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

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.

Download Full-text

TBK1 regulates YAP/TAZ and fibrogenic fibroblast activation

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00324.2019 ◽

2020 ◽

Vol 318 (5) ◽

pp. L852-L863 ◽

Cited By ~ 1

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.

Download Full-text

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

10.1101/159459 ◽

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.

Download Full-text

CXXC5 Attenuates Pulmonary Fibrosis in a Bleomycin-Induced Mouse Model and MLFs by Suppression of the CD40/CD40L Pathway

BioMed Research International ◽

10.1155/2020/7840652 ◽

2020 ◽

Vol 2020 ◽

pp. 1-15 ◽

Cited By ~ 1

Author(s):

Wei Cheng ◽

Fangfang Wang ◽

Airan Feng ◽

Xiaodan Li ◽

Wencheng Yu

Keyword(s):

Pulmonary Fibrosis ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Feedback Regulation ◽

Collagen Type I ◽

Mouse Lung ◽

Type I ◽

Negative Feedback Regulation ◽

Protein Levels ◽

Proliferation And Apoptosis

Objective. To investigate the role of CXXC5 and the CD40/CD40L pathway in lung fibrosis. Methods. (1) We constructed mouse models of bleomycin-induced pulmonary fibrosis and transfected them with a CXXC5 overexpression vector to evaluate the severity of pulmonary fibrosis. (2) Mouse lung fibroblast (MLF) models stably overexpressed or knockout of CXXC5 vector were constructed. After transforming growth factor-β1 (TGF-β1) stimulation, we examined the proliferation and apoptosis of the MLF model and evaluated the expression of mesenchymal markers and the CXXC5/CD40/CD40L pathway. Results. (1) Compared with other groups, the overexpressed CXXC5 group had less alveolar structure destruction, thinner alveolar septum, and lower Ashcroft score. (2) In bleomycin-induced mice, the expression of CD40 and CD40L increased at both transcriptional and protein levels, and the same changes were observed in α-smooth muscle actin (α-SMA) and collagen type I (Colla I). After upregulation of CXXC5, the increase in CD40, CD40L, α-SMA, and Colla I was attenuated. (3) Stimulated with TGF-β1, MLF proliferation was activated, apoptosis was suppressed, and the expression of CD40, CD40L, α-SMA, and Colla I was increased at both transcriptional and protein levels. After upregulation of CXXC5, these changes were attenuated. Conclusion. CXXC5 inhibits pulmonary fibrosis and transformation to myofibroblasts by negative feedback regulation of the CD40/CD40L pathway.

Download Full-text

Cell size, cell cycle, and α-smooth muscle actin expression by primary human lung fibroblasts

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1998.275.5.l998 ◽

1998 ◽

Vol 275 (5) ◽

pp. L998-L1005 ◽

Cited By ~ 10

Author(s):

Bruce D. Uhal ◽

Carlos Ramos ◽

Iravati Joshi ◽

Antonio Bifero ◽

Annie Pardo ◽

...

Keyword(s):

Human Lung ◽

Smooth Muscle Actin ◽

Lung Fibroblasts ◽

Light Scatter ◽

Forward Angle ◽

Muscle Actin ◽

Cell Sorter ◽

Human Lung Fibroblasts ◽

Group Iii

Primary human lung fibroblasts were separated into small ( group I), intermediate ( group II), and large ( group III) subpopulations by unit gravity sedimentation (1 G). The three subsets retained differences in cell size for up to 15 days of primary culture. Flow cytometric (fluorescence-activated cell sorter) measurements of forward-angle light scatter agreed well with fibroblast volume measured by image analysis and confirmed the utility of forward-angle light scatter for discriminating size subpopulations. Group II fibroblasts accumulated most rapidly by 8 days of culture and also contained the greatest proportion of S and G2/M phase cells as determined by fluorescence-activated cell sorter. Fibroblasts that were immunoreactive with antibodies to α-smooth muscle actin (α-SMA) were found only in group III. In situ end labeling of fragmented DNA detected apoptotic cells in both groups II and III, but double labeling for in situ end labeling and α-SMA revealed apoptotic cells in both the α-SMA-positive and -negative populations. These results demonstrate that primary human lung fibroblasts behave as predicted by classic models of cell cycle progression and differentiation. However, they do not support the hypothesis that the expression of α-actin is related to apoptosis. We also describe a simple and reproducible method for the high-yield isolation of human lung fibroblast subsets of differing proliferative potential and phenotype.

Download Full-text

HDAC8 inhibition ameliorates pulmonary fibrosis

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00551.2017 ◽

2019 ◽

Vol 316 (1) ◽

pp. L175-L186 ◽

Cited By ~ 9

Author(s):

Shigeki Saito ◽

Yan Zhuang ◽

Takayoshi Suzuki ◽

Yosuke Ota ◽

Marjorie E. Bateman ◽

...

