The circadian clock protein REVERBα inhibits pulmonary fibrosis development.

Pulmonary inflammatory responses lie under circadian control; however, the importance of circadian mechanisms in the underlying fibrotic phenotype is not understood. Here, we identify a striking change to these mechanisms resulting in a gain of amplitude and lack of synchrony within pulmonary fibrotic tissue. These changes result from an infiltration of mesenchymal cells, an important cell type in the pathogenesis of pulmonary fibrosis. Mutation of the core clock protein REVERBα in these cells exacerbated the development of bleomycin-induced fibrosis, whereas mutation of REVERBα in club or myeloid cells had no effect on the bleomycin phenotype. Knockdown of REVERBα revealed regulation of the little-understood transcription factor TBPL1. Both REVERBα and TBPL1 altered integrinß1 focal-adhesion formation, resulting in increased myofibroblast activation. The translational importance of our findings was established through analysis of 2 human cohorts. In the UK Biobank, circadian strain markers (sleep length, chronotype, and shift work) are associated with pulmonary fibrosis, making them risk factors. In a separate cohort, REVERBα expression was increased in human idiopathic pulmonary fibrosis (IPF) lung tissue. Pharmacological targeting of REVERBα inhibited myofibroblast activation in IPF fibroblasts and collagen secretion in organotypic cultures from IPF patients, thus suggesting that targeting of REVERBα could be a viable therapeutic approach.

Transcriptome analysis of IPF fibroblastic foci identifies key pathways involved in fibrogenesis.

INTRODUCTION: Fibroblastic foci represent the cardinal pathogenic lesion in idiopathic pulmonary fibrosis (IPF) and comprise activated fibroblasts and myofibroblasts, the key effector cells responsible for dysregulated extracellular matrix deposition in multiple fibrotic conditions. The aim of this study was to define the major transcriptional programmes involved in fibrogenesis in IPF by profiling unmanipulated myofibroblasts within fibrotic foci in situ by laser capture microdissection. METHODS: The challenges associated with deriving gene calls from low amounts of RNA and the absence of a meaningful comparator cell type were overcome by adopting novel data mining strategies and by using weighted gene co-expression network analysis (WGCNA), as well as an eigengene-based approach to identify transcriptional signatures, which correlate with fibrillar collagen gene expression. RESULTS: WGCNA identified prominent clusters of genes associated with cell cycle, inflammation/differentiation, translation and cytoskeleton/cell adhesion. Collagen eigengene analysis revealed that transforming growth factor ß1 (TGF-ß1), RhoA kinase and the TSC2/RHEB axis formed major signalling clusters associated with collagen gene expression. Functional studies using CRISPR-Cas9 gene-edited cells demonstrated a key role for the TSC2/RHEB axis in regulating TGF-ß1-induced mechanistic target of rapamycin complex 1 activation and collagen I deposition in mesenchymal cells reflecting IPF and other disease settings, including cancer-associated fibroblasts. CONCLUSION: These data provide strong support for the human tissue-based and bioinformatics approaches adopted to identify critical transcriptional nodes associated with the key pathogenic cell responsible for fibrogenesis in situ and further identify the TSC2/RHEB axis as a potential novel target for interfering with excessive matrix deposition in IPF and other fibrotic conditions.

A randomised, placebo-controlled study of omipalisib (PI3K/mTOR) in idiopathic pulmonary fibrosis.

