Low density lipoprotein receptor-related protein 1 couples ß1 integrin activation to degradation.

Low density lipoprotein receptor-related protein (LRP) 1 modulates cell adhesion and motility under normal and pathological conditions. Previous studies documented that LRP1 binds several integrin receptors and mediates their trafficking to the cell surface and endocytosis. However, the mechanism by which LRP1 may regulate integrin activation remains unknown. Here we report that LRP1 promotes the activation and subsequent degradation of ß1 integrin and thus supports cell adhesion, spreading, migration and integrin signaling on fibronectin. LRP1 interacts with surface ß1 integrin, binds the integrin activator kindlin2 and stimulates ß1 integrin-kindlin2 complex formation. Specifically, serine 76 in the LRP1 cytoplasmic tail is crucial for the interaction with kindlin2, ß1 integrin activation and cell adhesion. Interestingly, a loss of LRP1 induces the accumulation of several integrin receptors on the cell surface. Following internalization, intracellular trafficking of integrins is driven by LRP1 in a protein kinase C- and class II myosin-dependent manner. Ultimately, LRP1 dictates the fate of endocytosed ß1 integrin by directing it down the pathway of lysosomal and proteasomal degradation. We propose that LRP1 mediates cell adhesion by orchestrating a multi-protein pathway to activate, traffic and degrade integrins. Thus, LRP1 may serve as a focal point in the integrin quality control system to ensure a firm connection to the extracellular matrix.

Transcriptome profiling reveals the complexity of pirfenidone effects in idiopathic pulmonary fibrosis.

Despite the beneficial effects of pirfenidone in treating idiopathic pulmonary fibrosis (IPF), it remains unclear if lung fibroblasts (FB) are the main therapeutic target.To resolve this question, we employed a comparative transcriptomic approach and analysed lung homogenates (LH) and FB derived from IPF patients treated with or without pirfenidone.In FB, pirfenidone therapy predominantly affected growth and cell division pathways, indicating a major cellular metabolic shift. In LH samples, pirfenidone treatment was mostly associated with inflammation-related processes. In FB and LH, regulated genes were over-represented in the Gene Ontology node "extracellular matrix". We identified lower expression of cell migration-inducing and hyaluronan-binding protein (CEMIP) in both LH and FB from pirfenidone-treated IPF patients. Plasma levels of CEMIP were elevated in IPF patients compared to healthy controls and decreased after 7 months of pirfenidone treatment. CEMIP expression in FB was downregulated in a glioma-associated oncogene homologue-dependent manner and CEMIP silencing in IPF FB reduced collagen production and attenuated cell proliferation and migration.Cumulatively, our approach indicates that pirfenidone exerts beneficial effects via its action on multiple pathways in both FB and other pulmonary cells, through its ability to control extracellular matrix architecture and inflammatory reactions.

Pirfenidone exerts antifibrotic effects through inhibition of GLI transcription factors.

Pirfenidone is an antifibrotic drug, recently approved for the treatment of patients with idiopathic pulmonary fibrosis (IPF). Although pirfenidone exhibits anti-inflammatory, antioxidant, and antifibrotic properties, the molecular mechanism underlying its protective effects remains unknown. Here, we link pirfenidone action with the regulation of the profibrotic hedgehog (Hh) signaling pathway. We demonstrate that pirfenidone selectively destabilizes the glioma-associated oncogene homolog (GLI)2 protein, the primary activator of Hh-mediated gene transcription. Consequently, pirfenidone decreases overall Hh pathway activity in patients with IPF and in patient-derived primary lung fibroblasts and leads to diminished levels of Hh target genes, such as GLI1, Hh receptor Patched-1, α-smooth muscle actin, and fibronectin, and to reduced cell migration and proliferation. Interestingly, Hh-triggered TGF-ß1 expression potentiated Hh responsiveness of primary lung fibroblasts by elevating the available pool of glioma-associated oncogene homolog (GLI)1/GLI2, thus creating a vicious cycle of amplifying fibrotic processes. Because GLI transcription factors are not only crucial for Hh-mediated changes but are also required as mediators of TGF-ß signaling, our findings suggest that pirfenidone exerts its clinically beneficial effects through dual Hh/TGF-ß inhibition by targeting the GLI2 protein.-Didiasova, M., Singh, R., Wilhelm, J., Kwapiszewska, G., Wujak, L., Zakrzewicz, D., Schaefer, L., Markart, P., Seeger, W., Lauth, M., Wygrecka, M. Pirfenidone exerts antifibrotic effects through inhibition of GLI transcription factors.

