Reexpression of Cartilage-Specific Genes by Dedifferentiated Human Articular Chondrocytes Cultured in Alginate Beads

370

322

Objective. Osteoarthritic (OA) cartilage destruction depends on collagen-and aggrecan-degrading proteases such as collagenases (MMP-1 and MMP-13), stromelysin (MMP-3), MMP-14, as well as the so-called aggrecanases (ADAM-TS4 and ADAM-TS5). In this study, we tried to clarify whether these proteases are expressed in vivo in human normal and OA cartilage (and whether they are up-regulated or down-regulated during the disease process) and in interleukin-1␤ (IL-1␤)-stimulated chondrocytes in vitro.Methods. Quantitative polymerase chain reaction assays were developed and performed on RNA isolated directly from normal and degenerative cartilage tissue as well as from primary human articular chondrocytes cultured with and without IL-1␤.Results. In vivo, MMP-1 was detectable only at very low levels in any condition. MMP-13 expression was low in normal and early degenerative cartilage but was strongly up-regulated in late-stage OA specimens. MMP-1 and MMP-13 were expressed much higher in vitro than in vivo and were up-regulated by IL-1␤. Among all proteases, MMP-3 was by far the most strongly expressed, although it was strongly downregulated in late-stage OA specimens. Expression of MMP-3 was higher in vitro than in vivo and was up-regulated by IL-1␤. ADAM-TS5 and MMP-14 were expressed in all sample groups. Expression of ADAM-TS4 was very low in vivo and was induced in vitro after stimulation by IL-1␤.Conclusion. Our expression data clearly support MMP-13 as the major collagenase in OA cartilage. The most strongly expressed aggrecanase was ADAM-TS5. ADAM-TS4 was expressed only at a very low level in normal cartilage and was only slightly up-regulated in OA cartilage, casting doubt on this enzyme being the relevant aggrecanase of articular cartilage. Results of our study show that expression of many enzymes is significantly different in vitro and in vivo and suggest that IL-1␤ stimulation of articular chondrocytes might not be a good model for the matrix catabolism in OA cartilage.Osteoarthritic (OA) cartilage degeneration as well as cartilage destruction in rheumatoid arthritis depend, at least to a significant degree, on enzymatic degradation of matrix components. Two types of molecules, collagen fibrils and the proteoglycan aggrecan, represent the major targets in terms of enzymatic activity and functional loss of the cartilage matrix as a result of damage to these molecules. Whereas degradation of collagen fibrils leads to matrix instability with tissue swelling, degradation of proteoglycans leads to cartilage softening and loss of fixed charges (1), both of which are classic features of cartilage destruction.Catabolism of both types of molecules is supposed to involve different types of enzymes, largely from 2 protease families: matrix metalloproteinases (MMPs) and the "A disintegrin and metalloproteinases with thrombospondin type 1 motif" (ADAM-TS). The initial degradation of collagen fibrils (within the triple-helical region) depends on cleavage at the collagenase site, for which there exist 2 major candidate enzymes...

Section: Discussionmentioning

confidence: 99%

Relative messenger RNA expression profiling of collagenases and aggrecanases in human articular chondrocytes in vivo and in vitro

Bau¹,

Gebhard²,

Haag³

et al. 2002

370

322

“…Chondrocyte dedifferentiation is characterized by the loss of chondrocyte phenotype, which is represented by the decline in expression of type II procollagen and aggrecan, which are 2 major matrix genes expressed by chondrocytes (10,29,30). To examine the involvement of the respective integrins in dedifferentiation, we suppressed the expression of the 11 dominant integrins listed above in chondrocytes one by one by RNAi and observed whether any increase (or recovery) occurred in the expression of type II procollagen and aggrecan.…”

Section: Determination Of Dominant Integrins In Human Articular Chondmentioning

confidence: 99%

“…To date, several methods are known to prevent or delay the dedifferentiation of chondrocytes. The cellular phenotype can be preserved when chondrocytes are cultured in 3-dimensional matrices such as agarose (7), alginate (8)(9)(10), or collagen gels (11). Other researchers have found that the metabolic and morphologic changes could be reversed by the disruption of the cytoskeletal complex (12,13).…”

mentioning

confidence: 99%

αvβ5 Integrin promotes dedifferentiation of monolayer-cultured articular chondrocytes

