Identification of FGFR4 as a regulator of myofibroblast differentiation in Pulmonary Fibrosis.

Introduction IPF is a devastating lung disease with limited therapeutic options. FGFR4 is a known receptor for several paracrine Fibroblast growth factors (FGFs). FGFR4 is also the main receptor for FGF19, an endocrine FGF that was demonstrated by our group to have anti-fibrotic properties in the lung. We aimed to determine whether FGFR4 could modulate pulmonary fibrogenesis. Methods We assessed FGFR4 mRNA and protein levels in IPF and control lungs. In vitro, we determined the effect of TGF-b, Endothelin-1 and PDGF on FGFR4 expression in human lung fibroblasts. We determined the effect FGFR4 inhibition, using a specific pharmacological inhibitor (FGF401), or genetic deletion in murine embryonic fibroblasts (MEFs) on TGF-b-induced myofibroblastic differentiation. In vivo, we evaluated the development of bleomycin-induced lung fibrosis in Fgfr4-deficient (Fgfr4-/-) mice compared to Wild Type littermates (WT), and after FGF401 treatment in WT mice compared to a control group receiving the solvent only. Results FGFR4 was decreased in IPF lungs as compared to control lungs, at mRNA and protein levels. In vitro, FGFR4 was downregulated after treatment by TGF- ß, Endothelin-1 and PDGF. In vitro, FGFR4 inhibition by FGF401 prevented TGF-b1-induced collagen and ACTA2 increase in lung fibroblasts. Similar results were observed in Fgfr4-/- MEFs. In vivo, FGFR4 genetic deficiency or FGFR4 pharmacological inhibition did not modulate bleomycin-induced pulmonary fibrosis. Conclusion Our data suggest that FGFR4 exerts pro-fibrotic properties by enhancing TGF- ß signaling in vitro. However, the inhibition of FGFR4 is not sufficient to prevent the development of pulmonary fibrosis in vivo.

Anti-fibrotic effects of nintedanib on lung fibroblasts derived from patients with Progressive Fibrosing Interstitial Lung Diseases (PF-ILDs).

The tyrosine kinase inhibitor nintedanib has been recently approved for the treatment of Interstitial Lung Diseases (ILDs) that manifest a progressive fibrosis phenotype other than Idiopathic pulmonary Fibrosis (IPF). Nintedanib reduces the development of lung fibrosis in various animal models resembling features of PF-ILD and in vitro, it inhibits the fibrosing phenotype of human lung fibroblasts (HLFs) isolated from patients with IPF. To get insight on the cellular and molecular mechanisms that drive the clinical efficiency of nintedanib in patients with non-IPF PF-ILD, we investigated its effects on the fibrosing functions of HLFs derived from patients with PF-hypersensitivity pneumonitis (PF-HP, n = 7), PF-sarcoidosis (n = 5) and pleuroparenchymal fibroelastosis (PPFE, n = 4). HLFs were treated with nintedanib (10 nM-1 µM) and then stimulated with PDGF-BB (25-50 ng/ml) or TGF-ß1 (1 ng/ml) for 24-72 h to assess proliferation and migration or differentiation. At nanomolar concentrations, nintedanib reduced the levels of PDGF receptor and ERK1/2 phosphorylation, the proliferation and the migration of PF-HP, PF-sarcoidosis and PPFE HLFs stimulated with PDGF-BB. Moreover, nintedanib also attenuates the myofibroblastic differentiation driven by TGF-ß1 but only when it is used at 1 µM. The drug reduced the phosphorylation of SMAD2/3 and decreased the induction of collagen, fibronectin and α-smooth muscle actin expression induced by TGF-ß1. In conclusion, our results demonstrate that nintedanib counteracts fundamental fibrosing functions of lung fibroblasts derived from patients with PF-HP, PF-sarcoidosis and PPFE, at concentrations previously reported to inhibit control and IPF HLFs. Such effects may contribute to its clinical benefit in patients suffering from these irreversible ILDs.