Antihistone Properties of C1 Esterase Inhibitor Protect against Lung Injury.

RATIONALE: Acute respiratory distress syndrome is characterized by alveolar epithelial cell injury, edema formation, and intraalveolar contact phase activation. OBJECTIVES: To explore whether C1 esterase inhibitor (C1INH), an endogenous inhibitor of the contact phase, may protect from lung injury in vivo and to decipher the possible underlying mechanisms mediating protection. METHODS: The ability of C1INH to control the inflammatory processes was studied in vitro and in vivo. MEASUREMENTS AND MAIN RESULTS: Here, we demonstrate that application of C1INH alleviates bleomycin-induced lung injury via direct interaction with extracellular histones. In vitro, C1INH was found to bind all histone types. Interaction with histones was independent of its protease inhibitory activity, as demonstrated by the use of reactive-center-cleaved C1INH, but dependent on its glycosylation status. C1INH sialylated-N- and -O-glycans were not only essential for its interaction with histones but also to protect against histone-induced cell death. In vivo, histone-C1INH complexes were detected in bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome and multiple models of lung injury. Furthermore, reactive-center-cleaved C1INH attenuated pulmonary damage evoked by intravenous histone instillation. CONCLUSIONS: Collectively, C1INH administration provides a new therapeutic option for disorders associated with histone release.

FXII promotes proteolytic processing of the LRP1 ectodomain.

BACKGROUND: Factor XII (FXII) is a serine protease that is involved in activation of the intrinsic blood coagulation, the kallikrein-kinin system and the complement cascade. Although the binding of FXII to the cell surface has been demonstrated, the consequence of this event for proteolytic processing of membrane-anchored proteins has never been described. METHODS: The effect of FXII on the proteolytic processing of the low-density lipoprotein receptor-related protein 1 (LRP1) ectodomain was tested in human primary lung fibroblasts (hLF), alveolar macrophages (hAM) and in human precision cut lung slices (hPCLS). The identity of generated LRP1 fragments was confirmed by MALDI-TOF-MS. Activity of FXII and gelatinases was measured by S-2302 hydrolysis and zymography, respectively. RESULTS: Here, we demonstrate a new function of FXII, namely its ability to process LRP1 extracellular domain. Incubation of hLF, hAM, or hPCLS with FXII resulted in the accumulation of LRP1 ectodomain fragments in conditioned media. This effect was independent of metalloproteases and required FXII proteolytic activity. Binding of FXII to hLF surface induced its conversion to FXIIa and protected FXIIa against inactivation by a broad spectrum of serine protease inhibitors. Preincubation of hLF with collagenase I impaired FXII activation and, in consequence, LRP1 cleavage. FXII-triggered LRP1 processing was associated with the accumulation of gelatinases (MMP-2 and MMP-9) in conditioned media. CONCLUSIONS: FXII controls LRP1 levels and function at the plasma membrane by modulating processing of its ectodomain. GENERAL SIGNIFICANCE: FXII-dependent proteolytic processing of LRP1 may exacerbate extracellular proteolysis and thus promote pathological tissue remodeling.

TGF-ß directs trafficking of the epithelial sodium channel ENaC which has implications for ion and fluid transport in acute lung injury.