Keyword(s):

Smooth Muscle ◽

Pulmonary Fibrosis ◽

Transforming Growth Factor ◽

Therapeutic Potential ◽

Smooth Muscle Actin ◽

Lung Fibroblasts ◽

Peroxisome Proliferator ◽

Collagen Gels ◽

Cell Contractility

Idiopathic pulmonary fibrosis (IPF) is a fibroproliferative lung disease, and fibroblast-myofibroblast differentiation (FMD) is thought to be a key event in the pathogenesis of IPF. Histone deacetylase-8 (HDAC8) has been shown to associate with α-smooth muscle actin (α-SMA; a marker of FMD) and regulates cell contractility in vascular smooth muscle cells. However, the role of HDAC8 in FMD or pulmonary fibrosis has never been reported. This study investigated the role of HDAC8 in pulmonary fibrosis with a focus on FMD. We observed that HDAC8 expression was increased in IPF lung tissue as well as transforming growth factor (TGF)β1-treated normal human lung fibroblasts (NHLFs). Immunoprecipitation experiments revealed that HDAC8 was associated with α-SMA in TGFβ1-treated NHLFs. HDAC8 inhibition with NCC170 (HDAC8-selective inhibitor) repressed TGFβ1-induced fibroblast contraction and α-SMA protein expression in NHLFs cultured in collagen gels. HDAC8 inhibition with HDAC8 siRNA also repressed TGFβ1-induced expression of profibrotic molecules such as fibronectin and increased expression of antifibrotic molecules such as peroxisome proliferator-activated receptor-γ (PPARγ). Chromatin immunoprecipitation quantitative PCR using an antibody against H3K27ac (histone H3 acetylated at lysine 27; a known HDAC8 substrate and a marker for active enhancers) suggested that HDAC8 inhibition with NCC170 ameliorated TGFβ1-induced loss of H3K27ac at the PPARγ gene enhancer. Furthermore, NCC170 treatment significantly decreased fibrosis measured by Ashcroft score as well as expression of type 1 collagen and fibronectin in bleomycin-treated mouse lungs. These data suggest that HDAC8 contributes to pulmonary fibrosis and that there is a therapeutic potential for HDAC8 inhibitors to treat IPF as well as other fibrotic lung diseases.

Download Full-text

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

Clinical Science ◽

10.1042/cs20180435 ◽

2018 ◽

Vol 132 (14) ◽

pp. 1565-1580 ◽

Cited By ~ 6

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.

Download Full-text

ERK1/2 Signaling Pathway Activated by EGF Promotes Proliferation, Transdifferentiation, and Migration of Cultured Primary Newborn Rat Lung Fibroblasts

BioMed Research International ◽

10.1155/2020/7176169 ◽

2020 ◽

Vol 2020 ◽

pp. 1-9

Author(s):

Yu Hu ◽

JianHua Fu ◽

XueYan Liu ◽

XinDong Xue

Keyword(s):

Western Blotting ◽

Cell Cycle Progression ◽

Cellular Proliferation ◽

Smooth Muscle Actin ◽

S Phase ◽

Cell Counting ◽

Lung Fibroblasts ◽

Newborn Rats ◽

Protein Levels ◽

And Migration

Background. Bronchopulmonary dysplasia (BPD) is a common and serious complication in premature infants. Lung fibroblasts (LFs) are present in the extracellular matrix and participate in pulmonary development in response to BPD. The aim of this study was to investigate the effect of extracellular signal-regulated kinase (ERK) on LFs cultured from newborn rats. Material and Methods. Primary LFs were isolated and treated with epidermal growth factor (EGF, 20 ng/mL) in the presence or absence of an ERK inhibitor, PD98059 (10 μmol/L). Phosphorylated ERK1/2 (p-ERK1/2) protein levels were determined using immunocytochemistry, western blotting, and real-time reverse transcription quantitative (RT–q)PCR. LF proliferation was examined by flow cytometry and a cell counting kit-8 assay. LF transdifferentiation was examined by protein and mRNA expression of α-smooth muscle actin (α-SMA) by immunocytochemistry, western blotting, and RT–qPCR. LF migration was examined by the transwell method. Results. Phosphorylated ERK1/2, which was activated by EGF, promoted LF proliferation by accelerating cell-cycle progression from the G1 to S phase. After treatment with PD98059, the expression of p-ERK1/2 in LFs, cellular proliferation, and the percentage of cells in S phase were significantly decreased. Phosphorylated ERK1/2 also promoted the differentiation of LFs into myofibroblasts through increased α-SMA synthesis and migration. Conclusion. The activation of ERK promotes proliferation, transdifferentiation, and migration of lung fibroblasts from newborn rats.

