A. K. T. Wann

Primary cilia mediate mechanotransduction through control of ATP-induced Ca2+ signaling in compressed chondrocytes

We investigated the role of the chondrocyte primary cilium in mechanotransduction events related to cartilage extracellular matrix synthesis. We generated conditionally immortalized wild‐type (WT) and IFT88orpk (ORPK) mutant chondrocytes that lack primary cilia and assessed intracellular Ca2+ signaling, extracellular matrix synthesis, and ATP release in response to physiologically relevant compressive strains in a 3‐dimensional chondrocyte culture system. All conditions were compared to unloaded controls. We found that cilia were required for compression‐induced Ca2+ signaling mediated by ATP release, and an associated up‐regulation of aggrecan mRNA and sulfated glycosaminosglycan secretion. However, chondrocyte cilia were not the initial mechanoreceptors, since both WT and ORPK cells showed mechanically induced ATP release. Rather, we found that primary cilia were required for downstream ATP reception, since ORPK cells did not elicit a Ca2+ response to exogenous ATP even though WT and ORPK cells express similar levels of purine receptors. We suggest that purinergic Ca2+ signaling may be regulated by polycystin‐1, since ORPK cells only expressed the C‐terminal tail. This is the first study to demonstrate that primary cilia are essential organelles for cartilage mechanotransduction, as well as identifying a novel role for primary cilia not previously reported in any other cell type, namely cilia‐mediated control of ATP reception.—Wann, A. K. T., Zuo, N., Haycraft, C. J., Jensen, C. G., Poole, C. A., McGlashan, S. R., Knight, M. M. Primary cilia mediate mechanotransduction through control of ATP‐induced Ca2+ signaling in compressed chondrocytes. FASEB J. 26, 1663‐1671 (2012). www.fasebj.org

Eicosapentaenoic acid and docosahexaenoic acid reduce interleukin-1β-mediated cartilage degradation

IntroductionIn inflammatory joint disease, such as osteoarthritis (OA), there is an increased level of proinflammatory cytokines, such as interleukin (IL)-1β. These cytokines stimulate the production of matrix metalloproteinases (MMPs), which leads to the degradation of the cartilage extracellular matrix and the loss of key structural components such as sulphated glycosaminoglycan (sGAG) and collagen II. The aim of this study was to examine the therapeutic potential of n-3 polyunsaturated fatty acids (PUFAs) in an in vitro model of cartilage inflammation.MethodsTwo specific n-3 compounds were tested, namely, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), each at 0.1, 1 and 10 μM. Full thickness bovine cartilage explants, 5 mm in diameter, were cultured for 5 days with or without IL-1β and in the presence or absence of each n-3 compound. The media were replaced every 24 hours and assayed for sGAG content using the 1,9-dimethylmethylene blue (DMB) method. Chondrocyte viability was determined at the end of the culture period using fluorescence microscopy to visualise cells labelled with calcein AM and ethidium homodimer.ResultsTreatment with IL-1β (10 ng.ml-1) produced a large increase in sGAG release compared to untreated controls, but with no effect on cell viability, which was maintained above 80% for all treatments. In the absence of IL-1β, both n-3 compounds induced a mild catabolic response with increased loss of sGAG, particularly at 10 μM. By contrast, in the presence of IL-1β, both EPA and DHA at 0.1 and 1 μM significantly reduced IL-1β-mediated sGAG loss. The efficacy of the EPA treatment was maintained at approximately 75% throughout the 5-day period. However, at the same concentrations, the efficacy of DHA, although initially greater, reduced to approximately half that of EPA after 5 days. For both EPA and DHA, the highest dose of 10 μM was less effective.ConclusionsThe results support the hypothesis that n-3 compounds are anti-inflammatory through competitive inhibition of the arachidonic acid oxidation pathway. The efficacy of these compounds is likely to be even greater at more physiological levels of IL-1β. Thus we suggest that n-3 PUFAs, particularly EPA, have exciting therapeutic potential for preventing cartilage degradation associated with chronic inflammatory joint disease.

