Search

Additional file 1. Supplemental Data.

Additional file 2. Supplementary tables.

Osteoblast differentiation and proliferation are regulated by several modulators, among which are adenosine A2A receptors (A2ARs) and Wingless/Integrated-β-catenin pathways. Cytosolic β-catenin stabilization promotes its nuclear translocation and transcriptional activity. In the present study, we seek to determine whether there is a connection between A2AR stimulation and cellular β-catenin levels in osteoblasts. Osteoblast precursor cell line (MC3T3-E1) and primary murine osteoblasts were treated with CGS21680, a highly selective A2AR agonist. We analyzed cellular content and nuclear translocation of phosphorylated (p)-serine 552 (S552) β-catenin in response to A2AR stimulation in MC3T3-E1 cells, in both wild-type and A2AR knockout (A2AKO) mice. Moreover, we measured cellular β-catenin levels in MC3T3-E1 cells transfected with scrambled or protein kinase B (Akt) small interfering RNA following A2AR activation. CGS21680 (1 μM) stimulated an increase in both the cellular content and nuclear translocation of p-S552 β-catenin after 15 min of incubation. A2AR activation had no tangible effect on the cellular β-catenin level either in A2AKO mice or in osteoblasts with diminished Akt content. Our findings demonstrate an interaction between A2AR, β-catenin, and Akt signaling in osteoblasts. The existence of such a crosstalk has significant repercussions in the development of novel therapeutic approaches targeting medical conditions associated with reduced bone density.-Borhani, S., Corciulo, C., Larranaga-Vera, A., Cronstein, B. N. Adenosine A2A receptor (A2AR) activation triggers Akt signaling and enhances nuclear localization of β-catenin in osteoblasts.

Osteopenia and fragility fractures have been associated with human immunodeficiency virus (HIV) infection. Tenofovir, a common antiviral in HIV treatment, also leads to increases in bone catabolism markers and decreased BMD in children and young adults. In murine models and human cell lines, tenofovir inhibits adenosine triphosphate release and decreases extracellular adenosine levels. Adenosine and adenosine A2A receptor inhibit osteoclast formation, and increase local adenosine concentration with dipyridamole, an agent that blocks adenosine cellular uptake and stimulates new bone formation as well as bone morphogenic protein 2. We hypothesized that tenofovir regulates bone resorption by diminishing endogenous adenosine levels and questioned whether dipyridamole may be a useful treatment to counteract the deleterous bone effects of tenofovir. Primary murine osteoclasts were induced by M-CSF/RANKL, and the number of TRAP-positive-cells was studied after challenge with tenofovir alone or in combination with dipyridamole. Differentiation markers were studied by RT-PCR and MAPK/NFkB expression by Western blot. Male C57Bl/6 mice were treated as follows: saline 0.9% (control), tenofovir 75 mg/kg/day, dipyridamole 25 mg/kg/day, combination tenofovir/dipyridamole (n = 10, 4 weeks). Calcein/Alizarin Red-labeling of newly formed bone was used, and long bones were prepared for micro-computed tomography (μCT)/histology. Tenofovir produced a dose-dependent increase in osteoclast differentiation (EC50 = 44.5nM) that was reversed by dipyridamole (IC50 = 0.3 μM). Tenofovir increased cathepsin K and NFATc1 mRNA levels and dipyridamole reversed the effect. Dipyridamole reversed the effect of tenofovir on pERK1/2, pp38, and NFkB nuclear translocation. Mice treated with tenofovir lost nearly 10% of their body weight (p < 0.001). μCT revealed decreased BMD and altered trabecular bone in tenofovir-treated mice, reversed by dipyridamole. TRAP-staining showed increased osteoclasts in tenofovir-treated mice (p < 0.005), an effect reversed by dipyridamole. Similar results were obtained for cathepsin K and CD68. RANKL-positive cells were increased in tenofovir-treated mice, whereas osteoprotegerin-positive cells were decreased; both effects were reversed by dipyridamole. These results suggest that treatment with agents that increase local adenosine concentrations, like dipyridamole, might prevent bone loss following tenofovir treatment. © 2019 American Society for Bone and Mineral Research.

