scholarly journals RXFP1 expression is regulated by miR-144-3p in Fibroblasts from Patients with Idiopathic Pulmonary Fibrosis

2017 ◽  
Author(s):  
Harinath Bahudhanapati ◽  
Jiangning Tan ◽  
Justin A Dutta ◽  
Stephen B Strock ◽  
Yingze Zhang ◽  
...  
Keyword(s):  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Smooth Muscle Actin ◽  
Negative Regulator ◽  
Lung Fibroblasts ◽  
Luciferase Reporter ◽  
Alpha Smooth Muscle Actin ◽  
Protein Levels ◽  
Donor Lung ◽  
Luciferase Reporter Vector

ABSTRACTRelaxin has been considered as a potential therapy for patients with pulmonary fibrosis. We have previously shown, however, that a potential limitation of relaxin-based therapy for Idiopathic Pulmonary Fibrosis (IPF) is the loss of expression of the relaxin receptor Relaxin/Insulin Like Receptor 1 (RXFP1) expression in fibroblasts. The molecular mechanism for RXFP1 down-regulation in IPF patients remains unclear. To determine whether microRNAs play a role in RXFP1 gene expression, we employed a bioinformatics approach to identify microRNAs (miRs) that are predicted to target RXFP1. By in silico analysis, we identified a putative target site in the RXFP1 mRNA for the miR-144 family. We found that miR-144-3p was upregulated in IPF fibroblasts compared to donor lung fibroblast controls. Forced miR-144-3p mimic expression reduced RXFP1 mRNA and protein levels and increased expression of the myofibroblast marker alpha-smooth muscle actin (α-SMA) in donor lung fibroblasts. IPF lung fibroblasts transfected with a miR-144-3p inhibitor increased RXFP1 expression and reduced α-SMA expression. A lentiviral luciferase reporter vector carrying the WT 3’UTR of RXFP1 was repressed more in lung fibroblasts whereas vector carrying a mutated miR-144-3p binding site exhibited less sensitivity to endogenous miR-144-3p expression, suggesting that RXFP1 is a direct target of miR-144-3p. Thus, miR-144-3p is highly expressed in IPF fibroblasts and acts as a negative regulator of RXFP1 protein expression.

scholarly journals Targeting MAP3K19 prevents human lung myofibroblast activation both in vitro and in a humanized SCID model of idiopathic pulmonary fibrosis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Isabelle C. Jones ◽  
Milena S. Espindola ◽  
Rohan Narayanan ◽  
Ana L. Coelho ◽  
David M. Habiel ◽  
...  
Keyword(s):  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Smooth Muscle Actin ◽  
Mitogen Activated Protein Kinase ◽  
Lung Fibroblasts ◽  
Alpha Smooth Muscle Actin ◽  
Biochemical Assays ◽  
Mitogen Activated Protein ◽  
Si Rna

AbstractIdiopathic Pulmonary Fibrosis (IPF) is a disease with a devastating prognosis characterized by unrelenting lung scarring. Aberrant activation of lung fibroblasts is a key feature of this disease, yet the key pathways responsible for this are poorly understood. Mitogen-activated protein kinase, kinase, kinase- 19 (MAP3K19) was recently shown to be upregulated in IPF and this MAPK has a key role in target gene transcription in the TGF-β pathway. Herein, we further investigate the role of MAP3K19 in cultured normal and IPF fibroblasts and in a humanized SCID mouse model of IPF employing both short interfering (si) RNA and novel small-molecule inhibitors directed at this kinase. Targeting MAP3K19 had significant inhibitory effects on the expression of both alpha smooth muscle actin and extracellular matrix in cultured human IPF fibroblasts. Quantitative protein and biochemical assays, as well as histological analysis, showed that MAP3K19 was required for the development of lung fibrosis in SCID mice humanized with IPF lung fibroblasts. MAP3K19 was required for IPF myofibroblast differentiation, and targeting its activity attenuated the profibrotic activity of these cells both in vitro and in an adoptive transfer SCID model of pulmonary fibrosis.

scholarly journals LTBP2 is secreted from lung myofibroblasts and is a potential biomarker for idiopathic pulmonary fibrosis

2018 ◽  
Vol 132 (14) ◽  
pp. 1565-1580 ◽  
Author(s):  
Yasunori Enomoto ◽  
Sayomi Matsushima ◽  
Kiyoshi Shibata ◽  
Yoichiro Aoshima ◽  
Haruna Yagi ◽  
...  
Keyword(s):  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Bootstrap Method ◽  
Transforming Growth Factor Β ◽  
Lung Fibroblasts ◽  
Potential Biomarker ◽  
Fibrotic Process ◽  
Mouse Lungs