Cyclic movement stimulates hyaluronan secretion into the synovial cavity of rabbit joints

The novel hypothesis that the secretion of the joint lubricant hyaluronan (HA) is coupled to movement has implications for normal function and osteoarthritis, and was tested in the knee joints of anaesthetized rabbits. After washing out the endogenous synovial fluid HA (miscibility coefficient 0.4), secretion into the joint cavity was measured over 5 h in static joints and in passively cycled joints. The net static secretion rate (11.2 ± 0.7 μg h−1, mean ±s.e.m., n= 90) correlated with the variable endogenous HA mass (mean 367 ± 8 μg), with a normalized value of 3.4 ± 0.2 μg h−1 (100 μg)−1. Cyclic joint movement approximately doubled the net HA secretion rate to 22.6 ± 1.2 μg h−1 (n= 77) and raised the normalized percentage to 5.9 ± 0.3 μg h−1 (100 μg)−1. Secretion was inhibited by 2‐deoxyglucose and iodoacetate, confirming active secretion. The net accumulation rate underestimated true secretion rate due to some trans‐synovial loss. HA turnover time (endogenous mass/secretion rate) was 17–30 h (static) to 8–15 h (moved) The results demonstrate for the first time that the active secretion of HA is coupled to joint usage. Movement–secretion coupling may protect joints against the damaging effects of repetitive joint use, replace HA lost during periods of immobility (overnight), and contribute to the clinical benefit of exercise therapy in moderate osteoarthritis.

The primary cilium influences interleukin-1β-induced NFκB signalling by regulating IKK activity

The primary cilium is an organelle acting as a master regulator of cellular signalling. We have previously shown that disruption of primary cilia assembly, through targeting intraflagellar transport, is associated with muted nitric oxide and prostaglandin responses to the inflammatory cytokine interleukin-1β (IL-1β). Here, we show that loss of the primary cilium disrupts specific molecular signalling events in cytosolic NFκB signalling. The induction of cyclooxygenase 2 (COX2) and inducible nitrous oxide synthase (iNOS) protein is abolished. Cells unable to assemble cilia exhibit unaffected activation of IκB kinase (IKK), but delayed and reduced degradation of IκB, due to diminished phosphorylation of inhibitor of kappa B (IκB) by IKK. This results in both delayed and reduced NFκB p65 nuclear translocation and nuclear transcript binding. We also demonstrate that heat shock protein 27 (hsp27), an established regulator of IKK, is localized to the ciliary axoneme and cellular levels are dramatically disrupted with loss of the primary cilium. These results suggest that the primary cilia compartment exerts influence over NFκB signalling. We propose that the cilium is a locality for regulation of the molecular events defining NFκB signalling events, tuning signalling as appropriate.

Interleukin-1β sequesters hypoxia inducible factor 2α to the primary cilium.

BackgroundThe primary cilium coordinates signalling in development, health and disease. Previously we have shown that the cilium is essential for the anabolic response to loading and the inflammatory response to interleukin-1β (IL-1β). We have also shown the primary cilium elongates in response to IL-1β exposure. Both anabolic phenotype and inflammatory pathology are proposed to be dependent on hypoxia-inducible factor 2 alpha (HIF-2α). The present study tests the hypothesis that an association exists between the primary cilium and HIFs in inflammatory signalling.ResultsHere we show, in articular chondrocytes, that IL-1β-induces primary cilia elongation with alterations to cilia trafficking of arl13b. This elongation is associated with a transient increase in HIF-2α expression and accumulation in the primary cilium. Prolyl hydroxylase inhibition results in primary cilia elongation also associated with accumulation of HIF-2α in the ciliary base and axoneme. This recruitment and the associated cilia elongation is not inhibited by blockade of HIFα transcription activity or rescue of basal HIF-2α expression. Hypomorphic mutation to intraflagellar transport protein IFT88 results in limited ciliogenesis. This is associated with increased HIF-2α expression and inhibited response to prolyl hydroxylase inhibition.ConclusionsThese findings suggest that ciliary sequestration of HIF-2α provides negative regulation of HIF-2α expression and potentially activity. This study indicates, for the first time, that the primary cilium regulates HIF signalling during inflammation.

