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
Mouse breast regression protein 39 (BRP-39; Chi3l1) and its human homologue YKL-40 are chitinase-like proteins that lack chitinase activity. Although YKL-40 is expressed in exaggerated quantities and correlates with disease activity in asthma and many other disorders, the biological properties of BRP-39/YKL-40 have only been rudimentarily defined. We describe the generation and characterization of BRP-39−/− mice, YKL-40 transgenic mice, and mice that lack BRP-39 and produce YKL-40 only in their pulmonary epithelium. Studies of these mice demonstrated that BRP-39−/− animals have markedly diminished antigen-induced Th2 responses and that epithelial YKL-40 rescues the Th2 responses in these animals. The ability of interleukin13 to induce tissue inflammation and fibrosis was also markedly diminished in the absence of BRP-39. Mechanistic investigations demonstrated that BRP-39 and YKL-40 play an essential role in antigen sensitization and immunoglobulin E induction, stimulate dendritic cell accumulation and activation, and induce alternative macrophage activation. These proteins also inhibit inflammatory cell apoptosis/cell death while inhibiting Fas expression, activating protein kinase B/AKT, and inducing Faim 3. These studies establish novel regulatory roles for BRP-39/YKL-40 in the initiation and effector phases of Th2 inflammation and remodeling and suggest that these proteins are therapeutic targets in Th2- and macrophage-mediated disorders.
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
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
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
Hyperoxic acute lung injury (HALI) is characterized by a cell death response with features of apoptosis and necrosis that is inhibited by IL-11 and other interventions. We hypothesized that Bfl-1/A1, an antiapoptotic Bcl-2 protein, is a critical regulator of HALI and a mediator of IL-11–induced cytoprotection. To test this, we characterized the expression of A1 and the oxygen susceptibility of WT and IL-11 Tg(+) mice with normal and null A1 loci. In WT mice, 100% O2 caused TUNEL+ cell death, induction and activation of intrinsic and mitochondrial-death pathways, and alveolar protein leak. Bcl-2 and Bcl-xl were also induced as an apparent protective response. A1 was induced in hyperoxia, and in A1-null mice, the toxic effects of hyperoxia were exaggerated, Bcl-2 and Bcl-xl were not induced, and premature death was seen. In contrast, IL-11 stimulated A1, diminished the toxic effects of hyperoxia, stimulated Bcl-2 and Bcl-xl, and enhanced murine survival in 100% O2. In A1-null mice, IL-11–induced protection, survival advantage, and Bcl-2 and Bcl-xl induction were significantly decreased. VEGF also conferred protection via an A1-dependent mechanism. In vitro hyperoxia also stimulated A1, and A1 overexpression inhibited oxidant-induced epithelial cell apoptosis and necrosis. A1 is an important regulator of oxidant-induced lung injury, apoptosis, necrosis, and Bcl-2 and Bcl-xl gene expression and a critical mediator of IL-11– and VEGF-induced cytoprotection.
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.
Prolonged exposure to hyperoxia in neonates can cause hyperoxic acute lung injury (HALI), which is characterized by increased pulmonary permeability and diffuse infiltration of various inflammatory cells. Disruption of the epithelial barrier may lead to altered pulmonary permeability and maintenance of barrier properties requires intact epithelial tight junctions (TJs). However, in neonatal animals, relatively little is known about how the TJ proteins are expressed in the pulmonary epithelium, including whether expression of TJ proteins is regulated in response to hyperoxia exposure. This study determines whether changes in tight junctions play an important role in disruption of the pulmonary epithelial barrier during hyperoxic acute lung injury.
Newborn rats, randomly divided into two groups, were exposed to hyperoxia (95% oxygen) or normoxia for 1–7 days, and the severity of lung injury was assessed; location and expression of key tight junction protein occludin and ZO-1 were examined by immunofluorescence staining and immunobloting; messenger RNA in lung tissue was studied by RT-PCR; transmission electron microscopy study was performed for the detection of tight junction morphology.
We found that different durations of hyperoxia exposure caused different degrees of lung injury in newborn rats. Treatment with hyperoxia for prolonged duration contributed to more serious lung injury, which was characterized by increased wet-to-dry ratio, extravascular lung water content, and bronchoalveolar lavage fluid (BALF):serum FD4 ratio. Transmission electron microscopy study demonstrated that hyperoxia destroyed the structure of tight junctions and prolonged hyperoxia exposure, enhancing the structure destruction. The results were compatible with pathohistologic findings. We found that hyperoxia markedly disrupted the membrane localization and downregulated the cytoplasm expression of the key tight junction proteins occludin and ZO-1 in the alveolar epithelium by immunofluorescence. The changes of messenger RNA and protein expression of occludin and ZO-1 in lung tissue detected by RT-PCR and immunoblotting were consistent with the degree of lung injury.
