Joanne Porte

The COP9/signalosome complex is conserved in fission yeast and has a role in S phase

The COP9/signalosome complex is conserved from plant to mammalian cells. In Arabidopsis, it regulates the nuclear abundance of COP1, a transcriptional repressor of photomorphogenic development [1] [2]. All COP (constitutive photomorphogenesis) mutants inappropriately express genes that are normally repressed in the dark. Eight subunits (Sgn1-Sgn8) of the homologous mammalian complex have been purified [3] [4]. Several of these have been previously identified through genetic or protein interaction screens. No coherent model for COP9/signalosome function has yet emerged, but a relationship with cell-cycle progression by transcriptional regulation, protein localisation or protein stability is possible. Interestingly, the COP9/signalosome subunits possess domain homology to subunits of the proteasome regulatory lid complex [5] [6]. Database searches indicate that only Sgn5/JAB1 is present in Saccharomyces cerevisiae, precluding genetic analysis of the complex in cell-cycle regulation. Here we identify a subunit of the signalosome in the fission yeast Schizosaccharomyces pombe through an analysis of the DNA-integrity checkpoint. We provide evidence for the conservation of the COP9/signalosome complex in fission yeast and demonstrate that it functions during S-phase progression.

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.

Tryptase activates TGFβ in human airway smooth muscle cells via direct proteolysis

Transforming growth factor beta (TGFbeta) is a key remodelling factor in asthma. It is produced as a latent complex and the main limiting step in TGFbeta bioavailability is its activation. Mast cell tryptase has been shown to stimulate the release of functionally active TGFbeta from human airway smooth muscle (ASM) cells [P. Berger, P.O. Girodet, H. Begueret, O. Ousova, D.W. Perng, R. Marthan, A.F. Walls, J.M. Tunon de Lara, Tryptase-stimulated human airway smooth muscle cells induce cytokine synthesis and mast cell chemotaxis, FASEB J. 17 (2003) 2139-2141]. The aim of this study was to determine if tryptase could cause TGFbeta activation as well as expression in ASM cells via its receptor, proteinase-activated receptor 2 (PAR2). Tryptase caused TGFbeta activation without affecting levels of total TGFbeta. This effect was inhibited by the selective tryptase inhibitor FUT175 and leupeptin but not mimicked by the PAR2 activating peptide SLIGKV-NH(2). Furthermore, the ASM cells used in the study did not express PAR2. The results indicate that tryptase activates TGFbeta via a PAR2-independent proteolytic mechanism in human ASM cells and may help understanding the role of tryptase in asthma.

αvβ6 integrin may be a potential prognostic biomarker in interstitial lung disease

Idiopathic pulmonary fibrosis (IPF) and fibrotic nonspecific interstitial pneumonitis are progressive interstitial lung diseases (ILDs) with limited treatment options and poor survival. However, the rate of disease progression is variable, implying there may be different endotypes of disease. We hypothesised that immunophenotyping biopsies from ILD patients might reveal distinct endotypes of progressive fibrotic disease, which may facilitate stratification when undertaking clinical trials of novel therapies for IPF. 43 paraffin-embedded, formalin-fixed lung tissue sections were immunostained for five molecules implicated in the pathogenesis of the fibrosis: α-smooth muscle actin (αSMA), αvβ6 integrin, pro-surfactant protein C (SP-C), hepatocyte growth factor (HGF) and tenascin-C (TenC). Levels of immunostaining and numbers of fibroblastic foci were quantified using operator-dependent and -independent methods. The relationship of all these markers to overall survival was analysed. Staining revealed high levels of αSMA, αvβ6 integrin, pro-SP-C, HGF and TenC, and fibroblastic foci. Immunostaining varied across samples for all molecules but only the extent of αvβ6 integrin immunostaining was associated with increased mortality. There was no association with the other markers measured. Our data suggest high levels of αvβ6 integrin may identify a specific endotype of progressive fibrotic lung disease. High levels of αvβ6 integrin immunostaining on VATS biopsy predict worse survival than low levels of immunostaining http://ow.ly/IyCfH

Novel approach for interleukin-23 up-regulation in human dendritic cells and the impact on T helper type 17 generation

