Dysregulated bidirectional epithelial-mesenchymal crosstalk: a core determinant of lung fibrosis progression.

Progressive lung fibrosis is characterised by dysregulated extracellular matrix (ECM) homeostasis. Understanding of disease pathogenesis remains limited and has prevented the development of effective treatments. While an abnormal wound healing response is strongly implicated in lung fibrosis initiation, factors that determine why fibrosis progresses rather than regular tissue repair occurs are not fully explained. Within human lung fibrosis there is evidence of altered epithelial and mesenchymal lung populations as well as cells undergoing epithelial-mesenchymal transition (EMT), a dynamic and reversible biological process by which epithelial cells lose their cell polarity and down-regulate cadherin-mediated cell-cell adhesion to gain migratory properties. This review will focus upon the role of EMT and dysregulated epithelial-mesenchymal crosstalk in progressive lung fibrosis.

LKB1 depletion-mediated epithelial-mesenchymal transition induces fibroblast activation in lung fibrosis.

The factors that determine fibrosis progression or normal tissue repair are largely unknown. We previously demonstrated that autophagy inhibition-mediated epithelial-mesenchymal transition (EMT) in human alveolar epithelial type II (ATII) cells augments local myofibroblast differentiation in pulmonary fibrosis by paracrine signalling. Here, we report that liver kinase B1 (LKB1) inactivation in ATII cells inhibits autophagy and induces EMT as a consequence. In IPF lungs, this is caused by downregulation of CAB39L, a key subunit within the LKB1 complex. 3D co-cultures of ATII cells and MRC5 lung fibroblasts coupled with RNA sequencing (RNA-seq) confirmed that paracrine signalling between LKB1-depleted ATII cells and fibroblasts augmented myofibroblast differentiation. Together these data suggest that reduced autophagy caused by LKB1 inhibition can induce EMT in ATII cells and contribute to fibrosis via aberrant epithelial-fibroblast crosstalk.

Pseudohypoxic HIF pathway activation dysregulates collagen structure-function in human lung fibrosis.

Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we provide evidence that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of the oxygen status (pseudohypoxia). Whilst TGFß increased the rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting pyridinoline cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knockdown of Factor Inhibiting HIF (FIH), which modulates HIF activity, or oxidative stress caused pseudohypoxic HIF activation in the normal fibroblasts. By contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF-mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of human lung fibrosis mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.

Bidirectional epithelial-mesenchymal crosstalk provides self-sustaining profibrotic signals in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the prototypic progressive fibrotic lung disease with a median survival of 2 to 4 years. Injury to and/or dysfunction of the alveolar epithelium is strongly implicated in IPF disease initiation, but the factors that determine whether fibrosis progresses rather than normal tissue repair occurs remain poorly understood. We previously demonstrated that zinc finger E-box-binding homeobox 1-mediated epithelial-mesenchymal transition in human alveolar epithelial type II (ATII) cells augments transforming growth factor-ß-induced profibrogenic responses in underlying lung fibroblasts via paracrine signaling. Here, we investigated bidirectional epithelial-mesenchymal crosstalk and its potential to drive fibrosis progression. RNA-Seq of lung fibroblasts exposed to conditioned media from ATII cells undergoing RAS-induced epithelial-mesenchymal transition identified many differentially expressed genes including those involved in cell migration and extracellular matrix regulation. We confirmed that paracrine signaling between RAS-activated ATII cells and fibroblasts augmented fibroblast recruitment and demonstrated that this involved a zinc finger E-box-binding homeobox 1-tissue plasminogen activator axis. In a reciprocal fashion, paracrine signaling from transforming growth factor-ß-activated lung fibroblasts or IPF fibroblasts induced RAS activation in ATII cells, at least partially through the secreted protein acidic and rich in cysteine, which may signal via the epithelial growth factor receptor via epithelial growth factor-like repeats. Together, these data identify that aberrant bidirectional epithelial-mesenchymal crosstalk in IPF drives a chronic feedback loop that maintains a wound-healing phenotype and provides self-sustaining profibrotic signals.

GRK6 Depletion Induces HIF Activity in Lung Adenocarcinoma.

G protein-coupled receptor kinase 6 (GRK6) is expressed in various tissues and is involved in the development of several diseases including lung cancer. We previously reported that GRK6 is down-regulated in lung adenocarcinoma patients, which induces cell invasion and metastasis. However, further understanding of the role of GRK6 in lung adenocarcinoma is required. Here we explored the functional consequence of GRK6 inhibition in lung epithelial cells. Analysis of TCGA data was coupled with RNA sequencing (RNA-seq) in alveolar epithelial type II (ATII) cells following depletion of GRK6 with RNA interference (RNAi). Findings were validated in ATII cells followed by tissue microarray analysis. Pathway analysis suggested that one of the Hallmark pathways enriched upon GRK6 inhibition is 'Hallmark_Hypoxia' (FDR = 0.014). We demonstrated that GRK6 depletion induces HIF1α (hypoxia-inducible factor 1 alpha) levels and activity in ATII cells. The findings were further confirmed in lung adenocarcinoma samples, in which GRK6 expression levels negatively and positively correlate with HIF1α expression (P = 0.015) and VHL expression (P < 0.0001), respectively. Mechanistically, we showed the impact of GRK6 on HIF activity could be achieved via regulation of VHL levels. Taken together, targeting the HIF pathway may provide new strategies for therapy in GRK6-depleted lung adenocarcinoma patients.

Quantitative Proteomic Analysis in Alveolar Type II Cells Reveals the Different Capacities of RAS and TGF-ß to Induce Epithelial-Mesenchymal Transition.