Phosphatidylinositol 3-kinases (PI3Ks) and mammalian target of rapamycin (mTOR) play a role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Omipalisib (GSK2126458) is a potent inhibitor of PI3K/mTOR.A randomised, placebo-controlled, double-blind, repeat dose escalation, experimental medicine study of omipalisib in subjects with IPF was conducted (NCT01725139) to test safety, tolerability, pharmacokinetics and pharmacodynamics. Omipalisib was dosed at 0.25 mg, 1 mg and 2 mg twice daily for 8 days in four cohorts of four subjects randomised 3:1 to receive omipalisib or placebo (two cohorts received 2 mg twice daily).17 subjects with IPF were enrolled. The most common adverse event was diarrhoea, which was reported by four participants. Dose-related increases in insulin and glucose were observed. Pharmacokinetic analysis demonstrated that exposure in the blood predicts lung exposure. Exposure-dependent inhibition of phosphatidylinositol 3,4,5 trisphosphate and pAKT confirmed target engagement in blood and lungs. 18F-2-fluoro-2-deoxy-d-glucose(FDG)-positron emission tomography/computed tomography scans revealed an exposure-dependent reduction in 18F-FDG uptake in fibrotic areas of the lung, as measured by target-to-background, ratio thus confirming pharmacodynamic activity.This experimental medicine study demonstrates acceptable tolerability of omipalisib in subjects with IPF at exposures for which target engagement was confirmed both systemically and in the lungs.

Evidence for chemokine synergy during neutrophil migration in ARDS.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a life-threatening condition characterised by pulmonary oedema, respiratory failure and severe inflammation. ARDS is further characterised by the recruitment of neutrophils into the lung interstitium and alveolar space. OBJECTIVES: The factors that regulate neutrophil infiltration into the inflamed lung and our understanding of the pathomechanisms in ARDS remain incomplete. This study aimed at determining the role of the chemokine (C-C motif) ligand (CCL)2 and CCL7 in ARDS. METHODS: CCL2 and CCL7 protein levels were measured in bronchoalveolar lavage (BAL) fluid obtained from lipopolysaccharide(LPS)-challenged human volunteers and two separate cohorts of patients with ARDS. Neutrophil chemotaxis to ARDS BAL fluid was evaluated and the contribution of each was assessed and compared with chemokine (C-X-C motif) ligand 8 (CXCL8). Chemokine receptor expression on neutrophils from blood or BAL fluid of patients with ARDS was analysed by flow cytometry. RESULTS: CCL2 and CCL7 were significantly elevated in BAL fluid recovered from LPS-challenged volunteers and patients with ARDS. BAL fluid from patients with ARDS was highly chemotactic for human neutrophils and neutralising either CCL2 or CCL7 attenuated the neutrophil chemotactic response. Moreover, CCL2 and CCL7 synergised with CXCL8 to promote neutrophil migration. Furthermore, neutrophils isolated from the blood or BAL fluid differentially regulated the cell surface expression of chemokine (C-X-C motif) receptor 1 and C-C chemokine receptor type 2 during ARDS. CONCLUSION: This study highlights important inflammatory chemokines involved in regulating neutrophil migration, which may have potential value as therapeutic targets for the treatment of ARDS.

Regulation of neutrophilic inflammation by proteinase-activated receptor 1 during bacterial pulmonary infection.

Neutrophils are key effector cells of the innate immune response to pathogenic bacteria, but excessive neutrophilic inflammation can be associated with bystander tissue damage. The mechanisms responsible for neutrophil recruitment to the lungs during bacterial pneumonia are poorly defined. In this study, we focus on the potential role of the major high-affinity thrombin receptor, proteinase-activated receptor 1 (PAR-1), during the development of pneumonia to the common lung pathogen Streptococcus pneumoniae. Our studies demonstrate that neutrophils were indispensable for controlling S. pneumoniae outgrowth but contributed to alveolar barrier disruption. We further report that intra-alveolar coagulation (bronchoalveolar lavage fluid thrombin-antithrombin complex levels) and PAR-1 immunostaining were increased in this model of bacterial lung infection. Functional studies using the most clinically advanced PAR-1 antagonist, SCH530348, revealed a key contribution for PAR-1 signaling in influencing neutrophil recruitment to lung airspaces in response to both an invasive and noninvasive strain of S. pneumoniae (D39 and EF3030) but that PAR-1 antagonism did not impair the ability of the host to control bacterial outgrowth. PAR-1 antagonist treatment significantly decreased pulmonary levels of IL-1ß, CXCL1, CCL2, and CCL7 and attenuated alveolar leak. Ab neutralization studies further demonstrated a nonredundant role for IL-1ß, CXCL1, and CCL7 in mediating neutrophil recruitment in response to S. pneumoniae infection. Taken together, these data demonstrate a key role for PAR-1 during S. pneumoniae lung infection that is mediated, at least in part, by influencing multiple downstream inflammatory mediators.

