Differential responses of chondrocytes from normal and osteoarthritic human articular cartilage to mechanical stimulation

Mechanical stimulation is critically important for the maintenance of normal articular cartilage integrity. Molecular events regulating responses of chondrocytes to mechanical forces are beginning to be defined. Chondrocytes from normal human knee joint articular cartilage show increased levels of aggrecan mRNA following 0.33 Hz mechanical stimulation whilst at the same time relative levels of MMP3 mRNA are decreased. This anabolic response, associated with membrane hyperpolarisation, is activated via an integrin-dependent interleukin (IL)-4 autocrine/paracrine loop. Work in our laboratory suggests that this chondroprotective response may be aberrant in osteoarthritis (OA). Chondrocytes from OA cartilage show no changes in aggrecan or MMP3 mRNA following 0.33 Hz mechanical stimulation. alpha5beta1 integrin is the mechanoreceptor in both normal and OA chondrocytes but downstream signalling pathways differ. OA chondrocytes show membrane depolarisation following 0.33 Hz mechanical stimulation consequent to activation of an IL1beta autocrine/paracrine loop. IL4 signalling in OA chondrocytes is preferentially through the type I (IL4alpha/cgamma) receptor rather than via the type II (IL4alpha/IL13R) receptor. Altered mechanotransduction and signalling in OA may contribute to changes in chondrocyte behaviour leading to increased cartilage breakdown and disease progression.     

NMDA receptor expression and roles in human articular chondrocyte mechanotransduction

Mechanical forces influence articular cartilage structure by regulating chondrocyte activity. Mechanical stimulation results in activation of an alpha5beta1 integrin dependent intracellular signal cascade involving focal adhesion kinase and protein kinase C, triggering the release of interleukin-4 from the cell. In normal HAC the response to physiological mechanical stimulation is characterised by increased levels of aggrecan mRNA and a decrease in levels of mRNA for matrix metalloproteinase 3 (MMP-3), the net result of which would be to maintain and optimise cartilage structure and function. This protective/anabolic response is not seen when chondrocytes from osteoarthritic cartilage are subjected to an identical mechanical stimulation regime. Following the observation that the neurotransmitter substance P is involved in chondrocyte mechanotransduction the present study was undertaken to establish potential roles for glutamate receptors in the control of chondrocyte mechanical responses. Using immunohistochemistry and RTPCR normal and OA chondrocytes are shown to express NR1 and NR2a subunits of the NMDA receptor. Addition of NMDA receptor agonists to chondrocytes in primary culture resulted in changes in membrane potential consistent with expression of functional receptors. NMDA receptor antagonists inhibited the hyperpolarisation response of normal chondrocytes to mechanical stimulation but had no effect on the depolarisation response of osteoarthritic chondrocytes to mechanical stimulation. These studies indicate that at least one subset of the NMDA receptor family of molecules is expressed in cartilage and may have important modulatory effects on mechanotransduction and cellular responses following mechanical stimulation. Indeed the results suggest that there is an alteration of NMDA receptor signalling in OA chondrocytes, which may be critical in the abnormal response of OA chondrocytes to mechanical stimulation. Thus NMDA receptors appear to be involved in the regulation of human articular chondrocyte responses to mechanical stimulation, and in OA, mechanotransduction pathways may be modified as a result of altered activation and function of these receptors.     

IL-1beta synthesis by chondrocyte analyzed by 3D microscopy and flow cytometry: effect of Rhein

