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1.  β-catenin is a central mediator of pro-fibrotic Wnt signaling in systemic sclerosis 
Annals of the rheumatic diseases  2012;71(5):761-767.
Pathologic fibroblast activation drives fibrosis of the skin and internal organs in patients with systemic sclerosis (SSc). β-catenin is an integral part of adherens junctions and a central component of canonical Wnt signaling. Here, the authors addressed the role of β-catenin in fibroblasts for the development of SSc dermal fibrosis.
Nuclear accumulation of β-catenin in fibroblasts was assessed by triple staining for β-catenin, prolyl-4-hydroxylase-β and 4′,6-diamidino-2-phenylindole (DAPI). The expression of Wnt proteins in the skin was analysed by real-time PCR and immunohistochemistry. Mice with fibroblast-specific stabilisation or fibroblast-specific depletion were used to evaluate the role of β-catenin in fibrosis.
The auhors found significantly increased nuclear levels of β-catenin in fibroblasts in SSc skin compared to fibroblasts in the skin of healthy individuals. The accumulation of β-catenin resulted from increased expression of Wnt-1 and Wnt-10b in SSc. The authors further showed that the nuclear accumulation of β-catenin has direct implications for the development of fibrosis: Mice with fibroblast-specific stabilisation of β-catenin rapidly developed fibrosis within 2 weeks with dermal thickening, accumulation of collagen and differentiation of resting fibroblasts into myofibroblasts. By contrast, fibroblast-specific deletion of β-catenin significantly reduced bleomycin-induced dermal fibrosis.
The present study findings identify β-catenin as a key player of fibroblast activation and tissue fibrosis in SSc. Although further translational studies are necessary to test the efficacy and tolerability of β-catenin/Wnt inhibition in SSc, the present findings may have clinical implications, because selective inhibitors of β-catenin/Wnt signaling have recently entered clinical trials.
PMCID: PMC3951949  PMID: 22328737
2.  miR-199a-5p Is Upregulated during Fibrogenic Response to Tissue Injury and Mediates TGFbeta-Induced Lung Fibroblast Activation by Targeting Caveolin-1 
PLoS Genetics  2013;9(2):e1003291.
As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in fibrotic lung diseases, especially the idiopathic form (IPF), remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls). In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFβ exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFβ signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl4-induced mouse models of liver fibrosis, suggesting that dysregulation of miR-199a-5p represents a general mechanism contributing to the fibrotic process. MiR-199a-5p thus behaves as a major regulator of tissue fibrosis with therapeutic potency to treat fibroproliferative diseases.
Author Summary
Fibrosis is the final common pathway in virtually all forms of chronic organ failure, including lung, liver, and kidney, and is a leading cause of morbidity and mortality worldwide. Fibrosis results from the excessive activity of fibroblasts, in particular a differentiated form known as myofibroblast that is responsible for the excessive and persistent accumulation of scar tissue and ultimately organ failure. Idiopathic Lung Fibrosis (IPF) is a chronic and often rapidly fatal pulmonary disorder of unknown origin characterized by fibrosis of the supporting framework (interstitium) of the lungs. Given the poor prognosis of IPF patients, new insights into the biology of (myo)fibroblasts is of major interest to develop new therapeutics aimed at reducing (myo)fibroblast activity to slow or even reverse disease progression, thereby preserving organ function and prolonging life. MicroRNAs (miRNAs), a class of non-coding RNA recently identified, are associated with normal cellular processes; and deregulation of miRNAs plays a causative role in a vast array of complex diseases. In this study, we identified a particular miRNA: miR-199a-5p that governs lung fibroblast activation and ultimately lung fibrosis. Overall we showed that miR-199a-5p is a major regulator of fibrosis with strong therapeutic potency to treat fibroproliferative diseases such as IPF.
PMCID: PMC3573122  PMID: 23459460
3.  Functional Wnt Signaling Is Increased in Idiopathic Pulmonary Fibrosis 
PLoS ONE  2008;3(5):e2142.
Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease, characterized by distorted lung architecture and loss of respiratory function. Alveolar epithelial cell injury and hyperplasia, enhanced extracellular matrix deposition, and (myo)fibroblast activation are features of IPF. Wnt/β-catenin signaling has been shown to determine epithelial cell fate during development. As aberrant reactivation of developmental signaling pathways has been suggested to contribute to IPF pathogenesis, we hypothesized that Wnt/β-catenin signaling is activated in epithelial cells in IPF. Thus, we quantified and localized the expression and activity of the Wnt/β-catenin pathway in IPF.
Methodology/Principal Findings
The expression of Wnt1, 3a, 7b, and 10b, the Wnt receptors Fzd1-4, Lrp5-6, as well as the intracellular signal transducers Gsk-3β, β-catenin, Tcf1, 3, 4, and Lef1 was analyzed in IPF and transplant donor lungs by quantitative real-time (q)RT-PCR. Wnt1, 7b and 10b, Fzd2 and 3, β-catenin, and Lef1 expression was significantly increased in IPF. Immunohistochemical analysis localized Wnt1, Wnt3a, β-catenin, and Gsk-3β expression largely to alveolar and bronchial epithelium. This was confirmed by qRT-PCR of primary alveolar epithelial type II (ATII) cells, demonstrating a significant increase of Wnt signaling in ATII cells derived from IPF patients. In addition, Western blot analysis of phospho-Gsk-3β, phospho-Lrp6, and β-catenin, and qRT-PCR of the Wnt target genes cyclin D1, Mmp 7, or Fibronectin 1 demonstrated increased functional Wnt/β-catenin signaling in IPF compared with controls. Functional in vitro studies further revealed that Wnt ligands induced lung epithelial cell proliferation and (myo)fibroblast activation and collagen synthesis.
Our study demonstrates that the Wnt/β-catenin pathway is expressed and operative in adult lung epithelium. Increased Wnt/β-catenin signaling may be involved in epithelial cell injury and hyperplasia, as well as impaired epithelial-mesenchymal cross-talk in IPF. Thus, modification of Wnt signaling may represent a therapeutic option in IPF.
PMCID: PMC2374879  PMID: 18478089
4.  Wnt/β-catenin signaling is hyperactivated in systemic sclerosis and induces Smad-dependent fibrotic responses in mesenchymal cells 
Arthritis and rheumatism  2012;64(8):2734-2745.
Fibrosis in human diseases and animal models is associated with aberrant Wnt/β-catenin pathway activation. The regulation, activity, mechanism of action and significance of Wnt/β-catenin signaling in the context of systemic sclerosis (SSc) has not been characterized.
Expression of Wnt signaling pathway components in SSc skin biopsies was analyzed. The regulation of profibrotic responses by canonical Wnt/ß-catenin was examined in explanted human mesenchymal cells. Fibrotic responses were studied by proliferation, migration and gel contraction assays. The fate specification of subcutaneous preadipocytes by canonical Wnt signaling was evaluated.
