Search

Myofibroblasts are critical to processes involved in normal wound healing and during pathological fibrosis. They transdifferentiate from fibroblasts, and in doing so become contractile and capable of secreting large amounts of extracellular matrix proteins. Transforming growth factor-beta (TGFβ) is a key cytokine involved in wound healing and fibrogenesis. TGFβ signaling has long been the subject of experimental therapeutic approaches to inhibit fibrosis in a variety of organ systems. Inhibition of TGFβ can reduce myofibroblast transdifferentiation, contractility, and matrix production. Importantly, TGFβ is released from cells and sequestered in the extracellular matrix in a latent form that requires activation for biological function. There have been multiple mechanisms of TGFβ activation described in a variety of cell types and in cell free systems; however, myofibroblasts have previously been shown to activate TGFβ via cell surface integrins, particularly αvβ5 integrins. This chapter will provide detailed protocols for accurately measuring activation of TGFβ by myofibroblasts in vitro. Levels of active TGFβ usually represent a small proportion of the total amount of latent TGFβ present in the matrix. Methods to measure active TGFβ therefore need to be sensitive and specific to detect the active cytokine only.

Rationale: We recently identified a polymorphism associated with the AKAP13 gene resulted in higher levels of AKAP13 expression in the lung, particularly epithelium, and increased IPF susceptibility. AKAP13 is a Rho-GEF for RhoA, a key intermediate signal in the activation of the TGF-β activating integrin, αvβ6, in lung epithelial cells. However, the role for AKAP13 in the lung is still not understood. Aim: to investigate the role of AKAP13 in lung epithelial cells Method: AKAP13 gene expression was assessed via qPCR in primary human lung fibroblasts (normal, n=3; IPF, n=4) and primary epithelial cell lines (small airway SAEC, n=9; human bronchial HBEC, n=4). Immortalised human bronchial epithelial cells (iHBECs) were treated with AKAP13 siRNA to knockdown expression of AKAP13 and assessed for changes to mRNA after 48hrs. iHBECs were treated with 10μM of A13, an inhibitor for AKAP13-RhoA interaction, to assess for functional changes to RhoA activation in response to LPA. Results: AKAP13 gene expression was found to be 19-fold higher in epithelial cells, compared to fibroblasts, which had very low expression for AKAP13 (both normal and IPF), confirming our previous immunohistochemistry findings. Knockdown of the AKAP13 gene in iHBECS also resulted in a significant decrease in ITGB6 expression, the gene for αvβ6 (n=4, p=0.03). In addition, treatment of iHBECS with 10μM of A13 was able to supress RhoA activation in response to LPA. Conclusion: AKAP13 expression is found predominantly in epithelial cells in the lung. AKAP13 appears to regulate RhoA activation in iHBECs and influence αvβ6 expression. This suggests that it is involved in the RhoA-αvβ6 pathway that drives TGF-β activation in epithelial cells.

Idiopathic Pulmonary Fibrosis (IPF) is a progressive, fatal disease with limited treatment options. Protease mediated transforming growth factor-β (TGF-β) activation has been proposed as a pathogenic mechanism of lung fibrosis. Protease activity in the lung is tightly regulated by protease inhibitors, particularly secretory leukocyte protease inhibitor (SLPI). The bleomycin model of lung fibrosis was used to determine the effect of increased protease activity in the lungs of Slpi−/− mice following injury. Slpi−/−, and wild-type, mice received oropharyngeal administration of bleomycin (30 IU) and the development of pulmonary fibrosis was assessed. Pro and active forms of matrix metalloproteinase-2 (MMP-2) and MMP-9 were measured. Lung fibrosis was determined by collagen subtype specific gene expression, hydroxyproline concentration, and histological assessment. Alveolar TGF-β activation was measured using bronchoalveolar lavage cell pSmad2 levels and global TGF-β activity was assessed by pSmad2 immunohistochemistry. The active-MMP-9 to pro-MMP-9 ratio was significantly increased in Slpi−/− animals compared with wild-type animals, demonstrating enhanced metalloproteinase activity. Wild-type animals showed an increase in TGF-β activation following bleomycin, with a progressive and sustained increase in collagen type I, alpha 1 (Col1α1), III, alpha 1(Col3α1), IV, alpha 1(Col4α1) mRNA expression, and a significant increase in total lung collagen 28 days post-bleomycin. In contrast Slpi−/− mice showed no significant increase of alveolar TGF-β activity following bleomycin, above their already elevated levels, although global TGF-β activity did increase. Slpi−/− mice had impaired collagen gene expression but animals demonstrated minimal reduction in lung fibrosis compared with wild-type animals. These data suggest that enhanced proteolysis does not further enhance TGF-β activation, and inhibits sustained Col1α1, Col3α1 and Col4α1 gene expression following lung injury. However, these changes do not prevent the development of lung fibrosis. Overall, these data suggest that the absence of Slpi does not dramatically modify the development of lung fibrosis following bleomycin-induced lung injury.

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.

Heterotrimeric guanine nucleotide–binding protein (G protein) signaling is a ubiquitous signaling system that links hundreds of G protein–coupled receptors (GPCRs) with four G protein signaling pathways. Two of these pathways, one mediated by Gq and G11 and the other by G12 and G13, 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. Here, we investigated whether the Gq/G11 or G12/G13 pathways in epithelial cells are required to generate TGFβ and suppress alveolar inflammation. Mice deficient in both Gαq and Gα11 developed inflammation primarily due to alternatively activated (M2-polarized) macrophages, enhanced production of matrix metalloprotease 12 (MMP12), and age-related alveolar airspace enlargement consistent with emphysema. We found that mice with impaired Gq/G11 signaling had reduced stretch-mediated generation of TGFβ by epithelial cells and elevated macrophage MMP12 synthesis, but were protected from the effects of ventilator-induced lung injury. Furthermore, synthesis of the pleiotropic 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/G11 signaling maintains alveolar homeostasis and is likely to independently upregulate mechanotransduced epithelial TGFβ activation and downregulate epithelial IL-33 synthesis. Together, these findings suggest that disruption of Gq/G11 signaling promotes inflammatory emphysema, but protects against mechanostransduced lung injury.

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.

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.

Activation of latent transforming growth factor β (TGF-β) by αvβ6 integrin is critical in the pathogenesis of lung injury and fibrosis. We have previously demonstrated that the stimulation of protease activated receptor 1 promotes αvβ6 integrin-mediated TGF-β activation via RhoA, which is known to modulate cell contraction. However, whether other G protein-coupled receptors can also induce αvβ6 integrin-mediated TGF-β activation is unknown; in addition, the αvβ6 integrin signaling pathway has not yet been fully characterized. In this study, we show that lysophosphatidic acid (LPA) induces αvβ6-mediated TGF-β activation in human epithelial cells via both RhoA and Rho kinase. Furthermore, we demonstrate that LPA-induced αvβ6 integrin-mediated TGF-β activity is mediated via the LPA2 receptor, which signals via Gα q . Finally, we show that the expression levels of both the LPA2 receptor and αvβ6 integrin are up-regulated and are spatially and temporally associated following bleomycin-induced lung injury. Furthermore, both the LPA2 receptor and αvβ6 integrin are up-regulated in the overlying epithelial areas of fibrosis in patients with usual interstitial pneumonia. These studies demonstrate that LPA induces αvβ6 integrin-mediated TGF-β activation in epithelial cells via LPA2, Gα q , RhoA, and Rho kinase, and that this pathway might be clinically relevant to the development of lung injury and fibrosis.

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.

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.

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.