Exploration of a potent PI3 kinase/mTOR inhibitor as a novel anti-fibrotic agent in IPF.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is the most rapidly progressive and fatal of all fibrotic conditions with no curative therapies. Common pathomechanisms between IPF and cancer are increasingly recognised, including dysfunctional pan-PI3 kinase (PI3K) signalling as a driver of aberrant proliferative responses. GSK2126458 is a novel, potent, PI3K/mammalian target of rapamycin (mTOR) inhibitor which has recently completed phase I trials in the oncology setting. Our aim was to establish a scientific and dosing framework for PI3K inhibition with this agent in IPF at a clinically developable dose. METHODS: We explored evidence for pathway signalling in IPF lung tissue and examined the potency of GSK2126458 in fibroblast functional assays and precision-cut IPF lung tissue. We further explored the potential of IPF patient-derived bronchoalveolar lavage (BAL) cells to serve as pharmacodynamic biosensors to monitor GSK2126458 target engagement within the lung. RESULTS: We provide evidence for PI3K pathway activation in fibrotic foci, the cardinal lesions in IPF. GSK2126458 inhibited PI3K signalling and functional responses in IPF-derived lung fibroblasts, inhibiting Akt phosphorylation in IPF lung tissue and BAL derived cells with comparable potency. Integration of these data with GSK2126458 pharmacokinetic data from clinical trials in cancer enabled modelling of an optimal dosing regimen for patients with IPF. CONCLUSIONS: Our data define PI3K as a promising therapeutic target in IPF and provide a scientific and dosing framework for progressing GSK2126458 to clinical testing in this disease setting. A proof-of-mechanism trial of this agent is currently underway. TRIAL REGISTRATION NUMBER: NCT01725139, pre-clinical.

Translational models of lung disease.

The 2nd Cross Company Respiratory Symposium (CCRS), held in Horsham, U.K. in 2012, brought together representatives from across the pharmaceutical industry with expert academics, in the common interest of improving the design and translational predictiveness of in vivo models of respiratory disease. Organized by the respiratory representatives of the European Federation of Pharmaceutical Industries and Federations (EFPIA) group of companies involved in the EU-funded project (U-BIOPRED), the aim of the symposium was to identify state-of-the-art improvements in the utility and design of models of respiratory disease, with a view to improving their translational potential and reducing wasteful animal usage. The respiratory research and development community is responding to the challenge of improving translation in several ways: greater collaboration and open sharing of data, careful selection of the species, complexity and chronicity of the models, improved practices in preclinical research, continued refinement in models of respiratory diseases and their sub-types, greater understanding of the biology underlying human respiratory diseases and their sub-types, and finally greater use of human (and especially disease-relevant) cells, tissues and explants. The present review highlights these initiatives, combining lessons from the symposium and papers published in Clinical Science arising from the symposium, with critiques of the models currently used in the settings of asthma, idiopathic pulmonary fibrosis and COPD. The ultimate hope is that this will contribute to a more rational, efficient and sustainable development of a range of new treatments for respiratory diseases that continue to cause substantial morbidity and mortality across the world.

Proteinase-activated receptor-1, CCL2, and CCL7 regulate acute neutrophilic lung inflammation.