Analysis of published genome-wide expression datasets revealed elevated expression of the Wnt receptor Fzd2 and the Wnt target Lef1, and decreased expression of Wnt antagonists Dkk2 and Wif1 in skin biopsies from subsets of dcSSc patients. Immunohistochemistry showed increased nuclear β-catenin expression in these biopsies. In vitro, Wnt3a induced ß-catenin activation, stimulated fibroblast proliferation, migration, gel contraction and myofibroblast differentiation, and profibrotic gene expression. Genetic and pharmacological approaches were used to demonstrate that these profibrotic responses involved autocrine TGF-β signaling via Smads. In contrast, in explanted subcutaneous preadipocytes Wnt3a repressed adipogenesis and promoted myofibroblast differentiation.
Canonical Wnt signaling was hyperactivated in SSc skin biopsies, and in explanted mesenchymal cells Wnt3a stimulated fibrogenic responses while suppressing adipogenesis. Together, these results indicate that Wnts have potent profibrotic effects and canonical Wnt signaling plays an important role in the pathogenesis of fibrosis and lipoatrophy in SSc.
PMCID: PMC3553791  PMID: 22328118
5.  Activation of WNT / β-Catenin Signaling in Pulmonary Fibroblasts by TGF-β1 Is Increased in Chronic Obstructive Pulmonary Disease 
PLoS ONE  2011;6(9):e25450.
Chronic obstructive pulmonary disease (COPD) is characterized by abnormal extracellular matrix (ECM) turnover. Recently, activation of the WNT/β-catenin pathway has been associated with abnormal ECM turnover in various chronic diseases. We determined WNT-pathway gene expression in pulmonary fibroblasts of individuals with and without COPD and disentangled the role of β-catenin in fibroblast phenotype and function.
We assessed the expression of WNT-pathway genes and the functional role of β-catenin, using MRC-5 human lung fibroblasts and primary pulmonary fibroblasts of individuals with and without COPD.
Pulmonary fibroblasts expressed mRNA of genes required for WNT signaling. Stimulation of fibroblasts with TGF-β1, a growth factor important in COPD pathogenesis, induced WNT-5B, FZD8, DVL3 and β-catenin mRNA expression. The induction of WNT-5B, FZD6, FZD8 and DVL3 mRNA by TGF-β1 was higher in fibroblasts of individuals with COPD than without COPD, whilst basal expression was similar. Accordingly, TGF-β1 activated β-catenin signaling, as shown by an increase in transcriptionally active and total β-catenin protein expression. Furthermore, TGF-β1 induced the expression of collagen1α1, α-sm-actin and fibronectin, which was attenuated by β-catenin specific siRNA and by pharmacological inhibition of β-catenin, whereas the TGF-β1-induced expression of PAI-1 was not affected. The induction of transcriptionally active β-catenin and subsequent fibronectin deposition induced by TGF-β1 were enhanced in pulmonary fibroblasts from individuals with COPD.
β-catenin signaling contributes to ECM production by pulmonary fibroblasts and contributes to myofibroblasts differentiation. WNT/β-catenin pathway expression and activation by TGF-β1 is enhanced in pulmonary fibroblasts from individuals with COPD. This suggests an important role of the WNT/β-catenin pathway in regulating fibroblast phenotype and function in COPD.
PMCID: PMC3184127  PMID: 21980461
6.  Induction of a Mesenchymal Expression Program in Lung Epithelial Cells by Wingless Protein (Wnt)/β-Catenin Requires the Presence of c-Jun N-Terminal Kinase–1 (JNK1) 
Recent studies suggest the importance of the transition of airway epithelial cells (EMT) in pulmonary fibrosis, and also indicate a role for Wingless protein (Wnt)/β-catenin signaling in idiopathic pulmonary fibrosis. We investigated the possible role of the Wnt signaling pathway in inducing EMT in lung epithelial cells, and sought to unravel the role of c-Jun–N-terminal-kinase–1 (JNK1). The exposure of C10 lung epithelial cells or primary mouse tracheal epithelial cells (MTECs) to Wnt3a resulted in increases in JNK phosphorylation and nuclear β-catenin content. Because the role of β-catenin as a transcriptional coactivator is well established, we investigated T-cell factor/lymphocyte-enhancement factor (TCF/LEF) transcriptional activity in C10 lung epithelial cells after the activation of Wnt. TCF/LEF transcriptional activity was enhanced after the activation of Wnt, and this increase in TCF/LEF transcriptional activity was diminished after the small interfering (si)RNA-mediated ablation of JNK. The activation of the Wnt pathway by Wnt3a, or the expression of either wild-type or constitutively active β-catenin (S37A), led to the activation of an EMT transcriptome, manifested by the increased mRNA expression of CArG box-binding factor–A, fibroblast-specific protein (FSP)–1, α–smooth muscle actin (α-SMA), and vimentin, increases in the content of α-SMA and FSP1, and the concomitant loss of zona occludens–1. The siRNA-mediated ablation of β-catenin substantially decreased Wnt3a-induced EMT. The siRNA ablation of JNK1 largely abolished Wnt3a, β-catenin, and β-catenin S37a-induced EMT. In MTECs lacking Jnk1, Wnt3a-induced increases in nuclear β-catenin, EMT transcriptome, and the content of α-SMA or FSP1 were substantially diminished. These data show that the activation of the Wnt signaling pathway is capable of inducing an EMT program in lung epithelial cells through β-catenin, and that this process is controlled by JNK1.
PMCID: PMC3488690  PMID: 22461429
lung; epithelium; Wnt3a; fibrosis; epithelial to mesenchymal transition
7.  β-catenin is overexpressed in hepatic fibrosis and blockage of Wnt/β-catenin signaling inhibits hepatic stellate cell activation 
Molecular Medicine Reports  2014;9(6):2145-2151.
β-catenin, a core component of Wnt/β-catenin signaling, has been shown to be an important regulator of cellular proliferation and differentiation. Abnormal activation of Wnt/β-catenin signaling promotes tissue fibrogenesis. In the present study, the role of β-catenin during liver fibrogenesis was analyzed and the functional effects of β-catenin gene silencing in hepatic stellate cells (HSCs) using small interfering (si)RNA were investigated. The expression of β-catenin in human hepatic fibrosis tissues of different grades and normal human hepatic tissues was examined using immunohistochemistry. To inhibit the Wnt/β-catenin signaling pathway, siRNA for β-catenin was developed and transiently transfected into HSC-T6 cells using Lipofectamine 2000. β-catenin expression was evaluated by quantitative polymerase chain reaction (qPCR) and western blot analysis. The expression of collagen types I and III was evaluated by qPCR and immunofluorescent staining. Cellular proliferation and the cell cycle were analyzed using a methyl thiazolyl tetrazolium assay. Apoptosis was assessed by Annexin V staining. A higher expression level of β-catenin was identified in the patients with high-grade hepatic fibrosis in comparison with that of the normal controls. Additionally, β-catenin siRNA molecules were successfully transfected into HSCs and induced inhibition of β-catenin expression in a time-dependent manner. β-catenin siRNA treatment also inhibited synthesis of collagen types I and I in transfected HSCs. Furthermore, compared with those of the control group, siRNA-mediated knockdown of β-catenin in HSC-T6 cells inhibited cell proliferation and resulted in cell apoptosis. This study suggests a significant functional role for β-catenin in the development of liver fibrosis and demonstrates that downregulation of the Wnt/β-catenin signaling pathway inhibits HSC activation. Thus, this study provides a novel strategy for the treatment of hepatic fibrosis.
