TNF-α In Chondrocytes Research Articles

Synovial osteoclastogenesis mediated by chondrocyte-secreted TNFα promotes TMJ condylar resorption.

Insufficient occlusal support (IOS) frequently causes subchondral bone absorption in temporomandibular joint osteoarthritis, and the underlying mechanism requires further investigation. An IOS model was established by abrading rat molars. Micro-computed tomography was used to evaluate subchondral bone changes. Osteoclastogenesis of synovium-derived macrophages (SDMs) was confirmed by TRAP staining. Cartilage-specific TNFα depletion was achieved by intra-articular injection of adeno-associated virus carrying shRNA against murine TNFα under control of collagen type II. Invitro, chondrocytes were mechanically compressed and conditioned medium (CM) was collected to detect its ability to induce osteoclastogenesis of SDMs. Synovial osteoclastogenesis and condyle resorption were observed following IOS. TNFα level was elevated in hypertrophic chondrocytes after IOS. Synovial Wnt5a level increased, but Wnt3a level decreased after IOS. Depletion of TNFα in chondrocytes alleviated the synovial osteoclastogenesis and condyle bone resorption. Invitro compression of chondrocytes potentiated TNFα expression and secretion. The CM promoted osteoclastogenesis of SDMs, which were partially prohibited by TNFα neutralizing antibody. Furthermore, inhibition of Wnt3a facilitated osteoclastogenesis, whereas inhibition of Wnt5a partially suppressed osteoclastogenesis, of SDMs cultured in CM. Chondrocyte-secreted TNFα induced by IOS is a critical regulator of synovial osteoclastogenesis and subsequent condylar resorption, partially through non-canonical Wnt5a pathway.

Infrapatellar Fat Pad Modulates Osteoarthritis-Associated Cytokine and MMP Expression in Human Articular Chondrocytes.

Osteoarthritis (OA) most frequently affects the knee joint and is associated with an elevated expression of cytokines and extracellular cartilage matrix (ECM), degrading enzymes such as matrix metalloproteinases (MMPs). Differences in gene expression of the intra-articularly located infrapatellar fat pad (IPFP) and other fatty tissue suggest its autonomous function, yet its role in OA pathogenesis remains unknown. Human IPFPs and articular cartilage were collected from OA patients undergoing total knee arthroplasty, and biopsies from the IPFP of healthy patients harvested during knee arthroscopy served as controls (CO). Isolated chondrocytes were co-cultured with either osteoarthritic (OA) or CO-IPFPs in a transwell system. Chondrocyte expression of MMP1, -3, -13, type 1 and 2 collagens, interleukin IL1β, IL6, IL10, and tumor necrosis factor TNFα was analyzed by RTD-PCR at day 0 and day 2, and TNFα secretion was analyzed by ELISA. The cytokine release in IPFPs was assessed by an array. Results: Both IPFPs (CO, OA) significantly reduced the expression of type 2 collagen and TNFα in chondrocytes. On the other hand, only CO-IPFP suppressed the expression of type 1 collagen and significantly induced the MMP13 expression. On the contrary, IL1β and IL6 were significantly induced when exposed to OA-IPFP. Conclusions: The partial loss of the suppressive effect on type 1 collagen gene expression found for OA-IPFP shows the pathological remodeling and dedifferentiation potential of the OA-IPFP on the chondrocytes. However, the significant suppression of TNFα implies that the OA- and CO-IPFP could also exhibit a protective role in the knee joint, preventing the progress of inflammation.

Knockdown of MKL1 ameliorates oxidative stress-induced chondrocyte apoptosis and cartilage matrix degeneration by activating TWIST1-mediated PI3K/AKT signaling pathway in rats

