Metabolic, biochemical and biomechanical differences between ankle and knee joint cartilage and chondrocytes including resistance to the effects of catabolic cytokines and fibronectin fragments may be relevant to differences in prevalence of OA in these joints. Although there is increasing information available on how chondrocytes from knee and hip joint cartilage recognise and respond to mechanical stimuli, knowledge of mechanotransduction in ankle joint chondrocytes is limited. This study was undertaken to (i) establish whether the response of normal ankle joint derived chondrocytes to mechanical stimulation in vitro was similar to that of normal and osteoarthritic knee joint derived chondrocytes and (ii) to investigate whether these chondrocytes showed differences in expression of integrin associated regulatory and signalling molecules. Unlike normal knee joint chondrocytes, ankle joint chondrocytes did not show an increase in relative levels of aggrecan mRNA when mechanically stimulated. No obvious change in protein tyrosine phosphorylation was seen in ankle chondrocytes subsequent to mechanical stimulation but these cells expressed elevated levels of tyrosine phosphorylated proteins at rest when compared to normal knee joint chondrocytes. Ankle joint chondrocytes showed an increase in protein kinase B phosphorylation following 1 min 0.33 Hz stimulation which was inhibited by the presence of antibodies to alpha5beta1 integrin. Ankle joint chondrocytes appeared to show significant differences in levels of the integrin-associated proteins CD98, CD147 and galectin 3, PKCgamma and differences in responses to glutamate were seen. Chondrocytes from ankle and knee joint cartilage respond differently to 0.33 Hz mechanical stimulation. This may be related to modified integrin-dependent mechanotransduction as a result of changes in expression of integrin regulatory molecules such as CD98 or differential expression and function of downstream components of the mechanotransduction pathway such as PKC or NMDA receptors.

