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

OBJECTIVE: Bone morphogenetic protein (BMP) and transforming growth factor beta (TGFbeta) are potent anabolic factors in adult articular chondrocytes. In this study, we investigated whether intracellular inhibitors of BMP and TGFbeta 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-1beta (IL-1beta) 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-1beta 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-1beta suggests a potentially important role of IL-1beta signaling in chondrocytes, via indirect influencing of the BMP/TGFbeta signaling cascade.     

Fibronectin fragments and blocking antibodies to alpha2beta1 and alpha5beta1 integrins stimulate mitogen-activated protein kinase signaling and increase collagenase 3 (matrix metalloproteinase 13) production by human articular chondrocytes

OBJECTIVE: To determine if integrin-mediated signaling results in activation of chondrocyte mitogen-activated protein (MAP) kinases that lead to increased expression of matrix metalloproteinase 13 (MMP-13; collagenase 3), a potent mediator of cartilage matrix degradation. METHODS: Human articular chondrocytes isolated from normal ankle and knee cartilage obtained from tissue donors were cultured in monolayers. The cells were treated with a 120-kd fibronectin fragment (FN-f) that binds the alpha5beta1 integrin or with antibodies to specific integrin receptors. Activation of MAP kinases was determined by immunoblotting with phosphospecific antibodies. MMP production was measured by gelatin zymography, and MMP-13 production and activation were determined by immunoblotting and by a fluorogenic peptide assay. RESULTS: Human articular chondrocytes were found to respond to the 120-kd FN-f and to adhesion-blocking antibodies to the alpha2beta1 and alpha5beta1 integrins with increased phosphorylation of the extracellular signal-regulated kinase 1 (ERK1)/ERK2, c-Jun N-terminal kinase (JNK), and p38 MAP kinases. Intact FN and integrin-blocking antibodies to alpha1, alpha3, and alphaVbeta3 and a nonblocking alpha5 antibody had no effect. After MAP kinase activation, increased phosphorylation of c-Jun and the nuclear factor kappaB inhibitor was noted, followed by increased pro- and activated MMP-13 in the conditioned media. Inhibitors of mitogen-activated protein kinase kinase, p38, and JNK were each able to inhibit increased MMP-13 production, while the interleukin-1 receptor antagonist (IL-1Ra) protein did not. However, the IL-1Ra partially inhibited FN-f-induced activation of MMP-13. CONCLUSION: Integrin-mediated MAP kinase signaling stimulated by FN-f is associated with increased production and release of pro- and active MMP-13. Autocrine production of IL-1 appears to result in additional MMP-13 activation. These processes may play a key role in feedback loops responsible for progressive cartilage degradation in arthritis.     

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.     

Biologic effects of an interleukin-1 receptor antagonist protein on interleukin-1-stimulated cartilage erosion and chondrocyte responsiveness

Recombinant human interleukin-1 alpha (IL-1 alpha) and recombinant human IL-1 beta stimulate matrix proteoglycan degradation and inhibit glycosaminoglycan synthesis in bovine nasal cartilage explants. A 17-kd human recombinant IL-1 receptor antagonist protein (IRAP) caused a concentration-dependent (0.2-200 ng/ml) suppression of the effects of IL-1 alpha and IL-1 beta in cartilage organ cultures. IRAP inhibited the binding of radiolabeled IL-1 alpha to rabbit articular chondrocytes. Matrix metalloproteinase (collagenase, gelatinase, and stromelysin) and prostanoid production by IL-1-activated rabbit articular chondrocytes was also suppressed by IRAP. These results could have potential significance in the development of a new antiarthritis therapy based on an IRAP.     