PAR1 plays a central role in mediating the interplay between coagulation and inflammation, but its role in regulating acute neutrophilic inflammation is unknown. We report that antagonism of PAR1 was highly effective at reducing acute neutrophil accumulation in a mouse model of LPS-induced lung inflammation. PAR1 antagonism also reduced alveolar-capillary barrier disruption in these mice. This protection was associated with a reduction in the expression of the chemokines, CCL2 and CCL7, but not the proinflammatory cytokines, TNF and IL-6, or the classic neutrophil chemoattractants, CXCL1 and CXCL2. Antibody neutralization of CCL2 and CCL7 significantly reduced LPS-induced total leukocyte and neutrophil accumulation, recovered from the bronchoalveolar lavage fluid of challenged mice. Immunohistochemical analysis revealed that CCL2 predominantly localized to alveolar macrophages and pulmonary epithelial cells, whereas CCL7 was restricted to the pulmonary epithelium. In keeping with these observations, the intranasal administration of recombinant CCL2 (rCCL2) and rCCL7 led to the accumulation of neutrophils within the lung airspaces of naive mice in the absence of any underlying inflammation. Flow cytometry analysis further demonstrated an increase in Ly6G(hi) neutrophils expressing the chemokine receptors, CCR1 and CCR2, isolated from mouse lungs compared with circulating neutrophils. Conversely, the expression of CXCR2 decreased on neutrophils isolated from the lung compared with circulating neutrophils. Furthermore, this switch in chemokine receptor expression was accentuated after acute LPS-induced lung inflammation. Collectively, these findings reveal a novel role for PAR1 and the chemokines, CCL2 and CCL7, during the early events of acute neutrophilic inflammation.

Coagulation and coagulation signalling in fibrosis.

Following tissue injury, a complex and coordinated wound healing response comprising coagulation, inflammation, fibroproliferation and tissue remodelling has evolved to nullify the impact of the original insult and reinstate the normal physiological function of the affected organ. Tissue fibrosis is thought to result from a dysregulated wound healing response as a result of continual local injury or impaired control mechanisms. Although the initial insult is highly variable for different organs, in most cases, uncontrolled or sustained activation of mesenchymal cells into highly synthetic myofibroblasts leads to the excessive deposition of extracellular matrix proteins and eventually loss of tissue function. Coagulation was originally thought to be an acute and transient response to tissue injury, responsible primarily for promoting haemostasis by initiating the formation of fibrin plugs to enmesh activated platelets within the walls of damaged blood vessels. However, the last 20years has seen a major re-evaluation of the role of the coagulation cascade following tissue injury and there is now mounting evidence that coagulation plays a critical role in orchestrating subsequent inflammatory and fibroproliferative responses during normal wound healing, as well as in a range of pathological contexts across all major organ systems. This review summarises our current understanding of the role of coagulation and coagulation initiated signalling in the response to tissue injury, as well as the contribution of uncontrolled coagulation to fibrosis of the lung, liver, kidney and heart. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.

Ex vivo micro-computed tomography analysis of bleomycin-induced lung fibrosis for preclinical drug evaluation.

Research into the pathogenesis underlying the development of idiopathic pulmonary fibrosis is hampered by a repertoire of animal models that fail to recapitulate all the features of the human disease. Better use and understanding of what the animal models represent may improve clinical predictability. We interrogated ex vivo micro-computed tomography (CT) as a novel end-point measure in the mouse model of bleomycin-induced lung fibrosis (BILF), and to evaluate a therapeutic dosing regimen for preclinical drug evaluation. A detailed characterisation of BILF was performed using standard end-point measures (lung hydroxyproline and histology). High resolution micro-CT (∼13.7 µm voxel size) was evaluated for quantifying the extent and severity of lung fibrosis. The period from 14 to 28 days following bleomycin instillation represents progression of established fibrosis. A therapeutic dosing regimen during this period was validated using a transforming growth factor-ß receptor-1 kinase inhibitor, and micro-CT provided a highly sensitive and quantitative measure of fibrosis. Moreover, fibrotic lesions did not completely resolve, but instead persisted for ≥6 months following a single insult with bleomycin. Ex vivo micro-CT analysis of BILF allows robust evaluation of therapeutic dosing once fibrosis is already well established, requiring fewer mice than conventional biochemical end-points.

