Podosomes, specialized actin‐rich structures in macrophages (Mfs), degrade the extra‐cellular matrix (ECM) and are involved in cell migration. On two‐dimensional (2D) surfaces Mfs form spot‐like podosomes at the ventral cell surface that develop into protrusive structures in a three‐dimensional (3D) environment resembling the ECM. We have shown that the tips of these protrusive podosomes are characterized by increased accumulation of cysteine cathepsins (Cts) B, X, S, H, and L, both in human blood Mfs and in human monocytic cell line U‐937. Monocyte‐to‐Mf differentiation induces an increase in cysteine cathepsin expression and activity, promoting their translocation to the cell surface, where they interact with ECM. This group of proteases is crucial for the extracellular as well as intracellular degradation of ECM, as demonstrated by quantitative monitoring of collagen IV degradation. Furthermore, inhibiting CtsB, X, and S significantly impairs Mf invasion through the 3D matrix. Time‐lapse live‐cell imaging of CtsB activity revealed that the extracellular and the intracellular ECM degradation are associated with extensive endocytosis at the tip of protrusive podosomes. The targeting of cysteine cathepsins, as the major mediators of human Mf 3D invasion, could be an approach to the treatment of inflammatory and cancerous diseases.
Patients with chronic obstructive pulmonary disease (COPD) frequently suffer from chronic bronchitis (CB) and display steroid-resistant inflammation with increased sputum neutrophils and macrophages. Recently, a causal link between mucus hyper-concentration and disease progression of CB has been suggested.In this study, we have evaluated the steroid sensitivity of purified, patient-derived sputum and alveolar macrophages and used a novel mechanistic cross-talk assay to examine how macrophages and bronchial epithelial cells cross-talk to regulate MUC5B production.We demonstrate that sputum plug macrophages isolated from COPD patients with chronic bronchitis (COPD/CB) are chronically activated and only partially respond to ex vivo corticosteroid treatment compared to alveolar macrophages isolated from lung resections. Further, we show that pseudo-stratified bronchial epithelial cells grown in air-liquid-interface are inert to direct bacterial lipopolysaccharide stimulation and that macrophages are able to relay this signal and activate the CREB/AP-1 transcription factor complex and subsequent MUC5B expression in epithelial cells through a soluble mediator. Using recombinant protein and neutralizing antibodies, we identified a key role for TNFα in this cross-talk.For the first time, we describe ex vivo pharmacology in purified human sputum macrophages isolated from chronic bronchitis COPD patients and identify a possible basis for the steroid resistance frequently seen in this population. Our data pinpoint a critical role for chronically activated sputum macrophages in perpetuating TNFα-dependent signals driving mucus hyper-production. Targeting the chronically activated mucus plug macrophage phenotype and interfering with aberrant macrophage-epithelial cross-talk may provide a novel strategy to resolve chronic inflammatory lung disease.
Cathepsin X is a lysosomal cysteine protease, found predominantly in cells of monocyte/macrophage lineage. It acts as a monocarboxypepidase and has a strict positional and narrower substrate specificity relative to the other human cathepsins. In our recent studies we identified β2 subunit of integrin receptors and α and γ enolase as possible substrates for cathepsin X carboxypeptidase activity. In both cases cathepsin X is capable to cleave regulatory motifs at C-terminus affecting the function of targeted molecules. We demonstrated that via activation of β2 integrin receptor Mac-1 (CD11b/CD18) active cathepsin X enhances adhesion of monocytes/macrophages to fibrinogen and regulates the phagocytosis. By activation of Mac-1 receptor cathepsin X may regulate also the maturation of dendritic cells, a process, which is crucial in the initiation of adaptive immunity. Cathepsin X activates also the other β2 integrin receptor, LFA-1 (CD11a/CD18) which is involved in the proliferation of T lymphocytes. By modulating the activity of LFA-1 cathepsin X causes cytoskeletal rearrangements and morphological changes of T lymphocytes enhancing ameboid-like migration in 2-D and 3-D barriers and increasing homotypic aggregation. The cleavage of C-terminal amino acids of α and γ enolase by cathepsin X abolishes their neurotrophic activity affecting neuronal cell survival and neuritogenesis.
Summary Cathepsin X is a lysosomal, cysteine dependent carboxypeptidase. Its expression is restricted to cells of the immune system, suggesting a function related to the processes of inflammatory and immune responses. It has been shown to stimulate macrophage antigen‐1 (Mac‐1) receptor‐dependent adhesion and phagocytosis via interaction with integrin β 2 subunit. Here its potential role in regulating lymphocyte proliferation via Mac‐1 and the other β 2 integrin receptor, lymphocyte function‐associated antigen‐1 (LFA‐1) has been investigated. Cathepsin X has been shown to suppress proliferation of human peripheral blood mononuclear cells, by activation of Mac‐1, known as a suppressive factor for lymphocyte proliferation. On the other hand, co‐localization of cathepsin X and LFA‐1 supports the role of cathepsin X in regulating LFA‐1 activity, which enhances lymphocyte proliferation. As shown by fluorescence resonance energy transfer, using U‐937 and Jurkat cells transfected with α L ‐mCFP and β 2 ‐mYFP, recombinant cathepsin X directly activates LFA‐1. The activation was confirmed by increased binding of monoclonal antibody 24, recognizing active LFA‐1. We demonstrate that cathepsin X is involved in the regulation of two β 2 integrin receptors, LFA‐1 and Mac‐1, which exhibit opposing roles in lymphocyte activation.
