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.

Notch1 Induces Defective Epithelial Surfactant Processing and Pulmonary Fibrosis.

Rationale: Although type II alveolar epithelial cells (AEC2s) are chronically injured in idiopathic pulmonary fibrosis (IPF), they contribute to epithelial regeneration in IPF. Objectives: We hypothesized that Notch signaling may contribute to AEC2 proliferation, dedifferentiation characterized by loss of surfactant processing machinery, and lung fibrosis in IPF. Methods: We applied microarray analysis, kinome profiling, flow cytometry, immunofluorescence analysis, western blotting, quantitative PCR, and proliferation and surface activity analysis to study epithelial differentiation, proliferation, and matrix deposition in vitro (AEC2 lines, primary murine/human AEC2s), ex vivo (human IPF-derived precision-cut lung slices), and in vivo (bleomycin and pepstatin application, Notch1 [Notch receptor 1] intracellular domain overexpression). Measurements and Main Results: We document here extensive SP-B and -C (surfactant protein-B and -C) processing defects in IPF AEC2s, due to loss of Napsin A, resulting in increased intra-alveolar surface tension and alveolar collapse and induction of endoplasmic reticulum stress in AEC2s. In vivo pharmacological inhibition of Napsin A results in the development of AEC2 injury and overt lung fibrosis. We also demonstrate that Notch1 signaling is already activated early in IPF and determines AEC2 fate by inhibiting differentiation (reduced lamellar body compartment, reduced capacity to process hydrophobic SP) and by causing increased epithelial proliferation and development of lung fibrosis, putatively via altered JAK (Janus kinase)/Stat (signal transducer and activator of transcription) signaling in AEC2s. Conversely, inhibition of Notch signaling in IPF-derived precision-cut lung slices improved the surfactant processing capacity of AEC2s and reversed fibrosis. Conclusions: Notch1 is a central regulator of AEC2 fate in IPF. It induces alveolar epithelial proliferation and loss of Napsin A and of surfactant proprotein processing, and it contributes to fibroproliferation.

Therapeutic induction of Bcl2-associated athanogene 3-mediated autophagy in idiopathic pulmonary fibrosis.

BACKGROUND: Exaggerated fibroblast proliferation is a well-known feature in idiopathic pulmonary fibrosis (IPF) which may be - in part - due to insufficient autophagy, a lysosome dependent cellular surveillance pathway. Bcl2-associated athanogene 3 (BAG3) is a pivotal co-chaperone of the autophagy pathway. Here, we studied whether therapeutic modulation of BAG3-mediated autophagy can rescue insufficient autophagy and impact IPF fibroblast proliferation. METHODS: Primary interstitial fibroblasts or precision cut lung slices (PCLS) of IPF lungs were treated with (1) the antifibrotic drug pirfenidone (Pirf), (2) the demethylating agent 5-azacytidine (Aza), (3) the BAG3 modulator cantharidin (Ctd). Autophagy flux was measured following pretreatment with the autophagy inhibitors or by GFP-RFP-LC3B transfection followed by drug treatments. Proliferation was measured by 5-bromo-2'-deoxyuridine assay. BAG3, filamin C (FLNC), proliferating-cell-nuclear-antigen (PCNA), collagen1A1 (COL1A1) and autophagy proteins were assessed by immunoblotting or immunofluorescence. Loss of function experiments were performed by siRNA mediated knockdown of BAG3. RESULTS: In comparison with healthy donors, increased BAG3 protein was observed in IPF lung homogenates and IPF fibroblasts. In addition, the substrate of BAG3-mediated autophagy, FLNC, was increased in IPF fibroblasts, implying insufficient activation of BAG3-dependent autophagy. Therapeutic modulation of this pathway using Aza and Ctd alone or in combination with the IPF therapy drug Pirf rescued the insufficient BAG3-mediated autophagy and decreased fibroblast proliferation. Such effects were observed upon therapeutic modulation of BAG3 but not upon knock down of BAG3 per se in IPF fibroblasts. Similarly, PCLS of IPF patients showed a significant decrease in collagen deposition in response to these drugs, either alone or in a more potent form in combination with Pirf. CONCLUSIONS: Our study reveals that repurposing drugs that modulate autophagy regulating proteins render therapeutic benefits in IPF. Fine tuning of this pathway may hence signify a promising therapeutic strategy to ameliorate antifibrotic properties and augment the efficacy of current IPF therapy.