Fukui¹,

Ikeda²,

Tanaka³

et al. 2011

Objective. When cultured in monolayers, articular chondrocytes undergo an obvious phenotypic change. Although the involvement of integrins has been suggested, the exact mechanisms of the change have not been determined. This study was undertaken to clarify the mechanisms underlying the loss of chondrocyte phenotype early after plating.Methods. Primary cultured human articular chondrocytes were used for the experiments. Involvement of respective integrins in the phenotypic change was investigated in RNA interference (RNAi) experiments. A signaling pathway involved in the change was identified in experiments using specific inhibitors and adenoviruses encoding mutated genes involved in the pathway. Adenoviruses carrying mutated GTPases were used to determine the involvement of small GTPases in the process.Results. In monolayer-cultured chondrocytes, suppression of ␣v or ␤5 integrin expression by RNAi inhibited morphologic changes in the cells and increased (or prevented a reduction in) the expression of various cartilage matrix genes. Consistent results were obtained in experiments using a blocking antibody and a synthetic inhibitor of ␣v␤5 integrin. The decrease in cartilage matrix gene expression in chondrocytes after plating was mediated by ERK signaling, which was promoted primarily by ␣v␤5 integrin. In articular chondrocytes, the affinity of ␣v␤5 integrin for ligands was regulated by the small GTPase R-Ras. R-Ras was gradually activated in monolayer-cultured chondrocytes after plating, which caused a gradual decline in cartilage matrix gene expression through enhanced ␣v␤5 integrin activation and the subsequent increase in ERK signaling.Conclusion. Our findings indicate that ␣v␤5 integrin may be involved in the change that occurs in monolayer-cultured chondrocytes after plating.

“…Studies that have examined the plasticity of the chondrocyte phenotype from many different species have consistently shown that culture of these cells in monolayers on plastic substrata for prolonged periods or upon repeated passages leads to the loss of their spherical shape and to the acquisition of an elongated fibroblast-like morphology (7,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). These morphologic alterations are accompanied by profound biochemical changes, including loss of the cartilage-specific phenotype, as evidenced by an arrest of the synthesis of the cartilage-specific collagens (types II, IX, and XI) and proteoglycans (aggrecan), initiation of synthesis of the interstitial collagens (types I, III, and V), and increase in the synthesis of fibroblasttype proteoglycans (versican) at the expense of aggrecan (7,17,(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29).…”

mentioning

confidence: 99%

“…The chondrocyte phenotype can be reexpressed in these cells by culturing them in suspension, in agarose, with alginate beads, or on a hydrogel substrate (17,19,25,28,(30)(31)(32)(33)(34)(35)(36). These changes in the biosynthetic profile of dedifferentiated chondrocytes resemble some of the phenotypic changes displayed by OA chondrocytes, and the matrix they produce is similar to that synthesized by chondroprogenitor cells (7)(8)(9)(10)(11)(12)(13)17,(37)(38)(39)(40).…”

mentioning

confidence: 99%

Assessment of the gene expression profile of differentiated and dedifferentiated human fetal chondrocytes by microarray analysis

Stokes¹,

Liu²,

Coimbra³

et al. 2002

152

106

Objective. To study the changes in patterns of gene expression exhibited by human chondrocytes as they dedifferentiate into fibroblastic cells in culture in order to better understand the mechanisms that control this process and its relationship to the phenotypic changes that occur in chondrocytes during the development of osteoarthritis (OA).Methods. Human fetal epiphyseal chondrocytes (HFCs) were cultured either on poly-(2-hydroxyethyl methacrylate)-coated plates (differentiated HFC cultures) or in plastic tissue culture flasks as monolayers (dedifferentiated HFC cultures). Following 11 days of culture under either condition, poly(A؉) RNA was isolated from the two cell populations and subjected to a gene expression analysis using a microarray containing ϳ5,000 known human genes and ϳ3,000 expressed sequence tags (ESTs).Results. A >2-fold difference in the expression of 62 known genes and 6 ESTs was observed between the two cell types. The differences in expression of several of the genes detected by the microarray hybridization were confirmed by Northern analyses. Two transcription factor genes, TWIST and HIF-1␣, and a cellular adhesion protein gene, cadherin 11, were markedly regulated in response to differentiation and dedifferentiation. Expression of these genes was also detected in adult normal and OA cartilage and chondrocytes. Analysis of the gene expression profile of HFCs revealed a complex pattern of gene expression, including many genes not yet reported to be expressed by chondrocytes.Conclusion. Chondrocytes in monolayer become dedifferentiated, acquiring a fibroblast-like appearance and changing their pattern of gene expression from one of expression of chondrocyte-specific genes to one that resembles a fibroblastic or chondroprogenitor-like pattern. Changes in gene expression associated with the process of dedifferentiation of HFCs in vitro were observed in a wide variety of genes, including genes encoding extracellular matrix proteins, transcription factors, and growth factors. At least 3 of the genes that were regulated in response to dedifferentiation were also found to be expressed in adult normal and OA articular cartilage and chondrocytes.