IFT88 influences chondrocyte actin organization and biomechanics

Summary Objectives Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. Methods The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88orpk). Confocal microscopy was used to quantify F-actin and myosin IIB organisation. Viscoelastic cell and actin cortex mechanics were determined using micropipette aspiration with actin dynamics visualised in live cells transfected with LifeACT-GFP. Results IFT88orpk cells exhibited a significant increase in acto-myosin stress fibre organisation relative to wild-type (WT) cells in monolayer and an altered response to cytochalasin D. Rounded IFT88orpk cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88orpk cells. Following membrane blebbing, IFT88orpk cells exhibited slower reformation of the actin cortex. IFT88orpk cells showed increased actin deformability and reduced cortical tension confirming that IFT regulates actin cortex mechanics. The reduced cortical tension is also consistent with the reduced bleb formation. Conclusions This study demonstrates for the first time that the ciliary protein IFT88 regulates fundamental actin organisation and the stiffness of the actin cortex leading to alterations in cell deformation, mechanical properties and blebbing in an IFT88 chondrocyte cell line. This adds to the growing understanding of the role of primary cilia and IFT in regulating cartilage biology.

Mechanosensitive hyaluronan secretion

Joint movement was recently shown to stimulate the secretion of the lubricant hyaluronan (HA); also, exercise therapy and intra‐articular hyaluronan injections are used to treat moderate osteoarthritis. The present study quantifies the stimulus–response curves for HA secretion in vivo and reports a role of transcription–translation–translocation in the secretory response. After washing out endogenous HA from anaesthetized, cannulated rabbit knees, the joints were cycled passively at various frequencies and durations, with or without intra‐articular inhibitors of protein synthesis and Golgi processing. Newly secreted HA was harvested for analysis after 5 h. Joints displayed graded, non‐linear stimulus–response curves to both duration and frequency of movement; 1 min duration per 15 min or a frequency of 0.17 Hz raised HA secretion by 42–54%, while rapid (1.5 Hz) or prolonged cycling (9 min per 15 min) raised it by 110–130%. Movement‐stimulated secretion and phorbol ester‐stimulated secretion were partly inhibited by the translation inhibitor cycloheximide, by the transcription–translation inhibitors actinomycin D and puromycin and by the Golgi translocation inhibitor brefeldin A. There is thus a graded coupling between HA secretion and cyclic joint movement that depends partly on new protein synthesis. This is likely to be important for joint homeostasis, providing protection during repetitive cycling and potentially contributing to exercise therapy for osteoarthritis.

Mechanosensitive hyaluronan secretion: stimulus–response curves and role of transcription–translation–translocation in rabbit joints

Joint movement was recently shown to stimulate the secretion of the lubricant hyaluronan (HA); also, exercise therapy and intra‐articular hyaluronan injections are used to treat moderate osteoarthritis. The present study quantifies the stimulus–response curves for HA secretion in vivo and reports a role of transcription–translation–translocation in the secretory response. After washing out endogenous HA from anaesthetized, cannulated rabbit knees, the joints were cycled passively at various frequencies and durations, with or without intra‐articular inhibitors of protein synthesis and Golgi processing. Newly secreted HA was harvested for analysis after 5 h. Joints displayed graded, non‐linear stimulus–response curves to both duration and frequency of movement; 1 min duration per 15 min or a frequency of 0.17 Hz raised HA secretion by 42–54%, while rapid (1.5 Hz) or prolonged cycling (9 min per 15 min) raised it by 110–130%. Movement‐stimulated secretion and phorbol ester‐stimulated secretion were partly inhibited by the translation inhibitor cycloheximide, by the transcription–translation inhibitors actinomycin D and puromycin and by the Golgi translocation inhibitor brefeldin A. There is thus a graded coupling between HA secretion and cyclic joint movement that depends partly on new protein synthesis. This is likely to be important for joint homeostasis, providing protection during repetitive cycling and potentially contributing to exercise therapy for osteoarthritis.