Although differentiation of lung fibroblasts into α-smooth muscle actin (αSMA)-positive myofibroblasts is important in the progression of idiopathic pulmonary fibrosis (IPF), few biomarkers reflecting the fibrotic process have been discovered. We performed microarray analyses between FACS-sorted steady-state fibroblasts (lineage (CD45, TER-119, CD324, CD31, LYVE-1, and CD146)-negative and PDGFRα-positive cells) from untreated mouse lungs and myofibroblasts (lineage-negative, Sca-1-negative, and CD49e-positive cells) from bleomycin-treated mouse lungs. Amongst several genes up-regulated in the FACS-sorted myofibroblasts, we focussed on Ltbp2, the gene encoding latent transforming growth factor-β (TGF-β) binding protein-2 (LTBP2), because of the signal similarity to Acta2, which encodes αSMA, in the clustering analysis. The up-regulation was reproduced at the mRNA and protein levels in human lung myofibroblasts induced by TGF-β1. LTBP2 staining in IPF lungs was broadly positive in the fibrotic interstitium, mainly as an extracellular matrix (ECM) protein; however, some of the αSMA-positive myofibroblasts were also stained. Serum LTBP2 concentrations, evaluated using ELISA, in IPF patients were significantly higher than those in healthy volunteers (mean: 21.4 compared with 12.4 ng/ml) and showed a negative correlation with % predicted forced vital capacity (r = −0.369). The Cox hazard model demonstrated that serum LTBP2 could predict the prognosis of IPF patients (hazard ratio for death by respiratory events: 1.040, 95% confidence interval: 1.026–1.054), which was validated using the bootstrap method with 1000-fold replication. LTBP2 is a potential prognostic blood biomarker that may reflect the level of differentiation of lung fibroblasts into myofibroblasts in IPF.

scholarly journals TBK1 regulates YAP/TAZ and fibrogenic fibroblast activation

2020 ◽  
Vol 318 (5) ◽  
pp. L852-L863 ◽  
Author(s):  
Aja Aravamudhan ◽  
Andrew J. Haak ◽  
Kyoung Moo Choi ◽  
Jeffrey A. Meridew ◽  
Nunzia Caporarello ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Large Scale ◽  
Smooth Muscle Actin ◽  
Lung Fibroblasts ◽  
Cell Contact ◽  
Actin Stress Fiber ◽  
Fibroblast Activation ◽  
Protein Levels ◽  
Major Cell Type ◽  
Interfering Rna

Idiopathic pulmonary fibrosis (IPF) results in scarring of the lungs by excessive extracellular matrix (ECM) production. Resident fibroblasts are the major cell type involved in ECM deposition. The biochemical pathways that facilitate pathological fibroblast activation leading to aberrant ECM deposition are not fully understood. Tank binding protein kinase-1 (TBK1) is a kinase that regulates multiple signaling pathways and was recently identified as a candidate regulator of fibroblast activation in a large-scale small-interfering RNA (siRNA) screen. To determine the effect of TBK1 on fibroblast activation, TBK1 was inhibited pharmacologically (MRT-68601) and genetically (siRNA) in normal and IPF human lung fibroblasts. Reducing the activity or expression of TBK1 led to reduction in α-smooth muscle actin stress fiber levels by 40–60% and deposition of ECM components collagen I and fibronectin by 50% in TGF-β-stimulated normal and IPF fibroblasts. YAP and TAZ are homologous mechanoregulatory profibrotic transcription cofactors known to regulate fibroblast activation. TBK1 knockdown or inhibition decreased the total and nuclear protein levels of YAP/TAZ. Additionally, low cell-cell contact and increased ECM substrate stiffness augmented the phosphorylation and activation of TBK1, consistent with cues that regulate YAP/TAZ. The action of TBK1 toward YAP/TAZ activation was independent of LATS1/2 and canonical downstream TBK1 signaling mediator IRF3 but dependent on proteasomal machinery of the cell. This study identifies TBK1 as a fibrogenic activator of human pulmonary fibroblasts, suggesting TBK1 may be a novel therapeutic target in pulmonary fibrosis.

scholarly journals Sirtuin 7 is decreased in pulmonary fibrosis and regulates the fibrotic phenotype of lung fibroblasts