Extracellular adenosine triphosphate (ATP) plays a central role in a wide variety of joint diseases. ATP is generated intracellularly, and the concentration of the extracellular ATP pool is determined by the regulation of its transport out of the cell. A variety of ATP transporters have been described, with connexins and pannexins the most commonly cited. Both form intercellular channels, known as gap junctions, that facilitate the transport of various small molecules between cells and mediate cell–cell communication. Connexins and pannexins also form pores, or hemichannels, that are permeable to certain molecules, including ATP. All joint tissues express one or more connexins and pannexins, and their expression is altered in some pathological conditions, such as osteoarthritis (OA) and rheumatoid arthritis (RA), indicating that they may be involved in the onset and progression of these pathologies. The aging of the global population, along with increases in the prevalence of obesity and metabolic dysfunction, is associated with a rising frequency of joint diseases along with the increased costs and burden of related illness. The modulation of connexins and pannexins represents an attractive therapeutic target in joint disease, but their complex regulation, their combination of gap-junction-dependent and -independent functions, and their interplay between gap junction and hemichannel formation are not yet fully elucidated. In this review, we try to shed light on the regulation of these proteins and their roles in ATP transport to the extracellular space in the context of joint disease, and specifically OA and RA.

BACKGROUND AND PURPOSE Patients with active rheumatoid arthritis (RA) have increased cardiovascular mortality, paradoxically associated with reduced circulating lipid levels. The Janus kinase (Jak) inhibitor tofacitinib ameliorates systemic and joint inflammation in RA with a concomitant increase in serum lipids. We analyzed the effect of tofacitinib on the lipid profile of hyperlipidemic rabbits with chronic arthritis (CA) and on the regulation of reverse cholesterol transport (RCT) during chronic inflammation. EXPERIMENTAL APPROACH CA was induced in previously immunized rabbits fed with a high-fat diet (HFD) by administering four intra-articular injections of ovalbumin. A group of rabbits received tofacitinib (10 mg kg-1 day-1) for two weeks. Systemic and synovial inflammation and lipid content were evaluated. For in vitro studies, THP-1-derived macrophages were exposed to high lipid concentrations, and then stimulated with interferon gamma (IFNγ) in the presence or absence of tofacitinib in order to study RCT mediators. KEY RESULTS Tofacitinib decreased systemic and synovial inflammation and increased circulating lipid levels. Although it did not modify synovial macrophage density, it was able to reduce the lipid content within synovial macrophages. In foam macrophages in culture, IFNγ further stimulated intracellular lipid accumulation, while Jak/STAT inhibition provoked by tofacitinib induced lipid release by increasing cellular liver X receptor alpha (LXRα) and ATP-binding cassette transporter (ABCA1) synthesis. CONCLUSION AND IMPLICATIONS Active inflammation could be associated with lipid accumulation within macrophages of CA rabbits. Jak inhibition induced lipid release though RCT activation, providing a plausible explanation for the effect of tofacitinib on the lipid profile of RA patients.

Background Metabolic syndrome (MetS) may be associated with knee osteoarthritis (OA), but the association between the individual components and OA are not well-understood. We aimed to study the effect of hypercholesterolemia on synovial inflammation in knee OA. Methods OA was surgically induced in rabbits fed with standard diet (OA group, n = 10) or in rabbits fed with high fat diet (OA-HFD, n = 10). Healthy rabbits receiving standard diet (Control, n = 10) or fed with HFD (HFD, n = 6) were also monitored. Twelve weeks after OA induction, synovial membranes were isolated and processed for studies. Results Animals fed HFD showed higher levels of total serum cholesterol, triglycerides and C-reactive protein than control rabbits. Twelve weeks after OA induction, synovial membrane inflammation and macrophage infiltration were increased in rabbits with OA, particularly in the OA-HFD group. Extensive decrease of synovial adipose tissue area, adipocyte size and perilipin-1A synthesis were observed in the OA-HFD group in comparison to the OA and control groups. The HFD further increased the proinflammatory mediators IL-1β, IL-6 and TNF in the OA synovium. However, the synovial gene expression of adipokines, such as leptin and adiponectin, were markedly decreased in the rabbits with OA, especially in the OA-HFD group, in correlation with adipose tissue loss. However, circulating leptin was upregulated in the HFD and OA-HFD groups. Conclusion Our results indicate that a HFD is an aggravating factor worsening synovial membrane inflammation during OA, guided by increased infiltration of macrophages and removal of the adipose tissue, together with a remarkable presence of proinflammatory factors. Synovial adipocytes and dyslipemia could probably play pivotal roles in OA joint deterioration in patients with MetS, supporting that the link between obesity and OA transcends mechanical loading. Electronic supplementary material The online version of this article (doi:10.1186/s13075-017-1473-z) contains supplementary material, which is available to authorized users.

