Journal: The FASEB Journal

Article Title: Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae –induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation

doi: 10.1096/fj.201601045R

Figure Lengend Snippet: TRPV4 is required for TGF-β1–mediated differentiation of NHLFs and DHLFs. A) mLFs isolated from WT and TRPV4-KO mice were treated with TGF-β1 (2 ng/ml) for 24, 48, and 96 h, and expression of α-SMA and FN was analyzed by Western blotting. Note that time-dependent TGF-β1 response seen in WT fibroblasts was attenuated in TRPV4-KO fibroblasts. B) Effect of TGF-β1 treatment on TRPV4-dependent calcium flux in NHLFs. NHLFs were loaded with Fluo-4, and calcium flux was measured as described in Materials and Methods in response to a specific TRPV4 activator, GSK1016790A (100 nM), and quantified. C–E) TRPV4 protein was knocked down in NHLFs by transfecting them with siRNA against TRPV4 (20 nM). NHLFs transfected with nonspecific (NS) siRNA were used as control. Twenty-four hours after transfection, NHLFs were treated with TGF-β1 (2 ng/ml; 48 h). C) TRPV4 transcript was analyzed by real-time PCR. D) α-SMA localization was determined by immunofluorescence (red). E) α-SMA expression was analyzed by Western blotting and quantified. F) TRPV4-mediated calcium flux in NHLFs and DHLFs. Calcium flux, indicative of TRPV4 activity, was analyzed in NHLFs and DHLFs from same passage in response to GSK and quantified. G–I) Differential responses of NHLFs and DHLFs to TGF-β1. G) α-SMA transcript levels both at basal and in response to TGF-β1 (2 ng/ml; 48 h) were analyzed by real-time PCR in NHLFs and DHLFs. H, I) Effect of RN1734, a selective TRPV4 antagonist on TGF-β1–induced fibroblast differentiation in NHLFs and DHLFs. H) Both NHLFs and DHLFs were stimulated with or without TGF-β1 (2 ng/ml; 48 h) in the presence or absence of a TRPV4 inhibitor RN1734 (10, 30, and 50 μM), and α-SMA protein expression was determined by Western blotting. Blots were stripped and reprobed with GAPDH Ab. I) Blots were quantified using AlphaView software. Results are means ± sem from 3 independent experiments (B, C, E–G, I). *P < 0.05; **P < 0.01.

Article Snippet: DHLFs express enhanced TRPV4 activity and fibroblast differentiation in response to TGF-β1 To further evaluate the role of TRPV4 in asthma, we examined TRPV4 activity and TGF-β1–induced differentiation potential in NHLFs obtained from normal human lungs and DHLFs obtained from lungs of patients with asthma exacerbation (validated from Lonza).

Techniques: Isolation, Expressing, Western Blot, Transfection, Control, Real-time Polymerase Chain Reaction, Immunofluorescence, Activity Assay, Software

Journal: The FASEB Journal

Article Title: Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae –induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation

doi: 10.1096/fj.201601045R

Figure Lengend Snippet: PI3K mediates TGF-β1–dependent activation of TRPV4, whereas p38 and Rho are activated downstream of TRPV4. A–C) NHLFs were preincubated with indicated concentrations of PI3K inhibitor (LY294002; A), p38 MAPK inhibitor (SB203580; B), and Rho kinase inhibitor (Y27632; C) for 30 min, followed by treatment with TGF-β1 (2 ng/ml; 48 h), and α-SMA was determined by Western blotting. Note that all the 3 inhibitors blocked TGF-β1–induced α-SMA induction. D, E) PI3K P110α protein (D) and p38 MAPK (E) were knocked down in NHLFs by transfecting them with corresponding siRNAs (100 nM). NHLFs transfected with nonspecific (NS) siRNA were used as control. Twenty-four hours after transfection, NHLFs were treated with TGF-β1 (2 ng/ml; 48 h) and α-SMA expression was analyzed by Western blotting. F, G, J) Effect of TRPV4 antagonist on the activation of AKT, p38 MAPK, and Rho. NHLFs were preincubated with RN1734 (30 μM) and treated with TGF-β1 for indicated time points. Western blot analysis showing the phosphorylation of AKT (F) and phosphorylation of p38 MAPK (30 min; G) and Rho activation (J). Rho activity was measured by RhoA activation assay kit (Cytoskeleton) 60 min after TGF-β1 stimulation. Total Rho in the lysates was determined by Western blotting. Blots were stripped and reprobed with GAPDH Ab and quantified by using AlphaView software. H, I), Fluoroscence images showing the effect of PI3K inhibitor on TGF-β1–mediated TRPV4 membrane translocation. NHLFs were transfected with GFP-tagged TRPV4 construct, and translocation of TRPV4 to the membrane was analyzed in response to TGF-β1 treatment (2 ng/ml) with or without LY (50 μM) over time (0, 0.5, and 2 h). Number of cells with TRPV4-GFP at the plasma membrane was counted without knowledge of the treatment groups and expressed as percentage. Results are means ± sem from at least 3 independent experiments (A–C, I, J). *P < 0.05; **P < 0.01.

