RESUMEN
BACKGROUND: The chitinase-like protein, Chi3L1, is associated with increased fibrotic activity as well as inflammatory processes. The capacity of skin cells from systemic sclerosis (SSc) patients to produce Chi3L1, and the stimulation of its synthesis by cytokines or growth factors known to be associated with SSc, was investigated. METHODS: Cells were isolated from forearm and/or abdomen skin biopsies taken from SSc patients and normal individuals and stimulated with cytokines and growth factors to assess Chi3L1 expression. Chi3L1-expressing cells were characterized by immunohistochemical staining. RESULTS: Chi3L1 was not secreted by skin cells from normal individuals nor was its synthesis induced by any of the cytokines or growth factors investigated. In contrast, Chi3L1 secretion was induced by OSM or IL-1 in cells from all forearm biopsies of SSc patients, and endogenous secretion in the absence of cytokines was detected in several specimens. Patients with Chi3L1-producing cells at both the arm and abdomen had a disease duration of less than 3 years. Endogenous Chi3L1 production was not a property of the major fibroblast population nor of myofibroblasts, but rather was related to the presence of stem-like cells not present in normal skin. Other cells, however, contributed to the upregulation of Chi3L1 by OSM. CONCLUSIONS: The emergence of cells primed to respond to OSM with increased Chi3L1 production appears to be associated with pathological processes active in SSc. GENERAL SIGNIFICANCE: The presence of progenitor cells expressing the chilectin Chi3L1 in SSc skin appears to play a role in the initiation of the disease process.
RESUMEN
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.
Asunto(s)
Adipoquinas/química , Adipoquinas/metabolismo , Lectinas/química , Lectinas/metabolismo , Acetilglucosamina/metabolismo , Adipoquinas/antagonistas & inhibidores , Adipoquinas/genética , Sustitución de Aminoácidos , Dominio Catalítico , Quitina/metabolismo , Proteína 1 Similar a Quitinasa-3 , Quitinasas/química , Quitinasas/metabolismo , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Lectinas/antagonistas & inhibidores , Lectinas/genética , Oligosacáridos/química , Oligosacáridos/metabolismo , Mutación Puntual , Conformación Proteica , Pliegue de Proteína , Triptófano/metabolismoRESUMEN
Chondroadherin is a leucine-rich repeat protein known to mediate adhesion of isolated cells via the integrin α(2)ß(1) and to interact with collagen. In this work, we show that cell adhesion to chondroadherin leads to activation of MAPKs but does not result in cell spreading and division. This is in contrast to the spreading and dividing of cells grown on collagen, although the binding is mediated via the same α(2)ß(1) receptor. We identified a cell binding motif, CQLRGLRRWLEAK(318) by mass spectrometry after protease digestion of chondroadherin. Cells adhering to the synthetic peptide CQLRGLRRWLEAK(318) remained round, as was observed when they bound to the intact protein. The peptide added in solution was able to inhibit cell adhesion to the intact protein in a dose-dependent manner and was also verified to bind to the α(2)ß(1) integrin. A cyclic peptide, CQLRGLRRWLEAKASRPDATC(326), mimicking the structural constraints of this sequence in the intact protein, showed similar efficiency in inhibiting binding to chondroadherin. The unique peptide motif responsible for cellular binding is primarily located in the octamer sequence LRRWLEAK(318). Binding of cells to the active peptide or to chondroadherin immobilized on cell culture plates rapidly induces intracellular signaling (i.e. ERK phosphorylation). Thus, chondroadherin interaction with cells may be central for maintaining the adult chondrocyte phenotype and cartilage homeostasis. The peptides, particularly the more stable cyclic peptide, open new opportunities to modulate cell behavior in situations of tissue pathology.