Background Patients with active rheumatoid arthritis (RA) have increased cardiovascular mortality, paradoxically associated with reduced serum lipid levels. In fact, an inverse relationship between C-reactive protein (CRP) and circulating lipid levels has been observed [1,2]. The Jak inhibitor tofacitinib ameliorates systemic and joint inflammation in RA with a concomitant increase in serum lipids [3]. Objectives Our aim was to analyze the effect of tofacitinib on the lipid and inflammatory profile of hyperlipidemic rabbits with chronic arthritis (CA), and on the regulation of reverse cholesterol transport (RCT) during chronic inflammation. Methods New Zealand male rabbits were randomly assigned to three groups: control group (n=6), chronic arthritic rabbits (CA, n=9) and CA rabbits receiving tofacitinib (CA+TOFA, n=9). All animals were fed ad libitum with a high-fat diet (0.5% cholesterol, 4% peanut oil). CA was induced in previously immunized rabbits by administering four intra-articular injections of ovalbumin. A group of rabbits received tofacitinib (10mg/kg/day) for two weeks. All animals were euthanized 6 weeks after challenge, when synovial and serum samples were collected. For in vitro studies, vehicle and ABCA1 siRNA-treated THP-1-derived macrophages were exposed to high lipid concentrations, and then stimulated with interferon gamma (IFNγ) in the presence or absence of tofacitinib, in order to assess their cholesterol efflux capacity and the involved mediators. Results CA rabbits showed lower levels of serum TC and LDL-C compared to controls (p=0.001, p=0.012), while TC/HDL-C ratio was higher in CA+TOFA rabbits when compared to CA animals (150±15 vs 231±17, p=0.004). Synovial inflammation and CRP levels were increased in CA animals, and a significant reduction was shown in CA+TOFA rabbits in both parameters. We also observed an inverse correlation between serum TC and CRP (R=-0.454, p=0.029). Tofacitinib was able to reduce the lipid content within synovial macrophages up to a 58% (p=0.041), although it did not modify synovial macrophage density. In foam macrophages in culture, an inflammatory milieu induced by IFNγ further stimulated the intracellular lipid accumulation (p=0.041) along with a decrease in the protein levels of the nuclear factor LXRα (liver X receptor α) and the cholesterol transporter ABCA1 (ATP-binding cassette transporter 1) (p=0.002 and p=0.0047, respectively). Tofacitinib prevented the lipid accumulation within macrophages (p=0.029) by increasing LXRα (p=0.047) and ABCA1 synthesis (p=0.004) in a Jak/STAT-dependent manner, while it was unable to reduce lipid accumulation in ABCA1 silenced macrophages. Conclusions Our results suggest that active inflammation could be associated with lipid accumulation within macrophages – in the synovium and probably in other tissues – thus inducing a decrease in serum lipid levels. Tofacitinib may prevent this phenomenon, at least partially, by increasing RCT pathways in macrophages. Taken together, these findings further explain how serum lipid levels are diminished in RA and partially justify the effect of tofacitinib on the lipid profile in RA patients. References Choy E, Sattar N. Ann Rheum Dis 2009;68:460–9. Johnsson H et al. Ann Rheum Dis 2013;73:1495–9. van Vollenhoven RF et al. N Engl J Med 2012;367:508–19. Disclosure of Interest None declared