Article Snippet: DHLFs express enhanced TRPV4 activity and fibroblast differentiation in response to TGF-β1 To further evaluate the role of TRPV4 in asthma, we examined TRPV4 activity and TGF-β1–induced differentiation potential in NHLFs obtained from normal human lungs and DHLFs obtained from lungs of patients with asthma exacerbation (validated from Lonza).

Techniques: Activation Assay, Western Blot, Transfection, Control, Expressing, Phospho-proteomics, Activity Assay, Software, Membrane, Translocation Assay, Construct, Clinical Proteomics

Journal: The FASEB Journal

Article Title: Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae –induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation

doi: 10.1096/fj.201601045R

Figure Lengend Snippet: TRPV4 mediates matrix synthesis in fibroblasts. A, B) NHLFs were treated with TGF-β1 (2 ng/ml; indicated time) with or without TRPV4 inhibitor RN1734 (30 μM preincubation) and collagen 1A1 (A) and FN (B) transcripts were analyzed by real-time PCR. C) FN protein accumulation was analyzed by immunofluorescence in cells without permeabilizing with Triton X-100. D, E) FN protein expression was determined by Western blotting (D) and quantified (E). F–I) DHLFs exhibit higher basal and TGF-β1–induced matrix transcripts and protein levels compared with NHLFs. F, G) NHLFs and DHLFs were treated with TGF-β1 (2 ng/ml; 72 h), and Col1A1 (F) and FN (G) transcripts were analyzed by real-time PCR. H, I) FN protein expression was determined by western blotting (H) and quantified in the presence or absence of RN1734 (30 μM; I). Blots were stripped and reprobed with GAPDH Ab. Blots were quantified using AlphaView software (ProteinSimple). Results are means ± sem from at least 3 independent experiments (A, B, E, G, I). *P < 0.05; **P < 0.01.

Article Snippet: DHLFs express enhanced TRPV4 activity and fibroblast differentiation in response to TGF-β1 To further evaluate the role of TRPV4 in asthma, we examined TRPV4 activity and TGF-β1–induced differentiation potential in NHLFs obtained from normal human lungs and DHLFs obtained from lungs of patients with asthma exacerbation (validated from Lonza).

Techniques: Real-time Polymerase Chain Reaction, Immunofluorescence, Expressing, Western Blot, Software

Journal: The FASEB Journal

Article Title: Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae –induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation

doi: 10.1096/fj.201601045R

Figure Lengend Snippet: TRPV4 regulates TGF-β1–mediated fibrotic gene expression. A–C) NHLFs were treated with TGF-β1 (2 ng/ml; 48 h) with or without RN1734 (30 μM) and the transcripts of SM22 (A, B) and MRTF-A (C) were analyzed by real-time PCR. D, F) MRTF-A protein was determined by Western blotting (D), and TRPV4-dependent MRTF-A nuclear translocation (F) to the membrane in response to TGF-β1 (2 ng/ml; 24 h) in the presence or absence of TRPV4 inhibitor RN1734 (30 μM) was analyzed by Western blotting in nuclear fractions. Blot was stripped and reprobed with lamin Ab to confirm equal loading. B, E) DHLFs exhibit higher basal and TGF-β1–mediated SM22 mRNA and MRTF-A protein expression. NHLFs and DHLFs were treated with TGF-β1 (2 ng/ml; 48 h); SM22 transcript was analyzed by real-time PCR (B), and MRTF-A protein was determined by Western blotting (E). G) TGF-β1–mediated MRTF-A expression is sensitive to Rho kinase inhibition. NHLFs were treated with TGF-β1 (2 ng/ml; 48 h) with or without Y27632 (10 μM), and MRTF-A protein was determined by Western blotting. Blots were stripped and reprobed with GAPDH Ab and were quantified by using AlphaView software. Graphs represent means ± sem from at least 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: DHLFs express enhanced TRPV4 activity and fibroblast differentiation in response to TGF-β1 To further evaluate the role of TRPV4 in asthma, we examined TRPV4 activity and TGF-β1–induced differentiation potential in NHLFs obtained from normal human lungs and DHLFs obtained from lungs of patients with asthma exacerbation (validated from Lonza).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Western Blot, Translocation Assay, Membrane, Expressing, Inhibition, Software

