Interleukin‐1 in combination with oncostatin M up‐regulates multiple genes in chondrocytes: Implications for cartilage destruction and repair

Objective

To identify the genes up‐regulated by interleukin‐1 (IL‐1) in combination with oncostatin M (OSM) in chondrocytes that may be involved in mechanisms of cartilage repair and degradation.

Methods

Gene microarray and real‐time polymerase chain reaction (PCR) experiments were performed using RNA from SW1353 chondrocytes and primary human articular chondrocytes. Sections prepared from murine joints, injected with adenovirus vectors overexpressing IL‐1 and/or OSM, were analyzed by immunohistochemistry for selected proteins.

Results

The combination of IL‐1 and OSM markedly up‐regulated the expression of various genes, including matrix metalloproteinases (MMPs), cytokines, chemokines, extracellular matrix components, and genes involved in signal transduction. Real‐time PCR confirmed a synergistic induction of several MMPs, activin A, pentraxin 3 (PTX‐3), and IL‐8. The in vivo findings further indicated that stimulation with IL‐1 plus OSM induced protein expression of activin A, PTX‐3, and KC (the murine homolog of IL‐8), as compared with the changes induced by individual cytokine treatment and unstimulated controls.

Conclusion

The results confirm that the potent proinflammatory cytokine combination of IL‐1 plus OSM synergistically and coordinately up‐regulates many genes and several MMPs. Moreover, chondrocytes exhibit a potential repair response following this procatabolic stimulus such that the repair mechanisms are ultimately overwhelmed by degradative processes in the cartilage. This gene‐profiling study provides insight into the complex processes that mediate joint disease in the inflammatory arthritides through the coordinated expression of multiple genes.

     

The modulation of matrix metalloproteinase and ADAM gene expression in human chondrocytes by interleukin‐1 and oncostatin M: A time‐course study using real‐time quantitative reverse transcription–polymerase chain reaction

Objective

Previous studies have reported elevated levels of interleukin‐1 (IL‐1) and oncostatin M (OSM) in rheumatoid joints, as well as the synergistic degradation of human articular cartilage by this cytokine combination. The present study was undertaken to investigate the ability of IL‐1 and OSM to modulate gene expression of matrix metalloproteinase (MMP), ADAM, and ADAM‐TS (ADAM with thrombospondin motifs) family members in human chondrocytes.

Methods

T/C28a4 human chondrocytes were stimulated for 2–48 hours with IL‐1 and/or OSM. Total RNA was harvested, reverse transcribed, and assessed by real‐time polymerase chain reaction for the expression of various MMP, ADAM, and ADAM‐TS messenger RNAs (mRNA). Results were normalized to 18S ribosomal RNA.

Results

IL‐1 and OSM synergized to markedly induce the expression of the collagenases MMP‐1, MMP‐8, and MMP‐13 as well as MMP‐3, an activator of proMMPs. Expression of mRNA for MMPs 1, 3, and 13 was induced early, whereas that of MMP‐8 mRNA occurred late. Gene expression of MMP‐14, an MMP that degrades collagen and activates proMMP‐13, was elevated by this combination. IL‐1 and OSM also synergized to induce gene expression of the aggrecanase ADAM‐TS4, but not ADAM‐TS5.

Conclusion

These data indicate that the potent cartilage‐degrading properties of the combination of IL‐1 and OSM are potentially mediated by a synergistic induction of the aggrecan‐degrading enzyme ADAM‐TS4 and the collagen‐degrading enzymes MMP‐1, MMP‐8, MMP‐13, and MMP‐14, although differences in the magnitude of response and in the time course of induction were observed. A role for MMPs 3 and 14 in the activation of proMMPs may also be implicated.

     

Cytokine‐induced increases in ADAMTS‐4 messenger RNA expression do not lead to increased aggrecanase activity in ADAMTS‐5–deficient mice

Objective

To compare the regulation of aggrecanase messenger RNA (mRNA) and enzyme activity by proinflammatory cytokines in primary mouse chondrocytes.

Methods

Primary chondrocytes were isolated from knee epiphyses of 6–8‐day‐old mice and cultured as monolayers. The cells were incubated with tumor necrosis factor α (TNFα), oncostatin M (OSM), or interleukin‐6 (IL‐6)/soluble IL‐6 receptor, and mRNA levels were measured by quantitative polymerase chain reaction at various time points. To measure aggrecanase activity, the cells were incubated with cytokine in the presence of exogenous aggrecan, and substrate cleavage was measured using antibodies to neoepitopes.