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 21-04-2017, La artrosis, es una patología multifactorial, extremadamente común, que afecta a las articulaciones diartrodiales provocando dolor e incapacidad funcional, y para la que no existe ningún tratamiento. La recomendación más extendida para mejorar el pronóstico, es la modificación del estilo de vida para evitar factores de riesgo asociados a desordenes metabólicos como la diabetes y el síndrome metabólico. Un mayor conocimiento de los mecanismos celulares responsables de dichas asociaciones, sería fundamental para una mejor caracterización de la patogenia de la artrosis, facilitando así, nuevos abordajes terapéuticos. Con el objetivo de profundizar en los mecanismos por los que la hiperglucemia y la hiperlipemia podrían modular la evolución de la artrosis, hemos desarrollado dos aproximaciones. Por un lado, pusimos a punto un sistema de cultivo en tres dimensiones de condrocitos primarios, artrósicos para estimularlos con altas concentraciones de glucosa. También, realizamos un estudio en pacientes artrósicos con alteraciones en el metabolismo de la glucosa. Como resultado, hemos demostrado que una concentración elevada de glucosa induce un fenotipo hipertrófico en condrocitos artrósicos en cultivo, proceso que se inhibe cuando se impide la O-β-N-Acetilglicosilación proteica. Observamos además, que los pacientes artrósicos con diabetes muestran un aumento en la expresión de marcadores de hipertrofia en el cartílago, superior al que observamos en los pacientes con artrosis sin alteraciones metabólicas. Por otro lado, hemos trabajado con un modelo de artrosis en conejos con hiperlipemia para evaluar el efecto de la combinación de estos factores en los tejidos articulares, especialmente en la membrana adiposa infrapatelar. La hiperlipemia agravó el daño histológico en la membrana sinovial de los conejos artrósicos produciéndose una invasión de macrófagos, tejido fibrótico, incrementó la destrucción del tejido adiposo, reducción en el tamaño y el número de los adipocitos y reducción de los niveles de adipoquinas en la sinovial. Este trabajo describe alguno de los mecanismos por los que la hiperlipemia y la hiperglucemia podrían agravar el desarrollo de la artrosis humana. Así, nuestros resultados apoyan la idea de la existencia de un fenotipo metabólico de artrosis. Asimismo, estos datos podrían abrir nuevas líneas de investigación sobre la O-β-N-Acetilglicosilación de proteínas, y su posible modificación en el tratamiento de la artrosis o sobre la identificación de nuevas dianas en los distintos tejidos articulares, más allá del cartílago en pacientes con características distintivas como la dislipemia., Osteoarthritis (OA) is an extremely common multifactorial disease that affects diartrodial articulations, causing pain and functional loss. There is not any available pharmacological treatment, and the most extended recommendation is to follow a healthy lifestyle in order to avoid metabolic disorder related risk factors, as obesity, diabetes or metabolic syndrome. A greater knowledge of the cellular mechanisms behind this association is essential for a better characterization of the pathogenesis of osteoarthritis, thus, facilitating new therapeutic approaches. With the objective of deepening on the mechanisms by hyperglycemia and hyperlipemia, modulate the evolution of OA, we have developed two different approaches. First, we have worked with a three-dimensional primary osteoarthritic chondrocyte culture system to study the influence of high glucose concentrationse. We also performed a study in patients with impaired glucose metabolism. As a result, we have shown that a high concentration of glucose induces a hypertrophic phenotype in cultured chondrocytes. Also, that this process is inhibited when protein O-β-N-Acetylglycosylation is impaired. Similarly, compared to osteoarthritic patients without identified metabolic alterations, patients with osteoarthritis and diabetes show an increase of hypertrophyc marker expression in the cartilage. Second, we developed an osteoarthritic, hypercholesterolemic rabbit model to evaluate the effect of the combination of these factors on the articular tissues, especially in the infrapatellar adipose membrane. Hyperlipemia aggravated histological damage of the synovial membrane of this rabbits leading to macrophage invasion, fibrotic tissue, increased destruction of adipose tissue with reduced adipocyte size and a decrease of synovial adipokines. This paper describes some of the mechanisms by which hyperlipemia and hyperglycemia aggravate the development of human OA. Therefore, our results support the idea of the existence of a metabolic phenotype of osteoarthritis. In addition, these data opens a field to research on protein O-β-N-Acetylglycosylation, and its possible modification in the osteoarthritis treatment or in the identification of new pharmacologic targets in the different articular tissues, beyond the cartilage.