Objective: To identify functional interleukin-4 (IL4) receptor (IL4R) subtypes and associated Janus kinase/signal transducers and activators of transcription (JAK/STAT) molecules in human articular chondrocytes and assess the role of JAK/STAT proteins in chondrocyte mechanotransduction.Methods: Expression of IL4R subunits and associated molecules was assessed by immunohistochemistry and western blotting. Functional IL4R were identified by chemical crosslinking of IL4-stimulated chondrocytes and western blotting. JAK and STAT phosphorylation was assessed by western blotting.Results: Chondrocytes from normal and osteoarthritic (OA) cartilage express IL4R alpha, gamma c and IL13R alpha 1 subunits (components of the Type I and Type II IL4R). In the presence of IL4 only functional Type II IL4Rs were identified in normal or OA chondrocytes. With the exception of STAT2, no differences in JAK/STAT expression were detected between normal and OA cartilage. STAT2 was expressed in OA but not normal chondrocytes. Mechanical stimulation (MS) resulted in an IL4R-dependent increase in phosphorylated Tyk2 in normal chondrocytes, which could be abolished by IL1 beta preincubation. No phosphorylation of STAT5 or STATE was detected in either normal or OA chondrocytes following mechanical stimulation (MS) IL4 stimulation resulted in a decrease in Tyk2 phosphorylation and an increase in phosphorylation of STATE in both normal and OA chondrocytes.Conclusion: Chondrocytes from normal and OA cartilage signal through a Type II IL4R. This signalling is via a STATE-independent pathway. Differences in IL4 signalling are likely due to crosstalk between integrin and cytokine signalling pathways, and not differences in IL4R expression. (C) 2006 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Mechanical stimuli are known to have major influences on chondrocyte function. The molecular events that regulate chondrocyte responses to mechanical stimulation have been the subject of much study. Using an in vitro experimental system we have identified mechanotransduction pathways that control molecular and biochemical responses of human articular chondrocytes to cyclical mechanical stimulation, and how these responses differ in cells isolated from diseased cartilage. We have previously shown that mechanical stimulation of normal articular chondrocytes leads to a cell membrane hyperpolarisation. Within 1 hour following mechanical stimulation there is an increase in aggrecan mRNA levels. These responses are mediated via alpha5beta1 integrins, the neuropeptides substance P and NMDA, and the cytokine interleukin-4. In OA chondrocytes mechanical stimulation leads to cell membrane depolarisation, but no change in aggrecan mRNA at 1 hour. The depolarisation response is mediated via alpha5beta1 integrins, substance P and interleukin-4, but the cells show an altered response to NMDA. Having identified that the NMDA receptor is present in human articular cartilage and may play an important role in a chondroprotective mechanotransduction pathway, we were interested in whether other components associated with NMDA signalling may be involved in the chondrocyte mechanotransduction pathways. One such component is calcium/calmodulin-dependent protein kinase II (CaMKII). CaMKII mediates many cellular responses to elevated Ca2+ in a wide variety of cells and tissues. It is involved in the regulation of ion channels, cytoskeletal dynamics, gene transcription, neurotransmitter synthesis, insulin secretion, and cell division. CaMKII also shows a broad substrate specificity and is abundant in brain tissue, indicating that this kinase may play a number of roles in the functioning of the central nervous system. This kinase has been studied extensively in brain, but there is only a limited understanding of CaMKII in other tissues. CAMKII has four subunit isoforms (alpha,beta,gamma,delta). The alpha- and beta-isoforms have narrow distributions restricted mainly to neuronal tissues, but the gamma- and delta-isoforms are ubiquitously expressed within neuronal and non-neuronal tissues. The aim of this study was to investigate the expression of CaMKII in normal and OA cartilage and chondrocytes, and whether this enzyme is involved in the response of chondrocytes to cyclical mechanical stimuli. Reverse transcriptase-polymerase chain reaction (RT-PCR), using primers specific for the different CaMKII isoforms, was carried out to assess which isoforms are expressed in human articular chondrocytes. To assess whether CaMKII is expressed in human articular chondrocytes at the protein level, cultured chondrocytes were extracted and analysed by Western blotting using a pan-CaMKII antibody. Immunohistochemistry was carried out to investigate whether CaMKII is expressed by human articular chondrocytes in vivo. Frozen sections of normal, OA and ankle cartilage were incubated for one hour with CaMKII antibody and visualised using ABC and DAB. To assess the role of CaMKII in the mechanotransduction responses of normal and OA chondrocytes, human normal and OA articular chondrocytes were mechanically stimulated at 0.33 Hz, or by addition of recombinant IL-4 for 20 minutes. Cell responses to these stimuli, in the absence or presence of an inhibitor of CaMKII were assessed by measuring changes in cell membrane potential or changes in relative levels of aggrecan mRNA compared with the housekeeping gene GAPDH. Normal, OA, and ankle chondrocytes expressed the gamma and delta isoforms of CaMKII mRNA, but not the alpha and beta isoforms as demonstrated by RT-PCR. Western blotting showed a band at approximately 60 kDa consistent with the expression of CaMKII. Immunohistochemistry revealed the positive staining in the middle and deep zones, but not the superficial zone, of normal, OA, and ankle cartilage. The presence of a CaMKII inhibitor inhibits the membrane hyperpolarisation response and upregulation of aggrecan mRNA in normal chondrocytes following mechanical stimulation, but has no effect on the hyperpolarisation response to recombinant IL4. The depolarisation response of OA chondrocytes to mechanical stimulation is unaffected by the presence of the CaMKII inhibitor. The CaMKII isoforms gamma and delta are expressed in both normal and OA chondrocytes, both in vitro and in vivo, but are only involved in the response of normal chondrocytes to mechanical stimulation. This response is upstream of the effect of IL4. These findings are consistent with previous findings for the NMDA receptor, and suggest that dysregulation of NMDA-CaMKII signalling may be important in onset and progression of osteoarthritis.