Alveolar type II (ATII) epithelial cells function as stem cells, contributing to alveolar renewal, repair and cancer. Therefore, they are a highly relevant model for studying a number of lung diseases, including acute injury, fibrosis and cancer, in which signals transduced by RAS and transforming growth factor (TGF)-ß play critical roles. To identify downstream molecular events following RAS and/or TGF-ß activation, we performed proteomic analysis using a quantitative label-free approach (LC-HDMSE) to provide in-depth proteome coverage and estimates of protein concentration in absolute amounts. Data are available via ProteomeXchange with identifier PXD023720. We chose ATIIER:KRASV12 as an experimental cell line in which RAS is activated by adding 4-hydroxytamoxifen (4-OHT). Proteomic analysis of ATII cells treated with 4-OHT or TGF-ß demonstrated that RAS activation induces an epithelial-mesenchymal transition (EMT) signature. In contrast, under the same conditions, activation of TGF-ß signaling alone only induces a partial EMT. EMT is a dynamic and reversible biological process by which epithelial cells lose their cell polarity and down-regulate cadherin-mediated cell-cell adhesion to gain migratory properties, and is involved in embryonic development, wound healing, fibrosis and cancer metastasis. Thus, these results could help to focus research on the identification of processes that are potentially driving EMT-related human disease.

Autophagy inhibition-mediated epithelial-mesenchymal transition augments local myofibroblast differentiation in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF), the prototypic progressive fibrotic interstitial lung disease, is thought to be a consequence of repetitive micro-injuries to an ageing, susceptible alveolar epithelium. Ageing is a risk factor for IPF and incidence has been demonstrated to increase with age. Decreased (macro)autophagy with age has been reported extensively in a variety of systems and diseases, including IPF. However, it is undetermined whether the role of faulty autophagy is causal or coincidental in the context of IPF. Here, we report that in alveolar epithelial cells inhibition of autophagy promotes epithelial-mesenchymal transition (EMT), a process implicated in embryonic development, wound healing, cancer metastasis and fibrosis. We further demonstrate that this is attained, at least in part, by increased p62/SQSTM1 expression that promotes p65/RELA mediated-transactivation of an EMT transcription factor, Snail2 (SNAI2), which not only controls EMT but also regulates the production of locally acting profibrogenic mediators. Our data suggest that reduced autophagy induces EMT of alveolar epithelial cells and can contribute to fibrosis via aberrant epithelial-fibroblast crosstalk.

Paracrine signalling during ZEB1-mediated epithelial-mesenchymal transition augments local myofibroblast differentiation in lung fibrosis.

The contribution of epithelial-mesenchymal transition (EMT) to human lung fibrogenesis is controversial. Here we provide evidence that ZEB1-mediated EMT in human alveolar epithelial type II (ATII) cells contributes to the development of lung fibrosis by paracrine signalling to underlying fibroblasts. Activation of EGFR-RAS-ERK signalling in ATII cells induced EMT via ZEB1. ATII cells had extremely low extracellular matrix gene expression even after induction of EMT, however conditioned media from ATII cells undergoing RAS-induced EMT augmented TGFß-induced profibrogenic responses in lung fibroblasts. This epithelial-mesenchymal crosstalk was controlled by ZEB1 via the expression of tissue plasminogen activator (tPA). In human fibrotic lung tissue, nuclear ZEB1 expression was detected in alveolar epithelium adjacent to sites of extracellular matrix (ECM) deposition, suggesting that ZEB1-mediated paracrine signalling has the potential to contribute to early fibrotic changes in the lung interstitium. Targeting this novel ZEB1 regulatory axis may be a viable strategy for the treatment of pulmonary fibrosis.

Structural Induction Effect of a Zwitterion Pyridiniumolate for Metal-Organic Frameworks.

It is still an enormous challenge to obtain the metal-organic frameworks (MOFs) with specific properties by tuning their structures. Here we first reported that the structures of MOFs could be tuned by adding certain amount of zwitterion pyridiniumolate. To demonstrate the inductive effect, two series of assembly experiments were performed using different metal ions, namely, Cd(II) and Mn(II). The experimental results revealed that the zwitterion pyridiniumloate only acted as a structural induction agent (SIA), which did not exist in the aimed compounds. The SIAs could effectively tune the framework aperture or promote coordination and further tune the properties of MOFs without any removal or exchange after the synthesis. Therefore, the results could not only immensely expand the syntheses and structural diversity of MOFs with the fixed metal ions and organic ligands but also afford the possibility and effective convenience for tuning the properties of MOFs in the functional material research fields.

Poly[tetrachlorido{µ4-tetrakis[(imidazol-1-yl)methyl]methane-κ4N3:N3':N3'':N3'''}dizinc(II)] exhibiting a three-dimensional diamond topology.

The title novel noncentrosymmetric metal-organic framework, [Zn2Cl4(C17H20N8)]n, was prepared solvothermally using the tetradentate tetrakis[(imidazol-1-yl)methyl]methane (tiym) linker in the presence of zinc nitrate under acidic conditions. The asymmetric unit contains one Zn(II) cation, two Cl(-) anions and a quarter of each of two symmetry-independent tiym ligands. Each Zn(II) cation is four-coordinated by two Cl(-) anions and two imidazole N atoms from two tiym ligands, forming a distorted tetrahedral coordination geometry. The tetrahedral tetradentate tiym linker has a quaternary C atom located on a crystallographic -4 axis. With its four peripheral imidazole N atoms, the linkers are bridged by four [ZnCl2] subunits to generate a three-dimensional diamond topological framework, which is represented by the Schläfli symbol {6(6)}. To the best of our knowledge, the title compound is the first example of a non-interpenetrating diamond net based on the tiym ligand.