Several factors are known to be involved in the destruction of the articular cartilage. Interleukin-1 (IL-1) plays an important role in the pathogenesis of osteoarthritis (OA) either directly or through the stimulation of catabolic factors. The action of IL-1 on articular cartilage is multifaceted and it most likely plays an important role in the mechanism of cartilage destruction. IL-1 suppresses the synthesis of the cartilage matrix components and promotes the degradation of cartilage matrix macromolecules. Diacerein is an anthraquinone molecule that has been shown to reduce the severity of OA, both in man and in animal models. The present study was designed to evaluate in vitro effects of diacerein on IL-1beta expression in LPS or IL-1alpha stimulated chondrocytes. Intracellular IL-1beta production was analysed in articular chondrocytes cultured in monolayer or in alginate 3D-biosystems in the presence of lipopolysaccharide (LPS) or IL-1alpha, with or without diacerein. The results show that LPS and IL-1alpha increase intracellular IL-1beta and Diacerein inhibited LPS-induced and IL-1alpha induced IL-1beta production by articular chondrocytes. Moreover, the effect of mechanical stimulation was analysed. An inhibitory effect of DAR at therapeutic concentrations on IL-1beta production in articular chondrocytes is suggested.     

Anti-inflammatory effects of IL-4 and dynamic compression in IL-1beta stimulated chondrocytes

Mechanical loading can counteract inflammatory pathways induced by IL-1beta by inhibiting *NO and PGE2, catabolic mediators known to be involved in cartilage degradation. The current study investigates the potential of dynamic compression, in combination with the anti-inflammatory cytokine, IL-4, to further abrogate the IL-1beta induced effects. The data presented demonstrate that IL-4 alone can inhibit nitrite release in the presence and absence of IL-1beta and partially reverse the IL-1beta induced PGE2 release. When provided in combination, IL-4 and dynamic compression could further abrogate the IL-1beta induced nitrite and PGE2 release. IL-1beta inhibited [3H]thymidine incorporation and this effect could be reversed by IL-4 or dynamic strain alone or both in combination. By contrast, 35SO4 incorporation was not influenced by IL-4 and/or dynamic strain in IL-1beta stimulated constructs. IL-4 and mechanical loading may therefore provide a potential protective mechanism for cartilage destruction as observed in OA.     

ATP in the mechanotransduction pathway of normal human chondrocytes

Extracellular nucleotides have been shown to have diverse effects on chondrocyte function, generally acting via P2 purinoceptors. We have previously shown that mechanical stimulation at 0.33 Hz of normal human chondrocyte cultures causes cellular hyperpolarisation, while chondrocytes derived from osteoarthritic (OA) cartilage depolarise. Experiments have been undertaken to establish whether ATP is involved in the response of the chondrocyte to mechanical stimulation. Chondrocytes, isolated from normal and OA cartilage obtained, with consent, from human knee joints following surgery, were cultured in non-confluent monolayer. Cells were mechanically stimulated at 0.33 Hz for 20 minutes at 37 degrees C in the presence or absence of inhibitors of ATP signalling, or were stimulated by the addition of exogenous ATP or derivatives, and electrophysiological measurements recorded. Samples of medium bathing the cells were collected before and after mechanical stimulation, and the concentration of ATP in the cell medium was measured. Total RNA was extracted from cultured chondrocytes, reverse-transcribed and used for RT-PCR with primers specific for P2Y2 purinoceptors. ATP, UTP 2-methylthioadenosine and alphabeta-methylene adenosine 5'-triphosphate all induced a hyperpolarisation response in normal human articular chondrocytes. No significant change was observed in the membrane potentials of chondrocytes isolated from OA cartilage following the addition of these nucleotides to the medium. In normal chondrocytes, the hyperpolarisation induced by ATP was blocked by the presence of apamin, indicating the involvement of small-conductance calcium-activated potassium channels. Following mechanical stimulation of normal chondrocytes, an increase was observed in ATP concentration in the cell culture medium bathing the cells. The presence within the culture medium of suramin or pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) prior to and during the period of mechanical stimulation abolished the hyperpolarisation response in normal chondrocytes. The presence of mRNA for P2Y2 purinoceptors was demonstrated in both normal and OA chondrocytes by RT-PCR. These results suggest that ATP has a role in the response of normal chondrocytes to mechanical stimulation, via P2Y2 purinoceptors. This response appears to be different in chondrocytes derived from OA cartilage, and may be important in the progression of this disease.     