FGF19 Is Downregulated in Idiopathic Pulmonary Fibrosis and Inhibits Lung Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with limited therapeutic possibilities. FGF19 (fibroblast growth factor 19), an endocrine FGF, was recently shown to decrease liver fibrosis. To ask whether FGF19 had antifibrotic properties in the lung and decipher its effects on common features associated with lung fibrogenesis, we assessed, by ELISA, FGF19 concentrations in plasma and BAL fluids obtained from control subjects and patients with IPF. In vivo, using an intravenously administered adeno11-associated virus, we overexpressed FGF19 at the fibrotic phase of two experimental models of murine lung fibrosis and assessed its effect on lung morphology, lung collagen content, fibrosis markers, and profibrotic mediator expression at mRNA and protein levels. In vitro, we investigated whether FGF19 could modulate the TGF-ß-induced differentiation of primary human lung fibroblasts into myofibroblasts and the apoptosis of murine alveolar type II cells. Although FGF19 was not detected in BAL fluid, FGF19 concentration was decreased in the plasma of patients with IPF compared with control subjects. In vivo, the overexpression of FGF19 was associated with a marked decrease of lung fibrosis and fibrosis markers, with a decrease of profibrotic mediator expression and lung collagen content. In vitro, FGF19 decreased alveolar type 2 epithelial cell apoptosis through the decrease of the proapoptotic BIM protein expression and prevented TGF-ß-induced myofibroblast differentiation through the inhibition of JNK phosphorylation. Altogether, these data identify FGF19 as an antifibrotic molecule with potential therapeutic interest in fibrotic lung disorders.

Arsenic trioxide inhibits the functions of lung fibroblasts derived from patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal interstitial lung disease. Currently, no treatment can block or reverse the development of lung fibrosis in patients suffering from IPF. Recent studies indicate that arsenic trioxide (ATO), a safe, effective anti-cancer pro-oxidant drug, prevents the differentiation of normal human lung fibroblasts (NHLFs) in vitro and reduces experimental pulmonary fibrosis in vivo. In this context, we investigated the anti-fibrotic effects of ATO on the main fibrosis functions of human lung fibroblasts (HLFs) isolated from patients with IPF. IPF and non-IPF (control) HLFs were incubated with 0.01-1 µM ATO and stimulated with pro-fibrotic factors (PDGF-BB or TGF-ß1). We measured their rates of proliferation, migration and differentiation and the cell stress response triggered by ATO. ATO did not affect cell viability but strongly inhibited the proliferation and migration of PDGF-BB-stimulated IPF and control HLFs. ATO also prevented myofibroblastic differentiation, as assessed by the expression of α-smooth muscle actin (α-SMA) and collagen-1, and the phosphorylation of SMAD2/3 in TGF-ß1-stimulated HLFs. These antifibrotic effects were associated with increased expression of the transcription factor NRF2 and its target genes NQO1 and HMOX1. Genetic silencing of NRF2 inhibited the ATO-induced cell stress response but did not prevent the ATO-dependent inhibition of α-SMA expression in TGF-ß1-stimulated HLFs. The results demonstrate that ATO, at concentrations similar to exposure in blood plasma of ATO-treated cancer patients, counteracted pro-fibrotic activities of HLFs from IPF patients. We propose to consider ATO for clinical exploration to define the therapeutic potential in patients with IPF.

TNF-α and IL-10 Control CXCL13 Expression in Human Macrophages.

