Cytosolic free Ca²⁺ concentration exhibits a characteristic temporal pattern during in vitro cartilage differentiation: A possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells

Summary

We measured changes of cytosolic Ca²⁺ concentration during chondrogenesis, which occurs in high-density cultures (HDC) of chondrifying chicken mesenchymal cells. A significant, transient elevation was detected in Fura-2-loaded cells on day 3 of culturing, when majority of chondrogenic cells of HDC become differentiated. This 140 nM peak of cytosolic Ca²⁺ concentration is a result of increased Ca-influx and is indispensable to proper chondrogenesis, because addition of 0.8 mM EGTA to culture medium on day 2 or 3 significantly decreased the intracellular Ca²⁺ concentration abolishing the Ca²⁺-peak of day 3 and inhibited cartilage formation. Uncontrolled Ca²⁺ influx evoked by a Ca²⁺ ionophore exerted dual effects on chondrogenesis in a concentration-dependent manner; 0.1 mg/L A23187 increased, whereas 5 mg/L A23187 almost totally blocked cartilage formation. Intracellular Ca-stores seemed not to have any significant participation in the regulation of changes of cytosolic Ca²⁺ concentration of chondrifying cells. Activity of Ca–calmodulin-dependent protein phosphatase, calcineurin responded to changes of intracellular Ca²⁺ concentration induced by EGTA or A23187 in a differentiation stage-dependent manner. Since inhibition of calcineurin with cyclosporine A eliminated the peak in the cytosolic Ca²⁺ concentration, an active regulatory role of calcineurin on Ca²⁺ influx of chondrifying cells can be supposed.

Introduction

Hyaline cartilage is an important element of the vertebrate skeletal system. It provides primordia of bones formed by endochondral ossification and remains the major shock-absorbing structure of the articular surfaces of joints. Chondrogenic mesenchymal cells can be derived from different embryonic structures: the cranial part of neural crest is the source of cartilage primordia of several craniofacial bones; sclerotome of somites differentiates into vertebrae; appendicular bones derive from mesenchymal cells of somatopleura [1].

High-density cell culture system (HDC) established from chondrogenic mesenchymal cells isolated from limb buds of 4-day-old chicken embryos is a well-known model of in vitro cartilage differentiation [2], [3], [4]. This simple model can provide information on the molecular steps leading to differentiation of chondroprogenitor cells to chondroblasts. In HDC, formation of cartilage starts with the recruitment of chondroprogenitor mesenchymal progenitor cells that after condensation and nodule formation, differentiate into chondroblasts and chondrocytes. Condensation and nodule formation take place on the first day of culturing and are partly regulated by transient appearance of Ca²⁺-dependent intercellular junctions like N-CAM (neural cell adhesion molecule) and N-cadherin [5]. Chondroprogenitor cells differentiate into chondroblasts on the second and third days of culturing [4], [6], controlled by numerous growth factors and other signal molecules, e.g. FGF, BMP, Wnt, IGF and members of Hedgehog and Sox transcription factor families [7]. In parallel to the intracellular changes, extracellular matrix (ECM) surrounding the differentiating chondrogenic cells is also subject to profound changes: differentiating cells start to secrete cartilage-specific matrix components, such as collagen type II and aggrecan on the third day of culturing period [8]. The unique composition and organization of ECM is crucial for maintenance of the proper morphology and function of these cells [9]. Expression of collagen type II and core protein of aggrecan is controlled by Sox9, a high-mobility-group domain containing transcription factor [10], [11], [12]. Detection of the expression level and the phosphorylation status of Sox9, as well as monitoring the expression of the core protein of aggrecan are reliable markers of chondrogenesis.

Calcium ion is a ubiquitous cellular signal. The concentration of intracellular free Ca²⁺ (∼10⁻⁷ M) is 10⁴ times lower than that of the extracellular fluid. This distribution provides the potential for the influx of Ca²⁺ into cells, where it can act as a second messenger. Various stimuli promote the movement of Ca²⁺ either from the extracellular space or from intracellular stores into the cytosol. The elevated level of cytosolic free Ca²⁺ exerts a variety of specific changes in cellular function, such as activation of protein kinases and protein phosphatases, which, in turn, regulate other processes, like proliferation or differentiation [13]. The molecular steps leading to cartilage differentiation, among other factors are regulated by Ca²⁺ sensitive enzymes like one of the Ser/Thr specific protein kinases, PKCalpha [14] or the Ser/Thr-specific protein phosphatase calcineurin [15], [16], that is unique among phosphatases for its ability to sense changes of intracellular Ca²⁺ concentration through its activation by its calcium binding subunit and calmodulin. Calcineurin is best known as a regulator of T-lymphocyte activation, since its pharmacological inhibitors, cyclosporine A (CsA), tacrolimus, pimecrolimus and rapamycin are all used in the clinical practice as immunosuppressants [17]. Calcineurin is also known to participate in several differentiation processes, such as development of different muscle tissues and the nervous system [18].

In this study we measured the cytosolic free Ca²⁺ concentration during cartilage differentiation in the chondrogenic cells of HDC. A characteristic temporal pattern in the changes of cytosolic Ca²⁺ concentration could be observed; there was a significant and transient elevation on the third culturing day, the crucial day of chondrocyte differentiation. Moreover, beside the changes of the basal cytosolic Ca²⁺ level, cells of chondrifying micromass cultures also exhibit spontaneous calcium events, a phenomenon characteristic to several other primary cell cultures [19], [20]. We provide evidence that the temporal pattern of the changes of cytosolic free Ca²⁺ concentration in chondrifying cells is indispensible to proper cartilage formation and depends on extracellular Ca²⁺ rather than the availability of intracellular Ca-stores. We also demonstrate that calcineurin can play a dual role in Ca-signalling of chondrogenic cells: its activity is modulated by cytosolic Ca²⁺ concentration and the inhibition of calcineurin with CsA eliminates the Ca²⁺ peak of HDC resulting in a pronounced decrease in cartilage formation. This second observation raises the possibility of the active regulatory effect of this enzyme on the enhancement of Ca²⁺ influx to chondrifying cells.