SOX gene expression in human osteoarthritic cartilage.

OBJECTIVE: While the developmental role of the SOX transcription factors in fetal chondrocyte differentiation is well documented, much less is known about the expression of SOX family members in normal and osteoarthritic adult cartilage. Therefore, the aim of the present study was to present a thorough analysis of SOX gene expression in normal and osteoarthritic human adult cartilage. METHODS: RNA from normal and osteoarthritic knee cartilage from human adults was analyzed by gene expression profiling using GeneChip technology (Affymetrix) and quantitative real time PCR. RESULTS: Most members of the SOX transcription factor family showed no or very low expression levels in normal and osteoarthritic cartilage from adults. In contrast, SOX9 expression was fairly high in normal cartilage, amounting to approximately 20% of GAPDH levels. SOX9 transcript levels were substantially reduced in osteoarthritis. SOX6 levels were reduced, albeit starting from a low basis expression in normal tissue. CONCLUSION: The presented data indicate that the role of the SOX transcription factor family in adult human cartilage is most probably restricted to a few members, with SOX9 being the most prominent. Furthermore, the reduction of SOX9 and SOX6 transcript levels in osteoarthritic chondrocytes might be responsible for the loss of phenotypic stability of osteoarthritic chondrocytes.

Mechanisms of disease: role of chondrocytes in the pathogenesis of osteoarthritis--structure, chaos and senescence.

The extracellular matrix of articular cartilage is the primary target of osteoarthritic cartilage degradation. However, cartilage cells have a pivotal role during osteoarthritis, as they are mainly responsible for the anabolic-catabolic balance required for matrix maintenance and tissue function. In addition to the severe changes in the extracellular matrix, the cells also display abnormalities during osteoarthritic cartilage degeneration, such as inappropriate activation of anabolic and catabolic activities, and alterations in cell number through processes like proliferation and (apoptotic) cell death. The cells are exposed to additional stimuli such as nonphysiologic loading conditions and byproducts of matrix destruction, as well as abnormal levels of cytokines and growth factors. This exposure can lead to a structured cellular response pattern that may be either beneficial or detrimental to the cartilage tissue. Potentially even more problematic for preserving tissue homeostasis, neighboring osteoarthritic chondrocytes display strong heterogeneity in their phenotype, gene expression patterns, and cellular responses. As the disease progresses, osteoarthritic chondrocytes can no longer maintain tissue integrity. Evidence suggests that cell aging is important in the pathogenesis of osteoarthritis. Thus, anti-aging strategies might complement existing therapeutic targets related to anabolism, catabolism, inflammation, and apoptosis-processes that are integral to the pathogenesis of osteoarthritis.

Large-scale gene expression profiling reveals major pathogenetic pathways of cartilage degeneration in osteoarthritis.

OBJECTIVE: Despite many research efforts in recent decades, the major pathogenetic mechanisms of osteoarthritis (OA), including gene alterations occurring during OA cartilage degeneration, are poorly understood, and there is no disease-modifying treatment approach. The present study was therefore initiated in order to identify differentially expressed disease-related genes and potential therapeutic targets. METHODS: This investigation consisted of a large gene expression profiling study performed based on 78 normal and disease samples, using a custom-made complementary DNA array covering >4,000 genes. RESULTS: Many differentially expressed genes were identified, including the expected up-regulation of anabolic and catabolic matrix genes. In particular, the down-regulation of important oxidative defense genes, i.e., the genes for superoxide dismutases 2 and 3 and glutathione peroxidase 3, was prominent. This indicates that continuous oxidative stress to the cells and the matrix is one major underlying pathogenetic mechanism in OA. Also, genes that are involved in the phenotypic stability of cells, a feature that is greatly reduced in OA cartilage, appeared to be suppressed. CONCLUSION: Our findings provide a reference data set on gene alterations in OA cartilage and, importantly, indicate major mechanisms underlying central cell biologic alterations that occur during the OA disease process. These results identify molecular targets that can be further investigated in the search for therapeutic interventions.

Gene expression profiling of serum- and interleukin-1 beta-stimulated primary human adult articular chondrocytes--a molecular analysis based on chondrocytes isolated from one donor.