PAR-1-dependent and PAR-independent pro-inflammatory signaling in human lung fibroblasts exposed to thrombin.

Proteinase-activated receptors (PARs) are crucial in orchestrating cellular responses to coagulation proteinases, such as thrombin and FXa. Four PARs have been characterized and have been shown to be differentially expressed in mice and humans and between tissues. We have previously shown that in murine lung fibroblasts, PAR-1 is solely responsible for all cellular responses to thrombin and FXa. In contrast, we report here that in primary human lung fibroblasts (pHLFs), known PARs fail to account for all of the cellular responses to thrombin, in particular in the presence of high, but physiologically achievable concentrations of thrombin. We report that pHLFs secrete CCL2 in a PAR-1-dependent manner at low thrombin concentration (∼0.3 nM). At or above 10 nM thrombin, pharmacological antagonism (RWJ-58259) fails to block thrombin-induced CCL2 release; whereas PAR-1 cleavage-blocking monoclonal antibodies (ATAP2 and WEDE15) only partially inhibit thrombin-induced CCL2 secretion. In addition, activation of PAR-3, PAR-4, and transactivation of either PAR-2 or EGFR were ruled out as being responsible for thrombin-mediated CCL2 secretion at high yet standard concentrations of the proteinase. We further provide evidence that PAR-1-dependent and PAR-independent signaling involves the rapid phosphorylation of ERK, which in turn is absolutely required for thrombin-induced CCL2 secretion at both low and standard concentration of the proteinase. Our findings suggest the existence of a PAR-independent signaling mechanism in human lung fibroblasts and have important implications for the design of therapeutic strategies aimed at blocking pro-inflammatory signaling responses associated with excessive thrombin generation.

Deficiency of tumour necrosis factor-related apoptosis-inducing ligand exacerbates lung injury and fibrosis.

BACKGROUND: The death receptor ligand tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) shows considerable clinical promise as a therapeutic agent. TRAIL induces leukocyte apoptosis, reducing acute inflammatory responses in the lung. It is not known whether TRAIL modifies chronic lung injury or whether TRAIL has a role in human idiopathic pulmonary fibrosis (IPF). We therefore explored the capacity of TRAIL to modify chronic inflammatory lung injury and studied TRAIL expression in patients with IPF. METHODS: TRAIL(-/-) and wild-type mice were instilled with bleomycin and inflammation assessed at various time points by bronchoalveolar lavage and histology. Collagen deposition was measured by tissue hydroxyproline content. TRAIL expression in human IPF lung samples was assessed by immunohistochemistry and peripheral blood TRAIL measured by ELISA. RESULTS: TRAIL(-/-) mice had an exaggerated delayed inflammatory response to bleomycin, with increased neutrophil numbers (mean 3.19±0.8 wild type vs 11.5±5.4×10(4) TRAIL(-/-), p<0.0001), reduced neutrophil apoptosis (5.42±1.6% wild type vs 2.47±0.5% TRAIL(-/-), p=0.0003) and increased collagen (3.45±0.2 wild type vs 5.8±1.3 mg TRAIL(-/-), p=0.005). Immunohistochemical analysis showed induction of TRAIL in bleomycin-treated wild-type mice. Patients with IPF demonstrated lower levels of TRAIL expression than in control lung biopsies and their serum levels of TRAIL were significantly lower compared with matched controls (38.1±9.6 controls vs 32.3±7.2 pg/ml patients with IPF, p=0.002). CONCLUSION: These data suggest TRAIL may exert beneficial, anti-inflammatory actions in chronic pulmonary inflammation in murine models and that these mechanisms may be compromised in human IPF.