Transcriptional Profiling of Insulin-like Growth Factor Signaling Components in Embryonic Lung Development and Idiopathic Pulmonary Fibrosis.

Insulin-like growth factor (IGF) signaling controls the development and growth of many organs, including the lung. Loss of function of Igf1 or its receptor Igf1r impairs lung development and leads to neonatal respiratory distress in mice. Although many components of the IGF signaling pathway have shown to be dysregulated in idiopathic pulmonary fibrosis (IPF), the expression pattern of such components in different cellular compartments of the developing and/or fibrotic lung has been elusive. In this study, we provide a comprehensive transcriptional profile for such signaling components during embryonic lung development in mice, bleomycin-induced pulmonary fibrosis in mice and in human IPF lung explants. During late gestation, we found that Igf1 is upregulated in parallel to Igf1r downregulation in the lung mesenchyme. Lung tissues derived from bleomycin-treated mice and explanted IPF lungs revealed upregulation of IGF1 in parallel to downregulation of IGF1R, in addition to upregulation of several IGF binding proteins (IGFBPs) in lung fibrosis. Finally, treatment of IPF lung fibroblasts with recombinant IGF1 led to myogenic differentiation. Our data serve as a resource for the transcriptional profile of IGF signaling components and warrant further research on the involvement of this pathway in both lung development and pulmonary disease.

Targeting Histone Deacetylases in Idiopathic Pulmonary Fibrosis: A Future Therapeutic Option.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with limited therapeutic options, and there is a huge unmet need for new therapies. A growing body of evidence suggests that the histone deacetylase (HDAC) family of transcriptional corepressors has emerged as crucial mediators of IPF pathogenesis. HDACs deacetylate histones and result in chromatin condensation and epigenetic repression of gene transcription. HDACs also catalyse the deacetylation of many non-histone proteins, including transcription factors, thus also leading to changes in the transcriptome and cellular signalling. Increased HDAC expression is associated with cell proliferation, cell growth and anti-apoptosis and is, thus, a salient feature of many cancers. In IPF, induction and abnormal upregulation of Class I and Class II HDAC enzymes in myofibroblast foci, as well as aberrant bronchiolar epithelium, is an eminent observation, whereas type-II alveolar epithelial cells (AECII) of IPF lungs indicate a significant depletion of many HDACs. We thus suggest that the significant imbalance of HDAC activity in IPF lungs, with a "cancer-like" increase in fibroblastic and bronchial cells versus a lack in AECII, promotes and perpetuates fibrosis. This review focuses on the mechanisms by which Class I and Class II HDACs mediate fibrogenesis and on the mechanisms by which various HDAC inhibitors reverse the deregulated epigenetic responses in IPF, supporting HDAC inhibition as promising IPF therapy.

PACS2-TRPV1 axis is required for ER-mitochondrial tethering during ER stress and lung fibrosis.

