Obesity is associated with alterations in adipose tissue, including the recruitment of macrophages and T cells. Adipose tissue is no longer considered to be an inert tissue, functioning solely for energy storage. Various secreted products of adipose tissue, called adipokines, have recently been characterized, including adiponectin, leptin, resistin, and visfatin (1–3). Adipokines are associated with a chronic inflammatory response syndrome characterized by abnormal cytokine production, increased acute-phase reactant synthesis, and activation of inflammation (1, 3, 4). Recent studies have shown that adipokines represent a potent risk factor for the development and progression of rheumatoid and osteoarthritic joint diseases (5–7).
Resistin is a 12.5-kd cysteine-rich polypeptide that belongs to a family of resistin-like molecules (RELMs) or found in inflammatory zone (FIZZ) molecules (1, 2). Resistin is not only expressed by human adipocytes, but it is also expressed in high levels by macrophages (1). Many aspects of the biologic effects and the regulation of resistin remain subjects of controversy, but studies have provided evidence of a role of resistin in inflammatory processes (1, 3, 8). In rheumatoid and osteoarthritic joint diseases, increased levels of resistin were observed in the synovial fluid and tissue of patients with rheumatoid arthritis (RA) or osteoarthritis (OA) (5, 9, 10), and plasma levels of resistin were significantly correlated with the erythrocyte sedimentation rate and the C-reactive protein level (9). Furthermore, resistin was shown to up-regulate interleukin-1 (IL-1), IL-6, and tumor necrosis factor α (TNFα) in the blood and synovial fluid of patients with RA. Intraarticular injection of resistin was shown to induce arthritis in healthy mouse joints (11).
Cytokines and chemokines are mediators of inflammation and are known to be important in inflammatory diseases, including RA and OA (12–14). Cytokines are a category of signaling molecules that are involved in cellular communication. Chemokines are a specific class of cytokines that mediate chemoattraction (chemotaxis). Chemokines all have a similar protein structure, being 8–10 kd, with the 2 major subclasses having conserved cysteine residues either adjacent to each other (CC) or separated by 1 amino acid (CXC) (15). Using genome-wide expression analysis of human articular chondrocytes, we previously showed that a large site of chemokines was up-regulated by the proinflammatory cytokine IL-1β (12).
Most studies with resistin have focused on cells in the inflammatory cascade. It has recently been shown that resistin is elevated following traumatic joint injury and causes the loss of proteoglycan, the production of prostaglandin E2, and the release of inflammatory cytokines from articular cartilage (16). In this study, we investigated the expression levels of cytokines and chemokines in human articular chondrocytes in response to resistin, and we generated an overall picture of their regulation at the levels of transcription and posttranscription.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
Resistin, the adipocyte-derived cytokine, is a potent link between adipokines and inflammatory diseases (1, 11), including rheumatoid and osteoarthritic joint diseases (9, 11). To provide a view of the effect of resistin on the expression of human articular chondrocyte genes, we analyzed 25 genes related to the inflammatory cascade, including 6 cytokines, 14 chemokines, and 5 matrix genes. We found that the levels of the tested chemokines and cytokines were dramatically increased in human adult articular chondrocytes by exposure to the adipokine resistin. One exception was the lack of effect on CXCL12, which is also known as stromal cell–derived factor 1 (SDF-1). A similar pattern of expression was previously observed for chemokines induced by IL-1β in human articular chondrocytes (12). The expression of mRNA for MMP-1, MMP-13, and ADAMTS-4 was also increased, while that of mRNA for COL2A1 and aggrecan was down-regulated in response to the resistin. The expression of ADAMTS-5 was also monitored, and its expression was reduced by resistin (data not shown).
In inflamed joints, cytokines and chemokines are produced by the synovium, macrophages, and fibroblast-like synoviocytes, and they are thought to be key regulators of the inflammatory process (12, 13, 15, 22, 23). Cytokines both enhance the migration of cells into the joint and stimulate matrix metalloproteinase production in synovial fibroblasts and chondrocytes (22). Chemokines function in the recruitment of neutrophils, monocytes, immature dendritic cells, B cells, and activated T cells (24). Furthermore, it has recently been reported that the CXC family of chemokines is important in the regulation of angiogenesis in RA, and CCL2, CCL3, and CCR2 stimulate osteoclastogenesis (25–27). The production of chemokines and cytokines under the influence of resistin could therefore significantly alter the metabolism of chondrocytes.
Cytokines and chemokines that are highly up-regulated by resistin in inflammation have not previously been shown to be regulated by resistin in human chondrocytes. IL-1α, IL-1β, IL-6, IL-8, CCL2, CCL3, CCL4, and TNFα have been identified in patient serum, synovial fluid, and blood cells following resistin stimulation (9, 11, 16). Lee and colleagues (16) also reported that resistin stimulated the secretion of CCL2 and IL-6 in mouse cartilage. Adipokines are expressed in the joint tissue and serum of patients with rheumatoid and osteoarthritic joint diseases (9, 10, 16, 28–31). Adiponectin is unable to modulate TNFα or IL-1β release in chondrocytes (30), but resistin can up-regulate them, especially IL-1β, which was increased more than 100-fold following treatment with 100 ng/ml of resistin. As an important cytokine in inflammatory joint disease, IL-1β can induce enzymes that degrade the extracellular matrix and reduce the synthesis of the primary cartilage components COL2A1 and aggrecan (12).
