The exaggerated expression of chitinase-like protein YKL-40, the human homologue of breast regression protein–39 (BRP-39), was reported in a number of diseases, including chronic obstructive pulmonary disease (COPD). However, the in vivo roles of YKL-40 in normal physiology or in the pathogenesis of specific diseases such as COPD remain poorly understood. We hypothesized that BRP-39/YKL-40 plays an important role in the pathogenesis of cigarette smoke (CS)–induced emphysema. To test this hypothesis, 10-week-old wild-type and BRP-39 null mutant mice (BRP-39−/−) were exposed to room air (RA) and CS for up to 10 months. The expression of BRP-39 was significantly induced in macrophages, airway epithelial cells, and alveolar Type II cells in the lungs of CS-exposed mice compared with RA-exposed mice, at least in part via an IL-18 signaling–dependent pathway. The null mutation of BRP-39 significantly reduced CS-induced bronchoalveolar lavage and tissue inflammation. However, CS-induced epithelial cell apoptosis and alveolar destruction were further enhanced in the absence of BRP-39. Consistent with these findings in mice, the tissue expression of YKL-40 was significantly increased in the lungs of current smokers compared with the lungs of ex-smokers or nonsmokers. In addition, serum concentrations of YKL-40 were significantly higher in smokers with COPD than in nonsmokers or smokers without COPD. These studies demonstrate a novel regulatory role of BRP-39/YKL-40 in CS-induced inflammation and emphysematous destruction. These studies also underscore that maintaining physiologic concentrations of YKL-40 in the lung is therapeutically important in preventing excessive inflammatory responses or emphysematous alveolar destruction.
YKL-40/BRP-39; COPD; emphysema; cigarette smoke
Rationale: Prolonged exposure to 100% O2 causes hyperoxic acute lung injury (HALI), characterized by alveolar epithelial cell injury and death. We previously demonstrated that the murine chitinase-like protein, breast regression protein (BRP)–39 and its human homolog, YKL-40, inhibit cellular apoptosis. However, the regulation and roles of these molecules in hyperoxia have not been addressed.
Objectives: We hypothesized that BRP-39 and YKL-40 (also called chitinase-3–like 1) play important roles in the pathogenesis of HALI.
Methods: We characterized the regulation of BRP-39 during HALI and the responses induced by hyperoxia in wild-type mice, BRP-39–null (−/−) mice, and BRP-39−/− mice in which YKL-40 was overexpressed in respiratory epithelium. We also compared the levels of tracheal aspirate YKL-40 in premature newborns with respiratory failure.
Measurements and Main Results: These studies demonstrate that hyperoxia inhibits BRP-39 in vivo in the murine lung and in vitro in epithelial cells. They also demonstrate that BRP-39−/− mice have exaggerated permeability, protein leak, oxidation, inflammatory, chemokine, and epithelial apoptosis responses, and experience premature death in 100% O2. Lastly, they demonstrate that YKL-40 ameliorates HALI, prolongs survival in 100% O2, and rescues the exaggerated injury response in BRP-39−/− animals. In accord with these findings, the levels of tracheal aspirate YKL-40 were lower in premature infants treated with hyperoxia for respiratory failure who subsequently experienced bronchopulmonary dysplasia or death compared with those that did not experience these complications.
Conclusions: These studies demonstrate that hyperoxia inhibits BRP-39/YKL-40, and that BRP-39 and YKL-40 are critical regulators of oxidant injury, inflammation, and epithelial apoptosis in the murine and human lung.
BRP-39; YKL-40; hyperoxygen; BPD; HALI
BRP-39 and its human homolog YKL-40 have been regarded as a prototype of chitinase-like proteins (CLP) in mammals. Exaggerated levels of YKL-40 protein and/or mRNA have been noted in a number of diseases characterized by inflammation, tissue remodeling, and aberrant cell growth. Asthma is an inflammatory disease characterized by airway hyperresponsiveness and airway remodeling. Recently, the novel regulatory role of BRP-39/YKL-40 in the pathogenesis of asthma has been demonstrated both in human studies and allergic animal models. The levels of YKL-40 are increased in the circulation and lungs from asthmatics where they correlate with disease severity, and CHI3L1 polymorphisms correlate with serum YKL-40 levels, asthma and abnormal lung function. Animal studies using BRP-39 null mutant mice demonstrated that BRP-39 was required for optimal allergen sensitization and Th2 inflammation. These studies suggest the potential use of BRP-39 as a biomarker as well as a therapeutic target for asthma and other allergic diseases. Here, we present an overview of chitin/chitinase biology and summarize recent findings on the role of BRP-39 in the pathogenesis of asthma and allergic responses.