2017 ◽  
Vol 312 (6) ◽  
pp. L945-L958 ◽  
Author(s):  
Anne E. Wyman ◽  
Zahid Noor ◽  
Rita Fishelevich ◽  
Virginia Lockatell ◽  
Nirav G. Shah ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Smooth Muscle Actin ◽  
Accelerated Aging ◽  
Lung Fibroblasts ◽  
Mrna Levels ◽  
Muscle Actin ◽  
Limited Data ◽  
Severe Condition ◽  
Protein Levels ◽  
Induced Changes

Pulmonary fibrosis is a severe condition with no cure and limited therapeutic options. A better understanding of its pathophysiology is needed. Recent studies have suggested that pulmonary fibrosis may be driven by accelerated aging-related mechanisms. Sirtuins (SIRTs), particularly SIRT1, SIRT3, and SIRT6, are well-known mediators of aging; however, limited data exist on the contribution of sirtuins to lung fibrosis. We assessed the mRNA and protein levels of all seven known sirtuins in primary lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-associated interstitial lung disease (SSc-ILD) in comparison with lung fibroblasts from healthy controls. These unbiased tests revealed a tendency for all sirtuins to be expressed at lower levels in fibroblasts from patients compared with controls, but the greatest decrease was observed with SIRT7. Similarly, SIRT7 was decreased in lung tissues of bleomycin-challenged mice. Inhibition of SIRT7 with siRNA in cultured lung fibroblasts resulted in an increase in collagen and α-smooth muscle actin (α-SMA). Reciprocally, overexpression of SIRT7 resulted in lower basal and TGF-β-induced levels of COL1A1, COL1A2, COL3A1, and α-SMA mRNAs, as well as collagen and α-SMA proteins. Induced changes in SIRT7 had no effect on endogenous TGF-β mRNA levels or latent TGF-β activation, but overexpression of SIRT7 reduced the levels of Smad3 mRNA and protein. In conclusion, the decline in SIRT7 in lung fibroblasts has a profibrotic effect, which is mediated by changes in Smad3 levels.

Inhibition of miR-182-5p attenuates pulmonary fibrosis via TGF-β/Smad pathway

2019 ◽  
Vol 39 (5) ◽  
pp. 683-695 ◽  
Author(s):  
Y Chen ◽  
Q Zhang ◽  
Y Zhou ◽  
Z Yang ◽  
M Tan
Keyword(s):  
Pulmonary Fibrosis ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Lung Fibroblasts ◽  
In Vitro Assays ◽  
Luciferase Reporter ◽  
High Morbidity ◽  
Human Embryonic Lung ◽  
Promising Therapeutic Approach ◽  
Tgf Β1

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease with high morbidity and mortality. miR-182-5p is overexpressed in several fibrosis-related diseases but its effect in pulmonary fibrosis has not been reported yet. To investigate the function of miR-182-5p in pulmonary fibrosis, we established bleomycin (BLM)-induced fibrotic mice model and transforming growth factor-β1 (TGF-β1)-treated human embryonic lung fibroblasts model. In this study, miR-182-5p was highly expressed in pulmonary tissues of BLM-induced fibrotic mice. The content of hydroxyproline and TGF-β1 was decreased by downregulating the expression of miR-182-5p, indicating that fibrosis was alleviated in mice treated with Lentivirus-anti-miR-182-5p.Quantification of fibrosis-related proteins demonstrated that downregulation of miR-182-5p inhibited the expression of profibrotic proteins (fibronectin, α-smooth muscle actin, p-Smad2/p-Smad3) as well as enhanced the level of Smad7. In vitro assays validated that miR-182-5p was induced by TGF-β1 with the function of promoting fibrosis. In dual-luciferase reporter assay, Smad7 was demonstrated to be negatively regulated by miR-182-5p. Moreover, the effect of knocking down miR-182-5p on inhibiting fibrosis was achieved by upregulating the expression of Smad7. Therefore, miR-182-5p can be regarded as a biomarker of IPF and its inhibition may be a promising therapeutic approach in treating IPF.

The Strain Response of Lung Myofibroblasts Cultured on Electrospun Polycaprolactone Nanofibers

2013 ◽  
Author(s):  
Timothy J. Fee ◽  
Yong Zhou ◽  
Lauren E. Marshall ◽  
Joel L. Berry
Keyword(s):  
Smooth Muscle ◽  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Smooth Muscle Actin ◽  
Strain Response ◽  
Muscle Actin ◽  
Alpha Smooth Muscle Actin