Results

Expression of both ADAMTS‐4 and ADAMTS‐5 mRNA was up‐regulated by TNFα and OSM. ADAMTS‐5 mRNA expression was also up‐regulated by IL‐6. Treatment of wild‐type mouse chondrocytes with each of the 3 cytokines increased cleavage of aggrecan at Glu³⁷³↓³⁷⁴Ala and Glu¹⁶⁷⁰↓¹⁶⁷¹Gly; in chondrocytes lacking ADAMTS‐5 activity, there was negligible cleavage at either site despite increased expression of ADAMTS‐4 mRNA in the presence of TNFα or OSM. None of the cytokines substantially altered mRNA expression of ADAMTS‐1 or ADAMTS‐9.

Conclusion

Despite substantial increases in the expression of ADAMTS‐4 mRNA induced by TNFα and OSM, these cytokines induced little if any increase in aggrecanolysis in ADAMTS‐5–deficient mouse chondrocytes. Our data show a poor correlation between the level of cytokine‐induced ADAMTS‐4 mRNA expression and the level of aggrecan‐degrading activity in cultured chondrocytes.

     

MicroRNA‐140 is expressed in differentiated human articular chondrocytes and modulates interleukin‐1 responses

Objective

MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue‐specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA‐140 (miR‐140).

Methods

To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR‐140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin‐1β (IL‐1β) on miR‐140 expression. Double‐stranded miR‐140 (ds–miR‐140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA.

Results

Microarray analysis showed that miR‐140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR‐140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR‐140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL‐1β suppressed miR‐140 expression. Transfection of chondrocytes with ds–miR‐140 down‐regulated IL‐1β–induced ADAMTS5 expression and rescued the IL‐1β–dependent repression of AGGRECAN gene expression.

Conclusion

This study shows that miR‐140 has a chondrocyte differentiation–related expression pattern. The reduction in miR‐140 expression in OA cartilage and in response to IL‐1β may contribute to the abnormal gene expression pattern characteristic of OA.

     

Bone morphogenetic protein and transforming growth factor β inhibitory Smads 6 and 7 are expressed in human adult normal and osteoarthritic cartilage in vivo and are differentially regulated in vitro by interleukin‐1β

Objective

Bone morphogenetic protein (BMP) and transforming growth factor β (TGFβ) are potent anabolic factors in adult articular chondrocytes. In this study, we investigated whether intracellular inhibitors of BMP and TGFβ signaling, inhibitory Smad6 (I‐Smad6) and I‐Smad7, are expressed in articular chondrocytes in normal and osteoarthritic (OA) cartilage, and whether their expression shows a correlation with the anabolic activity of OA chondrocytes in vivo and after interleukin‐1β (IL‐1β) stimulation in vitro.

Methods

RNA isolated directly from normal and OA human knee cartilage as well as from cultured articular chondrocytes was analyzed by (quantitative) polymerase chain reaction technology. Immunolocalization of the I‐Smads was performed on tissue sections and compared with the anabolic cellular activity as documented by in situ hybridization experiments for aggrecan and type II collagen.

Results

Both Smad6 and Smad7 were expressed in all samples of normal and OA cartilage. Immunostaining (including confocal microscopy) confirmed the presence of Smad6 and Smad7 in the majority of normal and degenerated articular chondrocytes; localization was mostly cytoplasmic. No correlation between expression of the main anabolic genes and expression of the I‐Smads was found. In cultured articular chondrocytes, stimulation with IL‐1β showed up‐regulation of Smad7, whereas Smad6 was down‐regulated.

Conclusion

Both Smad6 and Smad7 are expressed in adult human articular chondrocytes. The primarily cytoplasmic localization suggests permanent activation of the I‐Smads in articular cartilage in vivo. No evidence was found that up‐regulation or down‐regulation of I‐Smads in OA cartilage correlates directly with the anabolic (or catabolic) activity of articular chondrocytes. The regulation in chondrocytes of Smad6 and Smad7 expression by IL‐1β suggests a potentially important role of IL‐1β signaling in chondrocytes, via indirect influencing of the BMP/TGFβ signaling cascade.

     

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

Objective

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.

Methods

Expression of AMPK subunits was examined, and AMPKα activity was ascertained by the phosphorylation status of AMPKα Thr¹⁷² 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.