BRP-39; human CHI3L1 protein; asthma; hypersensitivity
While the presence of the chitinase-like molecule YKL40 has been reported in COPD and asthma, its relevance to inflammatory processes elicited by cigarette smoke and common environmental allergens, such as house dust mite (HDM), is not well understood. The objective of the current study was to assess expression and function of BRP-39, the murine equivalent of YKL40 in a murine model of cigarette smoke-induced inflammation and contrast expression and function to a model of HDM-induced allergic airway inflammation.
CD1, C57BL/6, and BALB/c mice were room air- or cigarette smoke-exposed for 4 days in a whole-body exposure system. In separate experiments, BALB/c mice were challenged with HDM extract once a day for 10 days. BRP-39 was assessed by ELISA and immunohistochemistry. IL-13, IL-1R1, IL-18, and BRP-39 knock out (KO) mice were utilized to assess the mechanism and relevance of BRP-39 in cigarette smoke- and HDM-induced airway inflammation.
Cigarette smoke exposure elicited a robust induction of BRP-39 but not the catalytically active chitinase, AMCase, in lung epithelial cells and alveolar macrophages of all mouse strains tested. Both BRP-39 and AMCase were increased in lung tissue after HDM exposure. Examining smoke-exposed IL-1R1, IL-18, and IL-13 deficient mice, BRP-39 induction was found to be IL-1 and not IL-18 or IL-13 dependent, while induction of BRP-39 by HDM was independent of IL-1 and IL-13. Despite the importance of BRP-39 in cellular inflammation in HDM-induced airway inflammation, BRP-39 was found to be redundant for cigarette smoke-induced airway inflammation and the adjuvant properties of cigarette smoke.
These data highlight the contrast between the importance of BRP-39 in HDM- and cigarette smoke-induced inflammation. While functionally important in HDM-induced inflammation, BRP-39 is a biomarker of cigarette smoke induced inflammation which is the byproduct of an IL-1 inflammatory pathway.
We previously reported that YKL-40, the human analog of mouse breast regression protein-39 (BRP-39; chitinase 3-like 1), is elevated in the cerebrospinal fluid of patients with a variety of neuroinflammatory conditions, such as multiple sclerosis and traumatic brain injury. YKL-40 expression in the CNS was predominantly associated with reactive astrocytes in the vicinity of inflammatory lesions. Because previous studies have shown that reactive astrocytes play a critical role in limiting immune infiltration in the mouse model of experimental autoimmune encephalomyelitis (EAE), we explored the role of BRP-39 in regulating neuroinflammation in EAE. Using BRP-39-deficient mice (BRP-39−/−), we demonstrate the importance of BRP-39 in modulating the severity of clinical EAE and CNS neuroinflammation. At disease onset, absence of BRP-39 had little effect on clinical disease or lymphocytic infiltrate, but by 14 days post-immunization (dpi), differences in clinical scores were evident. By 28 dpi, BRP-39−/− mice showed more severe and persistent clinical disease than BRP-39+/+ controls. Histopathological evaluation showed that BRP-39−/− mice had more marked lymphocytic and macrophage infiltrates and gliosis vs. BRP-39+/+ mice. These findings support the role of BRP-39 expression in limiting immune cell infiltration into the CNS and offer a new target to modulate neuroinflammation.
BRP-39; Chitinase-like proteins; Experimental autoimmune encephalomyelitis; Multiple sclerosis; Neuroimmunology; YKL-40
The chitinase-like protein YKL-40 was found to be increased in patients with severe asthma and chronic obstructive pulmonary disease (COPD), two disease conditions featuring neutrophilic infiltrates. Based on these studies and a previous report indicating that neutrophils secrete YKL-40, we hypothesized that YKL-40 plays a key role in cystic fibrosis (CF) lung disease, a prototypic neutrophilic disease. The aim of this study was (i) to analyze YKL-40 levels in human and murine CF lung disease and (ii) to investigate whether YKL-40 single-nucleotide polymorphisms (SNPs) modulate CF lung disease severity. YKL-40 protein levels were quantified in serum and sputum supernatants from CF patients and control individuals. Levels of the murine homologue BRP-39 were analyzed in airway fluids from CF-like βENaC-Tg mice. YKL-40SNPs were analyzed in CF patients. YKL-40 levels were increased in sputum supernatants and in serum from CF patients compared to healthy control individuals. Within CF patients, YKL-40 levels were higher in sputum than in serum. BRP-39 levels were increased in airways fluids from βENaC-Tg mice compared to wild-type littermates. In both CF patients and βENaC-Tg mice, YKL-40/BRP-39 airway levels correlated with the severity of pulmonary obstruction. Two YKL-40 SNPs (rs871799 and rs880633) were found to modulate age-adjusted lung function in CF patients. YKL-40/BRP-39 levelsare increased in human and murine CF airway fluids, correlate with pulmonary function and modulate CF lung disease severity genetically. These findings suggest YKL-40 as a potential biomarker in CF lung disease.
To determine the roles of breast regression protein-39 (BRP-39) in regulating dendritic cell maturation and in pathology of acute asthma.