Objectives. Methotrexate (MTX), which is prescribed in the treatment of malignancy and autoimmune disease, has detrimental effects on a number of organ systems, including bone. At present, the exact mechanism of action of MTX on bone at the cellular level is unclear. Mechanical stimuli imparted by stretch, pressure, fluid flow and shear stress result in a variety of biochemical responses that are important in bone metabolism. Cyclical mechanical stimulation at 0.33 Hz induces rapid cell membrane hyperpolarization of human bone cells (HBC) via an integrin-mediated pathway which includes an IL-1beta autocrine/paracrine loop. This study was undertaken to investigate the effect of MTX on responses of HBC to 0.33 Hz mechanical stimulation.Methods. Electrophysiological responses of HBC were measured before and after mechanical stimulation at 0.33 Hz in the presence or absence of MTX. Semiquantitative RT-PCR was used to investigate effects of MTX on relative levels of type-1 collagen and bone morphogenetic protein-4 (BMP-4) following 0.33 Hz mechanical stimulation.Results. MTX dose-dependently inhibited HBC hyperpolarization in response to 0.33 Hz mechanical stimulation. Production/release of IL-1beta was inhibited by MTX, whereas its effects on target cells were not. Mechanical stimulation of HBC at 0.33 Hz caused a significant decrease in relative levels of BMP-4 mRNA, whereas relative levels of type-1 collagen mRNA were consistently increased, although these increases did not reach statistical significance. These trends were unaffected by MTX.Conclusions. These studies show that MTX affects HBC mechanotransduction by interfering with integrin-mediated signalling. The data also suggest that the mechanotransduction pathway responsible for the regulation of type-1 collagen and BMP-4 gene expression may be distinct from the IL-1beta-mediated signalling pathway.

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.

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.

OBJECTIVE:To assess whether substance P and the corresponding neurokinin 1 (NK1) receptor are expressed in human articular cartilage, and whether these molecules have a role in chondrocyte mechanotransduction.METHODS:Transgenic studies, immunohistochemistry, Western blotting, and reverse transcriptase-polymerase chain reaction were used to assess the expression of the preprotachykinin (PPT) gene, substance P, and NK1 in developing mice, in adult human articular cartilage, and in human chondrocytes in culture. Chondrocytes obtained from PPT knockout mice and human articular chondrocytes were mechanically stimulated in the presence or absence of inhibitors of substance P signaling, and cell membrane potentials or relative levels of aggrecan messenger RNA (mRNA) were measured.RESULTS:Replacing a region of the PPT gene transcriptional site that contains a dominant repressor of the proximal promoter activity with the constitutive minimal promoter of the human beta-globin promoter allowed expression of a marker gene in areas of chondrogenesis during mouse development and in adult chondrocytes grown in culture. Adult human articular chondrocytes expressed endogenous PPT mRNA, substance P, and the corresponding NK1 receptor in vivo and in vitro. Blockade of substance P signaling by a chemical antagonist to the NK1 receptor inhibited chondrocyte responses to mechanical stimulation.CONCLUSION:Substance P is expressed in human articular cartilage and is involved in chondrocyte mechanotransduction via the NK1 receptor in an autocrine and paracrine manner. This suggests that substance P and the NK1 receptor have roles in the maintenance of articular cartilage structure and function that were previously unrecognized.

OBJECTIVE:The objective of this study was to examine PKC isozyme expression in human articular chondrocytes and assess roles for RACK1, a receptor for activated C kinase in the mechanotransduction process. METHODS:Primary cultures of human articular chondrocytes and a human chondrocyte cell line were studied for expression of PKC isozymes and RACK1 by western blotting. Following mechanical stimulation of chondrocytes in vitro in the absence or presence of anti-integrin antibodies and RGD containing oligopeptides, subcellular localization of PKCalpha and association of RACK1 with PKCalpha and beta1 integrin was assessed. RESULTS:Human articular chondrocytes express PKC isozymes alpha, gamma, delta, iota, and lambda. Following mechanical stimulation at 0.33Hz chondrocytes show a rapid, beta1 integrin dependent, translocation of PKCalpha to the cell membrane and increased association of RACK1 with PKCalpha and beta1 integrin. CONCLUSIONS:RACK1 mediated translocation of activated PKCalpha to the cell membrane and modulation of integrin-associated signaling are likely to be important in regulation of downstream signaling cascades controlling chondrocyte responses to mechanical stimuli.

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.

Mechanical stimuli are known to have major influences on chondrocyte function. The molecular events that regulate chondrocyte responses to mechanical stimulation are beginning to be understood. In vitro analyses have allowed identification of mechanotransduction pathways that control molecular and biochemical responses of human articular chondrocytes to cyclical mechanical stimulation. These studies have shown that human articular chondrocytes use alpha5beta1 integrin as a mechanoreceptor. After stimulation of this integrin by mechanical stimulation, there is activation of a signal cascade, involving stretch-activated ion channels, the actin cytoskeleton and tyrosine phosphorylation of the focal adhesion complex molecules pp125 focal adhesion kinase and paxillin, and beta-catenin. Subsequently, there is secretion of interleukin-4, which acts in an autocrine manner via Type II receptors, to induce membrane hyperpolarization, increase levels of aggrecan messenger ribonucleic acid, and decrease levels of matrix metalloproteinase 3 messenger ribonucleic acid. Chondrocytes from osteoarthritic cartilage also use alpha5beta1 integrin as a mechanoreceptor, but downstream signaling cascades and cell responses including changes in aggrecan messenger ribonucleic acid are different. Abnormalities of chondroprotective mechanotransduction pathways in osteoarthritis may contribute to disease progression.