Studies have reported that megakaryocytic leukemia 1 (MKL1) is closely related to the pathological process of a variety of inflammatory diseases, but its role in osteoarthritis (OA) needs to be clarified. This study aimed to investigate the regulatory role of MKL1 in oxidative stress-induced chondrocyte apoptosis and cartilage matrix degeneration. The expressions of target mRNAs and proteins were measured by using reverse transcription-quantitative polymerase chain reaction and western blotting. ELISA assay was used to measure the levels of IL-6, IL-8, and TNF-α in chondrocytes. And commercial kits based on different spectrophotometry or colorimetry methods were performed to validate oxidative stress. CCK-8 and apoptosis kits were used to determine cell viability and apoptosis. Rat OA model was established by anterior cruciate ligament transection (ACLT), and the expression of MKL1 was interfered by injecting sh-MKL1 lentiviral vector into caudal vein. The results showed that the expression of MKL1was induced by H2O2 in chondrocytes. Knockdown of MKL1 alleviated H2O2-induced inflammation and cell apoptosis, reduced H2O2-induced oxidative stress, and improved cartilage matrix degeneration of chondrocytes. Besides, inhibition of MKL1 regulated the activation of TWIST1-mediated PI3K/AKT signaling. Further studies have found that TWIST1-mediated PI3K/AKT signaling was involved in the regulation mechanism of MKL1 on chondrocyte apoptosis and cartilage matrix degeneration. Next, intervention with MKL1 inhibited the progression of OA in rats. These results demonstrated that MKL1 regulate the apoptosis and cartilage matrix degeneration of chondrocytes via TWIST1-mediated PI3K/AKT signaling.

Circular RNA CCDC66 Regulates Osteoarthritis Progression by Targeting miR-3622b-5p

Objective: CircCCDC66 is involved in cancer progression, but its role in osteoarthritis (OA) remains unknown. This study was carried out to explore the biological role of circCCDC66 in OA and its underlying mechanism. Methods: The expression levels of miR-3622b-5p and circCCDC66 in OA cartilage tissues were detected by qRT-PCR. Cell Counting Kit-8 (CCK8) and flow cytometry were used to detect the chondrocyte viability and apoptosis. The expression of chondrocyte inflammatory factors (IL-6 and TNF-α) was measured by ELISA. The target genes of circCCDC66 and miR-3622b-5p were analyzed by bioinformatics analysis and luciferase reporter gene assay. The relationship between circCCDC66 and miR-3622b-5p was analyzed by bioinformatics analysis and luciferase reporter gene assay. Results: It was found that circCCDC66 expression in OA cartilage tissues was upregulated. CircCCDC66 overexpression inhibited proliferation and promoted apoptosis of chondrocytes and increased IL-6 and TNF-α levels in chondrocytes. miR-3622b-5p was predicted to be a downstream target gene of circCCDC66, and circCCDC66 overexpression inhibited miR-3622b-5p expression in chondrocytes. Moreover, miR-3622b-5p expression was downregulated in OA cartilage tissues. miR-3622b-5p overexpression increased chondrocyte proliferation, inhibited chondrocyte apoptosis, and enhanced the expression of IL-6 and TNF-α in chondrocytes. In addition, circCCDC66 overexpression enhanced SIRT3 expression in chondrocytes, while miR-3622b-5p overexpression inhibited SIRT3 expression in chondrocytes. Conclusion: CircCCDC66 promoted OA chondrocyte apoptosis by regulating the miR-3622b-5p/SIRT3 axis. CircCCDC66 may be a new therapeutic target of OA.

Role of nuclear factor of activated T cells 1 in the pathogenesis of osteoarthritis

Osteoarthritis (OA) is the most common form of joint disease in middle-aged individuals and the elderly. Previous studies have shown that the overexpression of matrix-degrading proteinases and proinflammatory cytokines is associated with the degradation of osteoarthritic cartilage. However, the transcription factors involved remain unclear. The present study aimed to determine the expression levels of nuclear factor of activated T cells 1 (NFAT1), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in patients with OA, and to validate the role of NFAT1 in the pathogenesis of OA. The expression levels of NFAT1, IL-1β and TNF-α in chondrocytes in the cartilage of patients with OA and healthy individuals were evaluated using western blot analysis. A luciferase reporter assay was performed to determine the activity of NFAT1 in primary human chondrocytes that were transfected with pNFAT1-luc plasmid and stimulated by IL-1β. An enzyme-linked immunosorbent assay was performed to detect the levels of TNF-α, matrix metalloproteinase (MMP)-1, MMP-3 and MMP-9 in the supernatant of cultured chondrocytes in which the NFAT1 was silenced. The expression levels of NFAT1, IL-1β and TNF-α in the cartilage of patients with OA were higher than those of the controls. IL-1β induced the expression of NFAT1 in primary chondrocytes. The expression levels of TNF-α, MMP-1, -3 and -9 promoted by IL-1β were decreased in NFAT1-silenced chondrocytes. In conclusion, NFAT1 may be important in the pathogenesis of OA and calcineurin-NFAT inhibitors may be potential effective agents for the treatment of OA.

