Gisli Jenkins

The role of proteases in transforming growth factor-β activation

Transforming growth factor-beta (TGFbeta) plays a central role in a number of developmental and pathological processes. There are 3 isoforms of TGFbeta (1-3) and all are sequestered in the extracellular matrix as latent complexes. Activation of this complex is the key biological checkpoint controlling TGF-beta bioavailability. This process is tightly regulated in a temporal, spatial and isoform specific manner highlighting its importance. There are many different mechanisms by which TGF-beta can be activated. Both serine and metalloproteinases play an important role in TGF-beta activation, at least in vitro, and many of these proteases have been implicated in pathological conditions. The mechanism of activation is distinct between the different proteases, but is not conserved between the two groups. Both serine proteases, such as plasmin, and metalloproteases, such as MMP2, can directly cleave latent TGFbeta, whereas others, such as thrombin and MMP14, interact with integrin mediated TGFbeta activation pathways. However, further studies are still required to fully understand the relevance of all of these pathways in vivo. Currently, the best described mechanism of TGF-beta1 activation in vivo is by integrins, although this process can be modulated by proteases. The primary mechanism of TGF-beta2 and TGF-beta3 activation has yet to be defined in vivo, although it is likely that TGF-beta3 is activated in a similar manner to TGF-beta1. This review describes the mechanism of protease driven TGF-beta activation, and discusses the physiological and pathological relevance of this process.

The pathogenesis of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with an appalling prognosis. The failure of anti-inflammatory therapies coupled with the observation that deranged epithelium overlies proliferative myofibroblasts to form the fibroblastic focus has lead to the emerging concept that IPF is a disease of deregulated epithelial-mesenchymal crosstalk. IPF is triggered by an as yet unidentified alveolar injury that leads to activation of transforming growth factor-β (TGF-β) and alveolar basement membrane disruption. In the presence of persisting injurious pathways, or disrupted repair pathways, activated TGF-β can lead to enhanced epithelial apoptosis and epithelial-to-mesenchymal transition (EMT) as well as fibroblast, and fibrocyte, transformation into myofibroblasts which are resistant to apoptosis. The resulting deposition of excess disrupted matrix by these myofibroblasts leads to the development of IPF.

Acute Exacerbation of Idiopathic Pulmonary Fibrosis. An International Working Group Report

Acute exacerbation of idiopathic pulmonary fibrosis has been defined as an acute, clinically significant, respiratory deterioration of unidentifiable cause. The objective of this international working group report on acute exacerbation of idiopathic pulmonary fibrosis was to provide a comprehensive update on the topic. A literature review was conducted to identify all relevant English text publications and abstracts. Evidence-based updates on the epidemiology, etiology, risk factors, prognosis, and management of acute exacerbations of idiopathic pulmonary fibrosis are provided. Finally, to better reflect the current state of knowledge and improve the feasibility of future research into its etiology and treatment, the working group proposes a new conceptual framework for acute respiratory deterioration in idiopathic pulmonary fibrosis and a revised definition and diagnostic criteria for acute exacerbation of idiopathic pulmonary fibrosis.

Lysophosphatidic Acid Induces αvβ6 Integrin-Mediated TGF-β Activation via the LPA2 Receptor and the Small G Protein Gαq

Activation of latent transforming growth factor beta (TGF-beta) by alphavbeta6 integrin is critical in the pathogenesis of lung injury and fibrosis. We have previously demonstrated that the stimulation of protease activated receptor 1 promotes alphavbeta6 integrin-mediated TGF-beta activation via RhoA, which is known to modulate cell contraction. However, whether other G protein-coupled receptors can also induce alphavbeta6 integrin-mediated TGF-beta activation is unknown; in addition, the alphavbeta6 integrin signaling pathway has not yet been fully characterized. In this study, we show that lysophosphatidic acid (LPA) induces alphavbeta6-mediated TGF-beta activation in human epithelial cells via both RhoA and Rho kinase. Furthermore, we demonstrate that LPA-induced alphavbeta6 integrin-mediated TGF-beta activity is mediated via the LPA2 receptor, which signals via G alpha(q). Finally, we show that the expression levels of both the LPA2 receptor and alphavbeta6 integrin are up-regulated and are spatially and temporally associated following bleomycin-induced lung injury. Furthermore, both the LPA2 receptor and alphavbeta6 integrin are up-regulated in the overlying epithelial areas of fibrosis in patients with usual interstitial pneumonia. These studies demonstrate that LPA induces alphavbeta6 integrin-mediated TGF-beta activation in epithelial cells via LPA2, G alpha(q), RhoA, and Rho kinase, and that this pathway might be clinically relevant to the development of lung injury and fibrosis.

