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 microm 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.

Fig. 1

Mean percentage of cells showing Lucifer yellow (LY) uptake in the presence of absence of EGTA and with and without pretreatment with flufenamic acid (FFA). Bovine chondrocytes were cultured in agarose for 24 h prior to LY uptake analysis. Error bars represent SEM for n = 3 separate specimens. For each specimen the mean percentage of LY-positive cells was calculated from 10 fields of view. Statistically significant differences based on Student t-tests are indicated at P < 0.05 with and without EGTA (*) and with and without FFA (+).

Fig. 2

Expression of connexin 43 in the cytoplasm of an isolated bovine chondrocyte cultured for 24 h in agarose gel. Confocal z-series obtained with a ×100 objective. Inset shows 3D serial reconstruction. Scale bar represents 5 µm.

Fig. 3

Approximately 50% of bovine chondrocytes expressing a primary cilium showed connexin 43 present on the primary cilium following 24 h in agarose culture. Cells were dual labelled with antibodies for connexin 43 (green) and acetylated α-tubulin (red) and visualized using confocal microscopy with a ×100 objective. Arrows indicate the position of the primary cilium for cells with no co-localization with connexin 43 (top row, red arrow) and for cells with clear connexin 43 expression on the cilium (bottom row, yellow arrow).

Fig. 4

Expression of connexin 43 in normal (A) and osteoarthritic (B) human articular cartilage. Confocal images were obtained with a ×10 objective to show the entire tissue depth. Arrows indicate the articular surface. Boxed inset images show additional ×4 magnification.

Fig. 5

Expression of connexin 43 hemichannels in chondrocytes within human articular cartilage. Cells were labelled with antibodies to the intracellular domain of connexin 43 (green) and the extracellular or hemichannel domain (red) and visualized using confocal and brightfield imaging with a ×100 objective. Co-localization indicates the presence of uncoupled hemichannels.

Fig. 6

Expression of chondrocyte P2Y1 and P2Y2 purine receptors in the superficial and deep zones of human articular cartilage. Images show 3D serial reconstruction of confocal z-series with corresponding brightfield images, all obtained with a ×100 objective. Note the lack of expression of P2Y2 in the deep zone.

Fig. 7

Expression of chondrocyte P2X2, P2X4 and P2X7 purine receptors in the superficial and deep zones of human articular cartilage. Images show 3D serial reconstruction of confocal z-series with corresponding brightfield images, all obtained with a ×100 objective.