Patients with active rheumatoid arthritis (RA) have increased cardiovascular mortality, paradoxically associated with reduced circulating lipid levels. The JAK inhibitor tofacitinib ameliorates systemic and joint inflammation in RA with a concomitant increase in serum lipids. We analysed the effect of tofacitinib on the lipid profile of hyperlipidaemic rabbits with chronic arthritis (CA) and on the changes in reverse cholesterol transport (RCT) during chronic inflammation.CA was induced in previously immunized rabbits, fed a high-fat diet, by administering four intra-articular injections of ovalbumin. A group of rabbits received tofacitinib (10 mg·kgTofacitinib decreased systemic and synovial inflammation and increased circulating lipid levels. Although it did not modify synovial macrophage density, it reduced the lipid content within synovial macrophages. In foam macrophages in culture, IFNγ further stimulated intracellular lipid accumulation, while the JAK/STAT inhibition provoked by tofacitinib induced lipid release by increasing the levels of cellular liver X receptor α and ATP-binding cassette transporter (ABCA1) synthesis.Active inflammation could be associated with lipid accumulation within macrophages of CA rabbits. JAK inhibition induced lipid release through RCT activation, providing a plausible explanation for the effect of tofacitinib on the lipid profile of RA patients.

Summary Objective There is increasing evidence that the addition of O-linked N-acetylglucosamine (O-GlcNAc) to proteins plays an important role in cell signaling pathways. In chondrocytes, accumulation of O-GlcNAc-modified proteins induces hypertrophic differentiation. Osteoarthritis (OA) is characterized by cartilage degradation, and hypertrophic-like changes in hyaline chondrocytes. However, the mechanisms responsible for these changes have not been described. Our aim was to study whether O-GlcNAcylation and the enzymes responsible for this modification are dysregulated in the cartilage of patients with knee OA and whether interleukin-1 could induce these modifications in cultured human OA chondrocytes (HOC). Design Human cartilage was obtained from patients with knee OA and from age and sex-matched healthy donors. HOC were cultured and stimulated with the catabolic cytokine IL-1α. Global protein O-GlcNAcylation and the synthesis of the key enzymes responsible for this modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), were assessed by western blot. Results OA was associated with a 4-fold increase in the global O-GlcNAcylation in the cartilage. OA cartilage showed a re-distribution of the OGT and OGA isoforms, with a net increase in the presence of both enzymes, in comparison to healthy cartilage. In HOC, IL-1α stimulation rapidly increased O-GlcNAcylation and OGT and OGA synthesis. Conclusions Our results indicate that a proinflammatory milieu could favor the accumulation of O-GlcNAcylated proteins in OA cartilage, together with the dysregulation of the enzymes responsible for this modification. The increase in O-GlcNAcylation could be responsible, at least partially, for the re-expression of hypertrophic differentiation markers that have been observed in OA.

Insulin is an inducer of chondrocyte hypertrophy and growth plate chondrogenesis, although the specific molecular mechanisms behind these effects are mostly unknown. Our aim was to investigate whether insulin-induced chondrocyte hypertrophy occurs through a modification in the amount of O-linked N-acetylglucosamine (O-GlcNAc)-modified proteins and in the expression of the key enzymes of this pathway, O-GlcNAc transferase and O-GlcNAcase (OGA). We also studied if O-GlcNAc accumulation per se, induced by an OGA inhibitor, was able to induce pre-hypertrophic chondrocyte differentiation both in vitro and in vivo. Insulin-induced differentiation of ATDC5 pre-chondrocytes occurred alongside a gradual increase in the accumulation of O-GlcNac-modified proteins (O-GlcNAcylated proteins), as well as an increase in the expression of O-GlcNAc transferase and OGA. In the absence of insulin, O-GlcNAc accumulation induced by thiamet-G, a specific OGA inhibitor, was able to increase the gene expression of differentiation markers, as well as the activity of MMP-2 and -9. Thiamet-G also activated pERK, p-JNK, and p-p38 and the O-GlcNAcylation of Akt. Thiamet-G administration to C57/bl mice induced a significant expansion in the growth plate height and in the hypertrophic zone height. Therefore, our results show that O-GlcNAc glycosylation has chondromodulating activity.