Conference Abstract| February 01 2001 Integrins, Stretch-Activated ION Channels and Interleukin-4 are Involved in the Regulation of Aggrecan and MMP3 Mrna Levels in Human Articular Chondrocytes following Mechanical Stimulation S.J. Millward-Sadler; S.J. Millward-Sadler 1Department of Pathology, Edinburgh University Medical School, Edinburgh, EH8 9AG, UK3** Rheumatic Diseases Unit, Western General Hospital, Edinburgh, EH4 2XU, UK Search for other works by this author on: This Site PubMed Google Scholar M.O. Wright; M.O. Wright *Department of Biomedical Sciences, Edinburgh University Medical School, Edinburgh, EH8 9AG, UK4** Rheumatic Diseases Unit, Western General Hospital, Edinburgh, EH4 2XU, UK Search for other works by this author on: This Site PubMed Google Scholar G. Nuki; G. Nuki *Department of Biomedical Sciences, Edinburgh University Medical School, Edinburgh, EH8 9AG, UK5** Rheumatic Diseases Unit, Western General Hospital, Edinburgh, EH4 2XU, UK Search for other works by this author on: This Site PubMed Google Scholar D.M. Salter D.M. Salter 1Department of Pathology, Edinburgh University Medical School, Edinburgh, EH8 9AG, UK6** Rheumatic Diseases Unit, Western General Hospital, Edinburgh, EH4 2XU, UK Search for other works by this author on: This Site PubMed Google Scholar Clin Sci (Lond) (2001) 100 (s44): 14P. https://doi.org/10.1042/cs100014Pb Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation S.J. Millward-Sadler, M.O. Wright, G. Nuki, D.M. Salter; Integrins, Stretch-Activated ION Channels and Interleukin-4 are Involved in the Regulation of Aggrecan and MMP3 Mrna Levels in Human Articular Chondrocytes following Mechanical Stimulation. Clin Sci (Lond) 1 February 2001; 100 (s44): 14P. doi: https://doi.org/10.1042/cs100014Pb Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsClinical Science Search Advanced Search This content is only available as a PDF. © 2001 The Biochemical Society and the Medical Research Society2001 Article PDF first page preview Close Modal You do not currently have access to this content.

S49 Mechanical stimuli are known to have major influences on chondrocyte function. The molecular events that regulate chondrocyte responses to mechanical stimulation are beginning to be understood. In vitro analyses have allowed identification of mechanotransduction pathways that control molecular and biochemical responses of human articular chondrocytes to cyclical mechanical stimulation. These studies have shown that human articular chondrocytes use 5 1 integrin as a mechanoreceptor. After stimulation of this integrin by mechanical stimulation, there is activation of a signal cascade, involving stretchactivated ion channels, the actin cytoskeleton and tyrosine phosphorylation of the focal adhesion complex molecules pp125 focal adhesion kinase and paxillin, and -catenin. Subsequently, there is secretion of interleukin-4, which acts in an autocrine manner via Type II receptors, to induce membrane hyperpolarization, increase levels of aggrecan messenger ribonucleic acid, and decrease levels of matrix metalloproteinase 3 messenger ribonucleic acid. Chondrocytes from osteoarthritic cartilage also use 5 1 integrin as a mechanoreceptor, but downstream signaling cascades and cell responses including changes in aggrecan messenger ribonucleic acid are different. Abnormalities of chondroprotective mechanotransduction pathways in osteoarthritis may contribute to disease progression. Integrin–Interleukin-4 Mechanotransduction Pathways in Human Chondrocytes