Myofibroblasts, considered main drivers in IPF, gather in fibrotic foci where adjacent macrophages secrete mediators affecting their function. Of special interest is PGE2, which acutely peaks during early inflammation and reemerges with extended high levels in the post-resolution phase. Here, we mimic the resolution milieu by chronic high dose PGE2 to study transcriptomic, metabolic and phenotypic changes in IPF myofibroblasts and macrophages. TGF-β1-induced myofibroblasts were treated with 500 nM PGE2 and global transcription was studied by RNA-Seq. High content imaging was used to confirm results in a myofibroblast reversal assay and fibroblast metabolic changes were studied by Seahorse analysis. PGE2-treated IPF BAL macrophages were analyzed by a fibrosis and resolution-focused QuantiGene RNA assay. PGE2 reverted 156 of the 392 genes changed by TGF-β1 in fibroblasts, including genes encoding αSMA, Col I and PAI-1 and the phenotypic myofibroblast reversal assay showed a significant decrease of αSMA positive IPF myofibroblasts. Augmented glycolysis and increased fatty acid oxidation (FAO) was observed in TGF-β1-derived myofibroblasts. Both glycolysis and exogenous FAO rate was reduced upon prolonged PGE2 treatment. PGE2 also had a profound effect on macrophage phenotype with a reduction in key inflammatory and pro-fibrotic genes, while upregulating resolution-type markers. Our results show the importance of PGE2 in driving an anti-fibrotic phenotypic switch in IPF and mechanistic knock-down studies of PGE2-inducible genes are ongoing to identify the key factors involved. Activation of this process may lead to new therapies targeting fibrotic disease.
Cathepsin X is a lysosomal cysteine protease exhibiting carboxypeptidase activity. Its expression is high in the cells of immune system and its function has been related to the processes of inflammatory and immune responses. It regulates processes such as adhesion, T lymphocyte activation and phagocytosis through its interaction with beta2 integrins. To investigate the role of cathepsin X in the migration of T lymphocytes, Jurkat T lymphocytes were stably transfected with a pcDNA3 expression vector containing cathepsin X cDNA. The cathepsin-X-overexpressing T lymphocytes exhibited polarised migration-associated morphology, enhanced migration on 2D and 3D models using intercellular adhesion molecule 1 (ICAM1)- and Matrigel-coated surfaces, and increased homotypic aggregation. The increased invasiveness of cathepsin-X-overexpressing cells does not involve proteolytic degradation of extracellular matrix. Confocal microscopy showed that the active mature form of cathepsin X was colocalised in migrating cells together with lymphocyte-function-associated antigen 1 (LFA-1). The colocalisation was particularly evident at the trailing edge protrusion, the uropod, that has an important role in T lymphocyte migration and cell-cell interactions. We propose that cathepsin X causes cytoskeletal rearrangements and stimulates migration of T lymphocytes by modulating the activity of the beta2 integrin receptor LFA-1.
Smoking is a risk factor for idiopathic pulmonary fibrosis (IPF), but the mechanism of the association remains unknown. We show that cigarette smoke extract (CSE) induces extensive translocation of the RNA regulatory protein HuR/ELAVL1 from the nucleus to the cytoplasm in TGFβ1-activated lung fibroblasts. Increased levels of cytoplasmic HuR are associated with different pathological processes and our aim was to investigate the multi-level RNA regulative networks of CSE-induced cytoplasmic HuR in lung fibrosis. Identification of HuR-specific RNA targets, explored by CLIP (cross-linking and immunoprecipitation) and subsequent high-throughput sequencing, revealed several fibrotic genes, including COL1A1 and COL1A2, as some of the most frequently bound targets in the cytoplasm. Evaluation of HuR-bound RNA sequences showed a difference in 4-meric binding motifs between nuclear and cytoplasmic HuR fractions, with increased binding to polyU-rich RNA motifs in the cytoplasm. Interestingly, the polyU-rich binding was reduced upon TGFβ1/CSE stimulation. Additionally, the TGFβ1/CSE stimulation led to reduced binding of cytoplasmic HuR to 3'UTR RNA regions and increased interaction with the coding regions. This suggests a global shift of HuR function from 3'UTR-dependent RNA stabilization in the direction of potential translational regulation of genes associated with fibrosis.
Cathepsin B (EC 3.4.22.1) is a lysosomal cysteine protease with both endopeptidase and exopeptidase activity. The former is associated with the degradation of the extracellular matrix proteins, which is a process required for tumour cell invasion and metastasis. In the present study, we show that 2A2 monoclonal antibody, raised by our group, is able to regulate cathepsin B activity. The EPGYSP sequence, located between amino acid residues 133-138 of cathepsin B in the proximity of the occluding loop, was determined to be the epitope for 2A2 monoclonal antibody using SPOT analysis. By surface plasmon resonance, an equilibrium dissociation constant (Kd) of 4.7 nM was determined for the interaction between the nonapeptide CIAEPGYSP, containing the epitope sequence, and 2A2 monoclonal antibody. 2A2 monoclonal antibody potentiated cathepsin B exopeptidase activity with a activation constant (Ka) of 22.3 nM, although simultaneously inhibiting its endopeptidase activity. The median inhibitory concentration values for the inhibition of hydrolysis of protein substrates, BODIPY FL casein and DQ-collagen IV were 761 and 702 nM, respectively. As observed by native gel electrophoresis and gel filtration, the binding of 2A2 monoclonal antibody to the cathepsin B/cystatin C complex caused the dissociation of cystatin C from the complex. The results obtained in the present study suggest that, upon binding, the 2A2 monoclonal antibody induces a conformational change in cathepsin B, stabilizing its exopeptidase conformation and thus disabling its harmful action associated with its endopeptidase activity.
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