, Binding Assay, Gene Expression, Transfection, Control, Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: TGF-β-Activated Kinase 1 (TAK1) Signaling Regulates TGF-β-Induced WNT-5A Expression in Airway Smooth Muscle Cells via Sp1 and β-Catenin

doi: 10.1371/journal.pone.0094801

Figure Lengend Snippet: (A-B) Mithramycin A attenuates WNT-5A mRNA and protein expression. (A) Cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of Mithramycin A (300 nM) for 24 hours. WNT-5A mRNA was analyzed by qRT-PCR. Data represent mean ± SEM of 4 independent experiments. **p<0.01 compared to vehicle basal, ## p<0.01 compared to TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test. (B) Cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of Mithramycin A (300 nM) for 48 hours. Whole cell extracts were prepared and WNT-5A protein abundance was evaluated by western analysis. GAPDH was assessed as loading control. (C, D) Cells were transfected with Sp1-specific or a non-targeting siRNA as control. Subsequently, cells were stimulated with TGF-β (2 ng/ml) for 24 hours and analyzed for the expression of Sp1 mRNA (C) and WNT-5A mRNA (D) by qRT-PCR. Data represent mean ± SEM of 5 independent experiments. *p<0.05, ***p<0.001 compared to non-targeting siRNA-transfected untreated control, #p<0.05, ### p<0.001 compared to non-targeting siRNA-transfected, TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test. (E) Mithramycin A attenuates TGF-β-induced extracellular matrix expression. Cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of Mithramycin A (300 nM) for 24 hours. Collagen IαI and fibronectin mRNA was analyzed by qRT-PCR. Data represent mean ± SEM of 4 independent experiments. *p<0.05, **p<0.01 compared to vehicle basal, #p<0.05, ## p<0.01 compared to TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test. (F) Sp1 is recruited to WNT-5A promoter in response to TGF-β. Cells were left untreated or stimulated with TGF-β (2 ng/ml) for 16 hours. Chromatin was prepared and ChIP analysis was performed as described in the Materials and Methods section. PCR was carried out using primers specific for Sp1 binding region on WNT-5A promoter A after immunoprecipitation with anti-Sp1 or control IgG antibody. Input DNA from chromatin preparation before immunoprecipitation was amplified to ascertain the loading. Resulting PCR products were analyzed by DNA PAGE. (G) TAK1 mediates recruitment of Sp1 to WNT-5A promoter in response to TGF-β. Cells were left untreated or stimulated with TGF-β (2 ng/ml) in the presence or absence of LL-Z1640-2 (0.5 µM) for 16 hours. ChIP analysis was performed as described above.

Article Snippet: Recombinant human TGF-β 1 and rat anti-WNT-5A antibody were from R&D systems (Abingdon, UK). siRNAs specific for human TAK1, human CUTL1, human TCF4 and human ETS1, rabbit anti-Sp1 (PEP2) X TransCruz, mouse anti-GAPDH, mouse anti-β-actin, horseradish peroxidase (HRP)-conjugated chicken anti-rat antibody and Protein A-agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics, Western Blot, Control, Transfection, Binding Assay, Immunoprecipitation, Amplification

Journal: bioRxiv

Article Title: Wnt5a-mediated Adipo-Cardiac Interorgan Communication in HFpEF

doi: 10.1101/2025.10.29.685456

Figure Lengend Snippet: (A) Schematic outline of study. (B) Cellular localization of differentially expressed genes (DEGs) in Nprc -AKO visceral adipose tissue (VAT). (C) Increased and decreased gene ontologies in Nprc -AKO vs control VAT. (D) Volcano plot of DEGs in Nprc -AKO vs control VAT (red indicates p < 0.01 and 50%-fold-change compared to control). (E) Reverse transcriptase quantitative PCR of top 5 Wnt isoforms in Nprc -AKO vs control VAT. (F) Plasma circulating levels of Wnt5a in global (GKO), cardiomyocyte-specific (CKO), adipocyte-specific (AKO), and control (WT) mice after L-NAME and high fat diet. (G) Cell size in H9C2 cells after treatment with Wnt5a. (H) RT-qPCR for hypertrophic markers NPPA, NPPB, and MYH7 in H9C2 cells after Wnt5a treatment.

Article Snippet: For studies investigating the effects of Wnt5a treatment, cells were transfected with either pcDNA3.2-Wnt5a plasmid (Addgene 43813) or empty vector control (Addgene 29496).