Interleukin‐23 (IL‐23) is important for T helper type 17 (Th17) responses and strategies to regulate IL‐23 in human dendritic cells (DC) are limited. This study describes a novel means to control IL‐23 secretion by conditioning DC with a phosphatidyl inositol 3‐kinase inhibitor Wortmannin (WM). Treatment of monocyte‐derived DC with WM increased Toll‐like receptor (TLR) ‐dependent IL‐23 secretion 10‐fold and IL‐12p70 twofold, but IL‐27 was unaffected. The effect of WM was restricted to TLR3/4 pathways, did not occur through TLR2, TLR7/8 or Dectin‐1, and was characterized by increased p19, p35 and p40 transcription. These responses were not solely dependent on phosphatidyl inositol 3‐kinase as the alternative inhibitor LY294002 did not modulate IL‐23 production. The normal patterns of activation of mitogen‐activated protein kinase pathways were unaffected by WM‐conditioning but IL‐23 secretion required p38, ERK and JNK pathways. Importantly, this effect was manifest in populations of blood DC. Conditioning freshly isolated myeloid DC with WM before TLR3 or TLR4 triggering resulted in high levels of IL‐23 secretion and an absence of IL‐12p70. These WM‐conditioned myeloid DC were highly effective at priming Th17 responses from naive CD4+ T cells. Our findings provide a novel means to generate IL‐23‐rich environments and Th17 responses and suggest as yet unidentified regulatory factors, identification of which will provide new approaches to control IL‐23‐dependent immunity in infectious disease, autoimmunity and malignancy.

Influenza Promotes Collagen Deposition via αvβ6-integrin Mediated Transforming Growth Factor β Activation

Background: The mechanism of influenza mediated TGFβ activation, and its role in pathogenesis is unclear. Results: H1N1 infection induced αvβ6-dependent TGFβ activity in iHBECs and increased epithelial cell death and collagen deposition in vivo. Conclusion: αvβ6 integrin-mediated TGFβ activation is involved in cell death and fibrogenesis following virus-induced epithelial injury. Significance: Viral infection may promote acute exacerbations of fibrotic lung disease. Influenza infection exacerbates chronic pulmonary diseases, including idiopathic pulmonary fibrosis. A central pathway in the pathogenesis of idiopathic pulmonary fibrosis is epithelial injury leading to activation of transforming growth factor β (TGFβ). The mechanism and functional consequences of influenza-induced activation of epithelial TGFβ are unclear. Influenza stimulates toll-like receptor 3 (TLR3), which can increase RhoA activity, a key event prior to activation of TGFβ by the αvβ6 integrin. We hypothesized that influenza would stimulate TLR3 leading to activation of latent TGFβ via αvβ6 integrin in epithelial cells. Using H1152 (IC50 6.1 μm) to inhibit Rho kinase and 6.3G9 to inhibit αvβ6 integrins, we demonstrate their involvement in influenza (A/PR/8/34 H1N1) and poly(I:C)-induced TGFβ activation. We confirm the involvement of TLR3 in this process using chloroquine (IC50 11.9 μm) and a dominant negative TLR3 construct (pZERO-hTLR3). Examination of lungs from influenza-infected mice revealed augmented levels of collagen deposition, phosphorylated Smad2/3, αvβ6 integrin, and apoptotic cells. Finally, we demonstrate that αvβ6 integrin-mediated TGFβ activity following influenza infection promotes epithelial cell death in vitro and enhanced collagen deposition in vivo and that this response is diminished in Smad3 knock-out mice. These data show that H1N1 and poly(I:C) can induce αvβ6 integrin-dependent TGFβ activity in epithelial cells via stimulation of TLR3 and suggest a novel mechanism by which influenza infection may promote collagen deposition in fibrotic lung disease.

Genetic variants associated with susceptibility to idiopathic pulmonary fibrosis in people of European ancestry: a genome-wide association study