Wnt Inhibitory Factor 1 Deficiency Uncouples Cartilage and Bone Destruction in Tumor Necrosis Factor α–Mediated Experimental Arthritis

Wnt signaling plays a pivotal role in skeletal development and in the control of cartilage and bone turnover. We have recently shown that the secreted Wnt antagonist Wnt inhibitory factor 1 (WIF-1) is mainly expressed in the upper layers of epiphyseal and articular cartilage and, to a lesser extent, in bone. Nevertheless, WIF-1(-/-) mice develop normally. In light of these findings, we undertook this study to analyze the role of WIF-1 in arthritis. Expression analyses for WIF-1 were performed by real-time reverse transcription-polymerase chain reaction (RT-PCR). WIF-1(-/-) and tumor necrosis factor (TNF)-transgenic mice were crossbred, and the progression of arthritis in TNF-transgenic WIF-1(-/-) mice and littermate controls was evaluated. Structural joint damage was analyzed by histologic staining, histomorphometry, and micro-computed tomography. Wnt/β-catenin signaling was investigated by real-time RT-PCR and immunofluorescence on primary chondrocytes. WIF-1 expression was repressed by TNFα in chondrocytes and osteoblasts and down-regulated in experimental arthritis and in articular cartilage from patients with rheumatoid arthritis. WIF-1 deficiency partially protected TNF-transgenic mice against bone erosion and loss of trabecular bone, probably as a result of less osteoclast activity. In contrast, arthritis-related cartilage damage was aggravated by WIF-1 deficiency, while overexpression of WIF-1 attenuated cartilage degradation in TNF-transgenic mice. In chondrocytes, TNFα stimulated canonical Wnt signaling, which could be blocked by WIF-1, indicating a direct effect of TNFα and WIF-1 on Wnt signaling in this system. These data suggest that WIF-1 may take part in the fine-tuning of cartilage and bone turnover, promoting the balance of cartilage versus bone anabolism.

A selective estrogen receptor modulator inhibits tumor necrosis factor-α-induced apoptosis through the ERK1/2 signaling pathway in human chondrocytes

Tumor necrosis factor α (TNF-α) is a pleiotropic cytokine mediating inflammatory as well as cell death activities, and is thought to induce chondrocytic chondrolysis in inflammatory and degenerative joint diseases. Selective estrogen receptor modulators (SERMs), such as raloxifene, which are commonly used in clinical settings act as estrogen agonists or antagonists. It is assumed that estrogens have a potential role in cartilage protection; however, the precise molecular mechanism for the protective effects of estrogens is unclear. This study was designed to examine whether raloxifene inhibits TNF-α-induced apoptosis in human chondrocytes and to clarify the mechanisms involved. We also investigated the signaling pathways responsible for the anti-apoptotic effect of raloxifene. Apoptosis in chondrocytes was determined by DNA fragmentation assay and caspase-3 activation. Raloxifene significantly inhibited TNF-α-induced caspase-3 activation and cell DNA fragmentation levels in chondrocytes. The inhibitory effect of raloxifene was abolished by the estrogen receptor antagonist ICI 182,780. Extracellular signal-regulated kinase 1/2 (ERK1/2) regulates apoptosis, acting as an apoptotic or anti-apoptotic signal. TNF-α-induced apoptosis was significantly enhanced by the ERK1/2 pathway inhibitor PD98059. Raloxifene stimulated a further increase in ERK1/2 phosphorylation in TNF-α-treated chondrocytes. Furthermore, the anti-apoptotic effects of raloxifene were inhibited by PD98059. In addition, the anti-apoptotic effects of raloxifene were completely abolished in ERK1/2 siRNA-treated chondrocytes. These results suggest that raloxifene prevents caspase-3-dependent apoptosis induced by TNF-α in human chondrocytes by activating estrogen receptors and the ERK1/2 signaling pathway.