Journal: The FASEB Journal

Article Title: Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae –induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation

doi: 10.1096/fj.201601045R

Figure Lengend Snippet: TRPV4 controls TGF-β1–mediated PAI-1 expression. A, B, D, E) NHLFs were treated with TGF-β1 (2 ng/ml; 48 h or indicated periods of time) with (B, D, E) or without (A) RN1734 (30 μM); PAI-1 transcript was analyzed by real-time PCR (A, B), and PAI-1 protein was determined by Western blotting (D) and quantification (E). C, F, G) DHLFs exhibit higher basal and TGF-β1–mediated PAI-1 expression. NHLFs and DHLFs were treated with TGF-β1 (2 ng/ml; 48 h); PAI-1 transcript was analyzed by real-time PCR (C), and PAI-1 protein was determined by Western blotting (F) and quantification (G). Blots were stripped and reprobed with GAPDH Ab and were quantified by using AlphaView software. Graphs represent means ± sem from at least 3 independent experiments (A, C, E, G). *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: DHLFs express enhanced TRPV4 activity and fibroblast differentiation in response to TGF-β1 To further evaluate the role of TRPV4 in asthma, we examined TRPV4 activity and TGF-β1–induced differentiation potential in NHLFs obtained from normal human lungs and DHLFs obtained from lungs of patients with asthma exacerbation (validated from Lonza).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Software

Journal: The FASEB Journal

Article Title: Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae –induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation

doi: 10.1096/fj.201601045R

Figure Lengend Snippet: TRPV4-mediated signal transduction downstream of TGF-β1. A–E) NHLFs were treated with TGF-β1 (2 ng/ml) for 48 h or indicated times in the presence or absence of SB203580 (10 μM) and Y27632 (10 μM); α-SMA, SM22, and PAI-1 transcripts were analyzed by real-time PCR (A), and PAI-protein (B, C, E) and FN protein (B–D) were analyzed by Western blotting. F) p38 MAPK is downstream of Rho kinase. NHLFs were treated with TGF-β1 (2 ng/ml) for 30 min in the presence or absence of Y27632 (10 μM), and phosphorylation of p38 was determined and quantified. Blots were stripped and reprobed with GAPDH Ab and were quantified by using AlphaView software. Graphs represent means ± sem from at least 3 independent experiments (A, D, E, F). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: DHLFs express enhanced TRPV4 activity and fibroblast differentiation in response to TGF-β1 To further evaluate the role of TRPV4 in asthma, we examined TRPV4 activity and TGF-β1–induced differentiation potential in NHLFs obtained from normal human lungs and DHLFs obtained from lungs of patients with asthma exacerbation (validated from Lonza).

Techniques: Transduction, Real-time Polymerase Chain Reaction, Western Blot, Phospho-proteomics, Software

Journal: Redox Biology

Article Title: Identification of tyrosine brominated extracellular matrix proteins in normal and fibrotic lung tissues

doi: 10.1016/j.redox.2024.103102

Figure Lengend Snippet: PXDN and laminin expression are modulated in human lung fibroblasts (HLF) and bone-marrow-derived macrophages (BMDM) upon TGF-β1 stimulation. (A – C) HLFs were treated 5 ng/mL TGF-β1 for 48 h, and analyzed for LAMA1 (A) or PXDN (B) mRNA or PXDN protein expression (C). (D – G) BMDM from C57BL6/NJ mice were treated 5 ng/mL TGF-β1 for 48 h, and analyzed for Pxdn (D) , Lama1 (E) , or Lamb1 (F) mRNA, or Laminin α/β1 protein expression by immunofluorescence imaging (G). Scale bar: 50 μm. Values are the mean of at least three independent biological replicates ± SEM. Differences among groups were evaluated by Student's T-test.

Article Snippet: HLF (ATCC PCS-201-013) were cultured in fibroblast basal medium (ATCC PCS-201-030) with fibroblast growth kit-low serum (ATCC PCS-201-041) at 37 °C in a 5% CO 2 atmosphere.

Techniques: Expressing, Derivative Assay, Immunofluorescence, Imaging

Journal: Scientific Reports

Article Title: Plant miRNA osa-miR172d-5p suppressed lung fibrosis by targeting Tab1

doi: 10.1038/s41598-023-29188-6

Figure Lengend Snippet: Osa-miR172d-5p identified as a plant miR candidate with an anti-fibrotic effect. ( A ) Scheme of plant miR selection. ( B ) In silico analysis of the interaction between osa-miR172d-5p and TAB1. ( C ) Human lung fibroblast HFL1 cells were transfected with the osa-miR172d-5p for 48 h, and TAB1 expression was evaluated via western blot analysis ( n = 4). ( D ) HFL1 cells were transfected with the indicated concentration of osa-miR172d-5p for 48 h, and cDNA was evaluated via qRT-PCR ( n = 4). Data are shown as mean ± SEM. * P < 0.05. *** P < 0.001 versus control group.