Summary Background Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease with high mortality, uncertain cause, and few treatment options. Studies have identified a significant genetic risk associated with the development of IPF; however, mechanisms by which genetic risk factors promote IPF remain unclear. We aimed to identify genetic variants associated with IPF susceptibility and provide mechanistic insight using gene and protein expression analyses. Methods We used a two-stage approach: a genome-wide association study in patients with IPF of European ancestry recruited from nine different centres in the UK and controls selected from UK Biobank (stage 1) matched for age, sex, and smoking status; and a follow-up of associated genetic variants in independent datasets of patients with IPF and controls from two independent US samples from the Chicago consortium and the Colorado consortium (stage 2). We investigated the effect of novel signals on gene expression in large transcriptomic and genomic data resources, and examined expression using lung tissue samples from patients with IPF and controls. Findings 602 patients with IPF and 3366 controls were selected for stage 1. For stage 2, 2158 patients with IPF and 5195 controls were selected. We identified a novel genome-wide significant signal of association with IPF susceptibility near A-kinase anchoring protein 13 (AKAP13; rs62025270, odds ratio [OR] 1·27 [95% CI 1·18–1·37], p=1·32 × 10−9) and confirmed previously reported signals, including in mucin 5B (MUC5B; rs35705950, OR 2·89 [2·56–3·26], p=1·12 × 10−66) and desmoplakin (DSP; rs2076295, OR 1·44 [1·35–1·54], p=7·81 × 10−28). For rs62025270, the allele A associated with increased susceptibility to IPF was also associated with increased expression of AKAP13 mRNA in lung tissue from patients who had lung resection procedures (n=1111). We showed that AKAP13 is expressed in the alveolar epithelium and lymphoid follicles from patients with IPF, and AKAP13 mRNA expression was 1·42-times higher in lung tissue from patients with IPF (n=46) than that in lung tissue from controls (n=51). Interpretation AKAP13 is a Rho guanine nucleotide exchange factor regulating activation of RhoA, which is known to be involved in profibrotic signalling pathways. The identification of AKAP13 as a susceptibility gene for IPF increases the prospect of successfully targeting RhoA pathway inhibitors in patients with IPF. Funding UK Medical Research Council, National Heart, Lung, and Blood Institute of the US National Institutes of Health, Agencia Canaria de Investigación, Innovación y Sociedad de la Información, Spain, UK National Institute for Health Research, and the British Lung Foundation.

Assessment of the effect of potential antifibrotic compounds on total and αVβ6 integrin-mediated TGF-β activation.

Transforming growth factor‐β (TGF‐β) plays an important role in the development of tissue fibrosis, and molecules inhibiting this pathway are attractive therapeutic targets for fibrotic diseases such as idiopathic pulmonary fibrosis (IPF). Activation of TGF‐β is the rate‐limiting step in TGF‐β bioavailability, and activation by the αVβ6 integrin is important in fibrosis of the lung, liver, and kidney. Activation of TGF‐β by αVβ6 requires direct cell–cell contact and measurable release of active TGF‐β in extracellular fluid compartments does not reflect tissue specific activation. The aim of this study was to determine the effect of antifibrotic compounds on both total, and specific αVβ6 integrin‐mediated TGF‐β activity. Using a transformed mink lung cell (TMLC) TGF‐β reporter, the effects of potential antifibrotic therapies including an activin‐like kinase (Alk5) inhibitor, Dexamethasone, Pirfenidone, N‐acetylcysteine (NAC), and BIBF1120 were assessed. Effects due to αVβ6 integrin‐mediated TGF‐β activity were measured using reporter cells cocultured with cells expressing αVβ6 integrins. These high‐throughput studies were validated using a phosphorylated Smad2 Enzyme‐Linked Immunosorbent Assay. Alk5 inhibitors are potent inhibitors of TGF‐β activity, whereas the novel antifibrotics, Pirfenidone, BIBF1120, and NAC are only moderate inhibitors, and Dexamethasone does not specifically affect TGF‐βactivity, but inhibits TGF‐β‐induced gene expression. None of the current small molecular inhibitors inhibit αVβ6‐mediated TGF‐β activity. These results demonstrate the potential of this high‐throughput assay of αVβ6‐specific TGF‐β activity and illustrate that currently available antifibrotics have limited effects on αVβ6 integrin‐mediated TGF‐β activity.