The chemokine CXCL13 controls the normal organization of secondary lymphoid tissues and the neogenesis of ectopic lymphoid structures in nonlymphoid organs, particularly the lungs. The progression and severity of idiopathic pulmonary fibrosis (IPF), a fatal and irreversible interstitial lung disease, is predicted by the circulating blood concentrations of CXCL13. Although CXCL13 is produced by pulmonary tissues, it has not been determined which cells are involved. This study examines CXCL13 production by lung tissue macrophages from patients with IPF and the signaling pathways controlling CXCL13 gene expression in human alveolar macrophages (AM) and monocyte-derived macrophages (MoDM). CXCL13 is found in CD68- and CD206-positive AM from patients with IPF, and the CXCL13 gene is induced in these macrophages and MoDM when they are stimulated with LPS. We found that TNF-α and IL-10 control optimal CXCL13 gene expression in MoDM and possibly in AM by activating the NF-κB and JAK/STAT pathways, respectively. We also found that blood TNF-α and CXCL13 concentrations are significantly correlated in patients with IPF, suggesting that TNF-α contributes to CXCL13 production in humans. In conclusion, the results of this study demonstrate that AM from patients with IPF produces CXCL13 and that the NF-κB and JAK/STAT pathways are required to induce the expression of this major chemokine.

FGF9 prevents pleural fibrosis induced by intrapleural adenovirus injection in mice.

Fibroblast growth factor 9 (FGF9) is necessary for fetal lung development and is expressed by epithelium and mesothelium. We evaluated the role of FGF9 overexpression on adenoviral-induced pleural injury in vivo and determined the biological effects of FGF9 on mesothelial cells in vitro. We assessed the expression of FGF9 and FGF receptors by mesothelial cells in both human and mouse lungs. Intrapleural injection of an adenovirus expressing human FGF9 (AdFGF9) or a control adenovirus (AdCont) was performed. Mice were euthanized at days 3, 5, and 14 Expression of FGF9 and markers of inflammation and myofibroblastic differentiation was studied by qPCR and immunohistochemistry. In vitro, rat mesothelial cells were stimulated with FGF9 (20 ng/ml), and we assessed its effect on proliferation, survival, migration, and differentiation. FGF9 was expressed by mesothelial cells in human idiopathic pulmonary fibrosis. FGF receptors, mainly FGFR3, were expressed by mesothelial cells in vivo in humans and mice. AdCont instillation induced diffuse pleural thickening appearing at day 5, maximal at day 14 The altered pleura cells strongly expressed α-smooth muscle actin and collagen. AdFGF9 injection induced maximal FGF9 expression at day 5 that lasted until day 14 FGF9 overexpression prevented pleural thickening, collagen and fibronectin accumulation, and myofibroblastic differentiation of mesothelial cells. In vitro, FGF9 decreased mesothelial cell migration and inhibited the differentiating effect of transforming growth factor-ß1. We conclude that FGF9 has a potential antifibrotic effect on mesothelial cells.

FGF9 and FGF18 in idiopathic pulmonary fibrosis promote survival and migration and inhibit myofibroblast differentiation of human lung fibroblasts in vitro.

Idiopathic pulmonary fibrosis (IPF) is characterized by an accumulation of extracellular matrix proteins and fibroblasts in the distal airways. Key developmental lung signaling pathways are reactivated in IPF. For instance, fibroblast growth factor 9 (FGF9) and FGF18, involved in epithelial-mesenchymal interactions, are critical for lung development. We evaluated the expression of FGF9, FGF18, and FGF receptors (FGFRs) in lung tissue from controls and IPF patients and assessed their effect on proliferation, survival, migration, and differentiation of control and IPF human lung fibroblasts (HLFs). FGF9, FGF18, and all FGFRs were present in the remodeled alveolar epithelium close to the fibroblast foci in IPF lungs. FGFR3 was generally detected in fibroblast foci by immunohistochemistry. In vitro, HLFs mainly expressed mesenchyme-associated FGFR isoforms (FGFR1c and FGFR3c) and FGFR4. FGF9 did not affect fibroblast proliferation, whereas FGF18 inhibited cell growth in control fibroblasts. FGF9 and FGF18 decreased Fas-ligand-induced apoptosis in control but not in IPF fibroblasts. FGF9 prevented transforming growth factor ß1-induced myofibroblast differentiation. FGF9 and FGF18 increased the migratory capacities of HLF, and FGF9 actively modulated matrix metalloproteinase activity. In addition, FGFR3 inhibition by small interfering RNA impacted p-ERK activation by FGF9 and FGF18 and their effects on differentiation and migration. These results identify FGF9 as an antiapoptotic and promigratory growth factor on HLF, maintaining fibroblasts in an undifferentiated state. The biological effects of FGF9 and FGF18 were partially driven by FGFR3. FGF18 was a less potent molecule. Both growth factors likely contribute to the fibrotic process in vivo.