Background Tofacitinib (TOFA) is a Janus Kinase (JAK) inhibitor approved for the treatment of rheumatoid arthritis (RA) in different countries. TOFA treatment has recently been shown to selectively inhibit the expression of several chemokines from synovial biopsies from RA patients that were treated over 28 days (1). However, TOFA did not modify the expression of various pro-inflammatory cytokines, nor histopathological synovial inflammation, including macrophage infiltration. In this regard, animal models may allow a better understanding of the consequences of JAK inhibition in chronic arthritis (CA). Our group developed a model of CA in rabbits that mimics severe human RA, thereby allowing investigation of TOFA and its consequences in synovial tissue. Objectives We have analyzed the early treatment effect of TOFA in a model of CA, aiming to evaluate early tissue changes rather than the final therapeutic effect. Methods A well-established model of antigen-induced CA was induced in 20 rabbits via four, weekly intra-articular injections of ovalbumin in previously immunized rabbits (CA group). After two intra-articular injections, 10 CA rabbits were treated with TOFA (10mg/kg/day, orally) for two weeks (CA+TOFA) and 10 healthy rabbits were used as controls. Animals were euthanised four weeks after intra-articular challenge, when we collected serum and synovial samples for different studies. Results CA animals showed reduced weight gain compared to controls, which was partially prevented by TOFA (weight gain, kg; Control: 0.8±0.05; CA: -0.09±0.06*; CA+TOFA: 0.18±0.1*#; *p Conclusions In synovial tissue with intense inflammatory activity, TOFA treatment mainly inhibited the expression of MMPs and, to a lesser extent, the expression of other pro-inflammatory mediators. These data suggest that this drug could act as an immunomodulator of the chronic inflammatory response. TOFA does not induce a fast or drastic reduction in cellular recruitment within synovial tissue, but a moderate and generalized decrease of cytokines with a stronger effect on MMPs. References Boyle DL et al. The JAK inhibitor tofacitinib suppresses synovial JAK1-STAT signalling in rheumatoid arthritis. Ann Rheum Dis. 2014 [epub ahead of print] Disclosure of Interest None declared

Background Osteoarthritis (OA) is characterized by progressive destruction of articular cartilage and changes in chondrocyte phenotype. The signalling of RhoA, a small GTPase protein, has a critical role in a range of cellular functions, including cytoskeleton rearrangement, being essential in focal adhesions and cell-matrix interactions. Several studies described the involvement of RhoA signalling in the healthy chondrocyte phenotype induced by interleukin (IL)-1α, a pro-catabolic cytokine locally increased in OA. Dysregulation of this signalling pathway could play an important role in the development of OA. Objectives We aimed to study RhoA signalling pathway in human OA cartilage, and to establish the underlying mechanism involved in its regulation employing IL-1α in “in vitro” studies. Methods Human OA cartilage was isolated from the knee of OA patients during joint replacement surgery while healthy cartilage was obtained from the knee of age and sex-matched healthy donors. In vitro studies were also conducted on human OA chondrocytes in culture (HOC). HOC were stimulated with 10 ng/ml IL-1α at different periods of time. Results We observed increased protein levels of RhoA and its effector kinase ROCK1 in the cartilage of OA patients when compared to healthy cartilage. Cytoskeletal arrangement is regulated through MYPT-1 phosphorylation -a regulatory subunit of MLC-phosphatase- by different kinases, including ROCK. OA cartilage also showed an increase in both the total and the phosphorylated (Thr-853) form of 70 KDa MYPT-1, compared to healthy cartilage. In addition, we found an increased phosphorylation of the final effector MLC in OA cartilage. In order to further elucidate the mechanism underlying RhoA activation in OA cartilage, we first studied whether IL-1α was able to mimic the RhoA signalling activation in HOC in culture. IL-1α induced a significant increase in RhoA synthesis that was not linked to ROCK1 activation. We also observed an increase in the presence and phosphorylation of the 70KDa MYPT-1 form, with a simultaneous decrease in the 110 KDa MYPT-1 form. ROCK inhibition was unable to prevent MYPT-1 phosphorylation induced by IL-1α in HOC. However, we did observe lower phosphorylation levels when using an ERK1/2 inhibitor. IL-1α induced RhoA recruitment to the HOC membrane, assessed by immunofluorescence. Phalloidin staining revealed that IL-1α triggered formation of stress fibers and the loss of focal adhesion in HOC. Both stress fiber formation and focal adhesion loss were improved by the ERK1/2 inhibitor. Conclusions RhoA signalling is enhanced in the cartilage of OA patients, probably mediated by an increase in MYPT-1 phosphorylation. Stimulation of HOC with IL-1α is able to mimic these changes. However, MYPT-1 phosphorylation in HOC appeared to depend on ERK1/2, rather than on ROCK, as observed in OA cartilage. Further studies are needed to confirm the role of ROCK and ERK1/2 in RhoA activation in chondrocytes. Our results support the hypothesis that RhoA signalling pathway could be a potential therapeutic target in OA. Disclosure of Interest None declared