These data suggest that the disruption of the pulmonary epithelial barrier induced by hyperoxia is, at least in part, due to massive deterioration in the expression and localization of key TJ proteins.
Acute lung injury; Hyperoxia; Newborn; Permeability; Tight Junction
Supplemental oxygen is frequently prescribed. However, prolonged exposure to high concentrations of oxygen causes hyperoxic acute lung injury (HALI), which manifests as acute respiratory distress syndrome in adults and leads to bronchopulmonary dysplasia in newborns (NBs). Nitric oxide (NO), NO synthases (NOSs), and angiopoietin (Ang) 2 have been implicated in the pathogenesis of HALI. However, the mechanisms of the contributions of NOS/NO and the relationship(s) between NOS/NO and Ang2 have not been addressed. In addition, the relevance of these moieties in adults and NBs has not been evaluated. To address these issues, we compared the responses in hyperoxia of wild-type (NOS [+/+]) and NOS null (−/−) young adult and NB mice. When compared with NOS2+/+ adult controls, NOS2−/− animals manifest exaggerated alveolar–capillary protein leak and premature death. These responses were associated with enhanced levels of structural cell death, enhanced expression of proapoptotic regulatory proteins, and Ang2. Importantly, silencing RNA knockdown of Ang2 decreased the levels of cell death and the expression of proapoptotic mediators. These effects were at least partially NOS2 specific, and were development dependent, because survival was similar in adult NOS3+/+ and NOS3−/− mice and NB NOS2+/+ and NOS2−/− mice, respectively. These studies demonstrate that NOS2 plays an important protective role in HALI in adult animals. They also demonstrate that this response is mediated, at least in part, by the ability of NOS2 to inhibit hyperoxia-induced Ang2 production and thereby decrease Ang2-induced tissue injury.
cytokines; hyperoxia; lung
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.
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.
Prolonged breathing of very high FIO2 (FIO2 ≥ 0.9) uniformly causes severe hyperoxic acute lung injury (HALI) and, without a reduction of FIO2, is usually fatal. The severity of HALI is directly proportional to PO2 (particularly above 450 mm Hg, or an FIO2 of 0.6) and exposure duration. Hyperoxia produces extraordinary amounts of reactive O2 species that overwhelms natural antioxidant defenses and destroys cellular structures through several pathways. Genetic predisposition has been shown to play an important role in HALI among animals, and some genetics-based epidemiologic research suggests that this may be true for humans as well. Clinically, the risk of HALI likely occurs when FIO2exceeds 0.7, and may become problematic when FIO2 exceeds 0.8 for an extended period of time. Both high-stretch mechanical ventilation and hyperoxia potentiate lung injury and may promote pulmonary infection. During the 1960s, confusion regarding the incidence and relevance of HALI largely reflected such issues as the primitive control of FIO2, the absence of PEEP, and the fact that at the time both ALI and ventilator-induced lung injury were unknown. The advent of PEEP and precise control over FIO2, as well as lung-protective ventilation, and other adjunctive therapies for severe hypoxemia, has greatly reduced the risk of HALI for the vast majority of patients requiring mechanical ventilation in the 21st century. However, a subset of patients with very severe ARDS requiring hyperoxic therapy is at substantial risk for developing HALI, therefore justifying the use of such adjunctive therapies.