Endoplasmic reticulum (ER) and mitochondria (mito) play a vital role in alveolar type II cell (AEC2) homeostasis and are both stressed in patients with idiopathic pulmonary fibrosis (IPF). Up to now, no data are available with regard to ER-mito cross talk and tethering under conditions of IPF. We here demonstrate that ER-mitochondrial tethering is reduced upon experimental ER stress in vitro and in the IPF AECII ex vivo, and this is-at least in part-due to decreased phosphofurin acidic cluster sorting protein 2 (PACS-2, also called PACS2) protein levels. PACS2 levels are influenced by its interaction with the transient receptor potential cation channel subfamily V member 1 (TRPV1) and can be experimentally modified by the TRPV1-modulating drug capsaicin (CPS). Employing alveolar epithelial cells with overexpression of the terminal ER stress signaling factor Chop or the IPF-associated surfactant protein C mutation (SPCΔexon4) in vitro, we observed a restoration of PACS2 levels upon treatment with CPS. Similarly, treatment of precision cut lung slices from IPF patients with CPS ex vivo forwarded similar effects. Importantly, in all models such kind of intervention also greatly reduced the extent of alveolar epithelial apoptosis. We therefore conclude that therapeutic targeting of the PACS2-TRPV1 axis represents an interesting novel, epithelial-protective approach in IPF.

Clinical and Functional Characteristics of Patients with Unclassifiable Interstitial Lung Disease (uILD): Long-Term Follow-Up Data from European IPF Registry (eurIPFreg).

(1) Aim of the study: In spite of extensive research, up to 20% of interstitial lung diseases (ILD) patients cannot be safely classified. We analyzed clinical features, progression factors, and outcomes of unclassifiable ILD (uILD). (2) Methods: A total of 140 uILD subjects from the University of Giessen and Marburg Lung Center (UGMLC) were recruited between 11/2009 and 01/2019 into the European Registry for idiopathic pulmonary fibrosis (eurIPFreg) and followed until 01/2020. The diagnosis of uILD was applied only when a conclusive diagnosis could not be reached with certainty. (3) Results: In 46.4% of the patients, the uILD diagnosis was due to conflicting clinical, radiological, and pathological data. By applying the diagnostic criteria of usual interstitial pneumonia (UIP) based on computed tomography (CT), published by the Fleischner Society, 22.2% of the patients displayed a typical UIP pattern. We also showed that forced vital capacity (FVC) at baseline (p = 0.008), annual FVC decline ≥10% (p < 0.0001), smoking (p = 0.033), and a diffusing capacity of the lung for carbon monoxide (DLco) ≤55% of predicted value at baseline (p < 0.0001) were significantly associated with progressive disease. (4) Conclusions: The most important prognostic factors in uILD are baseline level and decline in lung function and smoking. The use of Fleischner diagnostic criteria allows further differentiation and accurate diagnosis.

The ageing lung under stress.

Healthy ageing of the lung involves structural changes but also numerous cell-intrinsic and cell-extrinsic alterations. Among them are the age-related decline in central cellular quality control mechanisms such as redox and protein homeostasis. In this review, we would like to provide a conceptual framework of how impaired stress responses in the ageing lung, as exemplified by dysfunctional redox and protein homeostasis, may contribute to onset and progression of COPD and idiopathic pulmonary fibrosis (IPF). We propose that age-related imbalanced redox and protein homeostasis acts, amongst others (e.g. cellular senescence), as a "first hit" that challenges the adaptive stress-response pathways of the cell, increases the level of oxidative stress and renders the lung susceptible to subsequent injury and disease. In both COPD and IPF, additional environmental insults such as smoking, air pollution and/or infections then serve as "second hits" which contribute to persistently elevated oxidative stress that overwhelms the already weakened adaptive defence and repair pathways in the elderly towards non-adaptive, irremediable stress thereby promoting development and progression of respiratory diseases. COPD and IPF are thus distinct horns of the same devil, "lung ageing".

Proteasome activator PA200 regulates myofibroblast differentiation.

The proteasome is essential for the selective degradation of most cellular proteins and is fine-tuned according to cellular needs. Proteasome activators serve as building blocks to adjust protein turnover in cell growth and differentiation. Understanding the cellular function of proteasome activation in more detail offers a new strategy for therapeutic targeting of proteasomal protein breakdown in disease. The role of the proteasome activator PA200 in cell function and its regulation in disease is unknown. In this study, we investigated the function of PA200 in myofibroblast differentiation and fibrotic tissue remodeling. PA200 was upregulated in hyperplastic basal cells and myofibroblasts of fibrotic lungs from patients with idiopathic pulmonary fibrosis. Increased expression of PA200 and enhanced formation of PA200-proteasome complexes was also evident in experimental fibrosis of the lung and kidney in vivo and in activated primary human myofibroblasts of the lung in vitro. Transient silencing and overexpression revealed that PA200 functions as a negative regulator of myofibroblast differentiation of human but not mouse cells. Our data thus suggest an unexpected and important role for PA200 in adjusting myofibroblast activation in response to pro-fibrotic stimuli, which fails in idiopathic pulmonary fibrosis.

