Anneliese D. Recklies

The chitinase 3-like protein human cartilage glycoprotein 39 inhibits cellular responses to the inflammatory cytokines interleukin-1 and tumour necrosis factor-alpha

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.

Regulation of cartilage oligomeric matrix protein synthesis in human synovial cells and articular chondrocytes

OBJECTIVE Cartilage oligomeric matrix protein (COMP) is a component of the extracellular matrix of articular cartilage. Its increased presence in synovial fluid and serum has been associated with accelerated joint damage in patients with rheumatoid arthritis (RA) and osteoarthritis. To fully understand the reasons for fluctuations of COMP levels, we studied the biosynthesis of this molecule in cells derived from joint tissues. METHODS Synovial cells were derived from synovial tissues of patients with RA, and human articular chondrocytes were prepared from normal articular cartilage. Analysis by Northern blotting was used to evaluate steady-state levels of COMP messenger RNA (mRNA), while secretion of the protein into culture media was analyzed by Western blotting. Expression of COMP in synovial tissues was studied by reverse transcriptase-polymerase chain reaction analysis and by in situ hybridization. RESULTS COMP was synthesized and secreted by synovial cells as well as by articular chondrocytes in culture. The basal rate of synthesis was very low; however, COMP biosynthesis in both cell populations was induced very strongly by transforming growth factor beta1 (TGFbeta1). Interleukin-1beta counteracted COMP induction by TGF-beta1. COMP was not detected in culture media of skin or fetal lung fibroblasts, either in the absence or the presence of TGFbeta1. COMP mRNA was also present in fresh synovial tissue specimens obtained from patients with RA. CONCLUSION COMP is synthesized and secreted not only by articular chondrocytes, but also by synovial fibroblasts. The demonstration of COMP expression in surgical specimens of synovial tissues suggests that the inflamed synovium may provide an additional source for the elevated levels of COMP observed in arthritis. Thus, increased COMP levels in body fluids may be indicative of active synovitis as well as of accelerated joint erosion.

Inflammatory Cytokines Induce Production of CHI3L1 by Articular Chondrocytes

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-α (TNF-α) and interleukin-1 potently induced steady-state levels of CHI3L1 mRNA and protein secretion. Treatment of chondrocytes with TNF-α 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-α and interleukin-1, only inhibition of NF-κB 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-κB signaling also abolished constitutive expression by human chondrocytes. Thus, induction and continued secretion of CHI3L1 in chondrocytes require sustained activation of NF-κB. Selective induction of CHI3L1 by cytokines acting through NF-κB 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.

Human YKL-39 is a pseudo-chitinase with retained chitooligosaccharide-binding properties

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.

Identification and Characterization of the Integrin α2β1 Binding Motif in Chondroadherin Mediating Cell Attachment

Chondroadherin is a leucine-rich repeat protein known to mediate adhesion of isolated cells via the integrin α2β1 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 α2β1 receptor. We identified a cell binding motif, CQLRGLRRWLEAK318 by mass spectrometry after protease digestion of chondroadherin. Cells adhering to the synthetic peptide CQLRGLRRWLEAK318 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 α2β1 integrin. A cyclic peptide, CQLRGLRRWLEAKASRPDATC326, 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 LRRWLEAK318. 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.

THE SUBTERRANEAN CRUSTACEAN FAUNA OF CASTLEGUARD CAVE, COLUMBIA ICEFIELIDS, ALBERTA, CANADA, AND ITS ZOOGEOGRAPHIC SIGNIFICANCE*

A biological survey of Castleguard Cave has shown that the inner cave is inhabited by two species of aquatic crustaceans, the asellid isopod Salmasellus steganothrix and the crangonyctid amphipod Stygobromus canadensis, both of which are blind and unpigmented. The isopod is abundant throughout the portions of the system containing sediment-rich pools and was found in areas of the cave which lie beneath the Columbia Icefields. The amphipod, which is unique to Castleguard Cave, was found only in one series of pools about 2 km from the entrance. The occurrence of these aquatic species so far inside an area fully glacierized during the Wisconsinan and even at present partially under a permanent icefield suggests that they may be remnants of very old preglacial distributions. Since it is known that the cave has remained intact and internally ice free for more than 720,000 yr, it is hypothesized that it has served as a subglacial refugium for these organisms.

Cells from the skin of patients with systemic sclerosis secrete chitinase 3-like protein 1☆

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.