OBJECTIVETo establish whether chondrocytes from normal and osteoarthritic human articular cartilage recognize and respond to pressure induced mechanical strain in a similar manner.DESIGNChondrocytes, extracted from macroscopically normal and osteoarthritic human articular cartilage obtained from knee joints at autopsy, were grown in monolayer culture and subjected to cyclical pressure-induced strain (PIS) in the absence or presence of anti-integrin antibodies, agents known to block ion channels and inhibitors of key molecules involved in the integrin-associated signalling pathways. The response of the cells to mechanical stimulation was assessed by measuring changes in membrane potential.RESULTSUnlike chondrocytes from normal articular cartilage, which showed a membrane hyperpolarization response to PIS, chondrocytes from osteoarthritic cartilage responded by membrane depolarization. The mechanotransduction pathway involves alpha5beta1 integrins, stretch-activated ion channels, tyrosine kinases and phospholipase C but the actin cytoskeleton and protein kinase C, which are important in the membrane hyperpolarization response in normal chondrocytes, are not necessary for membrane depolarization in osteoarthritic chondrocytes in response to PIS.CONCLUSIONChondrocytes derived from osteoarthritic cartilage show a different signalling pathway via alpha5beta1 integrin in response to mechanical stimulation which may be of importance in the production of phenotypic changes recognized to be present in diseased cartilage.

Mechanical forces influence chondrocyte metabolism and function. We have previously shown that 0.33 Hz cyclical pressure-induced strain (PIS) results in membrane hyperpolarization of normal human articular chondrocytes (HAC) by activation of Ca(2+)-dependent K+ small conductance potassium activated calcium (SK) channels. The mechanotransduction pathway involves alpha 5 beta 1-integrin, stretch-activated ion channels (SAC) actin cytoskeleton and tyrosine protein kinases, with subsequent release of the chondroprotective cytokine interleukin-4 (IL-4). The objective of this study was to examine in detail tyrosine phosphorylation events in the mechanotransduction pathway. The results show tyrosine phosphorylation of three major proteins, p125, p90, and p70 within 1 minute of onset of mechanical stimulation. Immunoblotting and immunoprecipitation show these to be focal adhesion kinase (pp125FAK), beta-catenin, and paxillin, respectively. Tyrosine phosphorylation of all three proteins is inhibited by RGD containing oligopeptides and gadolinium, which is known to block SAC. beta-catenin coimmunoprecipitates with FAK and is colocalized with alpha 5-integrin and pp125FAK. These results indicate a previously unrecognized role for an integrin-beta-catenin signaling pathway in human articular chondrocyte (HAC) responses to mechanical stimulation.

OBJECTIVETo determine molecular events in the regulation of messenger RNA (mRNA) of cartilage matrix molecules and proteases by mechanical stimulation of chondrocytes from normal human articular cartilage and to ascertain whether similar regulatory systems are present in chondrocytes from osteoarthritic (OA) cartilage.METHODSChondrocytes extracted from macroscopically and microscopically normal and OA cartilage were mechanically stimulated in the presence or absence of GRGDSP or GRADSP oligopeptides, neutralizing interleukin-4 (IL-4) antibodies, gadolinium, or apamin. The relative levels of mRNA for aggrecan, tenascin, matrix metalloproteinase 1 (MMP-1), MMP-3, and tissue inhibitor of metalloproteinases 1 (TIMP-1) were determined by semiquantitative reverse transcription-polymerase chain reaction at several time points up to 24 hours poststimulation, using GAPDH as a control.RESULTSNormal chondrocytes showed an increase in aggrecan mRNA and a decrease in MMP-3 mRNA within 1 hour following stimulation, with a return to baseline levels within 24 hours. These changes were blocked by GRGDSP, IL-4 antibodies, and gadolinium, but were unaffected by apamin. In contrast, chondrocytes isolated from OA cartilage showed no change in aggrecan or MMP-3 mRNA levels following mechanical stimulation. The mRNA levels of tenascin, MMP-1, and TIMP-1 were unaltered in mechanically stimulated normal and OA chondrocytes.CONCLUSIONMechanical stimulation of human articular chondrocytes in vitro results in increased levels of aggrecan mRNA and decreased levels of MMP-3 mRNA. The transduction process involves integrins, stretch-activated ion channels, and IL-4. This chondroprotective response is absent in chondrocytes from OA cartilage. Abnormalities of mechanotransduction leading to aberrant chondrocyte activity in diseased articular cartilage may be important in the progression of OA.