Osteoarthritis (OA) is the most prevalent form of arthritis in the elderly and is characterized primarily by the degeneration and loss of articular cartilage. OA is considered a heterogeneous group of disorders with a variety of pathogenic factors, all of which result in similar patterns of cartilage degeneration (1). In OA of the knee, the medial compartment of the articular cartilage is the most susceptible to degeneration, whereas the lateral compartment remains relatively unaffected (2). This phenomenon appears in a single joint despite the same OA susceptibility of the cartilage matrix and the same genetic background.
There is now abundant evidence that chondrocytes play a critical role in cartilage degeneration. For example, OA chondrocytes secrete a variety of matrix breakdown products and cytokines, and cleavage of type II collagen is observed primarily around chondrocytes (3). Therefore, changes in the gene expression patterns of chondrocytes in response to various exogenous stimuli could affect the integrity of articular cartilage. Based on this concept, many groups of investigators have reported that gene expression in certain molecules differ from region to region within a single OA joint (4–7).
Because comprehensive gene expression profiles in intact and damaged regions of human OA cartilage have never been compared, the following questions remain unresolved. What percentage of the expressed transcripts shows obvious differences in expression levels in intact and damaged regions? What percentage of the differentially expressed transcripts shows the same expression pattern in different patient samples? What are the molecular functions involved in such groups of genes?
To provide answers to these questions, we used Affymetrix high-density oligonucleotide array analysis to compare the gene expression profile of chondrocytes in intact regions of joint cartilage with the profile of chondrocytes in damaged regions of joint cartilage from the same knee. We also evaluated the validity of our oligonucleotide array data according to the results of real-time quantitative polymerase chain reaction (PCR) amplification as well as gene expression data reported by other groups of investigators (4–7).
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- MATERIALS AND METHODS
In this study, we compared the gene expression profiles in intact versus damaged regions of OA cartilage. Transcripts with a ≥2-fold difference in mRNA expression between these 2 regions accounted for an average of 8% of all expressed transcripts per OA cartilage tissue sample, ∼10% of which were commonly detected in the 5 patient samples. The former observation indicates that the gene expression profile of chondrocytes in the intact region is quite different from that of chondrocytes in the damaged region, even though both regions are in contact with the same synovial fluid and have the same genetic background. The latter finding suggests that the gene expression profiles of chondrocytes in OA cartilage change in a region-specific manner. This also suggests the presence of common molecular mechanisms of OA development, assuming that the changes in gene expression patterns of chondrocytes lead to cartilage degeneration.
Using our selection criteria, we identified 114 genes that were differentially expressed in the intact region versus damaged region of OA cartilage. Thirty-five of these genes were up-regulated in the intact region, and 79 genes were up-regulated in the damaged region. The validity of our data was confirmed using real-time quantitative PCR and comparing our findings with those of previous studies. The possible roles of these genes with significantly altered mRNA expression are discussed here in terms of the reaction patterns of chondrocytes in order to determine the underlying mechanisms that may participate in the pathogenesis or progression of OA. Sandell and Aigner (39) described 5 categories of cellular reaction patterns related to OA development: phenotypic modulation of articular chondrocytes, formation of osteophytes, chondrocyte proliferation and apoptosis, matrix synthetic activity of chondrocytes, and matrix degradation activity of chondrocytes. The latter 3 are discussed below.
With regard to chondrocyte proliferation and apoptosis, the CCND1, PSAT, and S100A4 genes are known to be involved in cell proliferation, as mentioned above (17, 18, 21). These genes were more highly expressed in damaged regions than in intact regions of OA cartilage. This finding is consistent with the pathologic feature showing some clusters of chondrocytes in the surface layer of damaged cartilage, and it supports the hypothesis that changes in gene expression patterns of chondrocytes lead to the pathologic condition of OA in cartilage. Yet, among the 114 genes we identified, no genes were clearly related to apoptosis. (Complete data for all of the selected genes are available upon request from the corresponding author.) However, we cannot suggest that there are no differences in the activation of the apoptosis signaling pathway between intact and damaged regions of OA cartilage, because the proteolytic cascade, rather than transcriptional regulation, is important in apoptotic signaling (40).