Diminished prostaglandin E2 contributes to the apoptosis paradox in idiopathic pulmonary fibrosis.

RATIONALE: Patients with idiopathic pulmonary fibrosis (IPF), a progressive disease with a dismal prognosis, exhibit an unexplained disparity of increased alveolar epithelial cell (AEC) apoptosis but reduced fibroblast apoptosis. OBJECTIVES: To examine whether the failure of patients with IPF to up-regulate cyclooxygenase (COX)-2, and thus the antifibrotic mediator prostaglandin (PG)E(2), accounts for this imbalance. METHODS: Fibroblasts and primary type II AECs were isolated from control and fibrotic human lung tissue. The effects of COX-2 inhibition and exogenous PGE(2) on fibroblast and AEC sensitivity to Fas ligand (FasL)-induced apoptosis were assessed. MEASUREMENTS AND MAIN RESULTS: IPF lung fibroblasts are resistant to FasL-induced apoptosis compared with control lung fibroblasts. Inhibition of COX-2 in control lung fibroblasts resulted in an apoptosis-resistant phenotype. Administration of PGE(2) almost doubled the rate of FasL-induced apoptosis in fibrotic lung fibroblasts compared with FasL alone. Conversely, in primary fibrotic lung type II AECs, PGE(2) protected against FasL-induced apoptosis. In human control and, to a greater extent, fibrotic lung fibroblasts, PGE(2) inhibits the phosphorylation of Akt, suggesting that regulation of this prosurvival protein kinase is an important mechanism by which PGE(2) modulates cellular apoptotic responses. CONCLUSIONS: The observation that PGE(2) deficiency results in increased AEC but reduced fibroblast sensitivity to apoptosis provides a novel pathogenic insight into the mechanisms driving persistent fibroproliferation in IPF.

Pulmonary epithelium is a prominent source of proteinase-activated receptor-1-inducible CCL2 in pulmonary fibrosis.

RATIONALE: Studies in patients and experimental animals provide compelling evidence of the involvement of the major thrombin receptor, proteinase-activated receptor-1 (PAR(1)), and the potent chemokine, chemokine (CC motif) ligand-2 (CCL2)/monocyte chemotactic protein-1, in the pathogenesis of idiopathic pulmonary fibrosis (IPF). PAR(1) knockout mice are protected from bleomycin-induced lung inflammation and fibrosis and this protection is associated with marked attenuation in CCL2 induction. OBJECTIVES: The aim of this study was to determine which cell types represent the major source of PAR(1)-inducible CCL2 in the fibrotic lung. METHODS: Using immunohistochemistry and dual immunofluorescence, we examined PAR(1) and CCL2 expression in the bleomycin model and human IPF lung. PAR(1) and CCL2 gene expression was also assessed in laser-captured alveolar septae from patients with IPF. The ability of PAR(1) to induce CCL2 production by lung epithelial cells was also examined in vitro. MEASUREMENTS AND MAIN RESULTS: We report for the first time that PAR(1) and CCL2 are coexpressed and co-up-regulated on the activated epithelium in fibrotic areas in IPF. Similar observations were found in bleomycin-induced lung injury. Furthermore, we show that thrombin is a potent inducer of CCL2 gene expression and protein release by cultured lung epithelial cells via a PAR(1)-dependent mechanism. CONCLUSIONS: These data support the notion that PAR(1) activation on lung epithelial cells may represent an important mechanism leading to increased local CCL2 release in pulmonary fibrosis. Targeting PAR(1) on the pulmonary epithelium may offer a unique opportunity for therapeutic intervention in pulmonary fibrosis and other inflammatory and fibroproliferative conditions associated with excessive local generation of thrombin and CCL2 release.

Author Correction: The mTORC1/4E-BP1 axis represents a critical signaling node during fibrogenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Translational pharmacology of an inhaled small molecule αvß6 integrin inhibitor for idiopathic pulmonary fibrosis.

