Lukasz Wujak

Heparan Sulfate Proteoglycans Mediate Factor XIIa Binding to the Cell Surface

Background: Factor XIIa (FXIIa) binds to the cell surface; however, the underlying mechanism remains underexplored. Results: Heparan sulfate (HS) enhances FXIIa binding capacity and consequently migration of human lung fibroblasts (HLF) isolated from fibrotic lungs. Conclusion: HS is responsible for local accumulation of FXIIa on the cell surface. Significance: Enhanced association of FXIIa with HLF derived from diseased lungs suggests its role in fibrogenesis. 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.

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‐β1 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‐β signaling, our findings suggest that pirfenidone exerts its clinically beneficial effects through dual Hh/TGF‐β 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. FASEB J. 31, 1916–1928 (2017). www.fasebj.org

Antihistone Properties of C1 Esterase Inhibitor Protect against Lung Injury

&NA; 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.

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.

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.

Low density lipoprotein receptor-related protein 1 couples β1 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 β1 integrin and thus supports cell adhesion, spreading, migration and integrin signaling on fibronectin. LRP1 interacts with surface β1 integrin, binds the integrin activator kindlin2 and stimulates β1 integrin–kindlin2 complex formation. Specifically, serine 76 in the LRP1 cytoplasmic tail is crucial for the interaction with kindlin2, β1 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 β1 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.

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.

Transcriptome profiling reveals the complexity of pirfenidone effects in IPF

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. Pirfenidones mode of action in human lungs involves a complex interactome comprising genes related to inflammation and extracellular matrix architecture http://ow.ly/26NN30lpGON

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.