Article Snippet: Human lung fibroblast HFL1 cells (JCRB, Osaka, Japan) were cultured in 10% fetal bovine serum (FBS) (Sigma-Aldrich), Dulbecco's Modified Eagle Medium (DMEM) (044-29765; Fujifilm Tokyo, Japan) supplemented with penicillin G (876111)–streptomycin (876161; Meiji Pharmaceutical Co., Tokyo, Japan) in 5% CO 2 and 100% humidity at 37 °C.

Techniques: Selection, In Silico, Transfection, Expressing, Western Blot, Concentration Assay, Quantitative RT-PCR, Control

Journal: Scientific Reports

Article Title: Plant miRNA osa-miR172d-5p suppressed lung fibrosis by targeting Tab1

doi: 10.1038/s41598-023-29188-6

Figure Lengend Snippet: TAB1 knockdown suppressed TGFβ-induced fibrotic gene expression. ( A ) Human lung fibroblast HFL1 cells were transfected with TAB1-siRNA (10 nM, 48 h) and TAB1 expression levels were determined by western blot analysis. ( B – D ) Human lung fibroblast HFL1 cells were transfected with TAB1-siRNA (10 nM, 48 h) and treated with TGFβ (5 ng/mL for B , D 48 h; C 24 h). mRNA expression levels were assessed via RT-qPCR. ( B ) ASMA (αSMA) (n = 4), ( C ) COL1A1 (n = 4), and ( D ) FN (fibronectin; n = 4). Data are shown as mean ± SEM. * P < 0.05. ** P < 0.01. *** P < 0.001 versus control group.

Article Snippet: Human lung fibroblast HFL1 cells (JCRB, Osaka, Japan) were cultured in 10% fetal bovine serum (FBS) (Sigma-Aldrich), Dulbecco's Modified Eagle Medium (DMEM) (044-29765; Fujifilm Tokyo, Japan) supplemented with penicillin G (876111)–streptomycin (876161; Meiji Pharmaceutical Co., Tokyo, Japan) in 5% CO 2 and 100% humidity at 37 °C.

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

Journal: Cancer Science

Article Title: Elevated expression of angiomodulin (AGM/IGFBP‐rP1) in tumor stroma and its roles in fibroblast activation

doi: 10.1111/j.1349-7006.2012.02203.x

Figure Lengend Snippet: Effect of transforming growth factor‐β1 (TGF‐β1) on expression of angiomodulin (AGM), fibronectin (FN) and α‐smooth muscle actin (α‐SMA) in two kinds of cultured human fibroblasts. Human natal dermal fibroblasts (HDFs) (a) and WI38 cells (b) were incubated with the indicated concentrations (ng/mL) of TGF‐β1 in serum‐free medium for 2 days. From each culture, the conditioned medium and cell lysates were prepared, as described in Materials and Methods. AGM and fibronectin were analyzed with the conditioned media, while α‐SMA and β‐actin as an internal loading control were done with the cell lysates. The results were reproduced in at least three separate experiments.

Article Snippet: Human fetal lung fibroblast cell line WI38 and human bladder carcinoma cell line T24 (EJ‐1 strain) were provided from Japanese Collection of Research and Bioresources (JCRB, Osaka, Japan).

Techniques: Expressing, Cell Culture, Incubation, Control

Journal: Cancer Science

Article Title: Elevated expression of angiomodulin (AGM/IGFBP‐rP1) in tumor stroma and its roles in fibroblast activation

doi: 10.1111/j.1349-7006.2012.02203.x

Figure Lengend Snippet: Effects of angiomodulin (AGM) and transforming growth factor‐β1 (TGF‐β1) on growth of human fibroblasts. (a,b) Human natal dermal fibroblasts (HDFs) were incubated with the indicated concentrations of TGF‐β1 for 5 days (a) or AGM for 4 days (β) in DMEM/F12+5% FCS medium on 24‐well plates. Each point represents the mean ± SD of the numbers of cells in triplicate wells. (c) Time course of HDF growth in presence (●) or absence (○) of 10 μg/mL AGM. (d) Effect of varied concentrations of AGM on the growth of HDFs was examined in the presence (●) or absence (○) of 10 μM Smad inhibitor SB431542 (Smad inh.) for 6 days on a 96‐well plate. The cell growth was measured by the crystal violet staining. Each point represents the mean ± SD in triplicate wells. (e,f) Effects of varied concentrations of TGF‐β1 (e) or ΑGΜ (f) on the growth of WI38 cells were examined for 5 days as described in (a) and (b). Other experimental conditions are described in Materials and Methods.

Article Snippet: Human fetal lung fibroblast cell line WI38 and human bladder carcinoma cell line T24 (EJ‐1 strain) were provided from Japanese Collection of Research and Bioresources (JCRB, Osaka, Japan).

Techniques: Incubation, Staining