Exhalative Breath Markers Do Not Offer for Diagnosis of Interstitial Lung Diseases: Data from the European IPF Registry (eurIPFreg) and Biobank.

Background: New biomarkers are urgently needed to facilitate diagnosis in Interstitial Lung Diseases (ILD), thus reducing the need for invasive procedures, and to enable tailoring and monitoring of medical treatment. Methods: In this study we investigated if patients with idiopathic pulmonary fibrosis (IPF; n = 21), non-IPF ILDs (n = 57) and other lung diseases (chronic obstructive pulmonary disease (COPD) n = 24, lung cancer (LC) n = 16) as well as healthy subjects (n = 20) show relevant differences in exhaled NO (FeNO; Niox MINO), or in eicosanoid (PGE2, 8-isoprostane; enzyme-linked immunosorbent assay (ELISA)) levels as measured in exhaled breath condensates (EBC) and bronchoalveolar lavage fluids (BALF). Results: There was no significant difference in FeNO values between IPF, non-IPF ILDs and healthy subjects, although some individual patients showed highly elevated FeNO. On the basis of the FeNO signal, it was neither possible to differentiate between the kind of disease nor to detect exacerbations. In addition, there was no correlation between FeNO values and lung function. The investigation of the eicosanoids in EBCs was challenging (PGE2) or unreliable (8-isoprostane), but worked out well in BALF. A significant increase of free 8-isoprostane was observed in BALF, but not in EBCs, of patients with IPF, hypersensitivity pneumonitis (HP) and sarcoidosis, possibly indicating severity of oxidative stress. Conclusions: FeNO-measurements are not of diagnostic benefit in different ILDs including IPF. The same holds true for PGE2 and 8-isoprostane in EBC by ELISA.

Comparison of the antifibrotic effects of the pan-histone deacetylase-inhibitor panobinostat versus the IPF-drug pirfenidone in fibroblasts from patients with idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with a poor prognosis. Pirfenidone is the first antifibrotic agent to be approved for IPF-treatment as it is able to slow down disease progression. However, there is no curative treatment other than lung transplantation. Because epigenetic alterations are associated with IPF, histone deacetylase (HDAC)-inhibitors have recently been proven to attenuate fibrotic remodeling in vitro and in vivo. This study compared the effects of pirfenidone with the pan-HDAC-inhibitor panobinostat/LBH589, a FDA-approved drug for the treatment of multiple myeloma, head-to-head on survival, fibrotic activity and proliferation of primary IPF-fibroblasts in vitro. METHODS: Primary fibroblasts from six IPF-patients were incubated for 24h with vehicle (0.25% DMSO), panobinostat (LBH589, 85 nM) or pirfenidone (2.7 mM), followed by assessment of proliferation and expression analyses for profibrotic and anti-apoptosis genes, as well as for ER stress and apoptosis-markers. In addition, the expression status of all HDAC enzymes was examined. RESULTS: Treatment of IPF-fibroblasts with panobinostat or pirfenidone resulted in a downregulated expression of various extracellular matrix (ECM)-associated genes, as compared to vehicle-treated cells. In agreement, both drugs decreased protein level of phosphorylated (p)-STAT3, a transcription factor mediating profibrotic responses, in treated IPF-fibroblasts. Further, an increase in histone acetylation was observed in response to both treatments, but was much more pronounced and excessive in panobinostat-treated IPF-fibroblasts. Panobinostat, but not pirfenidone, led to a significant suppression of proliferation in IPF-fibroblasts, as indicated by WST1- and BrdU assay and markedly diminished levels of cyclin-D1 and p-histone H3. Furthermore, panobinostat-treatment enhanced α-tubulin-acetylation, decreased the expression of survival-related genes Bcl-XL and BIRC5/survivin, and was associated with induction of ER stress and apoptosis in IPF-fibroblasts. In contrast, pirfenidone-treatment maintained Bcl-XL expression, and was neither associated with ER stress-induction nor any apoptotic signaling. Pirfenidone also led to increased expression of HDAC6 and sirtuin-2, and enhanced α-tubulin-deacetylation. But in line with its ability to increase histone acetylation, pirfenidone reduced the expression of HDAC enzymes HDAC1, -2 and -9. CONCLUSIONS: We conclude that, beside other antifibrotic mechanisms, pirfenidone reduces profibrotic signaling also through STAT3 inactivation and weak epigenetic alterations in IPF-fibroblasts, and permits survival of (altered) fibroblasts. The pan-HDAC-inhibitor panobinostat reduces profibrotic phenotypes while inducing cell cycle arrest and apoptosis in IPF-fibroblasts, thus indicating more efficiency than pirfenidone in inactivating IPF-fibroblasts. We therefore believe that HDAC-inhibitors such as panobinostat can present a novel therapeutic strategy for IPF.