Iron oxide labeling does not affect differentiation potential of human bone marrow mesenchymal stem cells exhibited by their differentiation into cardiac and neuronal cells

Osteoarthritis (OA) is characterized by articular cartilage degradation and joint inflammation. The purpose of the present study is to elucidate the role of the specific function of PRMT1 in chondrocytes and its association with the pathophysiology of OA. We observed that the expression of PRMT1 was apparently upregulated in OA cartilage, as well as in chondrocytes stimulated with IL-1β. Additionally, knockdown of PRMT1 suppressed interleukin 1 beta (IL-1β)-induced extracellular matrix (ECM) metabolic imbalance by regulating the expression of MMP-13, ADAMTS-5, COL2A1, and ACAN. Furthermore, silencing of PRMT1 dramatically declined the production of prostaglandin E2 (PGE2) and nitric oxide as well as the level of pro-inflammatory cytokine IL-6 and TNF-α. Mechanistic analyses further revealed that IL-1β-induced activation of the Hedgehog/Gli-1 signaling is suppressed upon PRMT1 knockdown. However, the effects of inhibition of PRMT1-mediated IL-1β-induced cartilage matrix degradation and inflammatory response in OA chondrocytes were obviously abolished by Hedgehog agonist Purmorphamine (Pur). Our data collectively suggest that silencing of PRMT1 exerts anti-catabolic and anti-inflammatory effects on IL-1β-induced chondrocytes via suppressing the Gli-1 mediated Hedgehog signaling pathway, indicating that PRMT1 plays a critical role in OA development and serves as a promising therapeutic target for OA.     

IL-1beta induction of IL-6 and LIF in normal articular human chondrocytes involves the ERK, p38 and NFkappaB signaling pathways

Interleukin-1 (IL-1) is an important catabolic cytokine in rheumatoid and osteoarthritic joint disease. Besides inducing a catabolic response in articular chondrocytes it also strongly induces synergistic mediators such as leukemia inhibitory factor (LIF) and interleukin-6 (IL-6). The molecular basis of this is so far hardly understood. The aim of our study was to evaluate in vitro and in vivo whether IL-6 and LIF are differentially expressed in normal human and osteoarthritic adult articular chondrocytes and to investigate the potential intracellular signaling pathways of IL-1 involved in these gene regulation events. IL-6 and LIF mRNA expressions were found only at low levels in normal adult articular cartilage. Neither IL-6 nor LIF was strongly over-expressed in osteoarthritic cartilage degeneration. Clearly, both IL-6 and LIF can be very efficiently induced by IL-1beta in articular chondrocytes in vitro. However, this induction was somewhat less in osteoarthritic cells, which were overall activated in terms of expression of both cytokines without stimulation. Experiments using pathway selective inhibitors showed that intracellular signaling of IL-1beta for IL-6 and LIF is mediated by a mixture of the IL-1 signaling cascades. However, the ERK-pathway appeared to be particularly important and might be, therefore, of particular potential if one intends to block induction of these molecules by IL-1 in arthritic joint disease.     

NF-kappa B mediates the stimulation of cytokine and chemokine expression by human articular chondrocytes in response to fibronectin fragments

Fibronectin fragments (FN-f) that bind to the alpha(5)beta(1) integrin stimulate chondrocyte-mediated cartilage destruction and could play an important role in the progression of arthritis. The objective of this study was to identify potential cytokine mediators of cartilage inflammation and destruction induced by FN-f and to investigate the mechanism of their stimulation. Human articular chondrocytes, isolated from normal ankle cartilage obtained from tissue donors, were treated with a 110-kDa FN-f in serum-free culture, and expression of various cytokine genes was analyzed by cDNA microarray and by a cytokine protein array. Compared with untreated control cultures, stimulation by FN-f resulted in a >2-fold increase in IL-6, IL-8, MCP-1, and growth-related oncogene beta (GRO-beta). Constitutive and FN-f-inducible expression of GRO-alpha and GRO-gamma were also noted by RT-PCR and confirmed by immunoblotting. Previous reports of IL-1beta expression induced by FN-f were also confirmed, while TNF expression was found to be very low. Inhibitor studies revealed that FN-f-induced stimulation of chondrocyte chemokine expression was dependent on NF-kappaB activity, but independent of IL-1 autocrine signaling. The ability of FN-f to stimulate chondrocyte expression of multiple proinflammatory cytokines and chemokines suggests that damage to the cartilage matrix is capable of inducing a proinflammatory state responsible for further progressive matrix destruction, which also includes the chemoattraction of inflammatory cells. Targeting the signaling pathways activated by FN-f may be an effective means of inhibiting production of multiple mediators of cartilage destruction.     