Download Full-text

MicroRNA-96 inhibits FoxO3a function in IPF fibroblasts on type I collagen matrix

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00127.2014 ◽

2014 ◽

Vol 307 (8) ◽

pp. L632-L642 ◽

Cited By ~ 37

Author(s):

Richard Seonghun Nho ◽

Jintaek Im ◽

Yen-Yi Ho ◽

Polla Hergert

Keyword(s):

Type I Collagen ◽

Collagen Matrix ◽

Lung Fibroblasts ◽

Normal Lung ◽

Type I ◽

Mrna Levels ◽

Protein Levels ◽

Dose Dependent Fashion ◽

Mrna And Protein Expression

Idiopathic pulmonary fibrosis (IPF) is a lethal and progressive lung disease characterized by persistent (myo)fibroblasts and the relentless accumulation of collagen matrix. Unlike normal lung fibroblasts, IPF lung fibroblasts have suppressed forkhead box O3a (FoxO3a) activity, which allows them to expand in this diseased environment. microRNA-96 (miR-96) has recently been found to directly bind to the 3′-untranslated region of FoxO3a mRNA, which subsequently inhibits its function. We examined whether aberrantly low FoxO3a expression is in part due to increased miR-96 levels in IPF fibroblasts on polymerized collagen, thereby causing IPF fibroblasts to maintain their pathological properties. miR-96 expression was upregulated in IPF fibroblasts compared with control fibroblasts when cultured on collagen. In contrast, FoxO3a mRNA levels were reduced in most IPF fibroblasts. However, when miR-96 function was inhibited, FoxO3a mRNA and protein expression were increased, suppressing IPF fibroblast proliferation and promoting their cell death in a dose-dependent fashion. Likewise, FoxO3a and its target proteins p21, p27, and Bim expression was also increased in the presence of a miR-96 inhibitor in IPF fibroblasts. However, when control fibroblasts were treated with miR-96 mimic, FoxO3a, p27, p21, and Bim mRNA and protein levels were decreased. In situ hybridization analysis further revealed the presence of enhanced miR-96 expression in cells within the fibroblastic foci of IPF lung tissue. Our results suggest that when IPF fibroblasts interact with collagen-rich matrix, pathologically altered miR-96 expression inhibits FoxO3a function, causing IPF fibroblasts to maintain their pathological phenotype, which may contribute to the progression of IPF.

Download Full-text

GATA-6 mediates human bladder smooth muscle differentiation: involvement of a novel enhancer element in regulating α-smooth muscle actin gene expression

AJP Cell Physiology ◽

10.1152/ajpcell.00225.2007 ◽

2007 ◽

Vol 293 (3) ◽

pp. C1093-C1102 ◽

Cited By ~ 17

Author(s):

Akihiro Kanematsu ◽

Aruna Ramachandran ◽

Rosalyn M. Adam

Keyword(s):

Gene Expression ◽

Smooth Muscle ◽

Smooth Muscle Actin ◽

Muscle Differentiation ◽

Mrna Levels ◽

Muscle Actin ◽

Bladder Smooth Muscle ◽

Human Bladder ◽

Enhancer Element ◽

Smooth Muscle Differentiation

Hollow organs exposed to pathological stimuli undergo phenotypic modulation characterized by altered expression of smooth muscle contractile proteins and loss of normal function. The molecular mechanisms that regulate smooth muscle differentiation, especially in organs other than the vasculature, are poorly understood. In this study, we describe a role for the GATA-6 transcription factor in regulation of human bladder smooth muscle differentiation. Knockdown of endogenous GATA-6 in primary human bladder smooth muscle cells (pBSMC) led to decreased mRNA levels of the differentiation markers α-smooth muscle actin (α-SMA), calponin, and smooth muscle myosin heavy chain. Similar effects were obtained following downregulation of GATA-6 by forskolin-induced elevation of intracellular cAMP levels. Forskolin treatment of pBSMC abolished recruitment of GATA-6 to the α-SMA promoter in vivo and reduced activity of human α-SMA promoter-directed gene expression by >60%. This inhibitory effect was rescued by enforced expression of wild-type GATA-6 but not by a zinc-finger-deleted mutant, GATA-6-ΔZF, which lacks DNA-binding ability. In silico analysis of a region of the human α-SMA promoter, described previously as a transcriptional enhancer, identified a putative GATA-binding site at position −919/−913. Point mutation of this site in SMA-Luc abrogated GATA-6-induced activation of promoter activity. Together, these results provide the first evidence for a functional role for GATA-6 in regulation of bladder smooth muscle differentiation. In addition, these findings demonstrate that GATA-6 regulates human α-SMA expression via a novel regulatory cis element in the α-SMA promoter-enhancer.

Download Full-text