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-1β (IL-1β) signalling. Isolated chondrocytes and cartilage explants were subjected to cyclic mechanical loading in the presence and absence of the cytokine IL-1β. Nitric oxide (NO) and prostaglandin E2 (PGE2) release were used to monitor IL-1β 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. Mechanical loading suppressed NO and PGE2 release and prevented cartilage degradation. Loading activated HDAC6 and disrupted tubulin acetylation and cilia elongation induced by IL-1β. HDAC6 inhibition with tubacin blocked the anti-inflammatory effects of loading and restored tubulin acetylation and cilia elongation. Hypomorphic mutation of IFT88 reduced IL-1β 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. This study reveals that mechanical loading suppresses inflammatory signalling, partially dependent on IFT, by activation of HDAC6 and post transcriptional modulation of tubulin.

Cartilage health is maintained in response to a range of mechanical stimuli including compressive, shear and tensile strains and associated alterations in osmolality. The osmotic-sensitive ion channel Transient Receptor Potential Vanilloid 4 (TRPV4) is required for mechanotransduction. Mechanical stimuli inhibit interleukin-1β (IL-1β) mediated inflammatory signalling, however the mechanism is unclear. This study aims to clarify the role of TRPV4 in this response. TRPV4 activity was modulated glycogen synthase kinase (GSK205 antagonist or GSK1016790 A (GSK101) agonist) in articular chondrocytes and cartilage explants in the presence or absence of IL-1β, mechanical (10% cyclic tensile strain (CTS), 0.33 Hz, 24hrs) or osmotic loading (200mOsm, 24hrs). Nitric oxide (NO), prostaglandin E2 (PGE2) and sulphated glycosaminoglycan (sGAG) release and cartilage biomechanics were analysed. Alterations in post-translational tubulin modifications and primary cilia length regulation were examined. In isolated chondrocytes, mechanical loading inhibited IL-1β mediated NO and PGE2 release. This response was inhibited by GSK205. Similarly, osmotic loading was anti-inflammatory in cells and explants, this response was abrogated by TRPV4 inhibition. In explants, GSK101 inhibited IL-1β mediated NO release and prevented cartilage degradation and loss of mechanical properties. Upon activation, TRPV4 cilia localisation was increased resulting in histone deacetylase 6 (HDAC6)-dependent modulation of soluble tubulin and altered cilia length regulation. Mechanical, osmotic or pharmaceutical activation of TRPV4 regulates HDAC6-dependent modulation of ciliary tubulin and is anti-inflammatory. This study reveals for the first time, the potential of TRPV4 manipulation as a novel therapeutic mechanism to supress pro-inflammatory signalling and cartilage degradation.

Lithium chloride (LiCl) exhibits significant therapeutic potential as a treatment for osteoarthritis. Hedgehog signaling is activated in osteoarthritis, where it promotes chondrocyte hypertrophy and cartilage matrix catabolism. Hedgehog signaling requires the primary cilium such that maintenance of this compartment is essential for pathway activity. Here we report that LiCl (50 mM) inhibits Hedgehog signaling in bovine articular chondrocytes such that the induction of GLI1 and PTCH1 expression is reduced by 71 and 55%, respectively. Pathway inhibition is associated with a 97% increase in primary cilia length from 2.09 ± 0.7 μm in untreated cells to 4.06 ± 0.9 μm in LiCl‐treated cells. We show that cilia elongation disrupts trafficking within the axoneme with a 38% reduction in Arl13b ciliary localization at the distal region of the cilium, consistent with the role of Arl13b in modulating Hedgehog signaling. In addition, we demonstrate similar increases in cilia length in human chondrocytes in vitro and after administration of dietary lithium to Wistar rats in vivo. Our data provide new insights into the effects of LiCl on chondrocyte primary cilia and Hedgehog signaling and shows for the first time that pharmaceutical targeting of the primary cilium may have therapeutic benefits in the treatment of osteoarthritis.—Thompson, C. L., Wiles, A., Poole, C. A., Knight, M. M. Lithium chloride modulates chondrocyte primary cilia and inhibits Hedgehog signaling. FASEB J. 30, 716‐726 (2016). www.fasebj.org

Breast and prostate cancers preferentially metastasise to bone tissue, with metastatic lesions forming in the skeletons of most patients. On arriving in bone tissue, disseminated tumour cells enter a mechanical microenvironment that is substantially different to that of the primary tumour and is largely regulated by bone cells. Osteocytes, the most ubiquitous bone cell type, orchestrate healthy bone remodelling in response to physical exercise. However, the effects of mechanical loading of osteocytes on cancer cell behaviour is still poorly understood. The aim of this study was to characterise the effects of osteocyte mechanical stimulation on the behaviour of breast and prostate cancer cells. To replicate an osteocyte-controlled environment, this study treated breast (MDA-MB-231 and MCF-7) and prostate (PC-3 and LNCaP) cancer cell lines with conditioned media from MLO-Y4 osteocyte-like cells exposed to mechanical stimulation in the form of fluid shear stress. We found that osteocyte paracrine signalling acted to inhibit metastatic breast and prostate tumour growth, characterised by reduced proliferation and invasion and increased migration. In breast cancer cells, these effects were largely reversed by mechanical stimulation of osteocytes. In contrast, conditioned media from mechanically stimulated osteocytes had no effect on prostate cancer cells. To further investigate these interactions, we developed a microfluidic organ-chip model using the Emulate platform. This new organ-chip model enabled analysis of cancer cell migration, proliferation and invasion in the presence of mechanical stimulation of osteocytes by fluid shear stress, resulting in increased invasion of breast and prostate cancer cells. These findings demonstrate the importance of osteocytes and mechanical loading in regulating cancer cell behaviour and the need to incorporate these factors into predictive in vitro models of bone metastasis.

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. 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. 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. 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.