TGF-β Activation and Lung Fibrosis

Lung fibrosis can affect the parenchyma and the airways, classically giving rise to idiopathic pulmonary fibrosis (IPF) in the parenchyma or airway remodeling in asthma and chronic obstructive pulmonary disease. TGF-β activation has been implicated in the fibrosis of both IPF and airway remodeling. However, the mechanisms of TGF-β activation appear to differ depending on the cellular and anatomical compartments, with implications on disease pathogenesis. Although it appears that epithelial cell activation of TGF-β by the αvβ6 integrin is central in IPF, mesenchymal activation of TGF-β by the αvβ5 and αvβ8 integrins appears to predominate in airway remodeling. Interestingly, the mechanism of TGF-β by the integrins αvβ6 and αvβ5 is shared, relying on cytoskeletal changes, whereas activation of TGF-β by the αvβ8 integrin is distinct, relying on proteolytic cleavage of the latency-associated peptide of TGF-β by matrix metalloproteinase 14. This article describes the mechanisms through which epithelial cells activate TGF-β by the αvβ6 integrin and mesenchymal cells activate TGF-β by the αvβ5 integrin, and highlights their roles in lung fibrosis.

Integrin αvβ5-Mediated TGF-β Activation by Airway Smooth Muscle Cells in Asthma

Severe asthma is associated with airway remodeling, characterized by structural changes including increased smooth muscle mass and matrix deposition in the airway, leading to deteriorating lung function. TGF-β is a pleiotropic cytokine leading to increased synthesis of matrix molecules by human airway smooth muscle (HASM) cells and is implicated in asthmatic airway remodeling. TGF-β is synthesized as a latent complex, sequestered in the extracellular matrix, and requires activation for functionality. Activation of latent TGF-β is the rate-limiting step in its bioavailability. This study investigated the effect of the contraction agonists LPA and methacholine on TGF-β activation by HASM cells and its role in the development of asthmatic airway remodeling. The data presented show that LPA and methacholine induced TGF-β activation by HASM cells via the integrin αvβ5. Our findings highlight the importance of the β5 cytoplasmic domain because a polymorphism in the β5 subunit rendered the integrin unable to activate TGF-β. To our knowledge, this is the first description of a biologically relevant integrin that is unable to activate TGF-β. These data demonstrate that murine airway smooth muscle cells express αvβ5 integrins and activate TGF-β. Finally, these data show that inhibition, or genetic loss, of αvβ5 reduces allergen-induced increases in airway smooth muscle thickness in two models of asthma. These data highlight a mechanism of TGF-β activation in asthma and support the hypothesis that bronchoconstriction promotes airway remodeling via integrin mediated TGF-β activation.

Role of integrin-mediated TGFβ activation in the pathogenesis of pulmonary fibrosis

IPF (idiopathic pulmonary fibrosis) is a chronic progressive disease of unknown aetiology without effective treatment. IPF is characterized by excessive collagen deposition within the lung. Recent evidence suggests that the lung epithelium plays a key role in driving the fibrotic response. The current paradigm suggests that, after epithelial injury, there is impaired epithelial proliferation and enhanced epithelial apoptosis. This in turn promotes lung fibrosis through impaired basement membrane repair and increased epithelial-mesenchymal transition. Furthermore, fibroblasts are recruited to the wounded area and adopt a myofibroblast phenotype, with the up-regulation of matrix-synthesizing genes and down-regulation of matrix-degradation genes. There is compelling evidence that the cytokine TGFbeta (transforming growth factor beta) plays a central role in this process. In normal lung, TGFbeta is maintained in an inactive state that is tightly regulated temporally and spatially. One of the major TGFbeta-activation pathways involves integrins, and the role of the (alpha)vbeta6 integrin has been particularly well described in the pathogenesis of IPF. Owing to the pleiotropic nature of TGFbeta, strategies that inhibit activation of TGFbeta in a cell- or disease-specific manner are attractive for the treatment of chronic fibrotic lung conditions. Therefore the molecular pathways that lead to integrin-mediated TGFbeta activation must be precisely defined to identify and fully exploit novel therapeutic targets that might ultimately improve the prognosis for patients with IPF.