TGF-ß is a pathogenic factor in patients with acute respiratory distress syndrome (ARDS), a condition characterized by alveolar edema. A unique TGF-ß pathway is described, which rapidly promoted internalization of the αßγ epithelial sodium channel (ENaC) complex from the alveolar epithelial cell surface, leading to persistence of pulmonary edema. TGF-ß applied to the alveolar airspaces of live rabbits or isolated rabbit lungs blocked sodium transport and caused fluid retention, which--together with patch-clamp and flow cytometry studies--identified ENaC as the target of TGF-ß. TGF-ß rapidly and sequentially activated phospholipase D1, phosphatidylinositol-4-phosphate 5-kinase 1α, and NADPH oxidase 4 (NOX4) to produce reactive oxygen species, driving internalization of ßENaC, the subunit responsible for cell-surface stability of the αßγENaC complex. ENaC internalization was dependent on oxidation of ßENaC Cys(43). Treatment of alveolar epithelial cells with bronchoalveolar lavage fluids from ARDS patients drove ßENaC internalization, which was inhibited by a TGF-ß neutralizing antibody and a Tgfbr1 inhibitor. Pharmacological inhibition of TGF-ß signaling in vivo in mice, and genetic ablation of the nox4 gene in mice, protected against perturbed lung fluid balance in a bleomycin model of lung injury, highlighting a role for both proximal and distal components of this unique ENaC regulatory pathway in lung fluid balance. These data describe a unique TGF-ß-dependent mechanism that regulates ion and fluid transport in the lung, which is not only relevant to the pathological mechanisms of ARDS, but might also represent a physiological means of acutely regulating ENaC activity in the lung and other organs.

Heparan sulfate proteoglycans mediate factor XIIa binding to the cell surface.

Hageman factor (FXIIa) initiates the intrinsic coagulation pathway and triggers the kallikrein-kinin and the complement systems. In addition, it functions as a growth factor by expressing promitogenic activities toward several cell types. FXIIa binds to the cell surface via a number of structurally unrelated surface receptors; however, the underlying mechanisms are not yet fully understood. Here, we demonstrate that FXIIa utilizes cell membrane-bound glycosaminoglycans to interact with the cell surface of human lung fibroblasts (HLF). The combination of enzymatic, inhibitory, and overexpression approaches identified a heparan sulfate (HS) component of proteoglycans as an important determinant of the FXIIa binding capacity of HLF. Moreover, cell-free assays and competition experiments revealed preferential binding of FXIIa to HS and heparin over dextran sulfate, dermatan sulfate, and chondroitin sulfate A and C. Finally, we demonstrate that fibroblasts isolated from the lungs of the patients suffering from idiopathic pulmonary fibrosis (IPF) exhibit enhanced FXIIa binding capacity. Increased sulfation of HS resulting from elevated HS 6-O-sulfotransferase-1 expression in IPF HLF accounted, in part, for this phenomenon. Application of RNA interference technology and inhibitors of intracellular sulfation revealed the cooperative action of cell surface-associated HS and urokinase-type plasminogen activator receptor in the accumulation of FXIIa on the cell surface of IPF HLF. Moreover, FXIIa stimulated IPF HLF migration, which was abrogated by pretreatment of cells with heparinase I. Collectively, our study uncovers a novel role of HS-type glycosaminoglycans in a local accumulation of FXIIa on the cell membrane. The enhanced association of FXIIa with IPF HLF suggests its contribution to fibrogenesis.

Glucocorticoids recruit Tgfbr3 and Smad1 to shift transforming growth factor-ß signaling from the Tgfbr1/Smad2/3 axis to the Acvrl1/Smad1 axis in lung fibroblasts.