Mouse bone marrow-derived dendritic cells (BMDCs) were prepared, and infected with adenovirus over-expressing BRP-39. Ovalbumin (OVA)-induced murine model of acute asthma was made in female BALB/c mice by sensitizing and challenging with chicken OVA and Imject Alum. The transfected BMDCs were adoptively transferred into OVA-treated mice via intravenous injection. Airway hyperresponsiveness (AHR), inflammation and pulmonary histopathology were characterized.
The expression of BRP-39 mRNA and protein was significantly increased in lung tissues of OVA-treated mice. The BMDCs infected with adenovirus BRP-39 exhibited greater maturation and higher activity in vitro. Adoptive transfer of the cells into OVA-treated mice significantly augmented OVA-induced AHR and eosinophilic inflammation. Meanwhile, BRP-39 further enhanced the production of OVA-induced Th2 cytokines IL-4, IL-5 and IL-13, but significantly attenuated OVA-induced IFN-γ production in bronchoalveolar lavage fluid.
In OVA-induced murine model of acute asthma, BRP-39 is over-expressed in lung tissue and augments Th2 inflammatory response and AHR. BRP-39 promotes dendritic cell maturation in vitro. Therefore, BRP-39 may be a potential therapeutic target of asthma.
asthma; ovalbumin; bone marrow-derived dendritic cells (BMDCs); breast regression protein-39 (BRP-39); YKL-40; Th2 inflammation; airway hyperresponsiveness; bronchoalveolar lavage fluid
The 18 glycosyl hydrolase family of chitinases is an ancient gene family that is widely expressed from prokaryotes to eukaryotes. In mammals, despite the absence of endogenous chitin, a number of chitinases and chitinase-like proteins (C/CLPs) have been identified. However, their roles have only recently begun to be elucidated. Acidic mammalian chitinase (AMCase) inhibits chitin-induced innate inflammation; augments chitin-free, allergen-induced Th2 inflammation; and mediates effector functions of IL-13. The CLPs BRP-39/YKL-40 (also termed chitinase 3-like 1) inhibit oxidant-induced lung injury, augments adaptive Th2 immunity, regulates apoptosis, stimulates alternative macrophage activation, and contributes to fibrosis and wound healing. In accord with these findings, levels of YKL-40 in the lung and serum are increased in asthma and other inflammatory and remodeling disorders and often correlate with disease severity. Our understanding of the roles of C/CLPs in inflammation, tissue remodeling, and tissue injury in health and disease is reviewed below.
asthma; fibrosis; BRP-39/YKL-40; AMCase; chitotriosidase
YKL-39 is a Glyco_18 domain containing chitinase-like protein which is currently recognized as a biomarker for the activation of chondrocytes and the progress of the osteoarthritis in human. YKL-39 was identified as an abundantly secreted protein in primary culture of human articular chondrocytes. Two biological activities of YKL-39 might contribute to the disease progression. One is the induction of autoimmune response and second is the participation in tissue remodeling. Other mammalian chitinase-like proteins including chitotriosidase, SI-CLP, YKL-40 and YM1 are expressed by macrophages in various pathological conditions. In contrast, YKL-39 was never reported to be produced by macrophages. We used in vitro model of human monocyte-derived macrophage differentiation to analyse regulation of YKL-39 expression. Expression of YKL-39 was examined by real-time RT-PCR. CD14+ MACS sorted human monocytes differentiated for 6 days under different stimulations including IFNγ, IL-4, dexamethasone and TGF-β. We found that both IL-4 and TGF-β have weak stimulatory effect on YKL-39 expression in all donors tested (3.2 ± 1.7 fold, p = 0.006 and 6.3 ± 3.1 fold, p = 0.014 respectively). However the combination of IL-4 and TGF-β had strong stimulatory effect on the expression of YKL-39 in all analysed individual macrophage cultures (34 ± 36 fold, p = 0.05). IFN-γ did not show statistically significant effect of YKL-39 mRNA expression. Presence of dexamethasone almost completely abolished the stimulatory effects of IL-4 and TGF-β. In summary, we show here for the first time, that human cells of monocyte origin are able to produce YKL-39. Maturation of monocyte derived macrophages in the presence of Th2 cytokine IL-4 and TGF-β leads to the strong activation of YKL-39 expression. Thus elevated levels of YKL-39 observed during chronic inflammations can not be attributed solely to the activity of chondrocytes. In perspective, YKL-39 might serve as a useful biomarker to detect macrophage-specific response in pathologies like tumour, atherosclerosis and Alzheimer disease.
osteoarthritis; chitinase; YKL-39; macrophage; TGF-beta; IL-4
Mice and humans produce chitinase-like proteins (CLPs), which are highly homologous to chitinases but lack chitinolytic activity. Mice express primarily three CLPs, including breast regression protein-39 (BRP-39) [chitinase 3-like-1 (Chi3l1) or 38-kDa glycoprotein (gp38k)], Ym1 (Chi3l3) and Ym2 (Chi3l4). Recently, CLPs have attracted considerable attention due to their increased expression in a number of pathological conditions, including asthma, allergies, rheumatoid arthritis and malignant tumors. Although the exact functions of CLPs are largely unknown, the significance of their increased expression levels during pathophysiological states needs to be determined. The quantification of BRP-39, Ym1 and Ym2 is an important step in gaining insight into the in vivo regulation of the CLPs.