Results

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.

Conclusion

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.

     

Microenvironment regulation of extracellular signal–regulated kinase activity in chondrocytes: Effects of culture configuration,interleukin‐1, and compressive stress

Objective

To compare extracellular signal–regulated kinase (ERK) activity in response to interleukin‐1 (IL‐1) in chondrocytes under various culture configurations designed for the study of cartilage biology and repair, and also in response to dynamic load for chondrocytes in cartilage.

Methods

Isolated bovine articular chondrocytes were maintained in serum‐supplemented medium under 4 culture configurations: high‐density monolayer, attached to a cut surface of cartilage, within tissue‐engineered constructs, or within intact cartilage explants. Samples were subjected to a change of medium with or without IL‐1. Cartilage explants were also subjected to dynamic compression.

Results

In chondrocyte monolayers, both basal and IL‐1–stimulated ERK activities were similarly elevated at 0.5 hours after medium change, diminishing by 74% after 16 hours. In contrast, chondrocytes in other culture configurations exhibited lower basal levels of ERK activity and a moderate activation of ERK in response to IL‐1 that was sustained over the 16‐hour treatment time. The dynamic component of loading of cartilage explants led to a 5‐fold activation of ERK, compared with free‐swelling controls, that was indistinguishable from the effects of IL‐1.

Conclusion

ERK signaling in response to IL‐1 in chondrocyte monolayers exhibited a pattern that was distinct from that in other culture systems, suggesting that the extracellular matrix plays an important regulatory role in modulating the response to extracellular stimuli. Since IL‐1 and dynamic loading have distinct effects on chondrocyte biosynthesis, signaling pathways other than ERK participate in the chondrocyte responses to these stimuli.

     

Induction of increased cAMP levels in articular chondrocytes blocks matrix metalloproteinase–mediated cartilage degradation,but not aggrecanase‐mediated cartilage degradation

Objective

Calcitonin has been suggested to have chondroprotective effects. One signaling pathway of calcitonin is via the second messenger cAMP. We undertook this study to investigate whether increased cAMP levels in chondrocytes would be chondroprotective.

Methods

Cartilage degradation was induced in bovine articular cartilage explants by 10 ng/ml oncostatin M (OSM) and 20 ng/ml tumor necrosis factor (TNF). In these cultures, cAMP levels were augmented by treatment with either forskolin (4, 16, or 64 μM) or 3‐isobutyl‐1‐methyl xanthine (IBMX; 4, 16, or 64 μM). Cartilage degradation was assessed by 1) quantification of C‐terminal crosslinking telopeptide of type II collagen fragments (CTX‐II), 2) matrix metalloproteinase (MMP)–mediated aggrecan degradation by ³⁴²FFGV‐ G2 assay, 3) aggrecanase‐mediated degradation by ³⁷⁴ARGS‐G2 assay, 4) release of sulfated glycosaminoglycans (sGAG) into culture medium, 5) immunohistochemistry with a monoclonal antibody recognizing the CTX‐II epitope, and 6) toluidine blue staining of proteoglycans. MMP expression and activity were assessed by gelatin zymography.

Results

OSM and TNF induced an 8,000% increase in CTX‐II compared with control (P < 0.001). Both forskolin and IBMX dose‐dependently inhibited release of CTX‐II (P < 0.001). OSM and TNF induced a 6‐fold increase in ³⁴²FFGV‐G2, which was abrogated by forskolin and IBMX (by >80%). OSM and TNF stimulated MMP expression as visualized by zymography, and MMP expression was dose‐dependently inhibited by forskolin and IBMX. The highest concentration of IBMX lowered cytokine‐induced release of sGAG by 72%.

Conclusion

Levels of cAMP in chondrocytes play a key role in controlling catabolic activity. Increased cAMP levels in chondrocytes inhibited MMP expression and activity and consequently strongly inhibited cartilage degradation. Specific cAMP modulators in chondrocytes may be potential treatments for cartilage degenerative diseases.

     

Synergistic effects of glycoprotein 130 binding cytokines in combination with interleukin‐1 on cartilage collagen breakdown

Objective

To determine whether other glycoprotein 130 (gp130) binding cytokines can mimic the effects of oncostatin M (OSM) in acting synergistically with interleukin‐1α (IL‐1α) to induce cartilage collagen breakdown and collagenase expression, and to determine which receptors mediate these effects.