Glucocorticoids represent the mainstay therapy for many lung diseases, providing outstanding management of asthma but performing surprisingly poorly in patients with acute respiratory distress syndrome, chronic obstructive pulmonary disease, lung fibrosis, and blunted lung development associated with bronchopulmonary dysplasia in preterm infants. TGF-ß is a pathogenic mediator of all four of these diseases, prompting us to explore glucocorticoid/TGF-ß signaling cross-talk. Glucocorticoids, including dexamethasone, methylprednisolone, budesonide, and fluticasone, potentiated TGF-ß signaling by the Acvrl1/Smad1/5/8 signaling axis and blunted signaling by the Tgfbr1/Smad2/3 axis in NIH/3T3 cells, as well as primary lung fibroblasts, smooth muscle cells, and endothelial cells. Dexamethasone drove expression of the accessory type III TGF-ß receptor Tgfbr3, also called betaglycan. Tgfbr3 was demonstrated to be a "switch" that blunted Tgfbr1/Smad2/3 and potentiated Acvrl1/Smad1 signaling in lung fibroblasts. The Acvrl1/Smad1 axis, which was stimulated by dexamethasone, was active in lung fibroblasts and antagonized Tgfbr1/Smad2/3 signaling. Dexamethasone acted synergistically with TGF-ß to drive differentiation of primary lung fibroblasts to myofibroblasts, revealed by acquisition of smooth muscle actin and smooth muscle myosin, which are exclusively Smad1-dependent processes in fibroblasts. Administration of dexamethasone to live mice recapitulated these observations and revealed a lung-specific impact of dexamethasone on lung Tgfbr3 expression and phospho-Smad1 levels in vivo. These data point to an interesting and hitherto unknown impact of glucocorticoids on TGF-ß signaling in lung fibroblasts and other constituent cell types of the lung that may be relevant to lung physiology, as well as lung pathophysiology, in terms of drug/disease interactions.

From plasminogen to plasmin: role of plasminogen receptors in human cancer.

Cell surface-associated proteolysis mediated by plasmin (PLA) is an essential feature of wound healing, angiogenesis and cell invasion, processes that are dysregulated in cancer development, progression and systemic spread. The generation of PLA, initiated by the binding of its precursor plasminogen (PLG) to the cell surface, is regulated by an array of activators, inhibitors and receptors. In this review, we will highlight the importance of the best-characterized components of the PLG/PLA cascade in the pathogenesis of cancer focusing on the role of the cell surface-PLG receptors (PLG-R). PLG-R overexpression has been associated with poor prognosis of cancer patients and resistance to chemotherapy. We will also discuss recent findings on the molecular mechanisms regulating cell surface expression and distribution of PLG-R.

Loss of LRP1 promotes acquisition of contractile-myofibroblast phenotype and release of active TGF-ß1 from ECM stores.

In healing tissue, fibroblasts differentiate to α-smooth muscle actin (SMA)-expressing contractile-myofibroblasts, which pull the wound edges together ensuring proper tissue repair. Uncontrolled expansion of the myofibroblast population may, however, lead to excessive tissue scarring and finally to organ dysfunction. Here, we demonstrate that the loss of low-density lipoprotein receptor-related protein (LRP) 1 overactivates the JNK1/2-c-Jun-Fra-2 signaling pathway leading to the induction of α-SMA and periostin expression in human lung fibroblasts (hLF). These changes are accompanied by increased contractility of the cells and the integrin- and protease-dependent release of active transforming growth factor (TGF)-ß1 from the extracellular matrix (ECM) stores. Liberation of active TGF-ß1 from the ECM further enhances α-SMA and periostin expression thus accelerating the phenotypic switch of hLF. Global gene expression profiling of LRP1-depleted hLF revealed that the loss of LRP1 affects cytoskeleton reorganization, cell-ECM contacts, and ECM production. In line with these findings, fibrotic changes in the skin and lung of Fra-2 transgenic mice were associated with LRP1 depletion and c-Jun overexpression. Altogether, our results suggest that dysregulation of LRP1 expression in fibroblasts in healing tissue may lead to the unrestrained expansion of contractile myofibroblasts and thereby to fibrosis development. Further studies identifying molecules, which regulate LRP1 expression, may provide new therapeutic options for largely untreatable human fibrotic diseases.

LRP1 promotes synthetic phenotype of pulmonary artery smooth muscle cells in pulmonary hypertension.