Loss of epithelial Gq and G11 signaling inhibits TGFβ production but promotes IL-33–mediated macrophage polarization and emphysema

Signaling by Gq/11 is required for optimal TGFβ activation in the lung to prevent inflammation. Gq/11 signaling maintains healthy lungs Loss of signaling by the cytokine transforming growth factor–β (TGFβ) in mice results in emphysema-like symptoms, whereas excessive TGFβ signaling results in pulmonary fibrosis and ventilator-associated lung injury. G proteins of the Gq/11 and G12/13 families mediate the integrin-dependent activation and release of latent TGFβ from the epithelial cells. John et al. found that mice deficient in Gq/11, but not those deficient in G12/13, in lung epithelial cells had defective TGFβ activation and emphysema-like symptoms. In addition, the Gq/11-deficient mice had lung inflammation associated with increased amounts of the cytokine IL-33. However, the mice were protected from ventilator-induced injury. Together, these data suggest that Gq/11 signaling is required for optimal TGFβ activation in the lung and the prevention of inflammation. Heterotrimeric guanine nucleotide–binding protein (G protein) signaling links hundreds of G protein–coupled receptors with four G protein signaling pathways. Two of these, one mediated by Gq and G11 (Gq/11) and the other by G12 and G13 (G12/13), are implicated in the force-dependent activation of transforming growth factor–β (TGFβ) in lung epithelial cells. Reduced TGFβ activation in alveolar cells leads to emphysema, whereas enhanced TGFβ activation promotes acute lung injury and idiopathic pulmonary fibrosis. Therefore, precise control of alveolar TGFβ activation is essential for alveolar homeostasis. We investigated the involvement of the Gq/11 and G12/13 pathways in epithelial cells in generating active TGFβ and regulating alveolar inflammation. Mice deficient in both Gαq and Gα11 developed inflammation that was primarily caused by alternatively activated (M2-polarized) macrophages, enhanced matrix metalloproteinase 12 (MMP12) production, and age-related alveolar airspace enlargement consistent with emphysema. Mice with impaired Gq/11 signaling had reduced stretch-mediated generation of TGFβ by epithelial cells and enhanced macrophage MMP12 synthesis but were protected from the effects of ventilator-induced lung injury. Furthermore, synthesis of the cytokine interleukin-33 (IL-33) was increased in these alveolar epithelial cells, resulting in the M2-type polarization of alveolar macrophages independently of the effect on TGFβ. Our results suggest that alveolar Gq/11 signaling maintains alveolar homeostasis and likely independently increases TGFβ activation in response to the mechanical stress of the epithelium and decreases epithelial IL-33 synthesis. Together, these findings suggest that disruption of Gq/11 signaling promotes inflammatory emphysema but protects against mechanically induced lung injury.

Amplification of TGFβ Induced ITGB6 Gene Transcription May Promote Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating, progressive disease with poor survival rates and limited treatment options. Upregulation of αvβ6 integrins within the alveolar epithelial cells is a characteristic feature of IPF and correlates with poor patient survival. The pro-fibrotic cytokine TGFβ1 can upregulate αvβ6 integrin expression but the molecular mechanisms driving this effect have not previously been elucidated. We confirm that stimulation with exogenous TGFβ1 increases expression of the integrin β6 subunit gene (ITGB6) and αvβ6 integrin cell surface expression in a time- and concentration-dependent manner. TGFβ1-induced ITGB6 expression occurs via transcriptional activation of the ITGB6 gene, but does not result from effects on ITGB6 mRNA stability. Basal expression of ITGB6 in, and αvβ6 integrins on, lung epithelial cells occurs via homeostatic αvβ6-mediated TGFβ1 activation in the absence of exogenous stimulation, and can be amplified by TGFβ1 activation. Fundamentally, we show for the first time that TGFβ1-induced ITGB6 expression occurs via canonical Smad signalling since dominant negative constructs directed against Smad3 and 4 inhibit ITGB6 transcriptional activity. Furthermore, disruption of a Smad binding site at -798 in the ITGB6 promoter abolishes TGFβ1-induced ITGB6 transcriptional activity. Using chromatin immunoprecipitation we demonstrate that TGFβ1 stimulation of lung epithelial cells results in direct binding of Smad3, and Smad4, to the ITGB6 gene promoter within this region. Finally, using an adenoviral TGFβ1 over-expression model of pulmonary fibrosis we demonstrate that Smad3 is crucial for TGFβ1-induced αvβ6 integrin expression within the alveolar epithelium in vivo. Together, these data confirm that a homeostatic, autocrine loop of αvβ6 integrin activated TGFβ1-induced ITGB6 gene expression regulates epithelial basal αvβ6 integrin expression, and demonstrates that this occurs via Smad-dependent transcriptional regulation at a single Smad binding site in the promoter of the β6 subunit gene. Active TGFβ1 amplifies this pathway both in vitro and in vivo, which may promote fibrosis.