Techniques: Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Clinical Proteomics, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Wnt5a-mediated Adipo-Cardiac Interorgan Communication in HFpEF

doi: 10.1101/2025.10.29.685456

Figure Lengend Snippet: (A) Schematic outline of study. (B) Histologic and wheat germ agglutinin-based hypertrophy, (C) plasma Wnt5a levels, (D) left ventricular systolic pressure, (E) left ventricular end-diastolic pressure, (F) diastolic relaxation of the left ventricle, (G) right ventricular systolic pressure, (H) right ventricular end-diastolic pressure, (I) diastolic relaxation of the right ventricle, (J) left atrial end-systolic volume by echocardiography, and (K) E/e’ ratio by echocardiography in C57/BL6J mice treated with 8 weeks of L-NAME and high fat diet, followed by LGK974 or carrier control intraperitoneal injections.

Article Snippet: For studies investigating the effects of Wnt5a treatment, cells were transfected with either pcDNA3.2-Wnt5a plasmid (Addgene 43813) or empty vector control (Addgene 29496).

Techniques: Clinical Proteomics, Control

Journal: Disease models & mechanisms

Article Title: The Meckel-Gruber syndrome protein TMEM67 controls basal body positioning and epithelial branching morphogenesis in mice via the non-canonical Wnt pathway.

doi: 10.1242/dmm.019083

Figure Lengend Snippet: Fig. 5. The receptor tyrosine kinase-like orphan receptor ROR2 colocalises and interacts with TMEM67, and is dependent on this interaction for phosphorylation. (A) Four-colour IF imaging showing that endogenous ROR2 (green) colocalizes with TMEM67 (blue) and RPGRIP1L (red) at the ciliary transition zone. Arrowheads indicate regions shown in magnified insets. DAPI is pseudocoloured in grey. Scale bar: 10 μm. (B) Anti-HA co-immunoprecipitations (IPs) demonstrating interaction between full-length exogenous HA-tagged TMEM67 (size 115 kDa) and FLAG-tagged ROR2 (size 105 kDa). Input whole-cell extracts (WCE) for the indicated transfected constructs are on the left. IP of an irrelevant protein (HA-tagged MCPH1) was a negative control. Results are shown for immunoblotting (IB) for anti-FLAG (upper panel) and anti-TMEM67 (lower panel). * indicates a non-specific band in IPs; see supplementary material Fig. S6 for full unprocessed images. (C) Upper panel: IPs demonstrating interaction between FLAG-tagged ROR2 and endogenous TMEM67. Input WCE is shown on the left, and negative control IPs include a no antibody (Ab) control and goat (Gt) and rabbit (Rb) irrelevant (irr.) polyclonal antibodies (PAb). Immunoblotting (IB) for anti-FLAG shows pulldown of FLAG-ROR2 by Gt anti-ROR2 and Rb anti-TMEM67. Lower panel: IPs with irrelevant protein (FLAG-MCPH1, size 93 kDa). (E) Loss of the active phosphorylated ROR2 isoform (labelled P) in mutant Tmem67−/−cells following Wnt5a treatment, compared with strong induction of the active isoform (upper band, as indicated) in wild-type Tmem67+/+ cells. Loading control is for β-actin.

Article Snippet: Protein expression and in vitro binding assay Purified recombinant Wnt3a and Wnt5a proteins (R&D Systems Inc.) and purified BSA as a negative control (Sigma-Aldrich Co. Ltd), were labelled with NHS-fluorescein (Thermo Fisher Scientific Inc.), as described by the manufacturer.

Techniques: Phospho-proteomics, Imaging, Transfection, Construct, Negative Control, Western Blot, Control, Mutagenesis

Journal: Disease models & mechanisms

Article Title: The Meckel-Gruber syndrome protein TMEM67 controls basal body positioning and epithelial branching morphogenesis in mice via the non-canonical Wnt pathway.