Dynamic expression of HOPX in alveolar epithelial cells reflects injury and repair during the progression of pulmonary fibrosis.

Mechanisms of injury and repair in alveolar epithelial cells (AECs) are critically involved in the progression of various lung diseases including idiopathic pulmonary fibrosis (IPF). Homeobox only protein x (HOPX) contributes to the formation of distal lung during development. In adult lung, alveolar epithelial type (AT) I cells express HOPX and lineage-labeled Hopx+ cells give rise to both ATI and ATII cells after pneumonectomy. However, the cell function of HOPX-expressing cells in adult fibrotic lung diseases has not been investigated. In this study, we have established a flow cytometry-based method to evaluate HOPX-expressing cells in the lung. HOPX expression in cultured ATII cells increased over culture time, which was accompanied by a decrease of proSP-C, an ATII marker. Moreover, HOPX expression was increased in AECs from bleomycin-instilled mouse lungs in vivo. Small interfering RNA-based knockdown of Hopx resulted in suppressing ATII-ATI trans-differentiation and activating cellular proliferation in vitro. In IPF lungs, HOPX expression was decreased in whole lungs and significantly correlated to a decline in lung function and progression of IPF. In conclusion, HOPX is upregulated during early alveolar injury and repair process in the lung. Decreased HOPX expression might contribute to failed regenerative processes in end-stage IPF lungs.