PMCID: PMC4055486  PMID: 24691643
hepatic fibrosis; β-catenin; hepatic stellate cells; RNA interference
8.  β-catenin signaling: a novel mediator of fibrosis and potential therapeutic target 
Current Opinion in Rheumatology  2011;23(6):562-567.
Purpose of review
The Wnt/β-catenin signaling pathway plays a critical role in development and adult tissue homeostasis. Recent investigations implicate Wnt/β-catenin signaling in abnormal wound repair and fibrogenesis. The purpose of this review is to highlight recent key studies that support a role for Wnt/β-catenin signaling in fibrosis.
Recent findings
Studies of patients with fibrotic diseases have demonstrated changes in components of the Wnt/β-catenin pathway. In animal models, perturbations in Wnt/β-catenin signaling appear to aggravate or ameliorate markers of injury and fibrosis in a variety of different tissues. Studies also suggest that fibroblasts from different tissue sources may have markedly divergent responses to Wnt/β-catenin signaling. Cross-talk between Wnt/β-catenin and transforming growth factor-β pathways is complex and context-dependent, and may promote fibrogenesis through coregulation of fibrogenic gene targets. High throughput screening has identified several novel chemical inhibitors of Wnt/β-catenin signaling that may be of therapeutic potential.
Wnt/β-catenin signaling appears important in normal wound healing and its sustained activation is associated with fibrogenesis. The mechanism by which Wnt/β-catenin signaling may modify the response to injury is cell-type and context-dependent. Better understanding of this signaling pathway may provide a promising new therapeutic approach for human fibrotic diseases.
PMCID: PMC3280691  PMID: 21885974
β-catenin; fibrosis; Wnt; wound repair
9.  Epithelial cell α3β1 integrin links β-catenin and Smad signaling to promote myofibroblast formation and pulmonary fibrosis 
Pulmonary fibrosis, in particular idiopathic pulmonary fibrosis (IPF), results from aberrant wound healing and scarification. One population of fibroblasts involved in the fibrotic process is thought to originate from lung epithelial cells via epithelial-mesenchymal transition (EMT). Indeed, alveolar epithelial cells (AECs) undergo EMT in vivo during experimental fibrosis and ex vivo in response to TGF-β1. As the ECM critically regulates AEC responses to TGF-β1, we explored the role of the prominent epithelial integrin α3β1 in experimental fibrosis by generating mice with lung epithelial cell–specific loss of α3 integrin expression. These mice had a normal acute response to bleomycin injury, but they exhibited markedly decreased accumulation of lung myofibroblasts and type I collagen and did not progress to fibrosis. Signaling through β-catenin has been implicated in EMT; we found that in primary AECs, α3 integrin was required for β-catenin phosphorylation at tyrosine residue 654 (Y654), formation of the pY654–β-catenin/pSmad2 complex, and initiation of EMT, both in vitro and in vivo during the fibrotic phase following bleomycin injury. Finally, analysis of lung tissue from IPF patients revealed the presence of pY654–β-catenin/pSmad2 complexes and showed accumulation of pY654–β-catenin in myofibroblasts. These findings demonstrate epithelial integrin–dependent profibrotic crosstalk between β-catenin and Smad signaling and support the hypothesis that EMT is an important contributor to pathologic fibrosis.
PMCID: PMC2613463  PMID: 19104148
10.  WNT1-inducible signaling protein–1 mediates pulmonary fibrosis in mice and is upregulated in humans with idiopathic pulmonary fibrosis 
Idiopathic pulmonary fibrosis (IPF) is characterized by distorted lung architecture and loss of respiratory function. Enhanced (myo)fibroblast activation, ECM deposition, and alveolar epithelial type II (ATII) cell dysfunction contribute to IPF pathogenesis. However, the molecular pathways linking ATII cell dysfunction with the development of fibrosis are poorly understood. Here, we demonstrate, in a mouse model of pulmonary fibrosis, increased proliferation and altered expression of components of the WNT/β-catenin signaling pathway in ATII cells. Further analysis revealed that expression of WNT1-inducible signaling protein–1 (WISP1), which is encoded by a WNT target gene, was increased in ATII cells in both a mouse model of pulmonary fibrosis and patients with IPF. Treatment of mouse primary ATII cells with recombinant WISP1 led to increased proliferation and epithelial-mesenchymal transition (EMT), while treatment of mouse and human lung fibroblasts with recombinant WISP1 enhanced deposition of ECM components. In the mouse model of pulmonary fibrosis, neutralizing mAbs specific for WISP1 reduced the expression of genes characteristic of fibrosis and reversed the expression of genes associated with EMT. More importantly, these changes in gene expression were associated with marked attenuation of lung fibrosis, including decreased collagen deposition and improved lung function and survival. Our study thus identifies WISP1 as a key regulator of ATII cell hyperplasia and plasticity as well as a potential therapeutic target for attenuation of pulmonary fibrosis.
PMCID: PMC2662540  PMID: 19287097
11.  siRNA against plasminogen activator inhibitor-1 ameliorates bleomycin-induced lung fibrosis in rats 
Acta Pharmacologica Sinica  2012;33(7):897-908.
Plasminogen activator inhibitor-1 (PAI-1) is involved in the progression of pulmonary fibrosis. The present study was undertaken to examine the effects on pulmonary fibrosis of silencing PAI-1 expression with small interfering RNA (siRNA) and to assess the possible underlying mechanisms.
Male Wistar rats were subjected to intratracheal injection of bleomycin (BLM, 5 mg/kg, 0.2 mL) to induce pulmonary fibrosis. Histopathological changes of lung tissue were examined with HE or Masson's trichrome staining. The expression levels of α-smooth muscle actin (α-SMA), collagen type-I and type-III, caspase-3, as well as p-ERK1/2 and PI3K/Akt in the lung tissue were evaluated using imunohistochemistry and Western blot analyses. The fibroblasts isolated from BLM-induced fibrotic lung tissue were cultured and transfected with pcDNA-PAI-1 or PAI-1siRNA. The expression level of PAI-1 in the fibroblasts was measured using real time RT-PCR and Western blot analysis. The fibroblast proliferation was evaluated using MTT assay.
Intratracheal injection of PAI-1-siRNA (7.5 nmoL/0.2 mL) significantly alleviated alveolitis and collagen deposition, reduced the expression of PAI-1, α-SMA, collagen type-I and collagen type-III, and increased the expression of caspase-3 in BLM-induced fibrotic lung tissue. In consistence with the in vivo results, the proliferation of the cultured fibroblasts from BLM-induced fibrotic lung tissue was inhibited by transfection with PAI-1-siRNA, and accelerated by overexpression of PAI-1 by transfection with pcDNA-PAI-1. The expression of caspase-3 was increased as a result of PAI-1 siRNA transfection, and decreased after transfection with pcDNA-PAI-1. In addition, the levels of p-ERK1/2 and PI3K/Akt in the fibrogenic lung tissue were reduced after treatment with PAI-1siRNA.