doi: 10.1242/dmm.019083

Figure Lengend Snippet: Fig. 6. Loss of Wnt5a-induced branching morphogenesis during Tmem67−/−embryonic lung ex vivo organogenesis. (A) Embryonic (E12.5) lungs were explanted and treated for 0, 6 and 24 h with either control-conditioned medium or medium containing Wnt5a. Magnified insets (black frames) under high power are shown for 24-h treatments. Epithelial branching is significantly induced by Wnt5a in Tmem67+/+ lungs, but this response is absent in Tmem67−/−lungs. The bar graph shows quantification of the total number of branches in one lung for each genotype. Values shown are means of three independent replicates and error bars indicate ±s.e.m. The statistical significance of the pair-wise comparisons are represented as *P<0.05 and n.s. for non-significant, Student’s two-tailed t-test. (B) H&E staining of ex-vivo-cultured embryonic lung sections, showing normal acini (ac) and mesenchymal tissue (ms, in green) for wild-type Tmem67+/+ lung, and the stimulation of normal epithelial branching by Wnt5a (green asterisk and arrowheads). In contrast, Tmem67−/−lungs have abnormal mesenchymal cell condensates (red arrowheads), suggesting defective epithelial-mesenchymal induction. The red asterisks indicate abnormal bronchiolar formation; cl indicates the direction of the central lung. (C) Rho activation pull-down assays of whole-cell extracts from wild-type Tmem67+/+ and mutant Tmem67−/−embryonic (E15.5) lungs. Total RhoA in input material is shown as the loading control, with the ratio indicating active:total RhoA levels. A positive control for the assay (+GTPγS; loading with non-hydrolyzable GTPγS) and a negative control (+GDP; loading with GDP) are also shown. (D) Quantitative real-time PCR assays of transcript expression levels in wild-type Tmem67+/+ and mutant Tmem67−/−embryonic (E15.5) lungs for Shh, downstream effectors of the Shh signalling pathway (Gli1 and Ptch1) and a downstream effector of the canonical Wnt signalling pathway (Axin2). Levels of transcripts were all significantly increased in Tmem67−/−embryonic lungs, with the indicated pair-wise comparisons represented as **P<0.01, Student’s two-tailed t-test for n=3 independent assays. Error bars indicate ±s.e.m.

Article Snippet: Protein expression and in vitro binding assay Purified recombinant Wnt3a and Wnt5a proteins (R&D Systems Inc.) and purified BSA as a negative control (Sigma-Aldrich Co. Ltd), were labelled with NHS-fluorescein (Thermo Fisher Scientific Inc.), as described by the manufacturer.

Techniques: Ex Vivo, Control, Two Tailed Test, Staining, Cell Culture, Activation Assay, Mutagenesis, Positive Control, Negative Control, Real-time Polymerase Chain Reaction, Expressing

Journal: Disease models & mechanisms

Article Title: The Meckel-Gruber syndrome protein TMEM67 controls basal body positioning and epithelial branching morphogenesis in mice via the non-canonical Wnt pathway.

doi: 10.1242/dmm.019083

Figure Lengend Snippet: Fig. 7. Rescue of normal embryonic lung-branching morphogenesis and polarity in mutant Tmem67−/−tissue by ex vivo treatment with the RhoA activator calpeptin. (A) Embryonic lungs (age E11.5) grown in culture for the indicated times after treatment with either vehicle control (0.1% DMSO) or calpeptin at final concentration 1 unit/ml for 3 h. Tmem67−/−lungs had abnormally dilated branches (arrowheads) surrounded by areas of condensed mesenchyme, in contrast to the fine distal branches visible in Tmem67+/+ lungs. Calpeptin treatment of mutant Tmem67−/−lungs resulted in more developed branch development and a general morphology that was similar to the wild-type lungs. Magnified insets are indicated by the black frames and shown on the right. (B) The bar graph shows the quantification of the total number of terminal branches per lung (total n=3) for each genotype and treatment condition. The statistical significance of the indicated pair-wise comparisons is *P<0.05 and **P<0.01, Student’s two-tailed t-test. Error bars indicate ±s.e.m. (C) The polarity of mitotic cell division is rescued by treatment with calpeptin from predominantly parallel (para.) in mutant alveoli to predominantly perpendicular (perp.) divisions, as observed in wild-type epithelia. The statistical significance of the indicated pair-wise comparisons is ***P<0.001, chi-squared test, with the total number of cells counted in ten fields of view indicated above each bar. Representative examples of mitotic divisions, visualised by γ-tubulin (green) and indicated by the fine dotted lines, are shown on the right. Apical surfaces are highlighted by the broad dotted lines, with asterisks indicating the alveolar lumen. Scale bar: 20 μm. (D) Schematic in which signalling through the Wnt5a-TMEM67-ROR2 axis normally represses Shh and canonical Wnt (Wnt3a) signalling to moderate levels (small green arrow) between embryonic ages E9.5 and E11.5. Loss or mutation of any component in this axis (red cross) causes loss of repression (dashed line) with Shh and canonical Wnt pathway de-regulation and ectopic expression of Shh at later gestation ages (large red arrow). This contributes to pulmonary hypoplasia with condensed mesenchyme and impaired development of the alveolar system in the ciliopathy disease state.

Article Snippet: Protein expression and in vitro binding assay Purified recombinant Wnt3a and Wnt5a proteins (R&D Systems Inc.) and purified BSA as a negative control (Sigma-Aldrich Co. Ltd), were labelled with NHS-fluorescein (Thermo Fisher Scientific Inc.), as described by the manufacturer.

Techniques: Mutagenesis, Ex Vivo, Control, Concentration Assay, Two Tailed Test, Expressing