Tubastatin ameliorates pulmonary fibrosis by targeting the TGFß-PI3K-Akt pathway.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal disease. Histone deacetylase 6 (HDAC6) alters function and fate of various proteins via deacetylation of lysine residues, and is implicated in TGF-ß1-induced EMT (epithelial-mesenchymal transition). However, the role of HDAC6 in pulmonary fibrosis is unknown. METHODS: HDAC6 expression in IPF and control lungs was assessed by quantitative real-time PCR (qRT-PCR) and immunoblots. Lung fibroblasts were treated with TGF-ß1 ± HDAC6 inhibitors (Tubacin, Tubastatin, ACY1215, or MC1568), and fibrotic markers such as type I collagen were assessed using qRT-PCR and immunoblots. Mice were treated with bleomycin (oropharyngeal aspiration; single dose) ± Tubastatin (intraperitoneally injection; daily for 21 days), and lung collagen expression was gauged using immunoblots and trichrome staining. In a separate experiment, HDAC6 wild-type (WT) and knockout (KO) mice were administered bleomycin, and lungs were evaluated in the same manner. RESULTS: HDAC6 expression was deregulated in IPF lungs. Among the HDAC6 inhibitors tested, only Tubastatin significantly repressed TGF-ß1-induced expression of type-1 collagen in lung fibroblasts, and this finding was coupled with decreased Akt phosphorylation and increased Akt-PHLPP (PH domain and Leucine rich repeat Protein Phosphatase) association. Tubastatin repressed TGF-ß1-induced S6K phosphorylation, HIF-1α expression, and VEGF expression. Tubastatin also repressed TGF-ß1-induced inhibition of LC3B-II (a marker of autophagosome formation). In bleomycin-treated mouse lungs, HDAC6 expression was increased, and Tubastatin repressed type-1 collagen expression. However, in HDAC6 KO mice, bleomycin-induced type-1 collagen expression was not repressed compared to WT mice. Knockdown of HDAC6, as well as HDAC10, another potential Tubastatin target, did not inhibit TGF-ß1-induced collagen expression in lung fibroblasts. CONCLUSIONS: HDAC6 expression is altered during lung fibrogenesis. Tubastatin represses TGF-ß1-induced collagen expression, by diminishing Akt phosphorylation and regulating downstream targets such as HIF-1α-VEGF axis and autophagy. Tubastatin-treated WT mice are protected against bleomycin-induced fibrosis, but HDAC6 KO mice are not. Our data suggest that Tubastatin ameliorates pulmonary fibrosis, by targeting the TGFß-PI3K-Akt pathway, likely via an HDAC6-independent mechanism.

Senolytic drugs target alveolar epithelial cell function and attenuate experimental lung fibrosis ex vivo.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with poor prognosis and limited therapeutic options. The incidence of IPF increases with age, and ageing-related mechanisms such as cellular senescence have been proposed as pathogenic drivers. The lung alveolar epithelium represents a major site of tissue injury in IPF and senescence of this cell population is probably detrimental to lung repair. However, the potential pathomechanisms of alveolar epithelial cell senescence and the impact of senolytic drugs on senescent lung cells and fibrosis remain unknown. Here we demonstrate that lung epithelial cells exhibit increased P16 and P21 expression as well as senescence-associated ß-galactosidase activity in experimental and human lung fibrosis tissue and primary cells.Primary fibrotic mouse alveolar epithelial type (AT)II cells secreted increased amounts of senescence-associated secretory phenotype (SASP) factors in vitro, as analysed using quantitative PCR, mass spectrometry and ELISA. Importantly, pharmacological clearance of senescent cells by induction of apoptosis in fibrotic ATII cells or ex vivo three-dimensional lung tissue cultures reduced SASP factors and extracellular matrix markers, while increasing alveolar epithelial markers.These data indicate that alveolar epithelial cell senescence contributes to lung fibrosis development and that senolytic drugs may be a viable therapeutic option for IPF.

Amplified canonical transforming growth factor-ß signalling via heat shock protein 90 in pulmonary fibrosis.

Interstitial lung fibroblast activation coupled with extracellular matrix production is a pathological signature of idiopathic pulmonary fibrosis (IPF), and is governed by transforming growth factor (TGF)-ß/Smad signalling. We sought to define the role of heat shock protein (HSP)90 in profibrotic responses in IPF and to determine the therapeutic effects of HSP90 inhibition in a murine model of pulmonary fibrosis.We investigated the effects of HSP90 inhibition in vitro by applying 17-AAG (17-allylamino-17-demethoxygeldanamycin) to lung fibroblasts and A549 cells and in vivo by administering 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) to mice with bleomycin-induced pulmonary fibrosis.HSP90 expression was increased in (myo)fibroblasts from fibrotic human and mouse lungs compared with controls. 17-AAG inhibited TGF-ß1-induced extracellular matrix production and transdifferentiation of lung fibroblasts and epithelial-mesenchymal transition of A549 cells. The antifibrotic effects were associated with TGF-ß receptor disruption and inhibition of Smad2/3 activation. Co-immunoprecipitation revealed that HSP90ß interacted with TGF-ß receptor II and stabilised TGF-ß receptors. Furthermore, 17-DMAG improved lung function and decreased fibrosis and matrix metalloproteinase activity in the lungs of bleomycin-challenged mice.In conclusion, this is the first study to demonstrate that HSP90 inhibition blocks pulmonary fibroblast activation and ameliorates bleomycin-induced pulmonary fibrosis in mice.