Idiopathic Pulmonary Fibrosis is a devastating condition characterized by excessive localized production of collagen in the lungs. Over 131,000 people are living with IPF in America (1, 2). There is currently no known treatment or cure for the disease. It has recently been shown that IPF myofibroblasts are sensitive to the stiffness of their substrate. Specifically, alpha Smooth Muscle Actin (alpha-SMA), a known indicator of IPF activity, was differentially produced on soft vs. stiff substrates (3). This suggests a mechanotransduction pathway within the IPF myofibroblasts.

scholarly journals Akt1 regulates pulmonary fibrosis via modulating IL-13 expression in macrophages

2019 ◽  
Vol 25 (7) ◽  
pp. 451-461 ◽  
Author(s):  
Yunjuan Nie ◽  
Yudong Hu ◽  
Kaikai Yu ◽  
Dan Zhang ◽  
Yinze Shi ◽  
...  
Keyword(s):  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Smooth Muscle Actin ◽  
In Vivo Studies ◽  
Muscle Actin ◽  
Alpha Smooth Muscle Actin ◽  
Fibrosis Progression ◽  
Matrix Components ◽  
Deficient Mice

Idiopathic pulmonary fibrosis is a progressive interstitial pneumonia characterised by fibroblast accumulation, collagen deposition and extracellular matrix (ECM) remodelling. It was reported that Akt1 mediated idiopathic pulmonary fibrosis progression through regulating the apoptosis of alveolar macrophage, while its effect on macrophage-produced cytokines remains largely unknown. In the present study, we first examined the phosphorylation of Akt1 in lung sections from idiopathic pulmonary fibrosis patients by immunohistochemistry before applying a bleomycin-induced idiopathic pulmonary fibrosis model using Akt1−/− mice and Akt1+/+ littermates. The results showed that Akt1 was remarkably up-regulated in idiopathic pulmonary fibrosis patients, while in vivo studies revealed that Akt1-deficient mice had well-preserved alveolar structure and fewer collagens, secreted fewer matrix components, including alpha smooth-muscle actin and fibronectin and survived significantly longer than Akt1+/+ littermates. Additionally, the pro-fibrogenic cytokine IL-13 was down-regulated at least twofold in Akt1−/−mice compared to the Akt1+/+group on d 3 and 7 after bleomycin treatment. Furthermore, it was found that Akt1–/– macrophages displayed down-regulation of IL-13 compared to Akt1+/+ macrophages in which Akt1 was phosphorylated in response to IL-33 stimulation. These findings indicate that Akt1 modulates pulmonary fibrosis through inducing IL-13 production by macrophages, suggesting that targeting Akt1 may simultaneously block the fibrogenic processes of idiopathic pulmonary fibrosis.

scholarly journals Interaction of long noncoding RNAs and microRNAs in the pathogenesis of idiopathic pulmonary fibrosis

2015 ◽  
Vol 47 (10) ◽  
pp. 463-469 ◽  
Author(s):  
Chaoqun Huang ◽  
Ye Yang ◽  
Lin Liu
Keyword(s):  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Smooth Muscle Actin ◽  
Noncoding Rnas ◽  
Long Noncoding Rnas ◽  
Lung Fibroblasts ◽  
Competitive Endogenous Rnas ◽  
New Research ◽  
Actin Expression ◽  
Regulatory Functions

Long noncoding RNAs (lncRNAs) are transcribed RNAs with more than 200 nucleotides in length. A growing body of evidence supports the notion that lncRNAs act as competitive endogenous RNAs for microRNAs and play roles in physiological and pathological processes. Several studies have demonstrated the roles of microRNAs in the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, it is unknown whether lncRNAs are involved in IPF. To investigate the roles of lncRNAs in IPF, we determined the interaction of lncRNAs and microRNAs by motif search and manual comparison. The sequences of the dysregulated microRNAs in IPF including miR-21, miR-31, miR-101, miR-29, miR-199, and let-7d were used to search NONCODE database containing 33,829 human lncRNAs. A total of 34 lncRNAs with potential binding sites to these microRNAs were identified. We then examined the expression levels of the identified lncRNAs in the lungs of IPF patients by real-time PCR. Of 34 lncRNAs, nine lncRNAs were dysregulated in the IPF lungs. Four of them were inversely correlated to the microRNA expression in IPF. Further studies revealed that silencing the lncRNA CD99 molecule pseudogene 1 (CD99P1) inhibited proliferation and α-smooth muscle actin expression of lung fibroblasts, while knockdown of the lncRNA n341773 increased collagen expression in lung fibroblasts. These results suggest that CD99P1 and n341773 may be involved in the regulation of lung fibroblast proliferation and differentiation. The identification of regulatory functions of lncRNAs in lung fibroblasts may provide new research directions for the therapy of IPF.