The data demonstrate that PAI-1 siRNA inhibits alveolitis and pulmonary fibrosis in BLM-treated rats via inhibiting the proliferation and promoting the apoptosis of fibroblasts. Suppression ERK and AKT signalling pathways might have at least partly contributed to this process. Targeting PAI-1 is a promising therapeutic strategy for pulmonary fibrosis.
PMCID: PMC4011146  PMID: 22659625
idiopathic pulmonary fibrosis; pulmonary fibroblast; bleomycin; plasminogen activator inhibitor-1; RNA interference; α-smooth muscle actin; collagen; caspase-3; extracellular signal-regulated kinase; phosphatidylinositol 3-phosphate kinase
12.  Yin Yang 1 Is a Novel Regulator of Pulmonary Fibrosis 
Rationale: The differentiation of fibroblasts into myofibroblasts is a cardinal feature of idiopathic pulmonary fibrosis (IPF). The transcription factor Yin Yang 1 (YY1) plays a role in the proliferation and differentiation of diverse cell types, but its role in fibrotic lung diseases is not known.
Objectives: To elucidate the mechanism by which YY1 regulates fibroblast differentiation and lung fibrosis.
Methods: Lung fibroblasts were cultured with transforming growth factor (TGF)-β or tumor necrosis factor-α. Nuclear factor (NF)-κB, YY1, and α-smooth muscle actin (SMA) were determined in protein, mRNA, and promoter reporter level. Lung fibroblasts and lung fibrosis were assessed in a partial YY1-deficient mouse and a YY1f/f conditional knockout mouse after being exposed to silica or bleomycin.
Measurements and Main Results: TGF-β and tumor necrosis factor-α up-regulated YY1 expression in lung fibroblasts. TGF-β–induced YY1 expression was dramatically decreased by an inhibitor of NF-κB, which blocked I-κB degradation. YY1 is significantly overexpressed in both human IPF and murine models of lung fibrosis, including in the aggregated pulmonary fibroblasts of fibrotic foci. Furthermore, the mechanism of fibrogenesis is that YY1 can up-regulate α-SMA expression in pulmonary fibroblasts. YY1-deficient (YY1+/−) mice were significantly protected from lung fibrosis, which was associated with attenuated α-SMA and collagen expression. Finally, decreasing YY1 expression through instilled adenovirus-cre in floxed-YY1f/f mice reduced lung fibrosis.
Conclusions: YY1 is overexpressed in fibroblasts in both human IPF and murine models in a NF-κB–dependent manner, and YY1 regulates fibrogenesis at least in part by increasing α-SMA and collagen expression. Decreasing YY1 expression may provide a new therapeutic strategy for pulmonary fibrosis.
PMCID: PMC3136995  PMID: 21169469
nuclear factor-κB; α-smooth muscle actin; idiopathic pulmonary fibrosis
13.  Beta-Catenin Signaling Plays a Disparate Role in Different Phases of Fracture Repair: Implications for Therapy to Improve Bone Healing 
PLoS Medicine  2007;4(7):e249.
Delayed fracture healing causes substantial disability and usually requires additional surgical treatments. Pharmacologic management to improve fracture repair would substantially improve patient outcome. The signaling pathways regulating bone healing are beginning to be unraveled, and they provide clues into pharmacologic management. The β-catenin signaling pathway, which activates T cell factor (TCF)-dependent transcription, has emerged as a key regulator in embryonic skeletogenesis, positively regulating osteoblasts. However, its role in bone repair is unknown. The goal of this study was to explore the role of β-catenin signaling in bone repair.
Methods and Findings
Western blot analysis showed significant up-regulation of β-catenin during the bone healing process. Using a β-Gal activity assay to observe activation during healing of tibia fractures in a transgenic mouse model expressing a TCF reporter, we found that β-catenin-mediated, TCF-dependent transcription was activated in both bone and cartilage formation during fracture repair. Using reverse transcription-PCR, we observed that several WNT ligands were expressed during fracture repair. Treatment with DKK1 (an antagonist of WNT/β-catenin pathway) inhibited β-catenin signaling and the healing process, suggesting that WNT ligands regulate β-catenin. Healing was significantly repressed in mice conditionally expressing either null or stabilized β-catenin alleles induced by an adenovirus expressing Cre recombinase. Fracture repair was also inhibited in mice expressing osteoblast-specific β-catenin null alleles. In stark contrast, there was dramatically enhanced bone healing in mice expressing an activated form of β-catenin, whose expression was restricted to osteoblasts. Treating mice with lithium activated β-catenin in the healing fracture, but healing was enhanced only when treatment was started subsequent to the fracture.
These results demonstrate that β-catenin functions differently at different stages of fracture repair. In early stages, precise regulation of β-catenin is required for pluripotent mesenchymal cells to differentiate to either osteoblasts or chondrocytes. Once these undifferentiated cells have become committed to the osteoblast lineage, β-catenin positively regulates osteoblasts. This is a different function for β-catenin than has previously been reported during development. Activation of β-catenin by lithium treatment has potential to improve fracture healing, but only when utilized in later phases of repair, after mesenchymal cells have become committed to the osteoblast lineage.
In a study in mice Benjamin Alman and colleagues show that β-catenin functions differently in different stages of fracture repair; moreover, activation of β-catenin by lithium improves fracture healing when used in later phases of repair.
Editors' Summary
Most people break at least one bone during their life. If the damaged bone is immobilized with a plaster cast or with metal plates and pins, most fractures heal naturally and quickly. Soon after a bone is damaged, cells called pluripotent mesenchymal cells collect at the injury site. Here, they multiply and change (differentiate) into osteoblasts (cells that make bone) and chondrocytes (cells that make cartilage, the dense connective tissue that covers joints). Osteoblasts and chondrocytes mend the fracture by making new bone, a process called ossification. Bone healing involves two types of ossification. In intramembranous ossification, mesenchymal cells and osteoblast progenitor cells make bone directly, forming a hard “callus” within the fracture. In endochondral ossification, mesenchymal cells differentiate into chondrocytes and make cartilage at the fracture site, which osteoblasts turn into bone. Finally, the bone made by both types of ossification is remodeled so that it closely resembles the damaged bone's original shape and strength.
Why Was This Study Done?
Unfortunately, fractures do not always heal efficiently. If healing is delayed, additional surgery may be needed to repair the break. But surgery can be risky, so drug-based ways of encouraging bone repair would be very useful. To develop such treatments, researchers need to understand what controls the differentiation and activity of osteoblasts and chondrocytes during normal healing. In this study, the researchers have investigated the role of the β-catenin signaling pathway in bone repair. This pathway regulates bone formation during embryonic development, a process that closely resembles bone healing. β-catenin is usually degraded rapidly in cells. However, if a member of a particular family of proteins known as the WNT family binds to a WNT receptor on the surface of a cell, β-catenin moves into the cell's nucleus where it interacts with a protein called T cell factor (TCF). This interaction activates the transcription (the copying of DNA into messenger RNA, which is used to make proteins) of numerous genes and alters the behavior of the cell.