Dynamic compression counteracts IL-1beta induced iNOS and COX-2 activity by human chondrocytes cultured in agarose constructs

*NO and PGE2 are inflammatory mediators derived from the inducible iNOS and COX enzymes and are potentially important pharmacological targets in OA. Both mechanical loading and IL-1beta will influence the release of *NO and PGE2. Accordingly, the current study examines the effect of dynamic compression on *NO and PGE2 release by human chondrocytes cultured in agarose constructs in the presence and absence of selective iNOS and COX-2 inhibitors. The current data demonstrate that IL-1beta induced nitrite and PGE2 release and inhibited [3H]-thymidine and 35SO4 incorporation. Inhibitor experiments indicate that 1400W and NS-398 either partially reversed or abolished IL-1beta induced nitrite and PGE2 release. IL-1beta induced inhibition of cell proliferation and proteoglycan synthesis was partially reversed with 1400W but was not influenced by NS-398. For the dynamic loading experiments, 1400W and NS-398 either reduced or abolished the compression-induced inhibition of *NO and PGE2 release in the presence of IL-1beta. The IL-1beta induced inhibition of cell proliferation was not influenced by 1400W or NS-398 whereas strain-induced stimulation of proteoglycan synthesis in the presence of IL-1beta was enhanced by 1400W. The data obtained using human chondrocytes demonstrate that IL-1beta induced *NO and PGE2 release via an iNOS-driven-COX-2 inter-dependent pathway. This response could be reversed by dynamic compression. These data indicate interactions exist between the NOS and COX pathways, a finding which will provide new insights in the development of pharmacological or biophysical treatments for cartilage disorders such as OA.     

Mechanical stress protects against osteoarthritis via regulation of the AMPK/NF-κB signaling pathway

Mechanical stress plays a key role in regulating cartilage degradation in osteoarthritis (OA). The aim of this study was to evaluate the effects and mechanisms of mechanical stress on articular cartilage. A total of 80 male Sprague-Dawley rats were randomly divided into eight groups (n = 10 for each group): control group (CG), OA group (OAG), and CG or OAG subjected to low-, moderate-, or high-intensity treadmill exercise (CL, CM, CH, OAL, OAM, and OAH, respectively). Chondrocytes were obtained from the knee joints of rats; they were cultured on Bioflex 6-well culture plates and subjected to different durations of cyclic tensile strain (CTS) with or without exposure to interleukin-1β (IL-1β). The results of the histological score, immunohistochemistry, enzyme-linked immunosorbent assay, and western-blot analyses indicated that there were no differences between CM and CG, but OAM showed therapeutic effects compared with OAG. However, CH and OAH experienced more cartilage damage than CG and OAG, respectively. CTS had no therapeutic effects on collagen II of normal chondrocytes, which is consistent with findings after treadmill exercise. However, CTS for 4 hr could alleviate the chondrocyte damage induced by IL-1β by activating AMP-activated protein kinase (AMPK) phosphorylation and suppressing nuclear translocation of nuclear factor (NF)-κB p65. Our findings indicate that mechanical stress had no therapeutic effects on normal articular cartilage and chondrocytes; mechanical stress only caused damage with excessive stimulation. Still, moderate biomechanical stress could reduce sensitization to the inflammatory response of articular cartilage and chondrocytes through the AMPK/NF-κB signaling pathway.     