The level of gene expression is regulated at both the transcriptional and posttranscriptional levels in eukaryotic cells, fibroblasts, and chondrocytes (17, 21, 32). Modulation of the mRNA decay rate is a strategy widely used by cells to adjust the intensity of expression (33). Recently, Hao and Baltimore (17) reported that mRNA stability influences the levels of genes encoding inflammatory molecules in mouse fibroblasts, providing a temporally controlled process of protein expression. The same trend was observed in our human chondrocyte samples over a 24-hour time period for the cytokine and chemokine genes, including TNFα, IL-1β, IL-6, CXCL1, CXCL2, CCL2, CCL20, CCL5, CX3CL1, and CXCL5. As Hao and Baltimore had reported, we found the expression of genes from group I that were highly related to mRNA stability contained a large number of AREs (Table 1), which are known to destabilize mRNA. The effect of mRNA stability was also important in genes from group II, but mRNA from group III genes was more stable, and mRNA stability did not significantly affect their expression.
In the present study, although IL-1β and CXCL1 were not among the group I genes expressed in human articular chondrocytes, the extension of mRNA stability in these genes indicated that the mRNA stability is also involved in the steady-state level of mRNA. For BMP-2, Fukui and colleagues (21) showed the up-regulation of BMP-2 in chondrocytes via both transcription and mRNA stability. Furthermore, the results of mRNA stability analyses revealed that mRNA stability is also involved in the up-regulation of group II and group III genes. Together, the findings of these studies support the view that mRNA stability is an important determinant in resistin-induced gene expression.
To explore potential transcriptional regulation of the chemokines and cytokines, they were subclassified according to the extent of their up-regulation at 24 hours and were subjected to a computational analysis for transcription factor binding sites that were highly represented in each set. It has been demonstrated that the computed scores are highly correlated with binding probability, such that promoters with higher combined scores were more likely to be bound by the transcription factor than were promoters with lower scores (19). In the genes that were highly up-regulated, binding sites for factors related to NF-κB had very high scores (>90%). The importance of the NF-κB signaling pathway for resistin-induced inflammation has been reported for blood cells (11). We also showed that the activity of the pNF-κB luciferase reporter in human chondrocytes was increased significantly after resistin treatment. Cotransfection of the IKK-2 expression vector established that IKK-2 could enhance the promoter activity of CCL3 and CCL4 with resistin stimulation. Together, these observations showed that NF-κB signaling in human chondrocytes is involved in cytokine and chemokine expression with resistin treatment.
It has been reported that the NF-κB inhibitor hypoestoxide reduced fibronectin fragment induction of IL-6, IL-8, CCL2, CXCL1, CXCL2, and CXCL3 in human articular chondrocytes (34). Amos and colleagues (35) also demonstrated that inhibition of NF-κB activity inhibited most, but not all, mediators of inflammation. Thus, to address the role of NF-κB in resistin-mediated cytokine and chemokine expression, we used the cell-permeable IKK-NBD peptide; this peptide prevents the association of NEMO/IKKγ with IKKα and IKKβ, which is required for NF-κB activation (36). We showed that IKK-NBD modestly inhibited the resistin-induced cytokine and chemokine mRNA expression, but did not inhibit all of the mRNA expression. However, since IKK-NBD is a potent inhibitor of only canonical IKK signaling, the resistin-induced cytokine and chemokine mRNA up-regulation could also be activating NF-κB subunits by an IKK-independent mechanism, which could be important in further studies.
To begin to account for the additional expression, we investigated the role of another transcription factor with a high binding score, C/EBPβ. Cotransfection of the C/EBPβ expression vector indicated that C/EBPβ could also enhance the promoter activity of CCL3 (group III gene) and CCL4 (group I gene) (37, 38). Since IKK-NBD inhibited ∼40% of the CCL3 and CCL4 mRNA expression, C/EBPβ could also be an important regulator.
C/EBPβ has previously been associated with IL-1β–induced and TNFα-induced changes in chondrocyte gene expression. C/EBPβ is increased in chondrocytes by IL-1β and TNFα, and down-regulates COL2A1 and cartilage-derived retinoic acid–sensitive protein (CD-RAP) (37–39). In addition, C/EBPβ plays an important role in repressing cartilage gene expression in noncartilaginous tissues (40). Hirata and colleagues (41) reported that C/EBPβ promoted the transition from proliferation to hypertrophy in growth plate chondrocytes. A cooperative interaction of C/EBPβ and NF-κB has been demonstrated in other genes. The involvement of both C/EBPβ and NF-κB was recently shown in the expression of IL-1β and IL-8 (42, 43). C/EBPβ regulates the basal transcription activity of IL-8, and C/EBPβ and NF-κB together mediate the IL-8 response to infection by Pseudomonas aeruginosa (43).
In summary, we have shown that many cytokines and chemokines are up-regulated by the adipokine resistin in human articular chondrocytes. These findings begin to provide a molecular mechanism by which the increased levels of resistin that occur following traumatic joint injury (16) could lead to matrix degradation. The mRNA stability of some cytokines and chemokines was increased by resistin, which indicated the potential involvement of a posttranscriptional mechanism in the induction of these genes in human chondrocytes. By computational analysis and experimental studies, NF-κB is the most highly represented transcription factor binding site, but we demonstrate that C/EBPβ is also involved. Considering this finding in combination with our IL-1β results in human chondrocytes (12), it can be expected that this high-level increase in such a wide range of cytokines and chemokines will have a significant impact on cartilage cells and should be considered in the pathophysiology of rheumatoid and osteoarthritic joint diseases. These studies provide the basis for further investigation into the function and regulation of chemokines in synovial joint disease.