The small heat-shock protein αB-crystallin is essential for the nuclear localization of Smad4: impact on pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by the proliferation of myofibroblasts and the accumulation of extracellular matrix (ECM) in the lungs. TGF-ß1 is the major profibrotic cytokine involved in IPF and is responsible for myofibroblast proliferation and differentiation and ECM synthesis. αB-crystallin is constitutively expressed in the lungs and is inducible by stress, acts as a chaperone and is known to play a role in cell cytoskeleton architecture homeostasis. The role of αB-crystallin in fibrogenesis remains unknown. The principal signalling pathway involved in this process is the Smad-dependent pathway. We demonstrate here that αB-crystallin is strongly expressed in fibrotic lung tissue from IPF patients and in vivo rodent models of pulmonary fibrosis. We also show that αB-crystallin-deficient mice are protected from bleomycin-induced fibrosis. Similar protection from fibrosis was observed in αB-crystallin KO mice after transient adenoviral-mediated over-expression of IL-1ß or TGF-ß1. We show in vitro in primary epithelial cells and fibroblasts that αB-crystallin increases the nuclear localization of Smad4, thereby enhancing the TGF-ß1-Smad pathway and the consequent activation of TGF-ß1 downstream genes. αB-crystallin over-expression disrupts Smad4 mono-ubiquitination by interacting with its E3-ubiquitin ligase, TIF1γ, thus limiting its nuclear export. Conversely, in the absence of αB-crystallin, TIF1γ can freely interact with Smad4. Consequently, Smad4 mono-ubiquitination and nuclear export are favoured and thus TGF-ß1-Smad4 pro-fibrotic activity is inhibited. This study demonstrates that αB-crystallin may be a key target for the development of specific drugs in the treatment of IPF or other fibrotic diseases.

Antifibrotic effects of vitamin D3 on human lung fibroblasts derived from patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. Up to now, no treatment can stop the progression of IPF. Vitamin D3 (VD) reduces experimental lung fibrosis in murine models and depletion of vitamin D3 might be associated with the reduced survival of patients with IPF. In this context, we determined if VD can prevent the pro-fibrotic functions of human lung fibroblasts (HLFs) isolated from patients with IPF. IPF and control HLFs were derived from surgical lung biopsies collected from patients with IPF or with primary lung cancer, respectively. VD (3-100 nM) markedly reduced the basal and PDGF-induced proliferation of HLFs. VD also altered cell cycle by increasing the percentage of IPF HLFs arrested in the G0/G1 phase, and by downregulating the expression of various cell cycle regulatory proteins. In addition, VD barely prevented the TGF-ß1-induced differentiation in HLFs. At 100 nM, VD slightly reduced the expression of the pro-fibrotic marker α-smooth muscle actin, and had no effect on fibronectin and collagen-1 expression. In contrast, 100 nM VD strongly inhibited the aerobic glycolytic metabolism induced by TGF- ß1. Finally, VD reduced both the secretion of lactate, the levels of lactate deshydrogenase mRNA and the activity of intracellular LDH in IPF HLFs. In conclusion, our study shows that VD reduced pro-fibrotic functions of HLFs. These findings suggest that it might be interesting to assess the potential clinical benefits of vitamin D supplementation in patients with IPF, especially on lung function decline.

ERS International Congress 2023: highlights from the Basic and Translational Sciences Assembly.

