Regulation of the Extracellular Matrix by Ciliary Machinery.

The primary cilium is an organelle involved in cellular signalling. Mutations affecting proteins involved in cilia assembly or function result in diseases known as ciliopathies, which cause a wide variety of phenotypes across multiple tissues. These mutations disrupt various cellular processes, including regulation of the extracellular matrix. The matrix is important for maintaining tissue homeostasis through influencing cell behaviour and providing structural support; therefore, the matrix changes observed in ciliopathies have been implicated in the pathogenesis of these diseases. Whilst many studies have associated the cilium with processes that regulate the matrix, exactly how these matrix changes arise is not well characterised. This review aims to bring together the direct and indirect evidence for ciliary regulation of matrix, in order to summarise the possible mechanisms by which the ciliary machinery could regulate the composition, secretion, remodelling and organisation of the matrix.

Mechanical loading inhibits cartilage inflammatory signalling via an HDAC6 and IFT-dependent mechanism regulating primary cilia elongation.

OBJECTIVE: Physiological mechanical loading reduces inflammatory signalling in numerous cell types including articular chondrocytes however the mechanism responsible remains unclear. This study investigates the role of chondrocyte primary cilia and associated intraflagellar transport (IFT) in the mechanical regulation of interleukin-1ß (IL-1ß) signalling. DESIGN: Isolated chondrocytes and cartilage explants were subjected to cyclic mechanical loading in the presence and absence of the cytokine IL-1ß. Nitric oxide (NO) and prostaglandin E2 (PGE2) release were used to monitor IL-1ß signalling whilst Sulphated glycosaminoglycan (sGAG) release provided measurement of cartilage degradation. Measurements were made of HDAC6 activity and tubulin polymerisation and acetylation. Effects on primary cilia were monitored by confocal and super resolution microscopy. Involvement of IFT was analysed using ORPK cells with hypomorphic mutation of IFT88. RESULTS: Mechanical loading suppressed NO and PGE2 release and prevented cartilage degradation. Loading activated HDAC6 and disrupted tubulin acetylation and cilia elongation induced by IL-1ß. HDAC6 inhibition with tubacin blocked the anti-inflammatory effects of loading and restored tubulin acetylation and cilia elongation. Hypomorphic mutation of IFT88 reduced IL-1ß signalling and abolished the anti-inflammatory effects of loading indicating the mechanism is IFT-dependent. Loading reduced the pool of non-polymerised tubulin which was replicated by taxol which also mimicked the anti-inflammatory effects of mechanical loading and prevented cilia elongation. CONCLUSIONS: This study reveals that mechanical loading suppresses inflammatory signalling, partially dependent on IFT, by activation of HDAC6 and post transcriptional modulation of tubulin.

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.

IFT88 influences chondrocyte actin organization and biomechanics.

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 (IFT88(orpk)). 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: IFT88(orpk) 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 IFT88(orpk) cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88(orpk) cells. Following membrane blebbing, IFT88(orpk) cells exhibited slower reformation of the actin cortex. IFT88(orpk) 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.

The primary cilium influences interleukin-1ß-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-1ß (IL-1ß). 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.

Surface topography regulates wnt signaling through control of primary cilia structure in mesenchymal stem cells.

The primary cilium regulates cellular signalling including influencing wnt sensitivity by sequestering ß-catenin within the ciliary compartment. Topographic regulation of intracellular actin-myosin tension can control stem cell fate of which wnt is an important mediator. We hypothesized that topography influences mesenchymal stem cell (MSC) wnt signaling through the regulation of primary cilia structure and function. MSCs cultured on grooves expressed elongated primary cilia, through reduced actin organization. siRNA inhibition of anterograde intraflagellar transport (IFT88) reduced cilia length and increased active nuclear ß-catenin. Conversely, increased primary cilia assembly in MSCs cultured on the grooves was associated with decreased levels of nuclear active ß-catenin, axin-2 induction and proliferation, in response to wnt3a. This negative regulation, on grooved topography, was reversed by siRNA to IFT88. This indicates that subtle regulation of IFT and associated cilia structure, tunes the wnt response controlling stem cell differentiation.

Primary cilia elongation in response to interleukin-1 mediates the inflammatory response.

Primary cilia are singular, cytoskeletal organelles present in the majority of mammalian cell types where they function as coordinating centres for mechanotransduction, Wnt and hedgehog signalling. The length of the primary cilium is proposed to modulate cilia function, governed in part by the activity of intraflagellar transport (IFT). In articular cartilage, primary cilia length is increased and hedgehog signaling activated in osteoarthritis (OA). Here, we examine primary cilia length with exposure to the quintessential inflammatory cytokine interleukin-1 (IL-1), which is up-regulated in OA. We then test the hypothesis that the cilium is involved in mediating the downstream inflammatory response. Primary chondrocytes treated with IL-1 exhibited a 50% increase in cilia length after 3 h exposure. IL-1-induced cilia elongation was also observed in human fibroblasts. In chondrocytes, this elongation occurred via a protein kinase A (PKA)-dependent mechanism. G-protein coupled adenylate cyclase also regulated the length of chondrocyte primary cilia but not downstream of IL-1. Chondrocytes treated with IL-1 exhibit a characteristic increase in the release of the inflammatory chemokines, nitric oxide and prostaglandin E2. However, in cells with a mutation in IFT88 whereby the cilia structure is lost, this response to IL-1 was significantly attenuated and, in the case of nitric oxide, completely abolished. Inhibition of IL-1-induced cilia elongation by PKA inhibition also attenuated the chemokine response. These results suggest that cilia assembly regulates the response to inflammatory cytokines. Therefore, the cilia proteome may provide a novel therapeutic target for the treatment of inflammatory pathologies, including OA.

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 Ca(2+) ionophore ionomycin, prostaglandin E(2), 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 (Gd(3+), 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.

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.

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 microg h(-1), mean +/- s.e.m., n = 90) correlated with the variable endogenous HA mass (mean 367 +/- 8 microg), with a normalized value of 3.4 +/- 0.2 microg h(-1) (100 microg)(-1) . Cyclic joint movement approximately doubled the net HA secretion rate to 22.6 +/- 1.2 microg h(-1) (n = 77) and raised the normalized percentage to 5.9 +/- 0.3 microg h(-1) (100 microg)(-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.