Repression of IP-10 by Interactions between Histone Deacetylation and Hypermethylation in Idiopathic Pulmonary Fibrosis

ABSTRACT Targeted repression of a subset of key genes involved in tissue remodeling is a cardinal feature of idiopathic pulmonary fibrosis (IPF). The mechanism is unclear but is potentially important in disease pathogenesis and therapeutic targeting. We have previously reported that defective histone acetylation is responsible for the repression of the antifibrotic cyclooxygenase-2 gene. Here we extended our study to the repression of another antifibrotic gene, the potent angiostatic chemokine gamma interferon (IFN-γ)-inducible protein of 10 kDa (IP-10), in lung fibroblasts from patients with IPF. We revealed that this involved not only histone deacetylation, as with cyclooxygenase-2 repression, but also histone H3 hypermethylation, as a result of decreased recruitment of histone acetyltransferases and increased presence of histone deacetylase (HDAC)-containing repressor complexes, histone methyltransferases G9a and SUV39H1, and heterochromatin protein 1 at the IP-10 promoter, leading to reduced transcription factor binding. More importantly, treatment of diseased cells with HDAC or G9a inhibitors similarly reversed the repressive histone deacetylation and hypermethylation and restored IP-10 expression. These findings strongly suggest that epigenetic dysregulation involving interactions between histone deacetylation and hypermethylation is responsible for targeted repression of IP-10 and potentially other antifibrotic genes in fibrotic lung disease and that this is amenable to therapeutic targeting.

Into the matrix: targeting fibroblasts in pulmonary fibrosis.

Purpose of reviewThis review describes the challenges created by the existence of multiple molecular pathways leading to fibrosis and proposes that attention be focused on targeting the fibroblasts and myofibroblasts which themselves produce multiple cytokines and growth factors as well as the extracellular matrix, which is the hallmark of fibrotic lung disease. Recent findingsThe last 20 years have seen remarkable progress in our understanding of the molecular pathogenesis of pulmonary fibrosis leading to multiple programmes in drug discovery, and there are currently 15 actively recruiting trials registered on http://www.clinicaltrials.gov. Unfortunately, at this time only one new drug, pirfenidone, has progressed to approval for use in patients. Part of the frustration is that drugs that are effective in targeting inflammatory pathways have been ineffective in lung fibrosis. This may result from the inability to treat patients early in the disease process but it is also likely that pathways independent of inflammation can drive fibrosis. SummaryWe further propose that this approach should inhibit fibrosis independent of cell type or the signalling cascade that is activating these cells. We are hopeful that the next 20 years will see many more therapeutic options for patients suffering with fibrotic lung disease.

A central role for G9a and EZH2 in the epigenetic silencing of cyclooxygenase-2 in idiopathic pulmonary fibrosis

Selective silencing of the cyclooxygenase‐2 (COX‐2) gene with the loss of the antifibrotic mediator prostaglandin E2 contributes to the fibrotic process in idiopathic pulmonary fibrosis (IPF). This study explored the role of G9a‐ and enhancer of zeste homolog 2 (EZH2)‐mediated methylation of histone H3 lysine 9 (H3K9me3) and histone H3 lysine 27 (H3K27me3) in COX‐2 silencing in IPF. Chromatin immunoprecipitation (ChIP) and re‐ChIP assays demonstrated marked increases in H3K9me3, H3K27me3, and DNA methylation, together with their respective modifying enzymes G9a, EZH2, and DNA methyltransferases (Dnmts) and respective binding proteins heterochromatin protein 1 (HP1), polycomb protein complex 1 (PRC1) and methyl CpG binding protein 2 (MeCP2), at the COX‐2 promoter in lung fibroblasts from patients with IPF (F‐IPFs) compared with fibroblasts from nonfibrotic lungs. HP1, EZH2, and MeCP2 in turn were associated with additional repressive chromatin modifiers in F‐IPFs. G9a and EZH2 inhibitors and small interfering RNAs and the Dnmt1 inhibitor markedly reduced H3K9me3 (49–79%), H3K27me3 (44–81%), and DNA methylation (61–97%) at the COX‐2 promoter. These reductions were correlated with increased histone H3 and H4 acetylation, resulting in COX‐2 mRNA and protein reexpression in F‐IPFs. Our results support a central role for G9a‐ and EZH2‐mediated histone hypermethylation and a model of bidirectional, mutually reinforcing, and interdependent crosstalk between histone hypermethylation and DNA methylation in COX‐2 epigenetic silencing in IPF.—Coward, W. R., Feghali‐Bostwick, C. A., Jenkins, G., Knox, A. J., Pang, L. A central role for G9a and EZH2 in the epigenetic silencing of cyclooxygenase‐2 in idiopathic pulmonary fibrosis. FASEB J. 28, 3183–3196 (2014). www.fasebj.org