acute lung injury; acute respiratory distress syndrome; hyperoxia; oxygen toxicity; reactive oxygen species; ventilator-induced lung injury
Aberrant tissue repair and persistent inflammation following oxidant-mediated acute lung injury (ALI) can lead to the development and progression of various pulmonary diseases, but the mechanisms underlying these processes remain unclear. Hyperoxia is widely used in the treatment of pulmonary diseases, but the effects of this oxidant exposure in patients undergoing recovery from ALI are not clearly understood. Nrf2 has emerged as a crucial transcription factor that regulates oxidant stress through the induction of several detoxifying enzymes and other proteins. Using an experimental model of hyperoxia-induced ALI (HALI), we have examined the role of oxidant stress in resolving lung injury and inflammation. We found that when exposed to sub-lethal (72 h) hyperoxia, Nrf2-deficient, but not wild-type mice, succumbed to death during recovery. When both genotypes were exposed to a shorter period of HALI (48 h), the lungs of Nrf2-deficient mice during recovery exhibited persistent cellular injury, impaired alveolar and endothelial cell regeneration, and persistent cellular infiltration by macrophages and lymphocytes. GSH supplementation in Nrf2-deficient mice immediately after hyperoxia remarkably restored their ability to recover from hyperoxia-induced damage in a manner similar to that of wild-type mice. Thus, the results of the present study indicate that the Nrf2-regulated transcriptional response, and particularly GSH synthesis, is critical for lung tissue repair and the resolution of inflammation in vivo and suggests that a dysfunctional Nrf2-GSH pathway may compromise these processes in vivo.
Oxidative stress; Nrf2; acute lung injury; DNA injury
YKL-40 (chitinase 3-like protein 1) is expressed in a broad spectrum of inflammatory conditions and cancers. We have previously reported that YKL-40 levels are elevated in the cerebrospinal fluid (CSF) of macaques and humans with lentiviral encephalitis, as well as multiple sclerosis (MS). The current study assessed temporal CSF YKL-40 levels in subjects with severe traumatic brain injury (TBI; Glasgow Coma Scale [GCS] score ≤8). We also evaluated temporal expression of YKL-40 after parasagittal controlled cortical impact (CCI) injury over the parietal cortex (2.8 mm deep, 4 m/sec). We demonstrate that CSF YKL-40 levels are elevated after acute TBI, and that YKL-40 levels are higher in patients who died following injury than in patients who survived. YKL-40 levels significantly correlate with CSF levels of inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), as well as the inflammatory marker C-reactive protein (CRP). After CCI, in situ hybridization (ISH) showed that YKL-40 transcription is primarily associated with reactive astrocytes in pericontusional cortex. Tissue YKL-40 transcription time course analysis after CCI showed that YKL40 transcription in astrocytes began 1 day after injury, remained elevated for several days, and then declined by day 12. Similarly to our temporal CSF measurements in humans, YKL-40 induction after CCI is coincident with IL-1β expression. Taken together these findings demonstrate that YKL-40 is induced in astrocytes during acute neuroinflammation, is temporally related to inflammatory mediator expression, and may be a useful biomarker for understanding secondary injury and for patient prognosis.
chitinase; controlled cortical impact; cytokine; gliosis; neuroinflammation; traumatic brain injury; YKL-40
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
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
This report explains how our studies of asthma and Th2 inflammation led us to investigate the roles of chitinase-like proteins (CLPs) in lung injury and repair and puts forth an overall hypothesis that can explain the roles that these moieties play in biology and a hypothesis regarding the ways that dysregulated CLP expression may contribute to the pathogenesis of a variety of diseases. We test this hypothesis by assessing the contributions of the CLP breast regression protein (BRP)-39 in the pathogenesis of malignant melanoma metastasis to the lung.
BRP-39/YKL-40; inflammation; injury; repair; metastasis
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.
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.
A hallmark of hyperoxic acute lung injury is the influx of inflammatory cells to lung tissue and the production of proinflammatory cytokines, such as IL-1β; however, the mechanisms connecting hyperoxia and the inflammatory response to lung damage is not clear. The inflammasome protein complex activates caspase-1 to promote the processing and secretion of proinflammatory cytokines. We hypothesized that hyperoxia-induced K+ efflux activates the inflammasome via the purinergic P2X7 receptor to cause inflammation and hyperoxic acute lung injury. To test this hypothesis, we characterized the expression and activation of inflammasome components in primary murine alveolar macrophages exposed to hyperoxia (95% oxygen and 5% CO2) in vitro, and in alveolar macrophages isolated from mice exposed to hyperoxia (100% oxygen). Our results showed that hyperoxia increased K+ efflux, inflammasome formation, release of proinflammatory cytokines, and induction of caspase-1 and IL-1β cleavage both in vitro and in vivo. The P2X7 agonist ATP enhanced hyperoxia-induced inflammasome activation, whereas the P2X7 antagonist, oxidized ATP, inhibited hyperoxia induced inflammasome activation. In addition, when ATP was scavenged with apyrase, hyperoxia-induced inflammasome activation was significantly decreased. Furthermore, short hairpin RNA silencing of inflammasome components abrogated hyperoxia-induced secretion of proinflammatory cytokines in vitro. These results suggest that hyperoxia induces K+ efflux through the P2X7 receptor, leading to inflammasome activation and secretion of proinflammatory cytokines. These events would affect the permeability of the alveolar epithelium and ultimately lead to epithelial barrier dysfunction and cell death.
