Articular chondrocytes express connexin 43 hemichannels and P2 receptors – a putative mechanoreceptor complex involving the primary cilium?

Abstract

Mechanical loading is essential for the health and homeostasis of articular cartilage, although the fundamental mechanotransduction pathways are unclear. Previous studies have demonstrated that cyclic compression up-regulates proteoglycan synthesis via an intracellular Ca 2+ signalling pathway, mediated by the release of ATP. However, the mechanism(s) of ATP release has not been elucidated. The present study examines expression of the putative mechanosensitive ATP-release channel, connexin 43 and whether it is expressed on the chondrocyte primary cilium, which acts as a mechanosensor in a variety of other cell types. In addition the study characterized the expression of a range of purine receptors through which ATP may activate downstream signalling events controlling cell function. Bovine articular chondrocytes were isolated by sequential enzyme digestion and seeded in agarose constructs.To verify the presence of functional hemichannels, Lucifer yellow (LY) uptake into viable cells was quantified following treatment with a hemichannel agonist (EGTA) and antagonist (flufenamic acid). LY uptake was observed in 45% of chondrocytes, increasing to 83% following EGTA treatment ( P < 0.001). Treatment with the hemichannel blocker, flufenamic acid, significantly decreased LY uptake to less than 5% with and without EGTA. Immunofluorescence and confocal microscopy confirmed the presence of primary cilia and the expression of connexin 43. Approximately 50% of bovine chondrocyte primary cilia were decorated with connexin 43. Human chondrocytes in situ within cartilage explants also expressed connexin 43 hemichannels. However, expression was confined to the upper 200 μ m of the tissue closest to the articular surface. Immunofluorescence revealed the expression of a range of P2X and P2Y receptor subtypes within human articular cartilage. In conclusion, the expression of functional hemichannels by articular chondrocytes may represent the mechanism through which mechanical loading activates ATP release as part of a purinergic mechanotransduction pathway. Furthermore, the expression of connexin 43 on the chondrocyte primary cilium suggests the possible involvement of the cilium in this pathway.

Purinergic receptors expressed in human skeletal muscle fibres

“…The following primary antibodies were used, either alone or two together (rabbit+mouse): Rabbit polyclonal anti-P2X 1 (APR-001, Alomone Labs, Israel; 1:500; previously used in the following studies [6,8,[13][14][15][16][17][18][19]); mouse polyclonal anti-P2X 4 (H00005025-B01P; Abnova, Taiwan; 1:500); rabbit polyclonal anti-P2Y 1 (APR-009, Alomone Labs; 1:500; previously used in the following studies [18,[20][21][22]); polyclonal anti-P2Y 2 (APR-010, Alomone Labs; 1:500; previously used in the following studies [6,8,18,[22][23][24]); rabbit polyclonal anti-P2Y 4 (P6497, Sigma-Aldrich, Denmark; 1:250); rabbit polyclonal anti-P2Y 11 (APR-015, Alomone Labs; 1:500; previously used in the following studies [18,[25][26][27][28][29][30]); rabbit polyclonal anti-P2Y 12 (HPA013796, Sigma-Aldrich; 1:30; previously used in the following study [31]). The P2X 1 antibody was highly specific and directed against the epitope corresponding to amino acid residues 382-399 of rat P2X 1 (human 15/18 residues identical); the P2Y 11 antibody was corresponding to amino acid residues 357-373 of human P2Y 11 ; western blots for anti-P2X 1 and anti-P2Y 11 are shown on the Alomone homepage.…”

Purinergic signalling-evoked intracellular Ca2+ concentration changes in the regulation of chondrogenesis and skeletal muscle formation

“…Mature articular chondrocytes have been shown to express various P1 and P2 purinergic receptors including A 1 , A 2A and A 2B , as well as different subtypes of P2X (P2X 1,2,3,4,7 ) and P2Y (P2Y 1,2,4,6 ) receptors [79,80]. Given that P1 and P2 receptors are important regulators of mechanosensation or mechanotransduction in mature chondrocytes [79], it is plausible to assume that they may also be involved in the regulation of chondrogenesis.…”

“…Given that P1 and P2 receptors are important regulators of mechanosensation or mechanotransduction in mature chondrocytes [79], it is plausible to assume that they may also be involved in the regulation of chondrogenesis.…”

Purinergic signalling in the musculoskeletal system

“…Subsequently, P2U (P2Y 2 and/or P2Y 4 ) receptors were identified on sheep chondrocytes [298,299]. Chondrocytes have now been shown to express many P2 receptors including the P2X1, P2X2, P2X3, P2X4, P2X7, P2Y 1 and P2Y 2 receptors [300][301][302][303]. Some of these receptors (P2X2, P2X4, P2X7 and P2Y 1 ) were expressed throughout all the zones of articular cartilage, whilst the P2Y 2 receptor showed more limited expression only being present on superficial zone cells [302].…”

“…It has been shown recently that primary cilia are essential for chondrocyte mechanotransduction and the control of extracellular matrix secretion; in particular, chondrocyte primary cilia are required for ATP release and purinoceptor-mediated Ca 2+ signalling [331]. ATP transport from chondrocyte primary cilium was, at least in part, via connexin 43 hemichannels [302,332]. Release of ATP via pannexin 3 hemichannels has also been proposed [333].…”