MAP1LC3B overexpression protects against Hermansky-Pudlak syndrome type-1-induced defective autophagy in vitro.

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder, and some patients with HPS develop pulmonary fibrosis, known as HPS-associated interstitial pneumonia (HPSIP). We have previously reported that HPSIP is associated with severe surfactant accumulation, lysosomal stress, and alveolar epithelial cell type II (AECII) apoptosis. Here, we hypothesized that defective autophagy might result in excessive lysosomal stress in HPSIP. Key autophagy proteins, including LC3B lipidation and p62, were increased in HPS1/2 mice lungs. Electron microscopy demonstrated a preferable binding of LC3B to the interior of lamellar bodies in the AECII of HPS1/2 mice, whereas in wild-type mice it was present on the limiting membrane in addition to the interior of the lamellar bodies. Similar observations were noted in human HPS1 lung sections. In vitro knockdown of HPS1 revealed increased LC3B lipidation and p62 accumulation, associated with an increase in proapoptotic caspases. Overexpression of LC3B decreased the HPS1 knockdown-induced p62 accumulation, whereas rapamycin treatment did not show the same effect. We conclude that loss of HPS1 protein results in impaired autophagy that is restored by exogenous LC3B and that defective autophagy might therefore play a critical role in the development and progression of HPSIP.

Aberrant expression and activity of histone deacetylases in sporadic idiopathic pulmonary fibrosis.

BACKGROUND: Activation and differentiation of fibroblasts into contractile protein-expressing myofibroblasts and their acquired apoptosis-resistant phenotype are critical factors towards the development of idiopathic pulmonary fibrosis (IPF), a fatal disease characterised by distorted pulmonary structure and excessive extracellular matrix (ECM) deposition. The molecular mechanisms underlying these processes in IPF remain incompletely understood. We investigated the possible implication of aberrant overexpression and activity of histone deacetylases (HDACs) in IPF. METHODS: We analysed lung tissues from patients with sporadic IPF (n=26) and non-diseased control lungs (n=16) for expression of class I and II HDACs. Primary IPF fibroblasts were treated with HDAC inhibitors (HDACi) LBH589 or valproic acid (VPA). RESULTS: Compared to control lungs, protein levels of class I (HDAC1, HDAC2, HDAC3, HDAC8) and class II HDACs (HDAC4, HDAC 5, HDAC 7, HDAC 9) were significantly elevated in IPF lungs. Using immunohistochemistry, strong induction of nearly all HDAC enzymes was observed in myofibroblasts of fibroblast foci and in abnormal bronchiolar basal cells at sites of aberrant re-epithelialisation in IPF lungs, but not in controls. Treatment of primary IPF fibroblasts with the pan-HDACi LBH589 resulted in significantly reduced expression of genes associated with ECM synthesis, proliferation and cell survival, as well as in suppression of HDAC7, and was paralleled by induction of endoplasmic reticulum stress and apoptosis. The profibrotic and apoptosis-resistant phenotype of IPF fibroblasts was also partly attenuated by the class I HDACi VPA. CONCLUSIONS: Aberrant overexpression of HDACs in basal cells of IPF lungs may contribute to the bronchiolisation process in this disease. Similarly, generation and apoptosis resistance of IPF fibroblasts are mediated by enhanced activity of HDAC enzymes. Therefore, pan-HDAC inhibition by LBH589 may present a novel therapeutic option for patients with IPF.

Increased FGF1-FGFRc expression in idiopathic pulmonary fibrosis.

