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

Abstract

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 chondrocyte dedifferentiation caused alterations in cilia expression that influence 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 median length. Cilia loss was associated with disruption of ligandinduced 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 in association 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.

Cilia protein IFT88 regulates extracellular protease activity by optimizing LRP‐1–mediated endocytosis

“…The IFT88 ORPK mutation, which results in changes to matrix patterning in cartilage in vivo (33), renders cells unable to express fulllength IFT88 and assemble a primary cilium. This has also previously been associated with the regulation of multiple chondrocyte responses (37)(38)(39)(47)(48)(49), most of which are proposed to be indicative of roles for the cilium. Importantly, however, IFT88, and indeed this mutation, are both associated with cell biology that is not proven to be attributable to cilia function-for example, migration (50), cell division (51), and planar cell polarity (52).…”

Mechanical loading induces primary cilia disassembly in tendon cells via TGFβ and HDAC6

“…Elongation of osteocyte primary cilia using fenoldopam or lithium has been shown to be associated with increased ciliary mechanosensitivity 37 . In chondrocytes, mechanically-induced cilia disassembly inhibits hedgehog signalling 29 , 38 . Previous studies have also shown that cilia set length regulates injection of intraflagellar transport (IFT) cargoes as part of the balance point model first proposed by Marshall 39 .…”

Adseverin, an actin binding protein, regulates articular chondrocyte phenotype

“…Actin depolymerization induced by cytochalasin D in chondroprogenitor cells resulted in cytoplasmic localization of YAP/TAZ and downregulation of collagen type I gene expression (Zhong et al, ). There is evidence suggesting that actin polymerization status may affect a plethora of cellular processes (Carpenter, ) and cellular structures such as primary cilia, which may influence levels of transforming growth factor, Wnt, hedgehog, and mechanosignalling (Thompson et al, ). These factors have been shown to affect chondrocyte proliferation and differentiation to influence cartilage growth and metabolism.…”