Hyperoxic acute lung injury (HALI) is characterized by a cell death response that is inhibited by IL-6. Suppressor of cytokine signaling-1 (SOCS-1) is an antiapoptotic negative regulator of the IL-6–mediated Janus kinase–signal transducer and activator of transcription signaling pathway. We hypothesized that SOCS-1 is a critical regulator and key mediator of IL-6–induced cytoprotection in HALI. To test this hypothesis, we characterized the expression of SOCS-1 and downstream apoptosis signal–regulating kinase (ASK)-1–Jun N-terminal kinase signaling molecules in small airway epithelial cells in the presence of H2O2, which induces oxidative stress. We also examined these molecules in wild-type and lung-specific IL-6 transgenic (Tg+) mice exposed to 100% oxygen for 72 hours. In control small airway epithelial cells exposed to H2O2 or in wild-type mice exposed to 100% oxygen, a marked induction of ASK-1 and pJun N-terminal kinase was observed. Both IL-6–stimulated endogenous SOCS-1 and SOCS-1 overexpression abolished H2O2-induced ASK-1 activation. In addition, IL-6 Tg+ mice exposed to 100% oxygen exhibited reduced ASK-1 levels and enhanced SOCS-1 expression compared with wild-type mice. Interestingly, no significant changes in activation of the key ASK-1 activator, tumor necrosis factor receptor-1/tumor necrosis factor receptor–associated factor-2 were observed between wild-type and IL-6 Tg+ mice. Furthermore, the interaction between SOCS-1 and ASK-1 promotes ubiquitin-mediated degradation both in vivo and in vitro. These studies demonstrate that SOCS-1 is an important regulator in IL-6–induced cytoprotection against HALI.
IL-6; apoptosis signal–regulating kinase-1; suppressor of cytokine signaling-1; lung injury; tumor necrosis factor receptor-1
The chitinase-like protein YKL-40 is involved in inflammation and tissue remodeling. We recently showed that serum YKL-40 levels were elevated in patients with asthma and were correlated with severity, thickening of the subepithelial basement membrane, and pulmonary function. We hypothesized that single-nucleotide polymorphisms (SNPs) that affect YKL-40 levels also influence asthma status and lung function.
We carried out a genomewide association study of serum YKL-40 levels in a founder population of European descent, the Hutterites, and then tested for an association between an implicated SNP and asthma and lung function. One associated variant was genotyped in a birth cohort at high risk for asthma, in which YKL-40 levels were measured from birth through 5 years of age, and in two populations of unrelated case patients of European descent with asthma and controls.
A promoter SNP (−131C→G) in CHI3L1, the chitinase 3–like 1 gene encoding YKL-40, was associated with elevated serum YKL-40 levels (P = 1.1×10−13), asthma (P = 0.047), bronchial hyperresponsiveness (P = 0.002), and measures of pulmonary function (P = 0.046 to 0.002) in the Hutterites. The same SNP could be used to predict the presence of asthma in the two case–control populations (combined P = 1.2×10−5) and serum YKL-40 levels at birth (in cord-blood specimens) through 5 years of age in the birth cohort (P = 8.9×10−3 to 2.5×10−4).
CHI3L1 is a susceptibility gene for asthma, bronchial hyperresponsiveness, and reduced lung function, and elevated circulating YKL-40 levels are a biomarker for asthma and decline in lung function.
Obstructive sleep apnea (OSA) is a common disorder affecting 15–24% of the adults and is associated with increased risk of hypertension and atherosclerosis. The exact mechanisms underlying hypertension in OSA are not entirely clear. YKL-40/Chitinase-3-like protein-1 is a circulating moiety with roles in injury, repair and angiogenesis that is dysregulated in atherosclerosis and a number of other diseases. We sought to determine the role of YKL-40 in endothelial dysfunction and hypertension in OSA.
We studies 23 normotensive OSA (N-OSA) and 14 hypertensive OSA (H-OSA) without diabetes and apparent cardiovascular disease. Endothelial-dependent nitric oxide-mediated vasodilatory capacity was assessed by flow-mediated vasodilation (FMD). YKL-40, vascular endothelial growth factor (VEGF) and the soluble form of VEGF receptor-1or sFlt-1 were measured in plasma using ELISA methodology.