In order to understand the cellular disease mechanisms of osteoarthritic cartilage degeneration it is of primary importance to understand both the anabolic and the catabolic processes going on in parallel in the diseased tissue. In this study, we have applied cDNA-array technology (Clontech) to study gene expression patterns of primary human normal adult articular chondrocytes isolated from one donor cultured under anabolic (serum) and catabolic (IL-1beta) conditions. Significant differences between the different in vitro cultures tested were detected. Overall, serum and IL-1beta significantly altered gene expression levels of 102 and 79 genes, respectively. IL-1beta stimulated the matrix metalloproteinases-1, -3, and -13 as well as members of its intracellular signaling cascade, whereas serum increased the expression of many cartilage matrix genes. Comparative gene expression analysis with previously published in vivo data (normal and osteoarthritic cartilage) showed significant differences of all in vitro stimulations compared to the changes detected in osteoarthritic cartilage in vivo. This investigation allowed us to characterize gene expression profiles of two classical anabolic and catabolic stimuli of human adult articular chondrocytes in vitro. No in vitro model appeared to be adequate to study overall gene expression alterations in osteoarthritic cartilage. Serum stimulated in vitro cultures largely reflected the results that were only consistent with the anabolic activation seen in osteoarthritic chondrocytes. In contrast, IL-1beta did not appear to be a good model for mimicking catabolic gene alterations in degenerating chondrocytes.

Quantification of mRNA expression levels in articular chondrocytes with PCR technologies.

Unlike any other technology in molecular biology, the polymerase chain reaction (PCR) has changed the technological armamentarium of molecular scientists working on cartilage, in terms of outstanding sensitivity and accuracy. Four approaches to determine mRNA expression levels by PCR amplification of specific cDNA sequences are currently in use and are discussed in this chapter: conventional PCR with end-point determination, conventional PCR in the logarithmic amplification phase, conventional PCR using internal competitive DNA fragments, and real-time PCR as offered by TaqMan technology and others. The determination of mRNA expression levels by real-time quantitative PCR appears to be the most reliable method for accurate determination of gene expression levels within cartilage and cultured chondrocytes, as in other tissues and cell types. This technology offers outstanding sensitivity and accuracy in terms of determination of the amount of cDNA molecules. However, this method cannot account for factors such as efficiency of RNA isolation and reverse transcription conditions. Thus, normalization of the acquired data is required, with all its limitations as described.

Analysis of differential gene expression in healthy and osteoarthritic cartilage and isolated chondrocytes by microarray analysis.

The regulation of chondrocytes in osteoarthritic cartilage and the expression of specific gene products by these cells during early-onset and late-stage osteoarthritis are not well characterized. With the introduction of cDNA array technology, the measurement of thousands of different genes in one small tissue sample can be carried out. Interpretation of gene expression analyses in articular cartilage is aided by the fact that this tissue contains only one cell type in both normal and diseased conditions. However, care has to be taken not to over- and misinterpret results, and some major challenges must be overcome in order to utilize the potential of this technology properly in the field of osteoarthritis.

Down-regulation of the GTPase RhoB might be involved in the pre-apoptotic phenotype of osteoarthritic chondrocytes.

Anabolic activity, phenotypic alterations, and in particular survival of the chondrocytes are essential for the maintenance of proper articular cartilage and appears to fail during osteoarthritic cartilage degeneration. In this study, we investigated the presence and expression of RhoB in adult human articular cartilage and its regulation in osteoarthritic cartilage as well as in chondrocytes in vitro. RhoB belongs to the family of small GTPases, which are thought to be involved in a large range of activities important for eukaryotic cells. Conventional and quantificative PCR analysis showed significant levels of RhoB expression in normal articular cartilage. Immunolocalization and confocal laser scanning microscopy showed strong cytoplasmic signals for RhoB in normal chondrocytes. In osteoarthritic cartilage, a significantly lower expression of RhoB was detectable. In vitro experiments showed a quick (and transient) up-regulation of RhoB after stimulation with interleukin-1beta and serum. Our study suggests that RhoB is constitutively expressed and essential for adult articular chondrocytes, but significantly down-regulated in osteoarthritic chondrocytes. One intriguing speculation might be that the down-regulation of RhoB in osteoarthritic chondrocytes is at least partly a prerequisite for the sustained pre- or para-apoptotic phenotype of osteoarthritic chondrocytes, because RhoB is known to be one important molecule in the induction of apoptotic cell death in response to DNA damage and osteoarthritic chondrocytes are known to have significant DNA damage. Alternatively, RhoB could be involved in the activation or deactivation and the destabilization of the functional phenotype of chondrocytes in osteoarthritic joint degeneration Thirdly, RhoB is associated with the cell cycle, which is re-initiated in osteoarthritis.

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

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-1beta (IL-1beta)-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-1beta. 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-1beta. Among all proteases, MMP-3 was by far the most strongly expressed, although it was strongly down-regulated in late-stage OA specimens. Expression of MMP-3 was higher in vitro than in vivo and was up-regulated by IL-1beta. 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-1beta. 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-1beta stimulation of articular chondrocytes might not be a good model for the matrix catabolism in OA cartilage.