With regard to the matrix synthetic activity of chondrocytes, our microarray data showed that although the signal intensities of type II collagen and aggrecan were very high in both intact and damaged regions of OA cartilage, there were no differences in gene expression between these 2 regions (data not shown). However, we found that 3 interstitial collagen genes (COL1A1, COL1A2, and COL5A1) and 4 genes for enzymes involved in the collagen biosynthetic pathway (P4HA3, LOXL2, LEPREL1, and LOXL3) were highly expressed in the damaged region of OA cartilage. Interestingly, hierarchical clustering analysis showed that 4 of these 7 genes (COL1A2, COL5A1, LOXL2, and LOXL3) belonged to the same cluster as proliferation-associated genes (S100A4 and PSAT1). This suggests that wound healing, including the process of cell proliferation and interstitial collagen synthesis, occurs in damaged OA cartilage, where the expression of genes related to wound healing might be regulated in the same manner.
With regard to the matrix degradation activity of chondrocytes, the most obvious pathologic feature in the damaged region is advanced cartilage destruction. Therefore, we predicted that expression levels of protease genes would be higher in damaged regions than in intact regions. Consistent with this expectation, we identified 4 proteases (DKFZP586H2123, ADAMTS6, ADAM12, and PRSS11) with high expression in the damaged region of OA cartilage among the selected 114 genes. However, MMP-2, which can degrade the extracellular matrix, is only one protease showing high expression in the intact region of OA cartilage. In particular, the PRSS11 (HtrA1) gene was recently reported to enhance cartilage degeneration via digestion of major cartilage components (41), and the single-nucleotide polymorphisms of the ADAM12 gene are associated with the progression of knee OA (28). However, there is a discrepancy between the function of the detected genes and the histopathologic features. The expression levels of 3 genes known to inhibit degradation of the extracellular matrix (TNFAIP6, SERPINE1, and TIMP3) were significantly high in the damaged region compared with the intact region of OA cartilage. These molecules are probably up-regulated to prevent the progression of cartilage destruction in the damaged areas of OA cartilage.
We subsequently examined whether the results of comparisons between intact and damaged regions of OA cartilage from the same joint resembled the results of comparisons between normal and OA cartilage obtained from different persons. We found that comparisons between normal and OA cartilage and between intact and damaged regions of OA cartilage yielded similar results with regard to the expression pattern of 7 of the 10 genes examined (Table 3). This suggests that during the transition from normal cartilage to OA lesional cartilage, the gene expression profile changes before there is any apparent damage to the cartilage. Because the expression levels of some genes probably change during the transitional period from normal to normal-appearing cartilage, our study design might have allowed us to miss some important genes that show no differences in expression levels between intact and damaged regions. To overcome this problem, further studies comparing these samples with normal cartilage samples from normal joints are needed.
What causes the OA-specific pattern of gene expression? Several possible mechanisms have been investigated, such as mechanical stress, cytokine stimulation, cell–matrix interaction, hypoxia, and reoxygenation. One of the strongest candidates is mechanical stress, because the damaged cartilage region is usually subjected to mechanical loading, whereas the intact region is not. In particular, chondrocytes residing in damaged regions are susceptible to mechanical stress because the tensile properties of the damaged cartilage are lost as a result of destruction of the collagen network (42). Proinflammatory cytokines, especially interleukin-1 (IL-1) and TNFα, are also closely related to the development of OA (3). We thought that these cytokines might be accessible to the chondrocytes in damaged cartilage. However, some IL-1–induced genes were detected in both regions (in the intact region, ETS1, GUCY1A3, C4BPA, PBEF, and APOD; in the damaged region, TNFAIP6, PTGES, FN1, NGFB, and TNFSF11). Furthermore, although MMP-2 mRNA is not significantly up-regulated by treatment with IL-1 (43), our microarray data showed that MMP-2 mRNA was expressed 7.3-fold higher in intact regions than in damaged regions of OA cartilage. Therefore, the effects of the cytokine alone could not account for these complex conditions of OA in vivo. Up-regulation of MMP-2 mRNA might be the result of other factors, including hypoxia/reoxygenation, cell–matrix interactions, and intermittent hydrostatic pressures (44–46).
In conclusion, our study demonstrated a clear difference in the gene expression profile in damaged regions of human OA cartilage compared with that in intact regions. The pattern of differences between these 2 regions was similar among 5 pairs of OA cartilage samples. This finding implies that there is a common mechanism responsible for the destruction and maintenance of the articular cartilage in OA. Elucidation of this mechanism is important for the development of effective treatments for OA.