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
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.
Section snippets
Cell culture
Distal parts of the limb buds of 4-day-old Ross hybrid chicken embryos (Hamburger–Hamilton stages 22–24 [21]) were removed and primary micromass cultures of chondrifying mesenchymal cells were established from a cell suspension with a density of 1.5 × 107 cells/mL. Fifteen microliters droplets of the suspension were inoculated on round coverglasses (diameter: 30 mm; Menzel-Gläser, Menzel GmbH, Braunschweig, Germany) placed into plastic Petri dishes (Nunc, Naperville, IL, USA). Cells were allowed to
Cytosolic free Ca2+ concentration of untreated cell cultures shows a characteristic age-dependent pattern
Cytosolic free Ca2+ concentration was determined in Fura-2-loaded cells on different days of culturing. Basal level of intracellular Ca2+ concentration of chondroblasts was found to have an age-dependent pattern (Fig. 1A). Initially, Ca2+ level is low, with a starting concentration of about 75 nM on day 0, then it slightly increases in parallel with the progression of differentiation. A 140 nM peak of the cytosolic free Ca2+ concentration was observed on day 3 of culturing in cells of untreated
Discussion
Changes of intracellular Ca2+ concentration are important signalling events in different cellular processes, including cell and tissue differentiation. The Ca2+ sensitive PKCalpha is reported to influence proliferation and differentiation of chondrifying cells, via modulation of MAPK-signalling [14] and we have described a positive regulatory role of calcineurin in the in vitro chondrogenesis occurring in chicken HDC either under physiological conditions or under the effect of oxidative stress
Acknowledgements
The authors thank Mrs. Júlia Bárány and Mrs. Krisztina Bíró of the Department of Anatomy, and Mrs. Ibolya Varga of the Department of Physiology for their skillful and excellent technical assistance, and Ms. Mónika Fehér and Ms. Beatrix Dienes for the confocal microscopic images and measurements. We also thank Prof. Dr. József Posta and István Nagy of the Department of Inorganic and Analytical Chemistry, Faculty of Sciences, University of Debrecen, Hungary for their kind assistance in the
References (47)
- et al.
Stage-related capacity for limb chondrogenesis in cell culture
Dev. Biol.
(1977) - et al.
N-CAM and N-cadherin expression during in vitro chondrogenesis
Exp. Cell Res.
(1994) - et al.
Chondrogenesis of limb bud mesenchyme in vitro: stimulation by cations
Dev. Biol.
(1986) - et al.
Sox9 expression during chondrogenesis in micromass cultures of embryonic limb mesenchyme
Exp. Cell Res.
(2000) - et al.
Calmodulin: a prototypical calcium sensor
Trends Cell Biol.
(2000) - et al.
Calcineurin and NFAT4 induce chondrogenesis
J. Biol. Chem.
(2002) - et al.
Hydrogen peroxide inhibits formation of cartilage in chicken micromass cultures and decreases the activity of calcineurin: implication of ERK1/2 and Sox9 pathways
Exp. Cell Res.
(2005) - et al.
Ca2+/calcineurin signalling in cells of the immune system
Biochem. Biophys. Res. Commun.
(2003) - et al.
A new generation of Ca2+ indicators with greatly improved fluorescence properties
J. Biol. Chem.
(1985) - et al.
Calcineurin is a calmodulin-dependent protein phosphatase
Biochem. Biophys. Res. Commun.
(1982)
Protein phosphatase inhibitor-1 and inhibitor-2 from rabbit skeletal muscle
Methods Enzymol.
Role of intracellular calcium mobilization and cell-density-dependent signaling in oxidative-stress-induced cytotoxicity in HaCaT keratinocytes
J. Invest. Dermatol.
ATP autocrine/paracrine signaling induces calcium oscillations and NFAT activation in human mesenchymal stem cells
Cell Calcium
Characterization of Ca(2+) signaling pathways in human mesenchymal stem cells
Cell Calcium
Protein kinase C regulates chondrogenesis of mesenchymes via mitogen-activated protein kinase signaling
J. Biol. Chem.
Calcium dynamics and endoplasmic reticular function in the regulation of protein synthesis: implications for cell growth and adaptability
Cell Calcium
The role of calcineurin in lymphocyte activation
Semin. Immunol.
Calcineurin signaling and NFAT activation in cardiovascular and skeletal muscle development
Dev. Biol.
Calcineurin directs the reciprocal regulation of calcium entry pathways in nonexcitable cells
J. Biol. Chem.
Calcineurin and intracellular Ca2+-release channels: regulation or association?
Biochem. Biophys. Res. Commun.
Signalling to transcription: store-operated Ca2+ entry and NFAT activation in lymphocytes
Cell Calcium
Regulation of Ca2+-dependent desensitization in the vanilloid receptor TRPV1 by calcineurin and cAMP-dependent protein kinase
J. Biol. Chem.
Ca(2+) oscillations regulated by Na(+)–Ca(2+) exchanger and plasma membrane Ca(2+) pump induce fluctuations of membrane currents and potentials in human mesenchymal stem cells
Cell Calcium
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These two authors contributed equally to this work.