Primary cilia disassembly down-regulates mechanosensitive hedgehog signalling: a feedback mechanism controlling ADAMTS-5 expression in chondrocytes

SummaryObjectiveHedgehog signalling is mediated by the primary cilium and promotes cartilage degeneration in osteoarthritis. Primary cilia are influenced by pathological stimuli and cilia length and prevalence are increased in osteoarthritic cartilage. This study aims to investigate the relationship between mechanical loading, hedgehog signalling and cilia disassembly in articular chondrocytes.MethodsPrimary bovine articular chondrocytes were subjected to cyclic tensile strain (CTS; 0.33 Hz, 10% or 20% strain). Hedgehog pathway activation (Ptch1, Gli1) and A Disintegrin And Metalloproteinase with Thrombospondin Motifs 5 (ADAMTS-5) expression were assessed by real-time PCR. A chondrocyte cell line generated from the Tg737ORPK mouse was used to investigate the role of the cilium in this response. Cilia length and prevalence were quantified by immunocytochemistry and confocal microscopy.ResultsMechanical strain upregulates Indian hedgehog expression and activates hedgehog signalling. Ptch1, Gli1 and ADAMTS-5 expression were increased following 10% CTS, but not 20% CTS. Pathway activation requires a functioning primary cilium and is not observed in Tg737ORPK cells lacking cilia. Mechanical loading significantly reduced cilium length such that cilia became progressively shorter with increasing strain magnitude. Inhibition of histone deacetylase 6 (HDAC6), a tubulin deacetylase, prevented cilia disassembly and restored mechanosensitive hedgehog signalling and ADAMTS-5 expression at 20% CTS.ConclusionsThis study demonstrates for the first time that mechanical loading activates primary cilia-mediated hedgehog signalling and ADAMTS-5 expression in adult articular chondrocytes, but that this response is lost at high strains due to HDAC6-mediated cilia disassembly. The study provides new mechanistic insight into the role of primary cilia and mechanical loading in articular cartilage

Depletion of chondrocyte primary cilia reduces the compressive modulus of articular cartilage

Primary cilia are slender, microtubule based structures found in the majority of cell types with one cilium per cell. In articular cartilage, primary cilia are required for chondrocyte mechanotransduction and the development of healthy tissue. Loss of primary cilia in Col2aCre;ift88(fl/fl) transgenic mice results in up-regulation of osteoarthritic (OA) markers and development of OA like cartilage with greater thickness and reduced mechanical stiffness. However no previous studies have examined whether loss of primary cilia influences the intrinsic mechanical properties of articular cartilage matrix in the form of the modulus or just the structural properties of the tissue. The present study describes a modified analytical model to derive the viscoelastic moduli based on previous experimental indentation data. Results show that the increased thickness of the articular cartilage in the Col2aCre;ift88(fl/fl) transgenic mice is associated with a reduction in both the instantaneous and equilibrium moduli at indentation strains of greater than 20%. This reveals that the loss of primary cilia causes a significant reduction in the mechanical properties of cartilage particularly in the deeper zones and possibly the underlying bone. This is consistent with histological analysis and confirms the importance of primary cilia in the development of a mechanically functional articular cartilage

Adipogenic Differentiation of hMSCs is Mediated by Recruitment of IGF-1r Onto the Primary Cilium Associated With Cilia Elongation

Funded by Marie Curie Intra European Fellowship (GENOMICDIFF) Wellcome Trust 08471

Lithium chloride modulates chondrocyte primary cilia and inhibits Hedgehog signaling

This work was supported in part by funding from the AO Foundation (project S-12-15K) and the Medical Research Council (Swindon, United Kingdom) (MR/L002876/1). C.A.P. was supported by the Royal Society of New Zealand through a James Cook Research Fellowshi

Cometary science after Rosetta

The European Space Agency’s Rosetta mission ended operations on 30 September 2016 having spent over 2 years in close proximity to its target comet, 67P/Churyumov–Gerasimenko. Shortly before this, in summer 2016, a discussion meeting was held to examine how the results of the mission could be framed in terms of cometary and solar system science in general. This paper provides a brief history of the Rosetta mission, and gives an overview of the meeting and the contents of this associated special issue

Mechanical Stimulation: A Crucial Element of Organ-on-Chip Models

Organ-on-chip (OOC) systems recapitulate key biological processes and responses in vitro exhibited by cells, tissues, and organs in vivo. Accordingly, these models of both health and disease hold great promise for improving fundamental research, drug development, personalized medicine, and testing of pharmaceuticals, food substances, pollutants etc. Cells within the body are exposed to biomechanical stimuli, the nature of which is tissue specific and may change with disease or injury. These biomechanical stimuli regulate cell behavior and can amplify, annul, or even reverse the response to a given biochemical cue or drug candidate. As such, the application of an appropriate physiological or pathological biomechanical environment is essential for the successful recapitulation of in vivo behavior in OOC models. Here we review the current range of commercially available OOC platforms which incorporate active biomechanical stimulation. We highlight recent findings demonstrating the importance of including mechanical stimuli in models used for drug development and outline emerging factors which regulate the cellular response to the biomechanical environment. We explore the incorporation of mechanical stimuli in different organ models and identify areas where further research and development is required. Challenges associated with the integration of mechanics alongside other OOC requirements including scaling to increase throughput and diagnostic imaging are discussed. In summary, compelling evidence demonstrates that the incorporation of biomechanical stimuli in these OOC or microphysiological systems is key to fully replicating in vivo physiology in health and disease

