Abstract
BackgroundUnderstanding the key elements of signaling of chondroprogenitor cells at the earliest steps of differentiation may substantially improve our opportunities for the application of mesenchymal stem cells in cartilage tissue engineering, which is a promising approach of regenerative therapy of joint diseases. Ion channels, membrane potential and Ca2+-signaling are important regulators of cell proliferation and differentiation. Our aim was to identify such plasma membrane ion channels involved in signaling during chondrogenesis, which may serve as specific molecular targets for influencing chondrogenic differentiation and ultimately cartilage formation.Methodology/Principal FindingsUsing patch-clamp, RT-PCR and Western-blot experiments, we found that chondrogenic cells in primary micromass cell cultures obtained from embryonic chicken limb buds expressed voltage-gated NaV1.4, KV1.1, KV1.3 and KV4.1 channels, although KV1.3 was not detectable in the plasma membrane. Tetrodotoxin (TTX), the inhibitor of NaV1.4 channels, had no effect on cartilage formation. In contrast, presence of 20 mM of the K+ channel blocker tetraethyl-ammonium (TEA) during the time-window of the final commitment of chondrogenic cells reduced KV currents (to 27±3% of control), cell proliferation (thymidine incorporation: to 39±4.4% of control), expression of cartilage-specific genes and consequently, cartilage formation (metachromasia: to 18.0±6.4% of control) and also depolarized the membrane potential (by 9.3±2.1 mV). High-frequency Ca2+-oscillations were also suppressed by 10 mM TEA (confocal microscopy: frequency to 8.5±2.6% of the control). Peak expression of TEA-sensitive KV1.1 in the plasma membrane overlapped with this period. Application of TEA to differentiated chondrocytes, mainly expressing the TEA-insensitive KV4.1 did not affect cartilage formation.Conclusions/SignificanceThese data demonstrate that the differentiation and proliferation of chondrogenic cells depend on rapid Ca2+-oscillations, which are modulated by KV-driven membrane potential changes. KV1.1 function seems especially critical during the final commitment period. We show the critical role of voltage-gated cation channels in the differentiation of non-excitable cells with potential therapeutic use.
Highlights
Due to the lack of blood supply and the postmitotic nature of fully differentiated adult chondrocytes, articular cartilage has very limited self-repair capability following tissue damage
The expression level of plasma membrane channels often changes with the differentiation state of the cells, which in turn may influence further steps of the process [27,28,29]
Identifying ion channels in differentiating chondrocytes and tracking the changes in their expression levels can lead to better channels at the beginning of the recording, of which KV channels were blocked by 20 mM TEA
Summary
Due to the lack of blood supply and the postmitotic nature of fully differentiated adult chondrocytes, articular cartilage has very limited self-repair capability following tissue damage. Non-excitable cells, such as endothelial cells [5] and osteoblasts [6] were shown to display calcium oscillations, where ion channels from both the plasma membrane and from intracellular stores were found to be associated with these phenomena [7] Such events have been detected in isolated mature articular chondrocytes cultured in agarose constructs [8]. In chicken embryonic chondrogenic cells, we have previously described characteristic changes of the free cytosolic [Ca2+]i, which was dependent on extracellular Ca2+ and was associated with calcineurin activity, as well as evidence for purinergic Ca2+-signaling via P2X4 receptors These phenomena were temporally synchronized with chondrocyte differentiation [9,10]. Our aim was to identify such plasma membrane ion channels involved in signaling during chondrogenesis, which may serve as specific molecular targets for influencing chondrogenic differentiation and cartilage formation
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