We constructed a standard DNA for quantitative real-time PCR (qPCR) by containing three CLPs target fragments and five reference genes cDNA in a one-to-one ratio. We evaluated this system by analyzing the eight target cDNA sequences. Tissue cDNAs obtained by reverse transcription from total RNA from four embryonic stages and eight adult tissues were analyzed using the qPCR system with the standard DNA.
We established a qPCR system detecting CLPs and comparing their expression levels with those of five reference genes using the same scale in mouse tissues. We found that BRP-39 and Ym1 were abundant in the mouse lung, whereas Ym2 mRNA was abundant in the stomach, followed by lung. The expression levels of BRP-39 and Ym1 in the mouse lung were higher than those of two active chitinases and were comparable to glyceraldehyde-3-phosphate dehydrogenase, a housekeeping gene which is constitutively expressed in all tissues.
Our results indicate that catalytically inactive BRP-39 and Ym1 are constitutive genes in normal mouse lung.
BRP-39; Chitinase; Chitinase-like protein; Gene expression analysis; Quantitative real-time PCR system; Ym1; Ym2
The aim of this study was to investigate the hepatoprotective effect of BRP, a polysaccharide fraction isolated from Boschniakia rossica, against galactosamine and lipopolysaccharide induced fulminant hepatic failure. Mice were injected with a single dose of galactosamine/lipopolysaccharide with or without pretreatment of BRP. Results showed marked reduction of hepatic necrosis, serum marker enzymes and levels of tumor necrosis factor-α and interleukin-6 in BRP pretreated mice when compared with galactosamine/lipopolysaccharide-challenged mice. Mice pretreated with BRP decreased the activation of caspases-3 and caspase-8, and showed a reduced level of DNA fragmentation of liver cells. BRP also reduced hepatic lipid peroxidation, increased potential of hepatic antioxidative defense system, and reduced hepatic nitric oxide level which was elevated by galactosamine/lipopolysaccharide injection. Immunoblot analysis showed down-regulation of inducible nitric oxide synthase and cyclooxygenase-2 proteins of liver tissues in BRP pretreated group when compared with galactosamine/lipopolysaccharide-challenged group. Furthermore, treatment with galactosamine/lipopolysaccharide markedly increased toll-like receptor 4, nuclear level of nuclear factor-κB, and phosphorylation of both extracellular signal-regulated kinase and c-Jun N-terminal kinase in liver tissues. However, these increases were attenuated by pretreatment with BRP. The results suggest that BRP alleviates galactosamine/lipopolysaccharide-induced liver injury by enhancing antioxidative defense system, suppressing inflammatory responses and reducing apoptotic signaling.
Boschniakia rossica; polysaccharide; hepatic failure; mice
YKL-40 (YKL for the first three N-terminal residues of a 40 kDa protein) belongs to a group of human chitinase-like proteins (CLPs), which are similar to chitinases but lack chitinolytic activity. YKL-40 mRNA and its protein levels have been reported elevated in multiple disorders including asthma, cystic fibrosis, rheumatoid arthritis and malignant tumors. Here, we quantified the YKL-40 mRNA levels and compared them with chitinases and housekeeping genes in normal human tissues. To establish the quantitative real-time PCR (qPCR) system for evaluation of relative YKL-40 mRNA levels, we constructed a human standard DNA molecule by ligating cDNAs of YKL-40, two mammalian chitinases and two housekeeping genes in a one-to-one ratio. We generated cDNAs from various normal human tissues and analyzed the YKL-40 mRNA expression levels using a qPCR system with the standard DNA. We found that YKL-40 mRNA is present widely in human tissues while its expression patterns exhibit clear tissue specificity. Highest YKL-40 mRNA levels were detected in the liver, followed by kidney, trachea and lung. The levels of YKL-40 mRNA in the kidney and liver were more than 100-times higher than those of chitotriosidase mRNA. Our study provides for the first time a comprehensive analysis of the relative expression levels of YKL-40 mRNA versus mammalian chitinases in normal human tissues.