Early career members of Assembly 3 (Basic and Translational Sciences) of the European Respiratory Society (ERS) summarise the key messages discussed during six selected sessions that took place at the ERS International Congress 2023 in Milan, Italy. Aligned with the theme of the congress, the first session covered is "Micro- and macro-environments and respiratory health", which is followed by a summary of the "Scientific year in review" session. Next, recent advances in experimental methodologies and new technologies are discussed from the "Tissue modelling and remodelling" session and a summary provided of the translational science session, "What did you always want to know about omics analyses for clinical practice?", which was organised as part of the ERS Translational Science initiative's aims. The "Lost in translation: new insights into cell-to-cell crosstalk in lung disease" session highlighted how next-generation sequencing can be integrated with laboratory methods, and a final summary of studies is presented from the "From the transcriptome landscape to innovative preclinical models in lung diseases" session, which links the transcriptome landscape with innovative preclinical models. The wide range of topics covered in the selected sessions and the high quality of the research discussed demonstrate the strength of the basic and translational science being presented at the international respiratory conference organised by the ERS.

Effects of Ruxolitinib on fibrosis in preclinical models of systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune fibrotic disorder notably characterized by the production of antinuclear autoantibodies, which have been linked to an excess of apoptotic cells, normally eliminated by a macrophagic efferocytosis. As interferon (IFN) signature and phosphorylation of JAK-STAT proteins are hallmarks of SSc tissues, we tested the hypothesis that a JAK inhibitor, ruxolitinib, targeting the IFN signaling, could improve efferocytosis of IFN-exposed human macrophages in vitro as well as skin and lung fibrosis. In vivo, BLM- and HOCl-induced skin thickness and fibrosis is associated with an increase of caspase-3 positive dermal cells and a significant increase of IFN-stimulated genes expression. In BLM-SSc model, ruxolitinib prevented dermal thickness, fibrosis and significantly decreased the number of cleaved caspase-3 cells in the dermis. Ruxolitinib also improved lung architecture and fibrosis although IFN signature was not entirely decreased by ruxolitinib. In vitro, ruxolitinib improves efferocytosis capacity of human monocyte-differentiated macrophages exposed to IFN-γ or IFN-ß. In human fibroblasts derived from lung (HLF) biopsies isolated from patients with idiopathic pulmonary fibrosis, the reduced mRNA expression of typical TGF-ß-activated markers by ruxolitinib was associated with a decrease of the phosphorylation of SMAD2 /3 and STAT3. Our finding supports the anti-fibrotic properties of ruxolitinib in a systemic SSc mouse model and in vitro in human lung fibroblasts.

ERS International Congress 2022: highlights from the Basic and Translational Science Assembly.

In this review, the Basic and Translational Science Assembly of the European Respiratory Society provides an overview of the 2022 International Congress highlights. We discuss the consequences of respiratory events from birth until old age regarding climate change related alterations in air quality due to pollution caused by increased ozone, pollen, wildfires and fuel combustion as well as the increasing presence of microplastic and microfibres. Early life events such as the effect of hyperoxia in the context of bronchopulmonary dysplasia and crucial effects of the intrauterine environment in the context of pre-eclampsia were discussed. The Human Lung Cell Atlas (HLCA) was put forward as a new point of reference for healthy human lungs. The combination of single-cell RNA sequencing and spatial data in the HLCA has enabled the discovery of new cell types/states and niches, and served as a platform that facilitates further investigation of mechanistic perturbations. The role of cell death modalities in regulating the onset and progression of chronic lung diseases and its potential as a therapeutic target was also discussed. Translational studies identified novel therapeutic targets and immunoregulatory mechanisms in asthma. Lastly, it was highlighted that the choice of regenerative therapy depends on disease severity, ranging from transplantation to cell therapies and regenerative pharmacology.

Identification of Paired-related Homeobox Protein 1 as a key mesenchymal transcription factor in pulmonary fibrosis.