What Did the Researchers Do and Find?
The researchers first measured β-catenin levels in mouse and human bones. In both species, much more β-catenin was made in bones undergoing repair than in intact bones. Then they studied TCF reporter mice—animals in which TCF controls the expression of a marker gene. β-catenin-mediated TCF-dependent transcription, they report, was activated during both bone and cartilage formation after a fracture in these mice. Next, the researchers made mice that could be induced to express an inactive form of β-catenin or a stabilized (permanently active) form of β-catenin in all the cells in a bone fracture. Expression of inactive β-catenin slowed the rate of healing but, unexpectedly, so did expression of stabilized β-catenin. Osteoblast-specific expression of inactive β-catenin also delayed bone healing, whereas osteoblast-specific expression of stabilized β-catenin enhanced the process. Finally, treatment of wild-type mice with lithium (which prevents the degradation of β-catenin) enhanced bone healing if given after a fracture, but interfered with it if given before.
What Do These Findings Mean?
These findings indicate that β-catenin signaling (which, the researchers show, is mainly activated by WNT signaling) has different effects at different stages of bone repair. Early in the process, it controls the ratio of osteoblasts and chondrocytes made from the pluripotent mesenchymal cells. Consequently, too much or too little β-catenin interferes with bone healing at this stage. Later on, β-catenin promotes the differentiation of osteoblasts and enhances their ability to make bone, and so too little β-catenin at this stage prevents healing, whereas increased β-catenin levels stimulate healing. These findings need to be confirmed in people before testing agents that affect β-catenin signaling for their effects on human bone healing. Nevertheless, the researchers' final discovery that lithium improves bone healing if given at the right time is particularly encouraging; lithium is widely used to treat one form of depression so could be readily tested in clinical trials.
Additional Information.
Please access these Web sites via the online version of this summary at
MedlinePlus encyclopedia contains pages on broken bones and on bone fracture repair (in English and Spanish)
Wikipedia has pages on bone fracture and on bone healing (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The UK National Health Service Direct encyclopedia provides pages on broken bones
Animations of intramembranous and endochondral ossification are available from the Ministry of Advanced Education, Training and Technology, Province of British Columbia, Canada
The American Academy of Orthopedic Surgeons has an informative discussion of fractures
The Hospital for Sick Children in Toronto (where the authors of this study are affiliated) has a Web site called SickKids, which contains a page on child physiology, including diagrams of bone development
PMCID: PMC1950214  PMID: 17676991
14.  An LRP5 Receptor with Internal Deletion in Hyperparathyroid Tumors with Implications for Deregulated WNT/β-Catenin Signaling 
PLoS Medicine  2007;4(11):e328.
Hyperparathyroidism (HPT) is a common endocrine disorder with incompletely understood etiology, characterized by enlarged hyperactive parathyroid glands and increased serum concentrations of parathyroid hormone and ionized calcium. We have recently reported activation of the Wnt signaling pathway by accumulation of β-catenin in all analyzed parathyroid tumors from patients with primary HPT (pHPT) and in hyperplastic parathyroid glands from patients with uremia secondary to HPT (sHPT). Mechanisms that may account for this activation have not been identified, except for a few cases of β-catenin (CTNNB1) stabilizing mutation in pHPT tumors.
Methods and Findings
Reverse transcription PCR and Western blot analysis showed expression of an aberrantly spliced internally truncated WNT coreceptor low-density lipoprotein receptor–related protein 5 (LRP5) in 32 out of 37 pHPT tumors (86%) and 20 out of 20 sHPT tumors (100%). Stabilizing mutation of CTNNB1 and expression of the internally truncated LRP5 receptor was mutually exclusive. Expression of the truncated LRP5 receptor was required to maintain the nonphosphorylated active β-catenin level, transcription activity of β-catenin, MYC expression, parathyroid cell growth in vitro, and parathyroid tumor growth in a xenograft severe combined immunodeficiency (SCID) mouse model. WNT3 ligand and the internally truncated LRP5 receptor strongly activated transcription, and the internally truncated LRP5 receptor was insensitive to inhibition by DKK1.
The internally truncated LRP5 receptor is strongly implicated in deregulated activation of the WNT/β-catenin signaling pathway in hyperparathyroid tumors, and presents a potential target for therapeutic intervention.
Gunnar Westin and colleagues report the expression of an aberrantly spliced LRP5 receptor in primary and spontaneous parathyroid tumors and implicate it in the deregulated activation of the Wnt/β-catenin signaling pathway.
Editors' Summary
The parathyroid glands—four rice-sized glands in the neck—maintain a normal calcium balance in the body, to maintain strong bones and essential cellular functions. The glands release parathyroid hormone as a response to a decrease in blood calcium level. By stimulating calcium release from bone and its absorption in the gut, parathyroid hormone restores the blood calcium level. However, 100,000 new individuals in the US develop hyperparathyroidism (HPT) annually, characterized by enlarged, overactive parathyroid glands and high blood levels of calcium. Primary HPT (pHPT) is usually caused by a benign tumor (a non-life-threatening growth) in one of the parathyroid glands. Secondary HPT (sHPT) occurs in response to calcium regulatory disturbances, linked to vitamin D deficiency, and more or less invariably develops in patients with uremic kidney disease.
Why Was This Study Done?
HPT is usually treated by surgical removal of the enlarged parathyroid glands, which is done with great efficiency. However, ideally, doctors would like to know what drives the overgrowth of the parathyroid glands to be able to develop drugs for treatment or disease prophylaxis. Researchers recently reported that the cells in enlarged parathyroid glands from patients with HPT contain high amounts of β-catenin. This protein is part of the Wnt signaling pathway, which has been found to be disrupted in many tumor entities in other organs. In the absence of Wnt proteins, a group of proteins called the β-catenin destruction complex marks β-catenin so that it is rapidly destroyed. When Wnt proteins bind to a cell-surface receptor called Frizzled and a coreceptor called LRP5, the destruction complex is inhibited and β-catenin accumulates. This accumulation induces the production of other proteins (in particular, c-Myc) that stimulate cell growth and division. The accumulation of β-catenin in the enlarged parathyroid glands of patients with HPT could, therefore, significantly contribute to the overgrowth of their glands—but what causes β-catenin accumulation? In this study, the researchers have investigated this question to try to identify a target for drugs to treat HPT.
What Did the Researchers Do and Find?
The researchers looked for genetic changes (mutations) in β-catenin that stabilize the protein and measured the expression of LRP5 in abnormal parathyroid gland tissue from 37 patients with pHPT and 20 with uremia and sHPT. All the samples contained high levels of β-catenin, but only four contained a β-catenin–stabilizing mutation. All the sHPT samples and 32 pHPT samples (but none of the samples containing the β-catenin stabilizing mutation) expressed a mutated LRP5, with the central region deleted. To investigate the functional consequences of this internally deleted LRP5 protein, the researchers used a technique called RNA interference to block its expression in a human parathyroid tumor cell line. They found that expression of the mutated, short LRP5 is required for accumulation of β-catenin, expression of c-Myc, and continued growth of the cell line in test tubes and in animals.
