Ionotropic purinergic receptor P2X4 is involved in the regulation of chondrogenesis in chicken micromass cell cultures

Abstract

a b s t r a c tWe have previously demonstrated that elevation of free cytosolic Ca 2+ concentration at the time of differentiation of chondroblasts was mainly due to a Ca 2+ influx and it was indispensable to cartilage formation in chicken high density mesenchymal cell cultures (HDC) [C. Matta, J. Fodor, Z. Szijgyarto, T. Juhasz, P. Q1 Gergely, L. Csernoch, R. Zakany, Cytosolic free Ca 2+ concentration exhibits a characteristic temporal pattern during in vitro cartilage differentiation: a possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells, Cell Calcium 44 (2008) 310-323]. Here, we report that chondrogenic cells secreted ATP and administration of ATP to the culture medium evoked Ca 2+ transients exclusively in the presence of extracellular Ca 2+ and only on day 3 of culturing, when the final commitment of chondroblasts occurs. Moreover, ATP caused elevated protein expression of the chondrogenic transcription factor Sox9 and stimulated cartilage matrix production. Expression pattern of different types of both ionotropic and metabotropic purinergic receptors was detected. Agonists of metabotropic receptors, ADP and UDP did not evoke any Ca 2+ transients and had no influence on cartilage formation, while UTP caused transient elevation of cytosolic Ca 2+ concentration in 3-day-old HDC without stimulating matrix production. Suramin, which blocks all P2X receptors but not P2X 4 did not impede the effects of ATP, furthermore, P2X 4 appeared in the plasma membrane fraction and gave signals with immunocytochemistry only from day 3. In summary, we suggest a role of ionotropic purinergic signalling of P2X 4 in the generation of ATP-dependent Ca 2+ transients of differentiating chondroblasts.

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

“…In these experiments, proliferation of the differentiating chondrogenic cells was extremely dependent on a well-defined range of cytosolic Ca 2+ concentration [73]. In further experiments, we have proved the dependence of the above changes in cytosolic Ca 2+ concentration on purinergic signalling via ionotropic P2X 4 receptors [74]. Besides the long term day-by-day changes of the cytosolic Ca 2+ concentration, we have also detected rapid Ca 2+ oscillations in the differentiating chondrocytes.…”

“…Earlier we reported that this receptor contributed to the elevation of cytosolic Ca 2+ levels of chondrogenic cells on day 3 of culturing [73]. The apparent discrepancy between these studies may be explained by the fact that whilst Meyer and colleagues used cultures established from mesenchymal cells derived from the forelimbs only [95], the micromass cultures used in our study were of mixed origin containing progenitor cells from both fore and hind limb buds [74]. It has long been known that chondrogenesis in HDC derived from wing and leg precartilage cells is affected differently by certain morphogens such as transforming growth factor-β1 (TGF-β1) or retinoic acid, and they also differ from each other in condensation morphology and organisation of fibronectin during the early phase of differentiation [96].…”

“…P2X 1,4,5 and P2X 7 of the P2X-family and P2Y 1,2,3,4 and P2Y 5 of P2Y receptors in cells of differentiating micromass cultures, the receptor primarily responsible for the rise of Ca 2+ concentration evoked by extracellular ATP seemed to be P2X 4 receptor [74]. Earlier we reported that this receptor contributed to the elevation of cytosolic Ca 2+ levels of chondrogenic cells on day 3 of culturing [73].…”

“…The first study on the involvement of purinergic signalling in chondrogenesis in vitro showed that ATP acting on the P2Y 1 receptor inhibited cartilage formation in HDCs of embryonic chick limb bud-derived mesenchymal cells [95]. In contrast, our group has reported that cells in HDC responded to extracellular ATP by elevating their intracellular Ca 2+ levels mainly at the time of chondroblast formation and administration of ATP to the culture medium stimulated chondrogenesis [74].…”

Voltage-Dependent Calcium Channels in Chondrocytes: Roles in Health and Disease

“…Due to the fact that chondrocytes reside within the peculiar milieu of the ECM (which is acidic, hypertonic and hyperosmotic [6][7][8]), and since their function and differentiation is strongly dependent on intracellular calcium homeostasis [9,10], they deploy a whole range of ion channels to enable ion transport across the plasma membrane; the collection of ion channels is referred to as the chondrocyte 'channelome' [11]. Albeit generally considered to be non-excitable cells, chondrocytes and chondroprogenitor cells have been reported to express various voltage-dependent potassium channels (KV), ATP dependent potassium channels (KATP), large and small conductance calcium-activated potassium channels (BK and SK), transient receptor potential channels (primarily TRPV1 and TRPV4), purinergic receptors (both P2X and P2Y subfamily members), voltage gated and epithelial sodium channels, chloride channels, and also voltage-dependent calcium channels (VDCCs) [11][12][13].…”