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: 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

Journal: Annals of the New York Academy of Sciences

Article Title: Deletion of Wnt5a in osteoclasts results in bone loss through decreased bone formation.

doi: 10.1111/nyas.14293

Figure Lengend Snippet: Figure 1. Deletion of Wnt5a in vitro decreases osteoclast formation and function. (A) Bone marrow macrophages (BMMs) from 10-week-old male C57BL/6J (WT) or Wnt5aF/F mice were transduced with Lenti-GFP (control) or Lenti-Cre in the presence of M-CSF (30 ng/mL) and sRANKL (30 ng/mL). Lenti-Cre–mediated recombination results in excision of exon 2 of Wnt5a. (B) Wnt5aF/F BMMs transduced with Lenti-Cre had significantly lower gene expression compared with cells transduced with Lenti- GFP. (C) Wnt5aF/F BMMs transduced with Lenti-Cre had fewer osteoclasts compared with cells transduced with Lenti-GFP at day 5. No significant differences were observed in osteoclast number between Lenti-GFP– and Lenti-Cre–transduced WT BMMs. (D) Wnt5aF/F BMMs transduced with Lenti-Cre had significantly less total pit area compared with cells transduced with Lenti- GFP. No significant differences in resorbed pit area were observed between Lenti-GFP– and Lenti-Cre–transduced WT BMMs. (E) Lenti-Cre–transduced Wnt5aF/F BMMs had lower expression of the osteoclast markers Cathepsin K (Ctsk) and calcitonin receptor (Calcr) compared with Lenti-GFP–transduced Wnt5aF/F BMMs. TRAP was not significantly different. Data represent mean ± SD (n = 3), ∗P < 0.05. GFP, green fluorescent protein; WT, wild-type; OC, osteoclast.

Article Snippet: Phosphorylation was also verified by ectopic expression of full-length C-terminus Myc-DDK–tagged mouse WNT5A (Origene; Cat# MR205939) transfected into RAW 264.7 and Saos2 cells using Viromer Red (Origene; Cat# TT100302) as per the manufacturer’s protocol.

Techniques: In Vitro, Transduction, Control, Gene Expression, Expressing

Journal: Annals of the New York Academy of Sciences

Article Title: Deletion of Wnt5a in osteoclasts results in bone loss through decreased bone formation.

doi: 10.1111/nyas.14293

Figure Lengend Snippet: Figure 2. Deletion of Wnt5a in mature osteoclasts does not affect osteoclast formation. (A) Wnt5a cKO mice were generated by crossing Cathepsin K-Cre mice with Wnt5aF/F mice. Cre-mediated recombination in mature osteoclasts results in excision of exon 2. (B) Representative μ-CT 3D reconstructions of femurs from 10-week-old male control and Wnt5a cKO mice. Wnt5a cKO did not influence femur length in male or female mice. (C) Wnt5a cKO resulted in a decrease in body weight in male mice but did not affect body weight in females. (D) BMMs isolated from 10-week-old male and female mice were cultured in the presence of M-CSF (25 ng/mL) alone or with sRANKL (50 ng/mL) for 5 days. Cathepsin K-Cre resulted in a significant decrease in Wnt5a gene expression from osteoclasts from both male and female mice. (E) BMMs from 10-week-old male mice were cultured in the presence of M-CSF (25 ng/mL) and sRANKL (50 ng/mL) for 3 and 5 days. The number of multinucleated osteoclasts was not significantly different between genotypes in male mice at day 3 or 5. (F) Deletion of Wnt5a resulted in a significant decrease in TRAP and calcitonin receptor gene expression at day 5 compared with controls in male and female mice. Data represent mean ± SD (n = 7–10), ∗P < 0.05. Ctsk, cathepsin K; cKO, conditional knockout; M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of nuclear factor kappa-B ligand; OC, osteoclast; Calcr, calcitonin receptor.

Article Snippet: Phosphorylation was also verified by ectopic expression of full-length C-terminus Myc-DDK–tagged mouse WNT5A (Origene; Cat# MR205939) transfected into RAW 264.7 and Saos2 cells using Viromer Red (Origene; Cat# TT100302) as per the manufacturer’s protocol.

Techniques: Generated, Control, Isolation, Cell Culture, Gene Expression, Knock-Out

Journal: Annals of the New York Academy of Sciences

Article Title: Deletion of Wnt5a in osteoclasts results in bone loss through decreased bone formation.

doi: 10.1111/nyas.14293

Figure Lengend Snippet: Figure 3. Conditional deletion of Wnt5a in mature osteoclasts decreased femur microarchitectural indices without influencing osteoclast number or activity. Wnt5a cKO in 10-week-old male mice significantly decreased femoral (A) trabecular and (B) cortical microarchitectural indices. (C) Wnt5a cKO did not influence osteoclast number in vivo in femurs of 10-week-old male mice (40 × magnification). (D) No differences in serum CTX were observed in 10-week-old male mice. Data represent mean ± SD (n = 7), ∗P < 0.05. cKO, conditional knockout; BV/TV, bone volume fraction; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; BS/TV, bone surface over total volume; vBMD, volumetric bone mineral density; Ct.Th, cortical thickness; Ct.Ar, cortical area; Ct.Po, cortical porosity; N.OC/B.Pm, number of osteoclast over bone perimeter; Oc.S/BS, osteoclast surface; CTX, C-terminal telopeptide of collagen type I.