Asunto(s)
Sitios de Unión , Adhesión Celular , Proteínas de la Matriz Extracelular/metabolismo , Integrina alfa2beta1/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/fisiología , Bovinos , Adhesión Celular/efectos de los fármacos , División Celular , Forma de la Célula , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Integrina alfa2beta1/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/farmacología , Fosforilación , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiologíaRESUMEN
Activation of toll-like receptors (TLR) in articular chondrocytes has been reported to increase the catabolic compartment, leading to matrix degradation, while the main consequence of TLR activation in monocytic cells is the expression and secretion of components of the innate immune response, particularly that of inflammatory cytokines. The objective of the work reported here was to obtain a more complete picture of the response repertoire of articular chondrocytes to TLR activation. Mass spectrometry was used to analyse the secretome of stimulated and unstimulated cells. Characterization of TLR expression in rat articular chondrocytes by RT/PCR indicated that TLR4 was the major receptor form. Exposure of these cells to lipopolysaccharide (LPS), the well-characterized TLR4 ligand, induced production not only of the matrix metalloproteinases MMP3 and 13, but also of components traditionally associated with the innate immune response, such as the complement components C1r, C3 and complement factor B, long pentraxin-3 and osteoglycin. Neither TNF-alpha nor IL-1 was detectable in culture media following exposure to LPS. One of the most prominently-induced proteins was the chitinase-like protein, Chi3L1, linking its expression to the innate immune response repertoire of articular chondrocytes. In intact femoral heads, LPS induced expression of Chi3L1 in chondrocytes close to the articular surface, suggesting that only these cells mount a stress response to LPS. Thus articular chondrocytes have a capacity to respond to TLR activation, which results in the expression of matrix metalloproteases as well as subsets of components of the innate immune response without significant increases in the production of inflammatory cytokines. This could influence the erosive processes leading to cartilage degeneration as well as the repair of damaged matrix.
Asunto(s)
Cartílago Articular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Lipopolisacáridos/farmacología , Animales , Animales Recién Nacidos , Cartílago Articular/inmunología , Cartílago Articular/metabolismo , Línea Celular , Células Cultivadas , Proteína 1 Similar a Quitinasa-3 , Condrocitos/inmunología , Condrocitos/metabolismo , Complemento C3/metabolismo , Medios de Cultivo Condicionados/metabolismo , Citocinas/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Cabeza Femoral/efectos de los fármacos , Cabeza Femoral/metabolismo , Expresión Génica/efectos de los fármacos , Glicoproteínas/metabolismo , Humanos , Inmunidad Innata/inmunología , Metaloproteinasas de la Matriz Secretadas/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/metabolismo , Proteómica/métodos , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptores Toll-Like/genéticaRESUMEN
Elevated levels of CHI3L1 (chitinase-3-like protein 1) are associated with disorders exhibiting increased connective tissue turnover, such as rheumatoid arthritis, osteoarthritis, scleroderma, and cirrhosis of the liver. This secreted protein is not synthesized in young healthy cartilage, but is produced in cartilage from old donors or patients with osteoarthritis. The molecular processes governing the induction of CHI3L1 are currently unknown. To elucidate the molecular events involved in CHI3L1 synthesis, we investigated two models of articular chondrocytes: neonatal rat chondrocytes, which do not express CHI3L1, and human chondrocytes, which express CHI3L1 constitutively. In neonatal rat chondrocytes, the inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 potently induced steady-state levels of CHI3L1 mRNA and protein secretion. Treatment of chondrocytes with TNF-alpha for as little as 1 h was sufficient for sustained induction up to 72 h afterward. Using inhibitors selective for the major signaling pathways implicated in mediating the effects of TNF-alpha and interleukin-1, only inhibition of NF-kappaB activation was effective in curtailing cytokine-induced expression, including after removal of the cytokine, indicating that induction and continued production of CHI3L1 are controlled mainly by this transcription factor. Inhibition of NF-kappaB signaling also abolished constitutive expression by human chondrocytes. Thus, induction and continued secretion of CHI3L1 in chondrocytes require sustained activation of NF-kappaB. Selective induction of CHI3L1 by cytokines acting through NF-kappaB coupled with the known restriction of the catabolic responses by CHI3L1 in response to these inflammatory cytokines represents a key regulatory feedback process in controlling connective tissue turnover.