N-OSA subjects aged 49.1±2.3 years and H-OSA aged 51.3±1.9 years with BMI 36.1±1.6 and 37.6±1.9 kg/m2, respectively. The apnea-hypopnea index (AHI) was 41±5 events/hr in N-OSA and 46±6 in H-OSA with comparable degree of oxygen desaturations during sleep. FMD was markedly impaired in H-OSA (8.3%±0.8) compared to N-OSA (13.2%±0.6, P<0.0001). Plasma YKL-40 was significantly elevated in H-OSA (55.2±7.9 ng/ml vs. 35.6±4.2 ng/ml in N-OSA, P = 0.02) and had an inverse relationship with FMD (r = −0.52, P = 0.013). There was a significant positive correlation between sFlt-1/VEGF, a measure of decreased VEGF availability, and YKL-40 (r = 0.42, P = 0.04).
The levels of plasma YKL-40 were elevated in H-OSA group and inversely correlated with the endothelial-dependent vasodilatory capacity whereas there was a positive correlation between sFlt-1/VEGF and YKL-40. These findings suggest that YKL-40 is dysregulated, in part, due to perturbation of VEGF signaling, and may contribute to endothelial dysfunction and hypertension in OSA.
A secreted glycoprotein YKL-40 also named chitinase-3-like-1 is normally expressed by multiple cell types such as macrophages, chondrocytes, and vascular smooth muscle cells. However, a prominently high level of YKL-40 was found in a wide spectrum of human diseases including cancers and chronic inflammatory diseases where it was strongly expressed by cancerous cells and infiltrating macrophages. Here, we summarized recent important findings of YKL-40 derived from cancerous cells and smooth muscle cells during tumor angiogenesis and development. YKL-40 is a potent angiogenic factor capable of stimulating tumor vascularization mediated by endothelial cells and maintaining vascular integrity supported by smooth muscle cells. In addition, YKL-40 induces FAK-MAPK signaling and up-regulates VEGF receptor 2 in endothelial cells; but a neutralizing antibody (mAY) against YKL-40 inhibits its angiogenic activity. While YKL-40 is essential for angiogenesis, little is known about its functional role in tumor-associated macrophage (TAM)-mediated tumor development. Therefore, significant efforts are urgently needed to identify pathophysiological function of YKL-40 in the dynamic interaction between tumor cells and TAMs in the tumor microenvironment, which may offer substantial mechanistic insights into tumor angiogenesis and metastasis, and also point to a therapeutic target for treatment of cancers and other diseases.
YKL-40; angiogenesis; VEGF; tumor cells; vascular endothelial cells; tumor-associated macrophages; tumor microenvironment; neutralizing anti-YKL-40 antibody
Rationale: Acute lung injury and the acute respiratory distress syndrome are characterized by increased lung oxidant stress and apoptotic cell death. The contribution of epithelial cell apoptosis to the development of lung injury is unknown.
Objectives: To determine whether oxidant-mediated activation of the intrinsic or extrinsic apoptotic pathway contributes to the development of acute lung injury.
Methods: Exposure of tissue-specific or global knockout mice or cells lacking critical components of the apoptotic pathway to hyperoxia, a well-established mouse model of oxidant-induced lung injury, for measurement of cell death, lung injury, and survival.
Measurements and Main Results: We found that the overexpression of SOD2 prevents hyperoxia-induced BAX activation and cell death in primary alveolar epithelial cells and prolongs the survival of mice exposed to hyperoxia. The conditional loss of BAX and BAK in the lung epithelium prevented hyperoxia-induced cell death in alveolar epithelial cells, ameliorated hyperoxia-induced lung injury, and prolonged survival in mice. By contrast, Cyclophilin D–deficient mice were not protected from hyperoxia, indicating that opening of the mitochondrial permeability transition pore is dispensable for hyperoxia-induced lung injury. Mice globally deficient in the BH3-only proteins BIM, BID, PUMA, or NOXA, which are proximal upstream regulators of BAX and BAK, were not protected against hyperoxia-induced lung injury suggesting redundancy of these proteins in the activation of BAX or BAK.
Conclusions: Mitochondrial oxidant generation initiates BAX- or BAK-dependent alveolar epithelial cell death, which contributes to hyperoxia-induced lung injury.
cell death; epithelium; Bcl-2 proteins; acute respiratory distress syndrome