asthma; chitinase; chitinase-like protein; cystic fibrosis; gene expression analysis; malignant tumors; normal human tissues; quantitative real-time PCR system; rheumatoid arthritis; YKL-40
Previous studies have shown that BrpA plays a major role in acid and oxidative stress tolerance and biofilm formation by Streptococcus mutans. Mutant strains lacking BrpA also display increased autolysis and decreased viability, suggesting a role for BrpA in cell envelope integrity. In this study, we examined the impact of BrpA deficiency on cell envelope stresses induced by envelope-active antimicrobials. Compared to the wild-type strain UA159, the BrpA-deficient mutant (TW14D) was significantly more susceptible to antimicrobial agents, especially lipid II inhibitors. Several genes involved in peptidoglycan synthesis were identified by DNA microarray analysis as downregulated in TW14D. Luciferase reporter gene fusion assays also revealed that expression of brpA is regulated in response to environmental conditions and stresses induced by exposure to subinhibitory concentrations of cell envelope antimicrobials. In a Galleria mellonella (wax worm) model, BrpA deficiency was shown to diminish the virulence of S. mutans OMZ175, which, unlike S. mutans UA159, efficiently kills the worms. Collectively, these results suggest that BrpA plays a role in the regulation of cell envelope integrity and that deficiency of BrpA adversely affects the fitness and diminishes the virulence of OMZ175, a highly invasive strain of S. mutans.
The chitinase-like proteins YKL-39 (chitinase 3-like-2) and YKL-40 (chitinase 3-like-1) are highly expressed in a number of human cells independent of their origin (mesenchymal, epithelial or haemapoietic). Elevated serum levels of YKL-40 have been associated with a negative outcome in a number of diseases ranging from cancer to inflammation and asthma. YKL-39 expression has been associated with osteoarthritis. However, despite the reported association with disease, the physiological or pathological role of these proteins is still very poorly understood. Although YKL-39 is homologous to the two family 18 chitinases in the human genome, it has been reported to lack any chitinase activity. In the present study, we show that human YKL-39 possesses a chitinase-like fold, but lacks key active-site residues required for catalysis. A glycan screen identified oligomers of N-acetylglucosamine as preferred binding partners. YKL-39 binds chitooligosaccharides and a newly synthesized derivative of the bisdionin chitinase-inhibitor class with micromolar affinity, through a number of conserved tryptophan residues. Strikingly, the chitinase activity of YKL-39 was recovered by reverting two non-conservative substitutions in the active site to those found in the active enzymes, suggesting that YKL-39 is a pseudo-chitinase with retention of chitinase-like ligand-binding properties.
chitinase; chitinase-like proteins; glycan; glycan array; glycobiology; protein structure; lectin; X-ray crystallography
Cystic fibrosis (CF) lung disease begins in early life and is progressive with the major risk factor being an exaggerated inflammatory response. Currently, assessment of neutrophilic inflammation in early cystic fibrosis (CF) lung disease relies on bronchoalveolar lavage (BAL). The chitinase-like protein YKL-40 is raised in sputum and serum of adults with CF. We investigated YKL-40 in BAL, serum and urine to determine whether this reflected inflammation and infection in young children with CF.
YKL-40 was measured in matched samples of BAL, serum and urine obtained from 36 infants and young children with CF participating in an early surveillance program. Levels were compared to clinical data and markers of inflammation detected in the lung.
YKL-40 in BAL correlated with pulmonary infection [β=1.30 (SE 0.34), p < 0.001] and BAL markers of inflammation [macrophage number: r2 = 0.34, p < 0.001; neutrophil number: r2 = 0.74, p < 0.001; neutrophil elastase: r2 = 0.47, p < 0.001; CXCL8: r2 = 0.45, p < 0.001; IL-β: r2 = 0.62, p < 0.001]. YKL-40 was detectable in serum but levels did not correlate with BAL levels in the same individuals (r2 = 0.04, p = 0.14) or with inflammatory markers. YKL-40 was below the limit of detection in urine (30 pg/ml).
This study demonstrates that levels of the chitinase-like protein YKL-40 reflect airway inflammation and infection in early CF lung disease. The lack of increased YKL-40 in serum in the absence of systemic inflammation limits the benefit of this potential biomarker in early disease.
Cystic fibrosis; YKL-40; Biomarker; Lung disease
Tumor angiogenesis is an essential process for supplying rapidly growing malignant tissues with essential nutrients and oxygen. An angiogenic switch allows tumor cells to survive and grow, and provides them access to vasculature resulting in metastatic disease. Monocyte-derived macrophages recruited and reprogrammed by tumor cells serve as a major source of angiogenic factors boosting the angiogenic switch. Tumor endothelium releases angiopoietin-2 and further facilitates recruitment of TIE2 receptor expressing monocytes (TEM) into tumor sites. Tumor-associated macrophages (TAM) sense hypoxia in avascular areas of tumors, and react by production of angiogenic factors such as VEGFA. VEGFA stimulates chemotaxis of endothelial cells (EC) and macrophages. In some tumors, TAM appeared to be a major source of MMP9. Elevated expression of MMP9 by TAM mediates extracellular matrix (ECM) degradation and the release of bioactive VEGFA. Other angiogenic factors released by TAM include basic fibroblast growth factor (bFGF), thymidine phosphorylase (TP), urokinase-type plasminogen activator (uPA), and adrenomedullin (ADM). The same factors used by macrophages for the induction of angiogenesis [like vascular endothelial growth factor A (VEGF-A) and MMP9] support lymphangiogenesis. TAM can express LYVE-1, one of the established markers of lymphatic endothelium. TAM support tumor lymphangiogenesis not only by secretion of pro-lymphangiogenic factors but also by trans-differentiation into lymphatic EC. New pro-angiogenic factor YKL-40 belongs to a family of mammalian chitinase-like proteins (CLP) that act as cytokines or growth factors. Human CLP family comprises YKL-40, YKL-39, and SI-CLP. Production of all three CLP in macrophages is antagonistically regulated by cytokines. It was recently established that YKL-40 induces angiogenesis in vitro and in animal tumor models. YKL-40-neutralizing monoclonal antibody blocks tumor angiogenesis and progression. The role of YKL-39 and SI-CLP in tumor angiogenesis and lymphangiogenesis remains to be investigated.