BACKGROUND: Recent clinical studies show that tyrosine kinase inhibitors slow the rate of lung function decline and decrease the number of acute exacerbations in patients with Idiopathic Pulmonary Fibrosis (IPF). However, in the murine bleomycin model of fibrosis, not all tyrosine kinase signaling is detrimental. Exogenous ligands Fibroblast Growth Factor (FGF) 7 and 10 improve murine lung repair and increase survival after injury via tyrosine kinase FGF receptor 2b-signaling. Therefore, the level and location of FGF/FGFR expression as well as the exogenous effect of the most highly expressed FGFR2b ligand, FGF1, was analyzed on human lung fibroblasts. METHODS: FGF ligand and receptor expression was evaluated in donor and IPF whole lung homogenates using western blotting and qPCR. Immunohistochemistry for FGF1 and FGFR1/2/3/4 were performed on human lung tissue. Lastly, the effects of FGF1, a potent, multi-FGFR ligand, were studied on primary cultures of IPF and non-IPF donor fibroblasts. Western blots for pro-fibrotic markers, proliferation, FACS for apoptosis, transwell assays and MetaMorph analyses on cell cultures were performed. RESULTS: Whole lung homogenate analyses revealed decreased FGFR b-isoform expression, and an increase in FGFR c-isoform expression. Of the FGFR2b-ligands, FGF1 was the most significantly increased in IPF patients; downstream targets of FGF-signaling, p-ERK1/2 and p-AKT were also increased. Immunohistochemistry revealed FGF1 co-localization within basal cell sheets, myofibroblast foci, and Surfactant protein-C positive alveolar epithelial type-II cells as well as co-localization with FGFR1, FGFR2, FGFR3, FGFR4 and myofibroblasts expressing the migratory marker Fascin. Both alone and in the presence of heparin, FGF1 led to increased MAPK-signaling in primary lung fibroblasts. While smooth muscle actin was unchanged, heparin + FGF1 decreased collagen production in IPF fibroblasts. In addition, FGF1 + heparin increased apoptosis and cell migration. The FGFR inhibitor (PD173074) attenuated these effects. CONCLUSIONS: Strong expression of FGF1/FGFRs in pathogenic regions of IPF suggest that aberrant FGF1-FGFR signaling is increased in IPF patients and may contribute to the pathogenesis of lung fibrosis by supporting fibroblast migration and increased MAPK-signaling.

Regulation of 26S Proteasome Activity in Pulmonary Fibrosis.

RATIONALE: The ubiquitin-proteasome system is critical for maintenance of protein homeostasis by degrading polyubiquitinated proteins in a spatially and temporally controlled manner. Cell and protein homeostasis are altered upon pathological tissue remodeling. Dysregulation of the proteasome has been reported for several chronic diseases of the heart, brain, and lung. We hypothesized that proteasome function is altered upon fibrotic lung remodeling, thereby contributing to the pathogenesis of idiopathic pulmonary fibrosis (IPF). OBJECTIVES: To investigate proteasome function during myofibroblast differentiation. METHODS: We treated lung fibroblasts with transforming growth factor (TGF)-ß and examined proteasome composition and activity. For in vivo analysis, we used mouse models of lung fibrosis and fibrotic human lung tissue. MEASUREMENTS AND MAIN RESULTS: We demonstrate that induction of myofibroblast differentiation by TGF-ß involves activation of the 26S proteasome, which is critically dependent on the regulatory subunit Rpn6. Silencing of Rpn6 in primary human lung fibroblasts counteracted TGF-ß-induced myofibroblast differentiation. Activation of the 26S proteasome and increased expression of Rpn6 were detected during bleomycin-induced lung remodeling and fibrosis. Importantly, Rpn6 is overexpressed in myofibroblasts and basal cells of the bronchiolar epithelium in lungs of patients with IPF, which is accompanied by enhanced protein polyubiquitination. CONCLUSIONS: We identified Rpn6-dependent 26S proteasome activation as an essential feature of myofibroblast differentiation in vitro and in vivo, and our results suggest it has an important role in IPF pathogenesis.