What Do These Findings Mean?
The accumulation of β-catenin in all the enlarged parathyroid glands examined so far strongly implicates abnormal Wnt/β-catenin signaling in the development of pHPT and sHPT. These new findings identify which part of the signaling pathway is altered. The expression data and functional data together suggest that an internally deleted LRP5 coreceptor is often responsible for the accumulation of β-catenin. The functional data also show that expression of shortened LRP5 is necessary for the abnormal growth of parathyroid tumor cells. Exactly how the internally deleted coreceptor activates β-catenin signaling in parathyroid gland cells, or why a shorter-than-normal LRP5 is made, are not yet known. However, because these findings indicate that internally deleted LRP5 has a fundamental role in activating Wnt signaling in HPT, drugs that inactivate this aberrant protein but leave the normal protein unscathed might provide a nonsurgical treatment for this common hormone disorder.
Additional Information.
Please access these Web sites via the online version of this summary at
edlinePlus has encyclopedia pages on hyperparathyroidism, primary hyperparathyroidism, and secondary hyperparathyroidim (in English and Spanish)
Information is available for patients from the US National Institute of Diabetes and Digestive and Kidney Diseases on hyperparathyroidism, which includes links to organizations that help people with hyperparathyroidism
Wikipedia maintains pages on Wnt signaling pathway and on β-catenin (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC2082644  PMID: 18044981
15.  Activation of the Wnt/β-Catenin Signaling Pathway by Mechanical Ventilation Is Associated with Ventilator-Induced Pulmonary Fibrosis in Healthy Lungs 
PLoS ONE  2011;6(9):e23914.
Mechanical ventilation (MV) with high tidal volumes (VT) can cause or aggravate lung damage, so-called ventilator induced lung injury (VILI). The relationship between specific mechanical events in the lung and the cellular responses that result in VILI remains incomplete. Since activation of Wnt/β-catenin signaling has been suggested to be central to mechanisms of lung healing and fibrosis, we hypothesized that the Wnt/β-catenin signaling plays a role during VILI.
Methodology/Principal Findings
Prospective, randomized, controlled animal study using adult, healthy, male Sprague-Dawley rats. Animals (n = 6/group) were randomized to spontaneous breathing or two strategies of MV for 4 hours: low tidal volume (VT) (6 mL/kg) or high VT (20 mL/kg). Histological evaluation of lung tissue, measurements of WNT5A, total β-catenin, non-phospho (Ser33/37/Thr41) β-catenin, matrix metalloproteinase-7 (MMP-7), cyclin D1, vascular endothelial growth factor (VEGF), and axis inhibition protein 2 (AXIN2) protein levels by Western blot, and WNT5A, non-phospho (Ser33/37/Thr41) β-catenin, MMP-7, and AXIN2 immunohistochemical localization in the lungs were analyzed. High-VT MV caused lung inflammation and perivascular edema with cellular infiltrates and collagen deposition. Protein levels of WNT5A, non-phospho (Ser33/37/Thr41) β-catenin, MMP-7, cyclin D1, VEGF, and AXIN2 in the lungs were increased in all ventilated animals although high-VT MV was associated with significantly higher levels of WNT5A, non-phospho (Ser33/37/Thr41) β-catenin, MMP-7, cyclin D1, VEGF, and AXIN2 levels.
Our findings demonstrate that the Wnt/β-catenin signaling pathway is modulated very early by MV in lungs without preexistent lung disease, suggesting that activation of this pathway could play an important role in both VILI and lung repair. Modulation of this pathway might represent a therapeutic option for prevention and/or management of VILI.
PMCID: PMC3174135  PMID: 21935365
16.  Caveolin-1, TGF-β receptor internalization, and the pathogenesis of systemic sclerosis 
Current opinion in rheumatology  2008;20(6):713-719.
To review the scientific literature supporting the participation of caveolin-1 in the pathogenesis of tissue fibrosis and that modulation of the caveolin-1 pathway may represent a novel treatment for systemic sclerosis (SSc) and other fibrotic diseases.
Recent Findings
Caveolin-1 plays an important role in the regulation of transforming growth factor β (TGF-β) signaling owing to its participation in TGF-β receptor (TβR) internalization. TβR internalized through caveolin-1 lipid rafts undergoes rapid degradation, effectively decreasing TGF-β signaling. Studies have shown that caveolin-1 knockdown in vitro markedly increased collagen gene expression in normal human lung fibroblasts. Caveolin-1 was reduced in affected SSc lungs and skin and in idiopathic pulmonary fibrosis (IPF) lung tissues and fibroblasts. Increasing caveolin-1 expression markedly improved bleomycin-induced pulmonary fibrosis. Restoration of caveolin bioavailability employing penetratin, a cell-permeable peptide carrier for a bioactive caveolin-1 fragment abrogated TGF-β activation of cultured human dermal fibroblasts. Systemic administration of penetratin-caveolin-1 peptide to mice with bleomycin-induced lung fibrosis reduced fibrosis.
Caveolin-1 plays an important role in the regulation of TGF-β signaling and participates in the pathogenesis of SSc and IPF. Restoration of caveolin function employing active caveolin-1 fragments coupled to cell-permeable carrier peptides may represent a novel approach for their treatment.
PMCID: PMC2732362  PMID: 18949888
Caveolin-1; TGF-β; fibrosis; collagen; systemic sclerosis; idiopathic pulmonary fibrosis
17.  Enhanced migration of fibroblasts derived from lungs with fibrotic lesions. 
Thorax  1995;50(9):984-989.
BACKGROUND--The migration and proliferation of fibroblasts may be important in the pathogenesis of pulmonary fibrosis. Considerable data are available on the proliferation of fibroblasts, but very few on their migration. METHODS--The migratory activity of fibroblasts obtained from lung biopsy specimens from 11 patients with idiopathic pulmonary fibrosis (IPF) was studied using a 96-well chemotaxis chamber. Fibroblasts from eight normal controls, seven patients with interstitial fibrosis associated with a collagen vascular disease (IP-CVD), and 13 patients with sarcoidosis were also examined. Migratory activity was tested in a serum-free medium in the presence and absence of platelet derived growth factor (PDGF), 30 ng/ml, as a chemoattractant. RESULTS--Migration of fibroblasts from patients with IPF was enhanced in serum-free maintenance medium alone (mean (SD) controls v IPF: 183 (86) v 689 (491) cells/field), and was also enhanced when cells were stimulated by PDGF (controls v IPF: 829 (222) v 1928 (600) cells/field). Fibroblasts from tissues with dense fibrosis had a greater capacity for migration than those from an earlier stage of fibrosis. No correlation was found between migratory activity and proliferative capacity of the individual cells. CONCLUSIONS--The fact that fibroblasts from fibrotic lungs migrate faster than those from controls suggests that migration is related to the initiation of the pulmonary fibrotic process. These in vitro studies suggest that fibroblasts derived from the lungs of patients with pulmonary fibrosis have a migratory phenotype. Such a change in fibroblast phenotype, if it occurred in vivo, may be important in the context of the pathogenesis of pulmonary fibrosis.