Methods

The release of collagen and proteoglycan was assessed in bovine and human cartilage explant cultures. Messenger RNA (mRNA) and protein production from immortalized human chondrocytes (T/C28a4) was analyzed by Northern blotting and specific enzyme‐linked immunosorbent assays. Collagenase activity was measured by bioassay. Cell surface receptors were detected by flow cytometry.

Results

OSM in combination with IL‐1α caused a rapid synergistic induction of matrix metalloproteinase 1 mRNA, which was sustained over a 72‐hour period. Flow cytometric analyses detected both the OSM‐specific receptor and the gp130 receptor at the chondrocyte cell surface, but failed to detect the leukemia inhibitory factor receptor (LIFR). Cartilage degradation assays revealed that, of the gp130 binding cytokines, only OSM and IL‐6, in the presence of its soluble receptor (sIL‐6R), were able to act synergistically with IL‐1α to promote collagen release.

Conclusion

This study demonstrates that IL‐6 can mimic OSM in synergizing with IL‐1α to induce chondrocyte‐mediated cartilage collagen breakdown and collagenase production. In order to have this effect, IL‐6 requires the presence of its soluble receptor. The apparent absence of LIFR explains why other gp130 binding cytokines do not act in synergy with IL‐1α. Since OSM, IL‐6, and sIL‐6R levels have all been shown to be elevated in the rheumatoid joint, our findings suggest that these cytokines may be key mediators of cartilage collagen catabolism in the inflammatory arthritides.

     

Oncostatin M in combination with tumor necrosis factor α induces cartilage damage and matrix metalloproteinase expression in vitro and in vivo

Objective

To determine the effects of the proinflammatory cytokine combination of oncostatin M (OSM) and tumor necrosis factor α (TNFα) on cartilage destruction in both in vitro and in vivo model systems.

Methods

The release of collagen and proteoglycan was assessed in bovine cartilage explant cultures, while messenger RNA (mRNA) from bovine chondrocytes was analyzed by Northern blotting. Immunohistochemistry was performed on sections prepared from murine joints following injection of adenovirus vectors encoding murine OSM and/or murine TNFα.

Results

The combination of OSM + TNFα induced significant collagen release from bovine cartilage, accompanied by high levels of active collagenolytic activity. Northern blot analysis indicated that this cytokine combination synergistically induced matrix metalloproteinase 1 (MMP‐1), MMP‐3, and MMP‐13 mRNA. The in vivo data clearly indicated that OSM + TNFα overexpression increased MMP levels and decreased levels of tissue inhibitor of metalloproteinases 1 (TIMP‐1). Specifically, OSM + TNFα induced marked synovial hyperplasia, inflammation, and cartilage and bone destruction with a concomitant increase in MMP expression in both cartilage and synovium and decreased TIMP‐1 expression in the articular cartilage. These effects were markedly greater than those seen with either cytokine alone.

Conclusion

This study demonstrates that OSM + TNFα represents a potent proinflammatory cytokine combination that markedly induces MMP production in both cartilage and synovium, thus promoting joint destruction.

     

Correlation of polymorphic variation in the promoter region of the interleukin‐1β gene with secretion of interleukin‐1β protein

Objective

Significant variation in interleukin‐1β (IL‐1β) protein secretion between subjects has been observed when using a lipopolysaccharide (LPS)/ATP–mediated ex vivo blood stimulation assay. To explore the potential relationships between genetic polymorphisms in the IL1B cytokine gene and cellular responses to inflammatory stimuli such as LPS, we investigated the hypothesis that polymorphisms within the promoter and exon 5 of the IL1B gene contribute to the observed differences in IL‐1β protein secretion.

Methods

The IL1B gene polymorphisms C−511T, T−31C, and C3954T were tested for association with LPS‐induced secretion of IL‐1β protein as measured by an ex vivo blood stimulation assay. Samples from 2 independent study populations (n = 31 and n = 25) were available for use in the ex vivo assay after consent was obtained to analyze the DNA.

Results

A specific haplotype, composed of the T allele at −511 and the C allele at −31, was significantly associated with a 2–3‐fold increase in LPS‐induced IL‐1β protein secretion. This association was observed in both of the independent study populations (P = 0.0084 and P = 0.0017).

Conclusion

These data suggest that polymorphisms within the promoter region of the IL1B gene contribute to observed differences in LPS‐induced IL‐1β protein secretion.