Signal pathways regulating hyaluronan secretion into static and cycled synovial joints of rabbits

Joint lubrication, synovial fluid conservation and many pathophysiological processes depend on hyaluronan (HA). Intra‐articular HA injection and exercise, which stimulates articular HA production, ameliorate osteoarthritis. We therefore investigated the pathways regulating movement‐stimulated articular HA secretion rate () in vivo. Endogenous HA was removed from the knee joint cavity of anaesthetised rabbits by washout. Joints were then cycled passively or remained static for 5 h, with/without intra‐articular agonist/inhibitor, after which newly secreted HA was harvested for analysis. Movement almost doubled . Similar or larger increases were elicited in static joints by the intra‐articular Ca2+ ionophore ionomycin, prostaglandin E2, cAMP‐raising agents, serine/threonine phosphatase inhibitor and activation of protein kinase C (PKC). PKC‐stimulated secretion was inhibited by the PKC inhibitor bisindolylmaleimide I and inhibitors of the downstream kinases MEK‐ERK (U0126, PD98059). These agents inhibited movement‐stimulated secretion of HA (MSHA) only when the parallel p38 kinase path was simultaneously inhibited by SB203580 (ineffective alone). The phospholipase C inhibitor U73122 almost fully blocked MSHA (P= 0.001, n= 10), without affecting static . The ENaC channel blocker amiloride inhibited MSHA, whereas other inhibitors of stretch‐activated channels (Gd3+, ruthenium red, SKF96365) did not. It is proposed that MSHA may be mediated by PLC activation, leading to activation of parallel PKC–MEK–ERK and p38 kinase pathways.

Chondrocyte expansion is associated with loss of primary cilia and disrupted hedgehog signalling.

Tissue engineering-based therapies targeting cartilage diseases, such as osteoarthritis, require in vitro expansion of articular chondrocytes. A major obstacle for these therapies is the dedifferentiation and loss of phenotype accompanying chondrocyte expansion. Recent studies suggest that manipulation of hedgehog signalling may be used to promote chondrocyte re-differentiation. Hedgehog signalling requires the primary cilium, a microtubule-based signalling compartment, the integrity of which is linked to the cytoskeleton. We tested the hypothesis that alterations in cilia expression occurred as consequence of chondrocyte dedifferentiation and influenced hedgehog responsiveness. In vitro chondrocyte expansion to passage 5 (P5) was associated with increased actin stress fibre formation, dedifferentiation and progressive loss of primary cilia, compared to primary (P0) cells. P5 chondrocytes exhibited ~50 % fewer cilia with a reduced mean length. Cilia loss was associated with disruption of ligand-induced hedgehog signalling, such that P5 chondrocytes did not significantly regulate the expression of hedgehog target genes (GLI1 and PTCH1). This phenomenon could be recapitulated by applying 24 h cyclic tensile strain, which reduced cilia prevalence and length in P0 cells. LiCl treatment rescued cilia loss in P5 cells, partially restoring hedgehog signalling, so that GLI1 expression was significantly increased by Indian hedgehog. This study demonstrated that monolayer expansion disrupted primary cilia structure and hedgehog signalling associated with chondrocyte dedifferentiation. This excluded the possibility to use hedgehog ligands to stimulate re-differentiation without first restoring cilia expression. Furthermore, primary cilia loss during chondrocyte expansion would likely impact other cilia pathways important for cartilage health and tissue engineering, including transforming growth factor (TGF), Wnt and mechanosignalling.