Pulmonary hypertension (PH) is characterized by a thickening of the distal pulmonary arteries caused by medial hypertrophy, intimal proliferation and vascular fibrosis. Low density lipoprotein receptor-related protein 1 (LRP1) maintains vascular homeostasis by mediating endocytosis of numerous ligands and by initiating and regulating signaling pathways. Here, we demonstrate the increased levels of LRP1 protein in the lungs of idiopathic pulmonary arterial hypertension (IPAH) patients, hypoxia-exposed mice, and monocrotaline-treated rats. Platelet-derived growth factor (PDGF)-BB upregulated LRP1 expression in pulmonary artery smooth muscle cells (PASMC). This effect was reversed by the PDGF-BB neutralizing antibody or the PDGF receptor antagonist. Depletion of LRP1 decreased proliferation of donor and IPAH PASMC in a ß1-integrin-dependent manner. Furthermore, LRP1 silencing attenuated the expression of fibronectin and collagen I and increased the levels of α-smooth muscle actin and myocardin in donor, but not in IPAH, PASMC. In addition, smooth muscle cell (SMC)-specific LRP1 knockout augmented α-SMA expression in pulmonary vessels and reduced SMC proliferation in 3D ex vivo murine lung tissue cultures. In conclusion, our results indicate that LRP1 promotes the dedifferentiation of PASMC from a contractile to a synthetic phenotype thus suggesting its contribution to vascular remodeling in PH.

Inactivation of nuclear histone deacetylases by EP300 disrupts the MiCEE complex in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and highly lethal lung disease with unknown etiology and poor prognosis. IPF patients die within 2 years after diagnosis mostly due to respiratory failure. Current treatments against IPF aim to ameliorate patient symptoms and to delay disease progression. Unfortunately, therapies targeting the causes of or reverting IPF have not yet been developed. Here we show that reduced levels of miRNA lethal 7d (MIRLET7D) in IPF compromise epigenetic gene silencing mediated by the ribonucleoprotein complex MiCEE. In addition, we find that hyperactive EP300 reduces nuclear HDAC activity and interferes with MiCEE function in IPF. Remarkably, EP300 inhibition reduces fibrotic hallmarks of in vitro (patient-derived primary fibroblast), in vivo (bleomycin mouse model), and ex vivo (precision-cut lung slices, PCLS) IPF models. Our work provides the molecular basis for therapies against IPF using EP300 inhibition.

Factor XII in coagulation, inflammation and beyond.

Factor XII (FXII) is a protease that is mainly produced in the liver and circulates in plasma as a single chain zymogen. Following contact with negatively charged surfaces, FXII is converted into the two-chain active form, FXIIa. FXIIa initiates the intrinsic blood coagulation pathway via activation of factor XI. Furthermore, it converts plasma prekallikrein to kallikrein (PK), which reciprocally activates FXII and liberates bradykinin from high molecular weight kininogen. In addition, FXIIa initiates fibrinolysis via PK-mediated urokinase activation and activates the classical complement pathway. Even though the main function of FXII seems to relate to the activation of the intrinsic coagulation pathway and the kallikrein-kinin system, a growing body of evidence suggests that FXII may also directly regulate cellular responses. In this regard, it has been found that FXII/FXIIa induces the expression of inflammatory mediators, promotes cell proliferation, and enhances the migration of neutrophils and lung fibroblasts. In addition, it has been reported that genetic ablation of FXII protects against neuroinflammation, reduces the formation of atherosclerotic lesions in Apoe-/- mice, improves wound healing, and inhibits postnatal angiogenesis. Although the aforementioned effects can be partially explained by the downstream products of FXII activation, the ability of FXII/FXIIa to directly regulate cellular responses has recently emerged as an alternative hypothesis. These direct cellular reactions to FXII/FXIIa will be discussed in the review.

LRP1: A chameleon receptor of lung inflammation and repair.

The lung displays a remarkable capability to regenerate following injury. Considerable effort has been made thus far to understand the cardinal processes underpinning inflammation and reconstruction of lung tissue. However, the factors determining the resolution or persistence of inflammation and efficient wound healing or aberrant remodeling remain largely unknown. Low density lipoprotein receptor-related protein 1 (LRP1) is an endocytic/signaling cell surface receptor which controls cellular and molecular mechanisms driving the physiological and pathological inflammatory reactions and tissue remodeling in several organs. In this review, we will discuss the impact of LRP1 on the consecutive steps of the inflammatory response and its role in the balanced tissue repair and aberrant remodeling in the lung.