tumor-associated macrophages; TIE2 receptor; VEGF; LYVE-1; stabilin-1; chitinase-like protein
The active zone (AZ) protein Bruchpilot (Brp) is essential for rapid glutamate release at Drosophila melanogaster neuromuscular junctions (NMJs). Quantal time course and measurements of action potential-waveform suggest that presynaptic fusion mechanisms are altered in brp null mutants (brp69). This could account for their increased evoked excitatory postsynaptic current (EPSC) delay and rise time (by about 1 ms). To test the mechanism of release protraction at brp69 AZs, we performed knock-down of Synaptotagmin-1 (Syt) via RNAi (sytKD) in wildtype (wt), brp69 and rab3 null mutants (rab3rup), where Brp is concentrated at a small number of AZs. At wt and rab3rup synapses, sytKD lowered EPSC amplitude while increasing rise time and delay, consistent with the role of Syt as a release sensor. In contrast, sytKD did not alter EPSC amplitude at brp69 synapses, but shortened delay and rise time. In fact, following sytKD, these kinetic properties were strikingly similar in wt and brp69, which supports the notion that Syt protracts release at brp69synapses. To gain insight into this surprising role of Syt at brp69 AZs, we analyzed the structural and functional differentiation of synaptic boutons at the NMJ. At ‘tonic’ type Ib motor neurons, distal boutons contain more AZs, more Brp proteins per AZ and show elevated and accelerated glutamate release compared to proximal boutons. The functional differentiation between proximal and distal boutons is Brp-dependent and reduced after sytKD. Notably, sytKD boutons are smaller, contain fewer Brp positive AZs and these are of similar number in proximal and distal boutons. In addition, super-resolution imaging via dSTORM revealed that sytKD increases the number and alters the spatial distribution of Brp molecules at AZs, while the gradient of Brp proteins per AZ is diminished. In summary, these data demonstrate that normal structural and functional differentiation of Drosophila AZs requires concerted action of Brp and Syt.
Bruchpilot; active zone; neurotransmitter release; synaptic delay; presynaptic differentiation; synaptotagmin; dSTORM
BACKGROUND AND PURPOSE
Leukotrienes (LTs) are inflammatory mediators produced via the 5-lipoxygenase (5-LOX) pathway and are linked to diverse disorders, including asthma, allergic rhinitis and cardiovascular diseases. We recently identified the benzimidazole derivative BRP-7 as chemotype for anti-LT agents by virtual screening targeting 5-LOX-activating protein (FLAP). Here, we aimed to reveal the in vitro and in vivo pharmacology of BRP-7 as an inhibitor of LT biosynthesis.
We analysed LT formation and performed mechanistic studies in human neutrophils and monocytes, in human whole blood (HWB) and in cell-free assays. The effectiveness of BRP-7 in vivo was evaluated in rat carrageenan-induced pleurisy and mouse zymosan-induced peritonitis.
BRP-7 potently suppressed LT formation in neutrophils and monocytes and this was accompanied by impaired 5-LOX co-localization with FLAP. Neither the cellular viability nor the activity of 5-LOX in cell-free assays was affected by BRP-7, indicating that a functional FLAP is needed for BRP-7 to inhibit LTs, and FLAP bound to BRP-7 linked to a solid matrix. Compared with the FLAP inhibitor MK-886, BRP-7 did not significantly inhibit COX-1 or microsomal prostaglandin E2 synthase-1, implying the selectivity of BRP-7 for FLAP. Finally, BRP-7 was effective in HWB and impaired inflammation in vivo, in rat pleurisy and mouse peritonitis, along with reducing LT levels.
CONCLUSIONS AND IMPLICATIONS
BRP-7 potently suppresses LT biosynthesis by interacting with FLAP and exhibits anti-inflammatory effectiveness in vivo, with promising potential for further development.