Oscillations of the circadian clock protein, BMAL-1, align to daily cycles of mechanical stimuli: a novel means to integrate biological time within predictive in vitro model systems

PURPOSE: In vivo, the circadian clock drives 24-h rhythms in human physiology. Isolated cells in vitro retain a functional clockwork but lack necessary timing cues resulting in the rapid loss of tissue-level circadian rhythms. This study tests the hypothesis that repeated daily mechanical stimulation acts as a timing cue for the circadian clockwork. The delineation and integration of circadian timing cues into predictive in vitro model systems, including organ-on-a-chip (OOAC) devices, represent a novel concept that introduces a key component of in vivo physiology into predictive in vitro model systems. METHODS: Quiescent bovine chondrocytes were entrained for 3 days by daily 12-h bouts of cyclic biaxial tensile strain (10%, 0.33 Hz, Flexcell) before sampling during free-running conditions. The core clock protein, BMAL-1, was quantified from normalised Western Blot signal intensity and the temporal oscillations characterised by Cosinor linear fit with 24-h period. RESULTS: Following entrainment, the cell-autonomous oscillations of the molecular clock protein, BMAL-1, exhibited circadian (24 h) periodicity (p < 0.001) which aligned to the diurnal mechanical stimuli. A 6-h phase shift in the mechanical entrainment protocol resulted in an equivalent shift of the circadian clockwork. Thus, repeated daily mechanical stimuli synchronised circadian rhythmicity of chondrocytes in vitro. CONCLUSION: This work demonstrates that daily mechanical stimulation can act as a timing cue that is sufficient to entrain the peripheral circadian clock in vitro. This discovery may be exploited to induce and sustain circadian physiology within into predictive in vitro model systems, including OOAC systems. Integration of the circadian clock within these systems will enhance their potential to accurately recapitulate human diurnal physiology and hence augment their predictive value as drug testing platforms and as realistic models of human (patho)physiology. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s44164-022-00032-x

Nanoscale Mapping Reveals Functional Differences in Ion Channels Populating the Membrane of Primary Cilia.

BACKGROUND/AIMS: The primary cilium is a nanoscale membrane protrusion believed to act as a mechano-chemical sensor in a range of different cell types. Disruptions in its structure and signalling have been linked to a number of medical conditions, referred to as ciliopathies, but remain poorly understood due to lack of techniques capable of investigating signal transduction in cilia at nanoscale. Here we set out to use latest advances in nanopipette technology to address the question of ion channel distribution along the structure of primary cilium. METHODS: We used glass nanopipettes and Scanning Ion Conductance Microscopy (SICM) to image 3D topography of intact primary cilia in inner medullary collecting duct (IMCD) cells with nanoscale resolution. The high-resolution topographical images were then used to navigate the nanopipette along the structure of each cilium and perform spatially resolved single-channel recordings under precisely controlled mechanical and chemical stimulation. RESULTS: We have successfully obtained first single-channel recordings at specific locations of intact primary cilia. Our experiments revealed significant differences between the populations of channels present at the ciliary base, tip and within extra-ciliary regions in terms of mean conductance and sensitivity to membrane displacement as small as 100 nm. Ion channels at the base of cilium, where mechanical strain is expected to be the highest, appeared particularly sensitive to the mechanical displacement. CONCLUSION: Our results suggest the distribution of ion channels in the membrane of primary cilia is non-homogeneous. The relationship between the location and function of ciliary ion channels could be key to understanding signal transduction in primary cilia

Mechanical Stimulation Modulates Osteocyte Regulation of Cancer Cell Phenotype

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

Whole genome sequencing identifies putative associations between genomic polymorphisms and clinical response to the antiepileptic drug levetiracetam

In the context of pharmacogenomics, whole genome sequencing provides a powerful approach for identifying correlations between response variability to specific drugs and genomic polymorphisms in a population, in an unbiased manner. In this study, we employed whole genome sequencing of DNA samples from patients showing extreme response (n=72) and non-response (n=27) to the antiepileptic drug levetiracetam, in order to identify genomic variants that underlie response to the drug. Although no common SNP (MAF>5%) crossed the conventional genome-wide significance threshold of 5e-8, we found common polymorphisms in genes SPNS3, HDC, MDGA2, NSG1 and RASGEF1C, which collectively predict clinical response to levetiracetam in our cohort with ~91% predictive accuracy. Among these genes, HDC, NSG1, MDGA2 and RASGEF1C are potentially implicated in synaptic neurotransmission, while SPNS3 is an atypical solute carrier transporter homologous to SV2A, the known molecular target of levetiracetam. Furthermore, we performed gene- and pathway-based statistical analysis on sets of rare and low-frequency variants (MAF<5%) and we identified associations between the following genes or pathways and response to levetiracetam: a) genes PRKCB and DLG2, which are involved in glutamatergic neurotransmission, a known target of anticonvulsants, including levetiracetam; b) genes FILIP1 and SEMA6D, which are involved in axon guidance and modelling of neural connections; and c) pathways with a role in synaptic neurotransmission, such as WNT5A-dependent internalization of FZD4 and disinhibition of SNARE formation. In summary, our approach to utilise whole genome sequencing on subjects with extreme response phenotypes is a feasible route to generate plausible hypotheses for investigating the genetic factors underlying drug response variability in cases of pharmaco-resistant epilepsy