Pharmacological treatment with diacerein combined with mechanical stimulation affects the expression of growth factors in human chondrocytes

BackgroundOsteoarthritis (OA) as the main chronic joint disease arises from a disturbed balance between anabolic and catabolic processes leading to destructions of articular cartilage of the joints. While mechanical stress can be disastrous for the metabolism of chondrocytes, mechanical stimulation at the physiological level is known to improve cell function. The disease modifying OA drug (DMOAD) diacerein functions as a slowly-acting drug in OA by exhibiting anti-inflammatory, anti-catabolic, and pro-anabolic properties on cartilage. Combining these two treatment options revealed positive effects on OA-chondrocytes.MethodsCells were grown on flexible silicone membranes and mechanically stimulated by cyclic tensile loading. After seven days in the presence or absence of diacerein, inflammation markers and growth factors were analyzed using quantitative real-time PCR and enzyme linked immune assays. The influence of conditioned medium was tested on cell proliferation and cell migration.ResultsTensile strain and diacerein treatment reduced interleukin-6 (IL-6) expression, whereas cyclooxygenase-2 (COX2) expression was increased only by mechanical stimulation. The basic fibroblast growth factor (bFGF) was down regulated by the combined treatment modalities, whereas prostaglandin E2 (PGE2) synthesis was reduced only under OA conditions. The expression of platelet-derived growth factor (PDGF) and vascular endothelial growth factor A (VEGF-A) was down-regulated by both.ConclusionsFrom our study we conclude that moderate mechanical stimulation appears beneficial for the fate of the cell and improves the pharmacological effect of diacerein based on cross-talks between different initiated pathways.General significanceCombining two different treatment options broadens the perspective to treat OA and improves chondrocytes metabolism.     

Fibroblast activation protein alpha is expressed by chondrocytes following a pro-inflammatory stimulus and is elevated in osteoarthritis

Arthritis is characterised by the proteolytic degradation of articular cartilage leading to a loss of joint function. Articular cartilage is composed of an extracellular matrix of proteoglycans and collagens. We have previously shown that serine proteinases are involved in the activation cascades leading to cartilage collagen degradation. The aim of this study was to use an active-site probe, biotinylated fluorophosphonate, to identify active serine proteinases present on the chondrocyte membrane after stimulation with the pro-inflammatory cytokines IL-1 and oncostatin M (OSM), agents that promote cartilage resorption. Fibroblast activation protein alpha (FAPalpha), a type II integral membrane serine proteinase, was identified on chondrocyte membranes stimulated with IL-1 and OSM. Real-time PCR analysis shows that FAPalpha gene expression is up-regulated by this cytokine combination in both isolated chondrocytes and cartilage explant cultures and is significantly higher in cartilage from OA patients compared to phenotypically normal articular cartilage. Immunohistochemistry analysis shows FAPalpha expression on chondrocytes in the superficial zone of OA cartilage tissues. This is the first report demonstrating the expression of active FAPalpha on the chondrocyte membrane and elevated levels in cartilage from OA patients. Its cell surface location and expression profile suggest that it may have an important pathological role in the cartilage turnover prevalent in arthritic diseases.     

The critical role of UDP-galactose-4-epimerase in osteoarthritis: Modulating proteoglycans synthesis of the articular chondrocytes

UDP-galactose-4-epimerase (GALE) is a key enzyme catalyzing the interconversion of UDP-glucose and UDP-galactose, as well as UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine, which are all precursors for the proteoglycans (PGs) synthesis. However, whether GALE is essential in cartilage homeostasis remains unknown. Therefore, we investigated the role of GALE in PGs synthesis of human articular chondrocytes, the GALE expression in OA, and the regulation of GALE expression by interleukin-1beta (IL-1β). Silencing GALE gene with specific siRNAs resulted in a markedly inhibition of PGs synthesis in human articular chondrocytes. GALE protein levels were also decreased in both human and rat OA cartilage, thus leading to losses of PGs contents. Moreover, GALE mRNA expression was stimulated by IL-1β in early phase, but suppressed in late phase, while the suppression of GALE expression induced by IL-1β was mainly mediated by stress-activated protein kinase/c-Jun N-terminal kinase pathway. These data indicated a critical role of GALE in maintaining cartilage homeostasis, and suggested that GALE inhibition might contribute to OA progress.     