Asunto(s)
Cartílago Articular/metabolismo , Condrocitos/citología , Citocinas/metabolismo , Proteínas de la Matriz Extracelular/fisiología , Glicoproteínas/fisiología , Adipoquinas , Animales , Animales Recién Nacidos , Sitios de Unión , Células Cultivadas , Proteína 1 Similar a Quitinasa-3 , Condrocitos/metabolismo , Medios de Cultivo/farmacología , Medios de Cultivo Condicionados/farmacología , Proteínas de la Matriz Extracelular/metabolismo , Fémur/patología , Regulación de la Expresión Génica , Genes Dominantes , Glicoproteínas/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , Inflamación , Interleucina-1/metabolismo , Lectinas , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Plásmidos/metabolismo , Regiones Promotoras Genéticas , ARN/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , Transfección , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Expression of the chitinase 3-like protein HC-gp39 (human cartilage glycoprotein 39) is associated with conditions of increased matrix turnover and tissue remodelling. High levels of this protein have been found in sera and synovial fluids of patients with inflammatory and degenerative arthritis. In order to assess the role of HC-gp39 in matrix degradation induced by inflammatory cytokines, we have examined its effect on the responses of connective tissue cells to TNF-alpha (tumour necrosis factor-alpha) and IL-1 (interleukin-1) with respect to activation of signalling pathways and production of MMPs (matrix metalloproteases) and chemokines. Stimulation of human skin fibroblasts or articular chondrocytes with IL-1 or TNF-alpha in the presence of HC-gp39 resulted in a marked reduction of both p38 mitogen-activated protein kinase and stress-activated protein kinase/Jun N-terminal kinase phosphorylation, whereas nuclear translocation of nuclear factor kappaB proceeded unimpeded. HC-gp39 suppressed the cytokine-induced secretion of MMP1, MMP3 and MMP13, as well as secretion of the chemokine IL-8. The suppressive effects of HC-gp39 were dependent on phosphoinositide 3-kinase activity, and treatment of cells with HC-gp39 resulted in AKT-mediated serine/threonine phosphorylation of apoptosis signal-regulating kinase 1. This process could therefore be responsible for the down-regulation of cytokine signalling by HC-gp39. These results suggest a physiological role for HC-gp39 in limiting the catabolic effects of inflammatory cytokines.
Asunto(s)
Glicoproteínas/fisiología , Adipoquinas , Adulto , Autopsia , Células Cultivadas , Preescolar , Proteína 1 Similar a Quitinasa-3 , Condrocitos/enzimología , Condrocitos/metabolismo , Condrocitos/patología , Regulación hacia Abajo/fisiología , Activación Enzimática/fisiología , Fibroblastos/enzimología , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Inmunidad Celular/fisiología , Inflamación/enzimología , Inflamación/metabolismo , Interleucina-1/metabolismo , Interleucina-8/metabolismo , Lectinas , MAP Quinasa Quinasa Quinasa 5 , Quinasas Quinasa Quinasa PAM/metabolismo , Masculino , Metaloproteinasas de la Matriz/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Transducción de Señal/fisiología , Piel/enzimología , Piel/metabolismo , Piel/patología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The 39-kDa human cartilage glycoprotein (HCGP39), a member of a novel family of chitinase-like lectins (Chilectins), is overexpressed in articular chondrocytes and certain cancers. Proposed functions of this protein include a role in connective tissue remodeling and defense against pathogens. Similar to other Chi-lectins, HCGP39 promotes the growth of connective tissue cells. The ability of HCGP39 to activate cytoplasmic signaling pathways suggests the presence of a ligand for this protein at the cell surface. There is currently no information regarding the identity of any physiological or pathological ligands of the Chi-lectins or the nature of the protein-ligand interaction. Here, we show that HCGP39 is able to bind chitooligosaccharides with micromolar affinity. Crystal structures of the native protein and a complex with GlcNAc8 show that the ligand is bound in identical fashion to family 18 chitinases. However, unlike the chitinases, binding of the oligosaccharide ligand to HCGP39 induces a large conformational change. Thus, HCGP39 could be a lectin that binds chitin-like oligosaccharide ligands and possibly plays a role in innate responses to chitinous pathogens, such as fungi and nematodes.