PMCID: PMC1021314  PMID: 8539681
18.  Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis 
The Journal of Experimental Medicine  2008;205(7):1659-1672.
Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive lung disease in which fibroblasts accumulate in the alveolar wall within a type I collagen–rich matrix. Although lung fibroblasts derived from patients with IPF display durable pathological alterations in proliferative function, the molecular mechanisms differentiating IPF fibroblasts from their normal counterparts remain unknown. Polymerized type I collagen normally inhibits fibroblast proliferation, providing a physiological mechanism to limit fibroproliferation after tissue injury. We demonstrate that β1 integrin interaction with polymerized collagen inhibits normal fibroblast proliferation by suppression of the phosphoinositide 3-kinase (PI3K)–Akt–S6K1 signal pathway due to maintenance of high phosphatase activity of the tumor suppressor phosphatase and tensin homologue (PTEN). In contrast, IPF fibroblasts eluded this restraint, displaying a pathological pattern of β1 integrin signaling in response to polymerized collagen that leads to aberrant activation of the PI3K–Akt–S6K1 signal pathway caused by inappropriately low PTEN activity. Mice deficient in PTEN showed a prolonged fibroproliferative response after tissue injury, and immunohistochemical analysis of IPF lung tissue demonstrates activation of Akt in cells within fibrotic foci. These results provide direct evidence for defective negative regulation of the proliferative pathway in IPF fibroblasts and support the theory that the pathogenesis of IPF involves an intrinsic fibroblast defect.
PMCID: PMC2442643  PMID: 18541712
19.  Regulation of Transforming Growth Factor-β1–driven Lung Fibrosis by Galectin-3 
Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic dysregulated response to alveolar epithelial injury with differentiation of epithelial cells and fibroblasts into matrix-secreting myofibroblasts resulting in lung scaring. The prognosis is poor and there are no effective therapies or reliable biomarkers. Galectin-3 is a β-galactoside binding lectin that is highly expressed in fibrotic tissue of diverse etiologies.
Objectives: To examine the role of galectin-3 in pulmonary fibrosis.
Methods: We used genetic deletion and pharmacologic inhibition in well-characterized murine models of lung fibrosis. Further mechanistic studies were performed in vitro and on samples from patients with IPF.
Measurements and Main Results: Transforming growth factor (TGF)-β and bleomycin-induced lung fibrosis was dramatically reduced in mice deficient in galectin-3, manifest by reduced TGF-β1–induced EMT and myofibroblast activation and collagen production. Galectin-3 reduced phosphorylation and nuclear translocation of β-catenin but had no effect on Smad2/3 phosphorylation. A novel inhibitor of galectin-3, TD139, blocked TGF-β–induced β-catenin activation in vitro and in vivo and attenuated the late-stage progression of lung fibrosis after bleomycin. There was increased expression of galectin-3 in the bronchoalveolar lavage fluid and serum from patients with stable IPF compared with nonspecific interstitial pneumonitis and controls, which rose sharply during an acute exacerbation suggesting that galectin-3 may be a marker of active fibrosis in IPF and that strategies that block galectin-3 may be effective in treating acute fibrotic exacerbations of IPF.
Conclusions: This study identifies galectin-3 as an important regulator of lung fibrosis and provides a proof of principle for galectin-3 inhibition as a potential novel therapeutic strategy for IPF.
PMCID: PMC3410728  PMID: 22095546
fibrosis; epithelial cells; fibroblasts
20.  β-Catenin in the Alveolar Epithelium Protects from Lung Fibrosis after Intratracheal Bleomycin 
Rationale: Alveolar epithelial cells (AECs) play central roles in the response to lung injury and the pathogenesis of pulmonary fibrosis.
Objectives: We aimed to determine the role of β-catenin in alveolar epithelium during bleomycin-induced lung fibrosis.
Methods: Genetically modified mice were developed to selectively delete β-catenin in AECs and were crossed to cell fate reporter mice that express β-galactosidase (βgal) in cells of AEC lineage. Mice were given intratracheal bleomycin (0.04 units) and assessed for AEC death, inflammation, lung injury, and fibrotic remodeling. Mouse lung epithelial cells (MLE12) with small interfering RNA knockdown of β-catenin underwent evaluation for wound closure, proliferation, and bleomycin-induced cytotoxicity.
Measurements and Main Results: Increased β-catenin expression was noted in lung parenchyma after bleomycin. Mice with selective deletion of β-catenin in AECs had greater AEC death at 1 week after bleomycin, followed by increased numbers of fibroblasts and enhanced lung fibrosis as determined by semiquantitative histological scoring and total collagen content. However, no differences in lung inflammation or protein levels in bronchoalveolar lavage were noted. In vitro, β-catenin–deficient AECs showed increased bleomycin-induced cytotoxicity as well as reduced proliferation and impaired wound closure. Consistent with these findings, mice with AEC β-catenin deficiency showed delayed recovery after bleomycin.
Conclusions: β-Catenin in the alveolar epithelium protects against bleomycin-induced fibrosis. Our studies suggest that AEC survival and wound healing are enhanced through β-catenin–dependent mechanisms. Activation of the developmentally important β-catenin pathway in AECs appears to contribute to epithelial repair after epithelial injury.
PMCID: PMC3733436  PMID: 23306543
apoptosis; idiopathic pulmonary fibrosis; wound healing
21.  Endothelin-1 and Transforming Growth Factor-β1 Independently Induce Fibroblast Resistance to Apoptosis via AKT Activation 
Myofibroblast apoptosis is critical for the normal resolution of wound repair responses, and impaired myofibroblast apoptosis is associated with tissue fibrosis. Lung expression of endothelin (ET)-1, a soluble peptide implicated in fibrogenesis, is increased in murine models of pulmonary fibrosis and in the lungs of humans with pulmonary fibrosis. Mechanistically, ET-1 has been shown to induce fibroblast proliferation, differentiation, contraction, and collagen synthesis. In this study, we examined the role ET-1 in the regulation of lung fibroblast survival and apoptosis. ET-1 rapidly activates the prosurvival phosphatidylinositol 3′-OH kinase (PI3K)/AKT signaling pathway in normal and fibrotic human lung fibroblasts. ET-1–induced activation of PI3K/AKT is dependent on p38 mitogen-activated protein kinase (MAPK), but not extracellular signal-regulated kinase (ERK) 1/2, JNK, or transforming growth factor (TGF)-β1. Activation of the PI3K/AKT pathway by ET-1 inhibits fibroblast apoptosis, and this inhibition is reversed by blockade of p38 MAPK or PI3K. TGF-β1 has been shown to attenuate myofibroblast apoptosis through the p38 MAPK–dependent secretion of a soluble factor, which activates PI3K/AKT. In this study, we show that, although TGF-β1 induces fibroblast synthesis and secretion of ET-1, TGF-β1 activation of PI3K/AKT is not dependent on ET-1. We conclude that ET-1 and TGF-β1 independently promote fibroblast resistance to apoptosis through signaling pathways involving p38 MAPK and PI3K/AKT. These findings suggest the potential for novel therapies targeting the convergence of prosurvival signaling pathways activated by these two profibrotic mediators.