5-lipoxygenase; 5-lipoxygenase activating protein; leukotriene; benzimidazole; inflammation
Induced overexpression of the secretory protein YKL-40 promotes tumor growth in xenograft experiments. We investigated if targeting YKL-40 with a monoclonal antibody could inhibit tumor growth. YKL-40 expressing human melanoma cells (LOX) were injected subcutenously in Balb/c scid mice. Animals were treated with intraperitoneal injections of anti-YKL-40, isoptype control or PBS. Non-YKL-40 expressing human pancreatic carcinoma cell line PaCa 5061 served as additional control. MR imaging was used for evaluation of tumor growth. Two days after the first injections of anti-YKL-40, tumor volume had increased significantly compared with controls, whereas no effects were observed for control tumors from PaCa 5061 cells lacking YKL-40 expression. After 18 days, mean tumor size of the mice receiving repeated anti-YKL-40 injections was 1.82 g, >4 times higher than mean tumor size of the controls (0.42 g). The effect of anti-YKL-40 on the increase of tumor volume started within hours after injection and was dose dependent. Intratumoral hemorrhage was observed in the treated animals. The strong effect on tumor size indicates important roles for YKL-40 in melanoma growth and argues for a careful evaluation of antibody therapy directed against YKL-40.
Rationale. Hyperoxia exposure to developing lungs—critical in the pathogenesis of bronchopulmonary dysplasia—may augment lung inflammation by inhibiting anti-inflammatory mediators in alveolar macrophages. Objective. We sought to determine the O2-induced effects on the polarization of macrophages and the role of anti-inflammatory BRP-39 in macrophage phenotype and neonatal lung injury. Methods. We used RAW264.7, peritoneal, and bone marrow derived macrophages for polarization (M1/M2) studies. For in vivo studies, wild-type (WT) and BRP-39−/− mice received continuous exposure to 21% O2 (control mice) or 100% O2 from postnatal (PN) 1 to PN7 days, along with intranasal lipopolysaccharide (LPS) administered on alternate days (PN2, -4, and -6). Lung histology, bronchoalveolar lavage (BAL) cell counts, BAL protein, and cytokines measurements were performed. Measurements and Main Results. Hyperoxia differentially contributed to macrophage polarization by enhancing LPS induced M1 and inhibiting interleukin-4 induced M2 phenotype. BRP-39 absence led to further enhancement of the hyperoxia and LPS induced M1 phenotype. In addition, BRP-39−/− mice were significantly more sensitive to LPS plus hyperoxia induced lung injury and mortality compared to WT mice. Conclusions. These findings collectively indicate that BRP-39 is involved in repressing the M1 proinflammatory phenotype in hyperoxia, thereby deactivating inflammatory responses in macrophages and preventing neonatal lung injury.
The glycoprotein YKL-40 (CHI3L1) is a secreted chitinase family protein that induces angiogenesis, cell survival, and cell proliferation, and plays roles in tissue remodeling and immune regulation. It is expressed primarily in cells of mesenchymal origin, is overexpressed in numerous aggressive carcinomas and sarcomas, but is rarely expressed in normal ectodermal tissues. Bone marrow-derived mesenchymal stem cells (MSCs) can be induced to differentiate into various mesenchymal tissues and trans-differentiate into some non-mesenchymal cell types. Since YKL-40 has been used as a mesenchymal marker, we followed YKL-40 expression as undifferentiated MSCs were induced to differentiate into bone, cartilage, and neural phenotypes. Undifferentiated MSCs contain significant levels of YKL-40 mRNA but do not synthesize detectable levels of YKL-40 protein. MSCs induced to differentiate into chondrocytes and osteocytes soon began to express and secrete YKL-40 protein, as do ex vivo cultured chondrocytes and primary osteocytes. In contrast, MSCs induced to trans-differentiate into neurons did not synthesize YKL-40 protein, consistent with the general absence of YKL-40 protein in normal CNS parenchyma. However, these trans-differentiated neurons retained significant levels of YKL-40 mRNA, suggesting the mechanisms which prevented YKL-40 translation in undifferentiated MSCs remained in place, and that these trans-differentiated neurons differ in at least this way from neurons derived from neuronal stem cells. Utilization of a differentiation protocol containing β-mercaptoethanol resulted in cells that expressed significant amounts of intracellular YKL-40 protein that was not secreted, which is not seen in normal cells. Thus the synthesis of YKL-40 protein is a marker for MSC differentiation into mature mesenchymal phenotypes, and the presence of untranslated YKL-40 mRNA in non-mesenchymal cells derived from MSCs reflects differences between differentiated and trans-differentiated phenotypes.