scholarly journals Transglutaminase 2: a novel therapeutic target for idiopathic pulmonary fibrosis using selective small molecule inhibitors

Amino Acids ◽  
2021 ◽  
Author(s):  
Shaun Fell ◽  
Zhuo Wang ◽  
Andy Blanchard ◽  
Carmel Nanthakumar ◽  
Martin Griffin
Keyword(s):  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Therapeutic Target ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Transglutaminase 2 ◽  
Transforming Growth Factor Β1 ◽  
Lung Fibroblasts ◽  
Myofibroblast Phenotype ◽  
Novel Therapeutic

AbstractThis study investigates the effects of a site-directed TG2-selective inhibitor on the lung myofibroblast phenotype and ECM deposition to elucidate TG2 as a novel therapeutic target in idiopathic pulmonary fibrosis (IPF)—an incurable progressive fibrotic disease. IPF fibroblasts showed increased expression of TG2, α smooth muscle actin (αSMA) and fibronectin (FN) with increased extracellular TG2 and transforming growth factor β1 (TGFβ1) compared to normal human lung fibroblasts (NHLFs) which do not express αSMA and express lower levels of FN. The myofibroblast phenotype shown by IPF fibroblasts could be reversed by selective TG2 inhibition with a reduction in matrix FN and TGFβ1 deposition. TG2 transduction or TGFβ1 treatment of NHLFs led to a comparable phenotype to that of IPF fibroblasts which was reversible following selective TG2 inhibition. Addition of exogenous TG2 to NHLFs also induced the myofibroblast phenotype by a mechanism involving TGFβ1 activation which could be ameliorated by selective TG2 inhibition. SMAD3-deleted IPF fibroblasts via CRISPR-cas9 genome editing, showed reduced TG2 protein levels following TGFβ1 stimulation. This study demonstrates a key role for TG2 in the induction of the myofibroblast phenotype and shows the potential for TG2-selective inhibitors as therapeutic agents for the treatment of fibrotic lung diseases like IPF.

scholarly journals Transforming growth factor (TGF)-β1-induced miR-133a inhibits myofibroblast differentiation and pulmonary fibrosis

2019 ◽  
Vol 10 (9) ◽  
Author(s):  
Peng Wei ◽  
Yan Xie ◽  
Peter W. Abel ◽  
Yapei Huang ◽  
Qin Ma ◽  
...  
Keyword(s):  
Growth Factor ◽  
Pulmonary Fibrosis ◽  
Transforming Growth Factor ◽  
Tissue Growth ◽  
Negative Regulator ◽  
Lung Fibroblasts ◽  
Luciferase Reporter ◽  
Myofibroblast Differentiation ◽  
Dependent Manner ◽  
Tgf Β1

Abstract Transforming growth factor (TGF)-β1, a main profibrogenic cytokine in the progression of idiopathic pulmonary fibrosis (IPF), induces differentiation of pulmonary fibroblasts to myofibroblasts that produce high levels of collagen, leading to concomitantly loss of lung elasticity and function. Recent studies implicate the importance of microRNAs (miRNAs) in IPF but their regulation and individual pathological roles remain largely unknown. We used both RNA sequencing and quantitative RT-PCR strategies to systematically study TGF-β1-induced alternations of miRNAs in human lung fibroblasts (HFL). Our data show that miR-133a was significantly upregulated by TGF-β1 in a time- and concentration-dependent manner. Surprisingly, miR-133a inhibits TGF-β1-induced myofibroblast differentiation whereas miR-133a inhibitor enhances TGF-β1-induced myofibroblast differentiation. Interestingly, quantitative proteomics analysis indicates that miR-133a attenuates myofibroblast differentiation via targeting multiple components of TGF-β1 profibrogenic pathways. Western blot analysis confirmed that miR-133a down-regulates TGF-β1-induced expression of classic myofibroblast differentiation markers such as ɑ-smooth muscle actin (ɑ-SMA), connective tissue growth factor (CTGF) and collagens. miRNA Target Searcher analysis and luciferase reporter assays indicate that TGF-β receptor 1, CTGF and collagen type 1-alpha1 (Col1a1) are direct targets of miR-133a. More importantly, miR-133a gene transferred into lung tissues ameliorated bleomycin-induced pulmonary fibrosis in mice. Together, our study identified TGF-β1-induced miR-133a as an anti-fibrotic factor. It functions as a feed-back negative regulator of TGF-β1 profibrogenic pathways. Thus, manipulations of miR-133a expression may provide a new therapeutic strategy to halt and perhaps even partially reverse the progression of IPF.

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