Matrix remodeling is a salient feature of idiopathic pulmonary fibrosis (IPF). Targeting cells driving matrix remodeling could be a promising avenue for IPF treatment. Analysis of transcriptomic database identified the mesenchymal transcription factor PRRX1 as upregulated in IPF. PRRX1, strongly expressed by lung fibroblasts, was regulated by a TGF-ß/PGE2 balance in vitro in control and IPF human lung fibroblasts, while IPF fibroblast-derived matrix increased PRRX1 expression in a PDGFR-dependent manner in control ones. PRRX1 inhibition decreased human lung fibroblast proliferation by downregulating the expression of S phase cyclins. PRRX1 inhibition also impacted TGF-ß driven myofibroblastic differentiation by inhibiting SMAD2/3 phosphorylation through phosphatase PPM1A upregulation and TGFBR2 downregulation, leading to TGF-ß response global decrease. Finally, targeted inhibition of Prrx1 attenuated fibrotic remodeling in vivo with intra-tracheal antisense oligonucleotides in bleomycin mouse model of lung fibrosis and ex vivo using human and mouse precision-cut lung slices. Our results identified PRRX1 as a key mesenchymal transcription factor during lung fibrogenesis.

Alteration of human macrophage phenotypes by the anti-fibrotic drug nintedanib.

The tyrosine kinase inhibitor, Nintedanib (NTD), has been approved for the treatment of idiopathic pulmonary fibrosis (IPF). In cell-free systems, NTD was recently shown to inhibit kinase activity of the human recombinant colony-stimulating factor 1 (CSF1) receptor (CSF1R) which mediates major functions of pulmonary macrophages. In the present study, we have investigated the effects of NTD on the phenotype of human monocyte-derived macrophages controlled by CSF1 in order to identify its anti-inflammatory properties via CSF1R inhibition. NTD (0.01 to 1 µM) prevented the CSF1-induced phosphorylation of CSF1R and activation of the downstream signaling pathways. NTD, like the CSF1R inhibitor GW2580, significantly decreased the adhesion of macrophages and production of the chemokine ligand (CCL) 2. NTD also altered the polarization of macrophages to classical M1 and alternative M2a macrophages. It reduced the secretion of several pro-inflammatory and/or pro-fibrotic cytokines (IL-1ß, IL-8, IL-10 and CXCL13) by M1 macrophages but did not prevent the expression of M1 markers. While NTD (50-200 nM) partially blocked the synthesis of M2a markers (CD11b, CD200R, CD206, and CD209), it did not reduce synthesis of the M2a pro-fibrotic cytokines CCL22 and PDGF-BB, and increased CCL18 release when used at its highest concentration (1 µM). The effects of NTD on macrophage polarization only was partially mimicked by GW2580, suggesting that the drug inhibits other molecules in addition to CSF1R. In conclusion, NTD alters the CSF1-controlled phenotype of human macrophages mainly by blocking the activation of CSF1R that thus constitutes a new molecular target of NTD, at least in vitro.

Identification of periplakin as a major regulator of lung injury and repair in mice.

Periplakin is a component of the desmosomes that acts as a cytolinker between intermediate filament scaffolding and the desmosomal plaque. Periplakin is strongly expressed by epithelial cells in the lung and is a target antigen for autoimmunity in idiopathic pulmonary fibrosis. The aim of this study was to determine the role of periplakin during lung injury and remodeling in a mouse model of lung fibrosis induced by bleomycin. We found that periplakin expression was downregulated in the whole lung and in alveolar epithelial cells following bleomycin-induced injury. Deletion of the Ppl gene in mice improved survival and reduced lung fibrosis development after bleomycin-induced injury. Notably, Ppl deletion promoted an antiinflammatory alveolar environment linked to profound changes in type 2 alveolar epithelial cells, including overexpression of antiinflammatory cytokines, decreased expression of profibrotic mediators, and altered cell signaling with a reduced response to TGF-ß1. These results identify periplakin as a previously unidentified regulator of the response to injury in the lung.

Anti-parietal cell autoimmunity is associated with an accelerated decline of lung function in IPF patients.