Several inflammatory cytokines are involved in vascular inflammation resulting in endothelial dysfunction which is the earliest event in the atherosclerotic process leading to manifest cardiovascular disease. YKL-40 is an inflammatory glycoprotein involved in endothelial dysfunction by promoting chemotaxis, cell attachment and migration, reorganization and tissue remodelling as a response to endothelial damage. YKL-40 protein expression is seen in macrophages and smooth muscle cells in atherosclerotic plaques with the highest expression seen in macrophages in the early lesion of atherosclerosis. Several studies demonstrate, that elevated serum YKL-levels are independently associated with the presence and extent of coronary artery disease and even higher YKL-40 levels are documented in patients with myocardial infarction. Moreover, elevated serum YKL-40 levels have also been found to be associated with all-cause as well as cardiovascular mortality. Finally, YKL-40 levels are elevated both in patients with type 1 and type 2 diabetes, known to be at high risk for the development of cardiovascular diseases, when compared to non-diabetic persons. A positive association between elevated circulating YKL-40 levels and increasing levels of albuminuria have been described in patients with type 1 diabetes indicating a role of YKL-40 in the progressing vascular damage resulting in microvascular disease.
This review describes the present knowledge about YKL-40 and discusses its relation to endothelial dysfunction, atherosclerosis, cardiovascular disease and diabetes and look ahead on future perspectives of YKL-40 research.
Human cartilage chitinase 3-like protein 2 (CHI3L2 or YKL-39) is a member of family-18 glycosyl hydrolases that lacks chitinase activity. YKL-39 is known as a potential marker for the activation of chondrocytes and the progression of osteoarthritis. In this study, we cloned and expressed a functional form of human YKL-39 in the bacterial system. The Escherichia coli expressed YKL-30 was used as immugen for production of anti YKL-39 polyclonal and monoclonal antibodies. Both antibody types were highly selective, reacting only with YKL-39. Isotype mapping identified two hybridoma clones (so called clones 6H11 and 8H3) to be IgM isotype. Dot blot assay showed that the monoclonal antibody was strongly active with the synovial fluid of an osteoarthritis patient, human monocyte, and T lymphocyte cell lines. Database search for protein binding partners gave high hits with several glycoproteins that play particular roles in cartilage tissue scaffolding, connective tissue formation, and cell-cell interactions. In conclusion, anti YKL-39 polyclonal and monoclonal antibodies were raised and tested to be suitable for immunological applications, such as the investigation of the YKL-39 regulating pathway and the development of an immunosensing tool for sensitive detection of cartilage tissue destruction.
Tumor angiogenesis is of paramount importance in solid tumor development. Elevated serum levels of YKL-40, a secreted heparin-binding glycoprotein have been associated with a worse prognosis from a variety of advanced human cancers. Yet the role of YKL-40 activity in these cancers is still missing. Here, we have shown that ectopic expression of YKL-40 in MDA-MB-231 breast cancer cells and HCT-116 colon cancer cells led to larger tumor formation with an extensive angiogenic phenotype than did control cancer cells in mice. Affinity purified recombinant YKL-40 protein promoted vascular endothelial cell angiogenesis in vitro, the effects similar to the activities observed using MDA-MB-231 and HCT-116 cell conditioned medium after transfection with YKL-40. Further, YKL-40 was found to induce the coordination of membrane-bound receptor syndecan-1 and integrin αvβ3 and activate an intracellular signaling cascade including focal adhesion kinase and MAP kinase Erk1/2 in endothelial cells. Also, blockade of YKL-40 using siRNA gene knockdown suppressed tumor angiogenesis in vitro and in vivo. Immunohistochemical analysis of human breast cancer revealed a correlation between YKL-40 expression and blood vessel density. These findings provide novel insights into angiogenic activities and molecular mechanisms of YKL-40 in cancer development.
Signal transduction modifiers that modulate the lysophosphatidic acid (LPA) pathway have potential as anticancer agents. Herein, we describe metabolically stabilized LPA analogues that reduce cell migration and invasion and cause regression of orthotopic breast tumors in vivo. Two diastereoisomeric α-bromophosphonates (BrP-LPA) were synthesized, and the pharmacology was determined for five LPA G protein–coupled receptors (GPCRs). The syn and anti diastereomers of BrP-LPA are pan-LPA GPCR antagonists and are also nanomolar inhibitors of the lysophospholipase D activity of autotaxin, the dominant biosynthetic source of LPA. Computational models correctly predicted the diastereoselectivity of antagonism for three GPCR isoforms. The anti isomer of BrP-LPA was more effective than syn isomer in reducing migration of MDA-MB-231 cells, and the anti isomer was superior in reducing invasion of these cells. Finally, orthotopic breast cancer xenografts were established in nude mice by injection of MB-231 cells in an in situ cross-linkable extracellular matrix. After 2 weeks, mice were treated with the BrP-LPA alone (10 mg/kg), Taxol alone (10 mg/kg), or Taxol followed by BrP-LPA. All treatments significantly reduced tumor burden, and BrP-LPA was superior to Taxol in reducing blood vessel density in tumors. Moreover, both the anti- and syn-BrP-LPA significantly reduced tumors at 3 mg/kg.