The αvß6 integrin plays a key role in the activation of transforming growth factor-ß (TGFß), a pro-fibrotic mediator that is pivotal to the development of idiopathic pulmonary fibrosis (IPF). We identified a selective small molecule αvß6 RGD-mimetic, GSK3008348, and profiled it in a range of disease relevant pre-clinical systems. To understand the relationship between target engagement and inhibition of fibrosis, we measured pharmacodynamic and disease-related end points. Here, we report, GSK3008348 binds to αvß6 with high affinity in human IPF lung and reduces downstream pro-fibrotic TGFß signaling to normal levels. In human lung epithelial cells, GSK3008348 induces rapid internalization and lysosomal degradation of the αvß6 integrin. In the murine bleomycin-induced lung fibrosis model, GSK3008348 engages αvß6, induces prolonged inhibition of TGFß signaling and reduces lung collagen deposition and serum C3M, a marker of IPF disease progression. These studies highlight the potential of inhaled GSK3008348 as an anti-fibrotic therapy.

mTORC1 amplifies the ATF4-dependent de novo serine-glycine pathway to supply glycine during TGF-ß1-induced collagen biosynthesis.

The differentiation of fibroblasts into a transient population of highly activated, extracellular matrix (ECM)-producing myofibroblasts at sites of tissue injury is critical for normal tissue repair. Excessive myofibroblast accumulation and persistence, often as a result of a failure to undergo apoptosis when tissue repair is complete, lead to pathological fibrosis and are also features of the stromal response in cancer. Myofibroblast differentiation is accompanied by changes in cellular metabolism, including increased glycolysis, to meet the biosynthetic demands of enhanced ECM production. Here, we showed that transforming growth factor-ß1 (TGF-ß1), the key pro-fibrotic cytokine implicated in multiple fibrotic conditions, increased the production of activating transcription factor 4 (ATF4), the transcriptional master regulator of amino acid metabolism, to supply glucose-derived glycine to meet the amino acid requirements associated with enhanced collagen production in response to myofibroblast differentiation. We further delineated the signaling pathways involved and showed that TGF-ß1-induced ATF4 production depended on cooperation between canonical TGF-ß1 signaling through Smad3 and activation of mechanistic target of rapamycin complex 1 (mTORC1) and its downstream target eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). ATF4, in turn, promoted the transcription of genes encoding enzymes of the de novo serine-glycine biosynthetic pathway and glucose transporter 1 (GLUT1). Our findings suggest that targeting the TGF-ß1-mTORC1-ATF4 axis may represent a novel therapeutic strategy for interfering with myofibroblast function in fibrosis and potentially in other conditions, including cancer.

The mTORC1/4E-BP1 axis represents a critical signaling node during fibrogenesis.

Myofibroblasts are the key effector cells responsible for excessive extracellular matrix deposition in multiple fibrotic conditions, including idiopathic pulmonary fibrosis (IPF). The PI3K/Akt/mTOR axis has been implicated in fibrosis, with pan-PI3K/mTOR inhibition currently under clinical evaluation in IPF. Here we demonstrate that rapamycin-insensitive mTORC1 signaling via 4E-BP1 is a critical pathway for TGF-ß1 stimulated collagen synthesis in human lung fibroblasts, whereas canonical PI3K/Akt signaling is not required. The importance of mTORC1 signaling was confirmed by CRISPR-Cas9 gene editing in normal and IPF fibroblasts, as well as in lung cancer-associated fibroblasts, dermal fibroblasts and hepatic stellate cells. The inhibitory effect of ATP-competitive mTOR inhibition extended to other matrisome proteins implicated in the development of fibrosis and human disease relevance was demonstrated in live precision-cut IPF lung slices. Our data demonstrate that the mTORC1/4E-BP1 axis represents a critical signaling node during fibrogenesis with potential implications for the development of novel anti-fibrotic strategies.