Interleukin 17 induced nitric oxide suppresses matrix synthesis and protects cartilage from matrix breakdown

OBJECTIVE: To investigate the role of nitric oxide (NO) in basal and cytokine induced cartilage matrix breakdown and synthesis across different species and in chondrocytes cultured as isolated cells or as tissue explants. METHODS: Articular cartilage from bovine, porcine, or human joints was cultured as explants in serum-free media. Explants or monolayer cultures of primary chondrocytes were treated with cytokines in the absence or presence of inhibitors [antibodies to leukemia inhibitory factor (anti-LIF) or tumor necrosis factor-alpha, dexamethasone, or inhibitors of aggrecanase or NO synthase]. NO production and matrix breakdown and synthesis were measured. RESULTS: At low concentrations, a novel interleukin 17 (IL-17) family member induced matrix breakdown without altering NO production. Treatment of articular cartilage explants with dexamethasone or anti-LIF blocked NO production by IL-17, but not by IL-1alpha. Inhibition of NO production in cytokine treated cartilage explants enhanced matrix breakdown and partially overcame suppression of matrix synthesis. In isolated chondrocytes, inhibition of NO production decreased expression of gelatinase and increased expression of stromelysin. CONCLUSION: Endogenous NO serves a dual function in cartilage: to protect the tissue from matrix breakdown and to mediate suppression of proteoglycan synthesis by cytokines. Despite the similarities in biological function between IL-I and IL-17, their downstream signaling pathways are distinct and appear to be affected by extracellular matrix degradation.     

Induction of alpha1-antitrypsin synthesis in human articular chondrocytes by interleukin-6-type cytokines: evidence for a local acute-phase response in the joint.

OBJECTIVE: We have previously shown that human articular chondrocytes synthesize large amounts of interleukin-6 (IL-6) upon stimulation with proinflammatory cytokines and that they express the IL-6 receptor. The present study was undertaken to analyze whether different IL-6-type cytokines can induce synthesis of the acute-phase protein alpha1-antitrypsin in human articular chondrocytes. METHODS: Chondrocytes from human articular cartilage, cultured in agarose, were stimulated with IL-6-type cytokines. Total RNA was isolated and analyzed by Northern blotting. Levels of alpha1-antitrypsin protein were determined by enzyme immunoassay. RESULTS: Stimulation of chondrocytes with oncostatin M (OSM) and IL-6 led to a 5-10-fold increase in alpha1-antitrypsin synthesis. This increase was dose and time dependent. Furthermore, OSM and IL-6 induced IL-6 synthesis in chondrocytes, resulting in an autocrine amplification loop. CONCLUSION: Our data strongly suggest the existence of a local acute-phase response in the joint. Synthesis of the acute-phase protein alpha1-antitrypsin, a major inhibitor of serine proteinases, may be an important protective mechanism of articular chondrocytes to prevent cartilage damage in inflammatory joint diseases.     

Implication of interleukin 18 in production of matrix metalloproteinases in articular chondrocytes in arthritis: direct effect on chondrocytes may not be pivotal

OBJECTIVE: To clarify the effect of interleukin (IL) 18 on cartilage degeneration by studying the profile of IL18 receptor (IL18R) on chondrocytes and the direct effect of IL18 on production of matrix metalloproteinases (MMPs), aggrecanases, and tissue inhibitors of metalloproteinases (TIMPs) in articular chondrocytes. METHODS: Monolayer cultured human articular chondrocytes were isolated from non-arthritic subjects and patients with rheumatoid arthritis or osteoarthritis. Gene expression of IL18, IL18Ralpha, IL18Rbeta, MMPs, and aggrecanases was detected by RT-PCR. Protein levels of IL18Ralpha were analysed by flow cytometry. Protein levels of IL18, MMPs, and TIMPs were measured by ELISA. Aggrecanase-2 mRNA expression was quantitatively analysed by real time RT-PCR. Protein levels of signalling molecules were assayed by western blotting. RESULTS: IL18 mRNA was constitutively expressed in chondrocytes, and was enhanced by IL1beta stimulation. Flow cytometric analysis showed that IL1beta, tumour necrosis factor alpha, and IL18 up regulated IL18Ralpha expression levels. The level of IL18Rbeta mRNA was much lower than that of IL18Ralpha, and was slightly up regulated by IL1beta. In chondrocytes responding to IL18, IL18 (1-100 ng/ml) slightly increased the production of MMP-1, MMP-3, and MMP-13, which was blocked by NF-kappaB inhibitor and p38 mitogen activated protein kinase inhibitor. IL18 up regulated mRNA expression of aggrecanase-2, but not aggrecanase-1. IL18 also slightly stimulated TIMP-1 production?through extracellular signal regulated kinase activation. CONCLUSION: IL18 induces production of MMPs from chondrocytes in inflammatory arthritis. Although the direct effect of IL18 on chondrocytes may not be pivotal for the induction of cartilage degeneration, IL18 seems to play some part in the degradation of articular cartilage in arthritis.     