Asunto(s)
Condrocitos/metabolismo , Lectinas/química , Lectinas/metabolismo , Ligandos , Adipoquinas , Proteína 1 Similar a Quitinasa-3 , Cristalografía por Rayos X , Electrones , Glicoproteínas , Humanos , Modelos Moleculares , Oligosacáridos/química , Unión Proteica , Conformación Proteica , Transducción de SeñalRESUMEN
Human cartilage glycoprotein 39 (HC-gp39) is a glycoprotein secreted by articular chondrocytes, synoviocytes and macrophages. Increased levels of HC-gp39 have been demonstrated in synovial fluids of patients with rheumatoid or osteoarthritis. The increased secretion of HC-gp39 under physiological and pathological conditions with elevated connective-tissue turnover suggests its involvement in the homoeostasis of these tissues. We report here that HC-gp39 promotes the growth of human synovial cells as well as skin and fetal lung fibroblasts. A dose-dependent growth stimulation was observed when each of the fibroblastic cell lines was exposed to HC-gp39 in a concentration range from 0.1 to 2 nM, which is similar to the effective dose of the well-characterized mitogen, insulin-like growth factor-1. At suboptimal concentrations, the two growth factors work in a synergistic fashion. The use of selective inhibitors of the mitogen-activated protein kinase and the protein kinase B (AKT) signalling pathways indicates that both are involved in mediating the mitogenic response to HC-gp39. Phosphorylation of both extracellular signal-regulated kinases 1/2 and AKT occurred in a dose- and time-dependent fashion upon addition of HC-gp39. Activation of these signalling pathways could also be demonstrated in human chondrocytes. Thus HC-gp39 initiates a signalling cascade in connective-tissue cells which leads to increased cell proliferation, suggesting that this protein could play a major role in the pathological conditions leading to tissue fibrosis.
Asunto(s)
Cartílago Articular/fisiología , Glicoproteínas/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Adipoquinas , Cartílago Articular/citología , Cartílago Articular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Proteína 1 Similar a Quitinasa-3 , Quitinasas/fisiología , Células del Tejido Conectivo/citología , Células del Tejido Conectivo/efectos de los fármacos , Células del Tejido Conectivo/fisiología , Glicoproteínas/fisiología , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Lectinas , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Proto-Oncogénicas c-akt , Transducción de SeñalRESUMEN
Data presented previously suggest that release of components of the cartilage matrix, in response to catabolic agents, cannot be accounted for by proteolytic mechanisms alone. In the present study, the release of glycosaminoglycan-containing components from bovine nasal cartilage cultured in the presence of interleukin-1beta, and from bovine nasal, fetal bovine epiphyseal and adult human articular cartilage cultured in the presence of retinoic acid, was accompanied by the loss of link protein and hyaluronate into the culture medium. Chromatographic analysis of the released hyaluronate showed it to be markedly reduced in size relative to that extracted from the corresponding tissue. It is proposed that, under stimulation by catabolic agents, two independent, but concurrent, mechanisms act to promote the release of aggrecan from the cartilage matrix. First, proteolytic cleavage of the aggrecan core protein results in the production of glycosaminoglycan-containing fragments that are free to diffuse from the tissue. Secondly, cleavage of hyaluronate renders portions of the proteoglycan aggregate small enough so that complexes of aggrecan (or fragments containing its G1 domain) and link protein are released from the tissue. It is likely that both mechanisms contribute to cartilage metabolism in normal physiology and pathology.