Article Snippet: Phosphorylation was also verified by ectopic expression of full-length C-terminus Myc-DDK–tagged mouse WNT5A (Origene; Cat# MR205939) transfected into RAW 264.7 and Saos2 cells using Viromer Red (Origene; Cat# TT100302) as per the manufacturer’s protocol.

Techniques: Activity Assay, In Vivo, Knock-Out

Journal: Annals of the New York Academy of Sciences

Article Title: Deletion of Wnt5a in osteoclasts results in bone loss through decreased bone formation.

doi: 10.1111/nyas.14293

Figure Lengend Snippet: Figure 4. Deletion of Wnt5a from mature osteoclasts decreased bone formation. (A) Calcein double label and dynamic histomor- phometry of femoral trabecular bone from 10-week-old male Wnt5a cKO and Wnt5aF/+ control mice shows significantly lower mineralizing surface (MS/BS) and bone formation rate (BFR) when Wnt5a is deleted from mature osteoclasts. No differences in mineral apposition rates (MARs) were observed in the femur trabecular bone. (B) Deletion of Wnt5a from mature osteoclasts significantly decreased serum P1NP levels in 10-week-old male mice. Data represent mean ± SD (n = 4–7), ∗P < 0.05. cKO, conditional knockout; MS/BS, mineralizing surface; BFR, bone formation rate; MAR, mineral apposition rate; P1NP, N-terminal propeptide of type I procollagen.

Article Snippet: Phosphorylation was also verified by ectopic expression of full-length C-terminus Myc-DDK–tagged mouse WNT5A (Origene; Cat# MR205939) transfected into RAW 264.7 and Saos2 cells using Viromer Red (Origene; Cat# TT100302) as per the manufacturer’s protocol.

Techniques: Control, Knock-Out

Journal: Annals of the New York Academy of Sciences

Article Title: Deletion of Wnt5a in osteoclasts results in bone loss through decreased bone formation.

doi: 10.1111/nyas.14293

Figure Lengend Snippet: Figure 5. Ctsk-Cre nonspecifically targets periosteal mesenchymal cells but does not influence osteogenic differentiation. (A) Periosteal cells (POCs) were isolated from control and experimental Wnt5a cKO mice from intact hindlimbs and subjected to osteogenic differentiation for 7, 14, and 21 days. (B) Ctsk is expressed in periosteal mesenchymal cells, with cells from Wnt5a cKO expressing 54% less. This expression resulted in significant recombination of Wnt5a in POC isolated from experimental (Wnt5a cKO) mice. (C) Expression of the osteogenic genes Runx2, Osx, and Col1a1 was not different between genotypes at day 7 or 14. At day 21 of osteogenic differentiation, POC from Wnt5a cKO mice expressed significantly higher levels of the osteogenic genes. (D) POC from Wnt5a cKO mice showed lower mineralization at day 7, but no differences in genotypes at day 14 or 21 of osteogenic differentiation. Data represent mean ± SD, ∗P < 0.05. cKO, conditional knockout; POC, periosteal cell; ARS, Alizarin red-S.

Article Snippet: Phosphorylation was also verified by ectopic expression of full-length C-terminus Myc-DDK–tagged mouse WNT5A (Origene; Cat# MR205939) transfected into RAW 264.7 and Saos2 cells using Viromer Red (Origene; Cat# TT100302) as per the manufacturer’s protocol.

Techniques: Isolation, Control, Expressing, Knock-Out

Journal: Annals of the New York Academy of Sciences

Article Title: Deletion of Wnt5a in osteoclasts results in bone loss through decreased bone formation.

doi: 10.1111/nyas.14293

Figure Lengend Snippet: Figure 6. RANKL-activated RAW 264.7 cells produce a unique phosphorylated WNT5A. (A) Cellular extracts were immuno- precipitated with anti-WNT5A antibody, and then probed by immunoblotting using multiple modification-specific antibodies as indicated. Only the phospho-specific Ser antibody cross-reacted with the immunoprecipitated WNT5A in RANKL-treated RAW 264.7 cells. (B) Phosphatase (Ppase)-treated WNT5A was not detected by anti-phospho-Ser antibody, verifying it as a phosphorylation-specific modification. (C) Ectopically expressed Myc-WNT5A is inducibly phosphorylated at a Ser residue in RANKL-activated RAW 264.7 cells. (D) WNT5A is secreted into media by Saos2 and Raw 264.7 cells treated with and without RANKL. (E) Only RANKL-treated Raw 264.7 cells secrete phosphorylated WNT5A. Full length western blots are presented in Figure S4 (online only). IP, immunoprecipation; IB, immunoblot; RANKL, receptor activator of nuclear factor kappa-B ligand; Ab, antibody; Ppase, phosphatase; MmWnt5a, Mus musculus Wnt5a.

Article Snippet: Phosphorylation was also verified by ectopic expression of full-length C-terminus Myc-DDK–tagged mouse WNT5A (Origene; Cat# MR205939) transfected into RAW 264.7 and Saos2 cells using Viromer Red (Origene; Cat# TT100302) as per the manufacturer’s protocol.

Techniques: Western Blot, Modification, Immunoprecipitation, Phospho-proteomics, Residue