Chondrocyte AMP-activated protein kinase activity suppresses matrix degradation responses to proinflammatory cytokines interleukin-1β and tumor necrosis factor α

Interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) stimulate chondrocyte matrix catabolic responses, thereby compromising cartilage homeostasis in osteoarthritis (OA). AMP-activated protein kinase (AMPK), which regulates energy homeostasis and cellular metabolism, also exerts antiinflammatory effects in multiple tissues. This study was undertaken to test the hypothesis that AMPK activity limits chondrocyte matrix catabolic responses to IL-1β and TNFα. Expression of AMPK subunits was examined, and AMPKα activity was ascertained by the phosphorylation status of AMPKα Thr(172) in human knee articular chondrocytes and cartilage by Western blotting and immunohistochemistry, respectively. Procatabolic responses to IL-1β and TNFα, such as release of glycosaminoglycan, nitric oxide, and matrix metalloproteinases 3 and 13 were determined by dimethylmethylene blue assay, Griess reaction, and Western blotting, respectively, in cartilage explants and chondrocytes with and without knockdown of AMPKα by small interfering RNA. Normal human knee articular chondrocytes expressed AMPKα1, α2, β1, β2, and γ1 subunits. AMPK activity was constitutively present in normal articular chondrocytes and cartilage, but decreased in OA articular chondrocytes and cartilage and in normal chondrocytes treated with IL-1β and TNFα. Knockdown of AMPKα resulted in enhanced catabolic responses to IL-1β and TNFα in chondrocytes. Moreover, AMPK activators suppressed cartilage/chondrocyte procatabolic responses to IL-1β and TNFα and the capacity of TNFα and CXCL8 (IL-8) to induce type X collagen expression. Our findings indicate that AMPK activity is reduced in OA cartilage and in chondrocytes following treatment with IL-1β or TNFα. AMPK activators attenuate dephosphorylation of AMPKα and procatabolic responses in chondrocytes induced by these cytokines. These observations suggest that maintenance of AMPK activity supports cartilage homeostasis by protecting cartilage matrix from inflammation-induced degradation.

The Pattern Recognition Receptor CD36 Is a Chondrocyte Hypertrophy Marker Associated with Suppression of Catabolic Responses and Promotion of Repair Responses to Inflammatory Stimuli

Multiple inflammatory mediators in osteoarthritis (OA) cartilage, including S100/calgranulin ligands of receptor for advanced glycation end products (RAGE), promote chondrocyte hypertrophy, a differentiation state associated with matrix catabolism. In this study, we observed that RAGE knockout was not chondroprotective in instability-induced knee OA in 8-wk-old mice. Hence, we tested the hypothesis that expression of the alternative S100/calgranulin and patterning receptor CD36, identified here as a marker of growth plate chondrocyte hypertrophy, mediates chondrocyte inflammatory and differentiation responses that promote OA. In rat knee joint destabilization-induced OA, RAGE expression was initially sparse throughout cartilage but increased diffusely by 4 wk after surgery. In contrast, CD36 expression focally increased at sites of cartilage injury and colocalized with developing chondrocyte hypertrophy and aggrecan cleavage NITEGE neoepitope formation. However, CD36 transfection in normal human knee-immortalized chondrocytes (CH-8 cells) was associated with decreased capacity of S100A11 and TNF-alpha to induce chondrocyte hypertrophy and ADAMTS-4 and matrix metalloproteinase 13 expression. S100A11 lost the capacity to inhibit proteoglycans synthesis and gained the capacity to induce proteoglycan synthesis in CD36-transfected CH-8 cells. Moreover, S100A11 required the p38 MAPK pathway kinase MKK3 to induce NITEGE development in mouse articular cartilage explants. However, CH-8 cells transfected with CD36 demonstrated decreased S100A11-induced MKK3 and p38 phosphorylation. Therefore, RAGE and CD36 patterning receptor expression were linked with opposing effects on inflammatory, procatabolic responses to S100A11 and TNF-alpha in chondrocytes.

IL-10 overexpression differentially affects cartilage matrix gene expression in response to TNF-α in human articular chondrocytes in vitro