BACKGROUND: Autoantibodies against lung epithelial antigens are often detected in patients with Idiopathic Pulmonary Fibrosis (IPF). Anti-Parietal Cell Antibodies (APCA) target the H+/K+ATPase (proton pump). APCA prevalence and lung H+/K+ATPase expression was never studied in IPF patients. METHODS: We retrospectively collected clinical, lung function and imaging data from APCA positive patients (APCA+IPF) and compared them with APCA negative IPF patients matched on the date of diagnostic assessment. H+/K+ATPase expression was assessed with immunohistochemistry and PCR. RESULTS: Among 138 IPF patients diagnosed between 2007 and 2014 and tested for APCA, 19 (13.7%) APCA+ patients were identified. APCA+IPF patients were 16 men and 3 women, mean age 71 years. The median titer of APCA was 1:160. A pernicious anemia was present in 5 patients and preceded the fibrosis in 3 cases. With a mean follow up of 31 months, 2 patients had an exacerbation and 7 patients died. As compared with 19 APCA- IPF patients, APCA+IPF patients had a less severe disease with better DLCO (57% vs 43% predicted), preserved PaO2 (85 ± 8 mmHg vs 74 ± 11 mmHg), a lower rate of honeycombing on HRCT (58% vs 89%), but they experienced an accelerated decline of FVC (difference 61.4 ml/year; p = .0002). The H+/K+ATPase was strongly expressed by hyperplastic alveolar epithelial cells in the fibrotic lung. CONCLUSION: Anti-parietal cell autoimmunity is detected in some IPF patients and is associated with an accelerated decline of lung function. Anti-parietal cell autoimmunity may promote lung fibrosis progression.

The pro-apoptotic BAX protein influences cell growth and differentiation from the nucleus in healthy interphasic cells.

It has become more and more evident that the BCL-2 family proteins mediate a wide range of non-apoptotic functions. The pro-apoptotic BAX protein has been reported in interphasic nuclei. Whether the nuclear form of BAX could be involved in non-apoptotic function is still unknown. Our study showed for the first time that BAX was associated with chromatin in vitro. Next, we used gain and loss of function approaches to decipher the potential role of nuclear BAX in non-apoptotic cells. In vitro, nuclear BAX promoted cell proliferation in lung epithelial cells and primary human lung fibroblasts by modulating CDKN1A expression. Interestingly, BAX occupancy of CDKN1A promoter was specifically enriched close to the transcription-starting site. Nuclear BAX also modulated the basal myofibroblastic differentiation and migration of primary human lung fibroblasts. Finally, BAX nuclear localization was associated in vivo with the remodelling of lung parenchyma during development, tumorigenesis as well as fibrosis compared to control adult human lungs. Hence, our study established for the first time, a strong link between the nuclear localization of the pro-apoptotic BAX protein and key basic cellular functions in the non-apoptotic setting.

Fhit regulates EMT targets through an EGFR/Src/ERK/Slug signaling axis in human bronchial cells.

UNLABELLED: In many cancers, including lung carcinomas, Fragile histidine triad (Fhit) is frequently decreased or lost. Fhit status has recently been shown to be associated with elevated in vitro and in vivo invasiveness in lung cancer. Tumor cell invasion is facilitated by epithelial-mesenchymal transition (EMT), a process by which tumor cells lose their epithelial features to acquire a mesenchymal cell-like phenotype. In this study, the mechanism underlying Fhit-regulated EMT was deciphered. Using Slug knockdown, pharmacologic inhibitors PD98059, PP1, and gefitinib as well as an anti-EGFR antibody, it was demonstrated that Fhit silencing in bronchial cells induced overexpression of two primary EMT-associated targets, MMP-9 and vimentin, to regulate cell invasion dependent on an EGFR/Src/ERK/Slug signaling pathway. Moreover, ectopic expression of Fhit in Fhit-deficient lung cancer cells downregulated this pathway. Finally, an inverse correlation was observed between Fhit and phospho-EGFR levels in a cohort of human squamous cell lung carcinoma specimens. These results demonstrate a Fhit-dependent mechanism in the control of EMT-regulated EGFR signaling. IMPLICATIONS: This study adds new insight into the regulatory mechanism of EMT, a process known to increase resistance to conventional and targeted therapies in lung cancer.