Biomarkers in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, debilitating, fibrotic lung disease leading to respiratory failure and ultimately to death. Being the prototype of interstitial lung diseases, IPF is characterized by marked heterogeneity regarding its clinical course. Despite significant progress in the understanding of its pathogenesis, we still cannot reliably predict the course of the disease and the response to treatment of an individual patient. Non-invasive biomarkers, in particular serum biomarkers, for the (early) diagnosis, differential diagnosis, prognosis and prediction of therapeutic response are urgently needed. Numerous molecules involved in alveolar epithelial cell injury, fibroproliferation and matrix remodeling as well as immune regulation have been proposed as potential biomarkers. Furthermore, genetic variants of TOLLIP, MUC5B, and other genes are associated with a differential response to treatment and with the development and/or the prognosis of IPF. Additionally, the bacterial signature in IPF lungs, as shown from microbiome analyses, as well as mitochondrial DNA seem to have promising roles as biomarkers. Moreover, combination of multiple biomarkers may identify comprehensive biomarker signatures in IPF patients. However, there is still a long way until these potential biomarkers complete or substitute for the clinical and functional parameters currently available for IPF.

Coagulation factor XII regulates inflammatory responses in human lungs.

Increased procoagulant activity in the alveolar compartment and uncontrolled inflammation are hallmarks of the acute respiratory distress syndrome (ARDS). Here, we investigated whether the contact phase system of coagulation is activated and may regulate inflammatory responses in human lungs. Components of the contact phase system were characterized in bronchoalveolar lavage fluids (BALF) from 54 ARDS patients and 43 controls, and their impact on cytokine/chemokine expression in human precision cut lung slices (PCLS) was assessed by a PCR array. Activation of the contact system, associated with high levels of coagulation factor XIIa (Hageman factor, FXIIa), plasma kallikrein and bradykinin, occurred rapidly in ARDS lungs after the onset of the disease and virtually normalized within one week from time of diagnosis. FXII levels in BALF were higher in ARDS non-survivors than survivors and were positively correlated with tumor necrosis factor (TNF)-α concentration. FXII induced the production and release of interleukin (IL)-8, IL-1ß, IL-6, leukemia inhibitory factor (LIF), CXCL5 and TNF-α in human PCLS in a kallikrein-kinin-independent manner. In conclusion, accumulation of FXII in ARDS lungs may contribute to the release of pro-inflammatory mediators and is associated with clinical outcome. FXII inhibition may thus offer a novel and promising therapeutic approach to antagonize overwhelming inflammatory responses in ARDS lungs without interfering with vital haemostasis.

FXYD1 negatively regulates Na(+)/K(+)-ATPase activity in lung alveolar epithelial cells.

Acute respiratory distress syndrome (ARDS) is clinical syndrome characterized by decreased lung fluid reabsorption, causing alveolar edema. Defective alveolar ion transport undertaken in part by the Na(+)/K(+)-ATPase underlies this compromised fluid balance, although the molecular mechanisms at play are not understood. We describe here increased expression of FXYD1, FXYD3 and FXYD5, three regulatory subunits of the Na(+)/K(+)-ATPase, in the lungs of ARDS patients. Transforming growth factor (TGF)-ß, a pathogenic mediator of ARDS, drove increased FXYD1 expression in A549 human lung alveolar epithelial cells, suggesting that pathogenic TGF-ß signaling altered Na(+)/K(+)-ATPase activity in affected lungs. Lentivirus-mediated delivery of FXYD1 and FXYD3 allowed for overexpression of both regulatory subunits in polarized H441 cell monolayers on an air/liquid interface. FXYD1 but not FXYD3 overexpression inhibited amphotericin B-sensitive equivalent short-circuit current in Ussing chamber studies. Thus, we speculate that FXYD1 overexpression in ARDS patient lungs may limit Na(+)/K(+)-ATPase activity, and contribute to edema persistence.