Interleukin 1 beta suppresses transforming growth factor-induced inorganic pyrophosphate (PPi) production and expression of the PPi-generating enzyme PC-1 in human chondrocytes.

Articular cartilage chondrocytes have the unique ability to elaborate large amounts of extracellular pyrophosphate (PPi), and transforming growth factor beta (TGF beta) appears singular among cartilage regulatory factors in stimulating PPi production. TGF beta caused a time and dose-dependent increase in intracellular and extracellular PPi in human articular chondrocyte cultures. TGF beta and interleukin 1 beta (IL-1 beta) antagonistically regulate certain chondrocyte functions. IL-1 beta profoundly inhibited basal and TGF beta-induced PPi elaboration. To address mechanisms involved with the regulation of PPi synthesis by IL-1 beta and TGF beta, we analyzed the activity of the PPi-generating enzyme NTP pyrophosphohydrolase (NTPPPH) and the PPi-hydrolyzing enzyme alkaline phosphatase. Human chondrocyte NTPPPH activity was largely attributable to plasma cell membrane glycoprotein 1, PC-1. Furthermore, TGF beta induced comparable increases in the activity of extracellular PPi, intracellular PPi, and cellular NTPPPH and in the levels of PC-1 protein and mRNA in chondrocytes as well as a decrease in alkaline phosphatase. All of these TGF beta-induced responses were completely blocked by IL-1 beta. Thus, IL-1 beta may be an important regulator of mineralization in chondrocytes by inhibiting TGF beta-induced PPi production and PC-1 expression.     

Effects of interleukin-1 beta on insulin-like growth factor-I autocrine/paracrine axis in cultured rat articular chondrocytes.

OBJECTIVE--To clarify the interaction of tissue destruction and repair of articular cartilage during inflammation, the effects of interleukin-1 beta (IL-1 beta) on the expression of insulin-like growth factor I (IGF-I), its receptor, and its binding proteins were examined. METHODS--Articular chondrocytes from five week rats were cultured in serum free medium treated with IL-1 beta (1-100 U/ml) for 24 hours. The concentration of IGF-1 in the conditioned medium was measured by RIA, and IGFBP were analysed by immunoligand blotting method. IGF-I receptors were also examined by [125I]IGF-I binding study. RESULTS--IL-1 beta induced the secretion of IGF-I and IGF-binding protein in chondrocytes; this was not inhibited by indomethacin (5 micrograms/ml). IL-1 beta also increased the number of IGF-I receptors but had no effect on receptor affinity. IL-1 beta inhibited chondrocyte proliferation, while exogenous IGF-I and growth hormone stimulated chondrocyte cell growth. IL-1 beta did not change IGF-I mRNA levels. CONCLUSION--IL-1 beta up-regulated the IGF-I autocrine/paracrine axis in cultured articular chondrocytes. These observations provide insight into the critical role played by IL-1 beta in tissue destruction and repair, and into the direct interaction between cytokines and growth factors associated with inflammatory arthropathy.