PMCID: PMC2746991  PMID: 19188658
myofibroblast; fibrosis; Fas; p38 mitogen-activated protein kinase; mesenchymal cells
22.  The Role of PPARs in Lung Fibrosis 
PPAR Research  2007;2007:71323.
Pulmonary fibrosis is a group of disorders characterized by accumulation of scar tissue in the lung interstitium, resulting in loss of alveolar function, destruction of normal lung architecture, and respiratory distress. Some types of fibrosis respond to corticosteroids, but for many there are no effective treatments. Prognosis varies but can be poor. For example, patients with idiopathic pulmonary fibrosis (IPF) have a median survival of only 2.9 years. Prognosis may be better in patients with some other types of pulmonary fibrosis, and there is variability in survival even among individuals with biopsy-proven IPF. Evidence is accumulating that the peroxisome proliferator-activated receptors (PPARs) play important roles in regulating processes related to fibrogenesis, including cellular differentiation, inflammation, and wound healing. PPARα agonists, including the hypolidipemic fibrate drugs, inhibit the production of collagen by hepatic stellate cells and inhibit liver, kidney, and cardiac fibrosis in animal models. In the mouse model of lung fibrosis induced by bleomycin, a PPARα agonist significantly inhibited the fibrotic response, while PPARα knockout mice developed more serious fibrosis. PPARβ/δ appears to play a critical role in regulating the transition from inflammation to wound healing. PPARβ/δ agonists inhibit lung fibroblast proliferation and enhance the antifibrotic properties of PPARγ agonists. PPARγ ligands oppose the profibrotic effect of TGF-β, which induces differentiation of fibroblasts to myofibroblasts, a critical effector cell in fibrosis. PPARγ ligands, including the thiazolidinedione class of antidiabetic drugs, effectively inhibit lung fibrosis in vitro and in animal models. The clinical availability of potent and selective PPARα and PPARγ agonists should facilitate rapid development of successful treatment strategies based on current and ongoing research.
PMCID: PMC1940051  PMID: 17710235
23.  WNT5A Is a Regulator of Fibroblast Proliferation and Resistance to Apoptosis 
Usual interstitial pneumonia (UIP) is a specific histopathologic pattern of interstitial lung fibrosis that may be idiopathic or secondary to autoimmune diseases and environmental exposures. In this study, we compared gene expression patterns in primary fibroblasts isolated from lung tissues with UIP histology and fibroblasts isolated from lung tissues with normal histology using expression microarrays. We found that WNT5A was significantly increased in fibroblasts obtained from UIP lung tissues compared with normal lung fibroblasts, an observation verified by quantitative real-time RT-PCR and Western blot. Because the role of WNT5A in UIP is unknown, we treated normal lung fibroblasts or UIP lung fibroblasts with WNT5A, and found that WNT5A increased proliferation as well as relative resistance to H2O2-induced apoptosis. This effect was not mediated through the canonical WNT/β-catenin pathway, as WNT5A induced a decrease in β-catenin levels in the same cells. In addition, WNT5A induced increases in fibronectin and α5-integrin in normal lung fibroblasts. Collectively, our data suggest that WNT5A may play a role in fibroblast expansion and survival characteristics of idiopathic pulmonary fibrosis and other fibrotic interstitial lung diseases that exhibit UIP histological patterns.
PMCID: PMC2778165  PMID: 19251946
gene expression; idiopathic pulmonary fibrosis; cell growth; apoptosis; extracellular matrix
24.  β-Catenin in the Fibroproliferative Response to Acute Lung Injury 
Resolution of alveolar epithelial/capillary membrane damage after acute lung injury requires coordinated and effective tissue repair to reestablish a functional alveolar epithelial/capillary membrane barrier. We hypothesized that signaling pathways important in lung alveolar bud ontogeny are activated in the recovery and remodeling phases after profound oxidant stress lung injury in a murine model. To test this, we characterized the expression of noncanonical β-catenin pathway proteins E-cadherin, integrin-linked kinase–1, and β-catenin in mice undergoing normoxic recovery after exposure to butylated hydroxytoluene (BHT, ionol) and concomitant sublethal (75% O2) hyperoxia. Mice developed early acute lung injury with subsequent inflammation, collagen deposition, interstitial cellular proliferation, and lung architectural distortion. Reduced E-cadherin expression after 6 d of BHT and hyperoxia was accompanied by enhanced expression and nuclear localization of β-catenin and increased integrin-linked kinase-1 expression during subsequent normoxic recovery. This resulted in increased expression of the cotranscriptional regulators TCF-1 and -3 and cyclin D1. Proliferation of murine lung epithelial-12 cells in vitro after 8 h of treatment with BHT quinone-methide and hyperoxia and 48 h of normoxic recovery was enhanced 2.7-fold compared with vehicle-treated control mice at the same time point. BHT/hyperoxia-exposed mice treated with the pan-caspase inhibitor z-ASP had increased acute lung injury and reduced survival despite the presence of TUNEL-positive cells, suggesting enhanced lung cell necrosis. β-Catenin expression was reduced in z-ASP–co-treated lungs after BHT/hyperoxia. The noncanonical cadherin–β-catenin axis is associated with fibroproliferative repair after BHT/hyperoxia exposure and may regulate epithelial proliferation and lung matrix remodeling and repair in response to lung injury.
PMCID: PMC2644193  PMID: 16272459
β-catenin; E-cadherin; lung injury repair; cell junctions; caspase
25.  Transforming growth factor β regulates β-catenin expression in lung fibroblast through NF-κB dependent pathway 
β-catenin contributes to the pathogenesis of lung fibrosis. However, the expression of β-catenin in fibroblasts under fibrotic conditions has not been studied. We investigated the expression of β-catenin in lung fibroblasts from bleomycin (BLM)-challenged mice and human lung fibroblasts treated with transforming growth factor β (TGF-β) or lysophosphatidic acid (LPA) by western blot analysis. The result showed that the expression of β-catenin was significantly increased in lung fibrotic foci and lung fibroblasts from bleomycin-challenged mice. TGF-β stimulated β-catenin expression and induced differentiation in human lung fibroblasts in vitro. Pretreatment of the NF-κB activation inhibitor attenuated the TGF-β-induced expression of β-catenin and differentiation in human lung fibroblasts. Similarly, LPA induced β-catenin expression in human lung fibroblasts, and pre-treatment of the neutralized anti-TGF-β antibody attenuated the LPA-induced expression of β-catenin and differentiation in human lung fibroblasts. The results suggested that β-catenin expression is upregulated in lung fibroblast during differentiation, and that TGF-β induced β-catenin expression in human lung fibroblasts through the activation of NF-κB.
PMCID: PMC4199410  PMID: 25175023
β-catenin; LPA; TGF-β; NF-κB; pulmonary fibrosis; fibroblast

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