Cartilage-specific extracellular matrix synthesis is the prerequisite for chondrocyte survival and cartilage function, but is affected by the pro-inflammatory cytokine TNF-α in arthritis. The aim of the present study was to characterize whether the immunoregulatory cytokine IL-10 might modulate cartilage matrix and cytokine expression in response to TNF-α. Primary human articular chondrocytes were treated with either recombinant IL-10, TNF-α or a combination of both (at 10 ng/mL each) or transduced with an adenoviral vector overexpressing human IL-10 and subsequently stimulated with 10 ng/ml TNF-α for 6 or 24 h. The effects of IL-10 on the cartilage-specific matrix proteins collagen type II, aggrecan, matrix-metalloproteinases (MMP)-3, -13 and pro-inflammatory cytokines were evaluated by real-time RT-PCR and immunohistochemistry. Transduced chondrocytes overexpressed high levels of IL-10 which significantly up-regulated collagen type II expression. TNF-α suppressed collagen type II and aggrecan, but increased MMP and cytokine expression in chondrocytes compared to the non-stimulated controls. The TNF-α mediated down-regulation of aggrecan expression was significantly antagonized by IL-10 overexpression, whereas the suppression of collagen type II was barely affected. The MMP-13 and IL-1β expression by TNF-α was slightly reduced by IL-10. These results suggest that IL-10 overexpression modulates some catabolic features of TNF-α in chondrocytes.

Interleukin-10 modulates pro-apoptotic effects of TNF-α in human articular chondrocytes in vitro

The aim of this study is to determine if there is an antagonistic effect between tumour necrosis factor (TNF)-α and the immunoregulatory interleukin (IL)-10 on chondrocytes survival. Serum-starved primary human articular chondrocytes were stimulated with either 10 ng/ml recombinant TNF-α, IL-10 or a combination of both (at 10 ng/ml each). Chondrocyte apoptosis was determined by measuring caspase-3/7, -8 and -9 activities using caspase assays. Mitochondrial apoptotic inducer bax, and the suppressor bcl-2 were evaluated using western blotting at 48 h. Results indicated that TNF-α increased caspase activities and resulted in a significant ( p = 0.001) increase in bax/bcl-2 ratio. Stimulation with IL-10 did not alter caspase activities, while co-treatment with IL-10 and TNF-α inhibited TNF-α induced caspase activities and significantly ( p > 0.004) impaired bax/bcl-2 ratio. At 24 h, mRNA levels for collagen type II, TNF-α and IL-10 were determined using real-time RT-PCR. Stimulation with TNF-α or TNF-α and IL-10 significantly inhibited collagen type II and increased IL-10 and TNF-α mRNA expression. IL-10 modulated the pro-apoptotic capacity of TNF-α in chondrocytes as shown by the decrease in caspase activities and bax/bcl-2 ratio compared to TNF-α stimulated chondrocytes, suggesting a mostly antagonistic interplay of IL-10 and TNF-α on mitochondrial apoptotic pathways.

TUMOUR NECROSIS FACTOR α ENHANCES THE EXPRESSION OF HYDROXYL LYASE, CYTOPLASMIC ANTIPROTEINASE-2 AND A DUAL SPECIFICITY KINASE TTK IN HUMAN CHONDROCYTE-LIKE CELLS

Tumour necrosis factor α (TNF-α) is a cytokine with pleiotropic effects on cells ranging from proliferation to apoptosis. These biological effects of TNF-α are believed to be elicited by the induction or enhancement of the expression of TNF-α responsive genes in the target cells. TNF-α is pro-inflammatory and a principal mediator in the pathogenesis of arthritis. The activation of an inflammatory cascade by TNF-α in arthritis results in the degradation of cartilage, joint destruction and loss of function. Because TNF-α is an important mediator in the pathogenesis of arthritis, the present study addresses the identification of novel TNF-α responsive genes in HTB-94 cell line which is of human origin and maintains a chondrocytic phenotype. The three identified cDNAs were previously not known to be induced or upregulated by TNF-α in chondrocytes or cells of chondrocytic lineage. One of the identified cDNAs had sequence similarity to human hydroxyl lyase mRNA (PLOD), an enzyme involved in collagen biosynthesis and its metabolism; the second cDNA had sequence similarity to the human cytoplasmic anti-proteinase-2 mRNA (CAP-2), a member of a group of proteins shown to be associated with protecting cells from TNF-α-induced apoptosis; and the third cDNA had sequence similarity to a dual specificity kinase, TTK, which is associated with cell proliferation. Relative gene expression level analysis by PCR and by Northern blotting revealed that treatment with TNF-α enhanced the expression of PLOD, CAP2 and TTK transcripts which confirmed the results obtained with display gels. Furthermore, TTK mRNA expression was also induced in human articular chondrocytes treated with TNF-α but not in untreated chondrocytes. Our results suggest that these genes may play a role in chondrocytic responses to TNF-α-mediated stimuli affecting the cartilage homeostasis.