Dynamic compression inhibits the synthesis of nitric oxide and PGE(2) by IL-1beta-stimulated chondrocytes cultured in agarose constructs

Both mechanical loading and interleukin-1beta (IL-1beta) are known to regulate metabolic processes in articular cartilage through pathways mediated by nitric oxide ((*)NO) and PGE(2). This study uses a well-characterized model system involving isolated chondrocytes cultured in agarose constructs to test the hypothesis that dynamic compression alters the synthesis of (*)NO and PGE(2) by IL-1beta-stimulated articular chondrocytes. The data presented demonstrate for the first time that dynamic compression counteracts the effects of IL-1beta on articular chondrocytes by suppressing both (*)NO and PGE(2) synthesis. Inhibitor experiments indicated that the dynamic compression-induced inhibition of PGE(2) synthesis and stimulation of proteoglycan synthesis were (*)NO mediated, while compression-induced stimulation of cell proliferation was (*)NO independent. The inhibition of (*)NO and PGE(2) by dynamic compression is a finding of major significance that could contribute to the development of novel strategies for the treatment of cartilage-degenerative disorders.     

Fibroblast activation protein alpha is expressed by chondrocytes following a pro-inflammatory stimulus and is elevated in osteoarthritis

Arthritis is characterised by the proteolytic degradation of articular cartilage leading to a loss of joint function. Articular cartilage is composed of an extracellular matrix of proteoglycans and collagens. We have previously shown that serine proteinases are involved in the activation cascades leading to cartilage collagen degradation. The aim of this study was to use an active-site probe, biotinylated fluorophosphonate, to identify active serine proteinases present on the chondrocyte membrane after stimulation with the pro-inflammatory cytokines IL-1 and oncostatin M (OSM), agents that promote cartilage resorption. Fibroblast activation protein alpha (FAPα), a type II integral membrane serine proteinase, was identified on chondrocyte membranes stimulated with IL-1 and OSM. Real-time PCR analysis shows that FAPα gene expression is up-regulated by this cytokine combination in both isolated chondrocytes and cartilage explant cultures and is significantly higher in cartilage from OA patients compared to phenotypically normal articular cartilage. Immunohistochemistry analysis shows FAPα expression on chondrocytes in the superficial zone of OA cartilage tissues. This is the first report demonstrating the expression of active FAPα on the chondrocyte membrane and elevated levels in cartilage from OA patients. Its cell surface location and expression profile suggest that it may have an important pathological role in the cartilage turnover prevalent in arthritic diseases.     

Chondroprotective and anti-inflammatory effects of sesamin

Osteoarthritis (OA) is a major disability of elderly people. Sesamin is the main compound in Sesamun indicum Linn., and it has an anti-inflammatory effect by specifically inhibiting Δ5-desaturase in polyunsaturated fatty acid biosynthesis. The chondroprotective effects of sesamin were thus studied in a porcine cartilage explant induced with interleukin-1beta (IL-1β) and in a papain-induced osteoarthritis rat model. With the porcine cartilage explant, IL-1β induced release of sulfated-glycosaminoglycan (s-GAG) and hydroxyproline release, and this induction was significantly inhibited by sesamin. This ability to inhibit these processes might be due to its ability to decrease expression of MMP-1, -3 and -13, which can degrade both PGs and type II collagen, both at the mRNA and protein levels. Interestingly, activation of MMP-3 might also be inhibited by sesamin. Moreover, in human articular chondrocytes (HACs), some pathways of IL-1β signal transduction were inhibited by sesamin: p38 and JNK. In the papain-induced OA rat model, sesamin treatment reversed the following pathological changes in OA cartilage: reduced disorganization of chondrocytes in cartilage, increased cartilage thickness, and decreased type II collagen and PGs loss. Sesamin alone might increase formation of type II collagen and PGs in the cartilage tissue of control rats. These results demonstrate that sesamin efficiently suppressed the pathological processes in an OA model. Thus, sesamin could be a potential therapeutic strategy for treatment of OA.