[Frontiers in Bioscience S5, 305-324, January 1, 2013]

Calcium signalling in chondrogenesis: implications for cartilage repair

Csaba Matta1, Roza Zakany1

1Department of Anatomy, Histology and Embryology, University of Debrecen, Medical and Health Science Centre, Nagyerdei krt. 98, H-4032 Debrecen, Hungary

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Adult mesenchymal stem cells in cartilage repair
4. Articular cartilage: from structure to function
5. Chondrogenesis is regulated by interplay between numerous intra- and extracellular factors
6. Calcium signalling: a single messenger with diverse functions
7. Ca2+ entry processes in MSCs and in differentiating or mature chondrocytes
7.1. Voltage-operated Ca2+ entry pathways
7.2. Ligand-operated Ca2+ entry pathways
7.2.1. Purinergic signalling pathways
7.2.2. N-methyl-D-aspartate receptor mediated pathways
7.2.3. Transient Receptor Potential (TRP) pathways
7.3. Ca2+ release from internal stores and store-operated Ca2+ entry
8. Ca2+ elimination processes in MSCs, differentiating and mature chondrocytes
9. Temporal characteristics of Ca2+ dependent signals
9.1. Day-by-day variation of cytosolic Ca2+ concentration
9.2. Ca2+ oscillations in mesenchymal stem cells and chondrocytes
10. Ca2+ signalling during mechanotransduction
11. Conclusions
12. Acknowledgements
13. References

1. ABSTRACT

Undifferentiated mesenchymal stem cells (MSCs) represent an important source for cell-based tissue regeneration techniques that require differentiation towards specific lineages, including chondrocytes. Chondrogenesis, the process by which committed mesenchymal cells differentiate into chondrocytes, is controlled by complex but not yet completely understood mechanisms that involve many components, including intracellular signalling pathways, as well as plasma membrane receptors and ion channels. Some of these signalling components are Ca2+ sensitive. Although the Ca2+-signalling toolkit of undifferentiated MSCs and mature chondrocytes are extensively studied, the adaptation of these components during differentiation and their role in chondrogenesis is not adequately established. In this review, various aspects of Ca2+ signalling are discussed in MSCs and in mature chondrocytes including spatial and temporal aspects, as well as Ca2+ entry and elimination processes, with implications for their involvement in chondrogenesis. A better understanding of these pathways is envisaged to provide a more efficient differentiation of MSCs towards chondrocytes that may lead to the development of better cartilage tissue engineering techniques.

2. INTRODUCTION

Prevalence of musculoskeletal disorders (comprised more than 100 conditions, including various rheumatic, arthritic and joint diseases) is constantly increasing owing to unfavourable changes in the population of developed countries, exerting an ever-growing burden on healthcare systems around the globe (1). Osteoarthritis (OA), characterised by inflammation and breakdown of the shock-absorbing articular cartilage within the joint, is the most common form of chronic musculoskeletal disorders. However, no effective or curative treatment is available for OA. Therefore, there is a pressing socio-economic need for the development of novel and innovative therapeutic strategies to preserve or regenerate the natural articular cartilage and its underlying bone. During the last decade, with an increase in our knowledge about the molecular and biological characteristics of embryonic or adult pluripotent mesenchymal stem cells (MSCs), their applicability in regenerative medicine has emerged. However, regeneration of proper articular cartilage is still a challenge, mainly due to the fact that albeit extensive research has been conducted in this field over the last 50 years, our current knowledge of the molecular steps and precise regulation of chondrogenesis is far from being complete.

In this review, after a brief overview of cartilage biology and the normal course of chondrogenesis, we will focus on Ca2+ dependent signalling pathways that regulate this process; in particular, Ca2+ signalling in MSCs and adult articular chondrocytes will be described, with implications for the involvement of these pathways in chondrogenesis and possible cartilage regeneration techniques.

3. ADULT MESENCHYMAL STEM CELLS IN CARTILAGE REPAIR

Originally isolated from bone marrow by Pittenger and colleagues in 1999 (2), somatic or adult MSCs have been described to differentiate into various connective tissue cells, including the chondrogenic lineage that can give rise to chondroblasts and chondrocytes (3). MSCs have been identified in various mature tissues, including skeletal tissues, such as bone and cartilage (4). Indeed, the intrinsic regeneration capacity of cartilage that is exploited during the treatment of early-stage osteoarthritic patients (e.g. Pridie-drilling, microfracture, etc.) can mainly be accounted for the cartilage-forming capacity of bone marrow-derived MSCs (5, 6). However, it is fibrous cartilage, rather than hyaline cartilage, that is formed in situ as a result of such bone marrow-opening techniques, which is characterised by less favourable biomechanical properties.

Owing to the apparent insufficiency of physiological repair mechanisms to regenerate the proper structure of articular hyaline cartilage, the concept of cell-based therapies has emerged. MSCs represent an ideal source of these cells, due to their capacity to differentiate into chondrocytes (7). MSC-based cartilage regeneration strategies hold much potential for treating large-scale lesions as the currently accepted and clinically used autologous chondrocyte implantation (ACI) techniques can only be successfully applied to treat smaller cartilage lesions (8). However, to overcome the apparent difficulties of articular cartilage regeneration, a better understanding of signalling mechanisms that govern chondrogenesis of MSCs is required.

4. ARTICULAR CARTILAGE: FROM STRUCTURE TO FUNCTION

For attempts in articular cartilage tissue regeneration to be eventually successful, an in-depth knowledge of the structure, components, as well as biomechanical characteristics of the mature tissue is essential. Hyaline cartilage provides the source of bones formed by endochondral ossification; enables bone elongation at epiphyseal growth plates; covers the articular surfaces of joints; and supports the wall of various internal organs (9). Articular cartilage is a special kind of hyaline cartilage that essentially comprises two main components: chondrocytes, the single cell type of cartilage, are located in cavities called lacunae and are embedded in a unique extracellular matrix (ECM). In hyaline cartilage, 3-4 chondrocytes usually form small clusters known as isogenous cell groups (10). Adult articular chondrocytes in mature articular cartilage are enigmatic cells: first, they are postmitotic cells, thus considered as immortalised cells that maintain the proper composition of cartilage ECM throughout a lifespan; second, they are able to withstand enormously high compressive forces in major weight bearing joints; third, due to lack of blood vessels in cartilage, these cells can produce ATP required for their metabolic functions by obligate anaerobic respiration and are able to synthesise large amounts of ECM components in spite of low oxygen tension and nutrient supply; and fourth, they are able to survive the harsh conditions (i.e., the low extracellular pH owing to lactate accumulation) brought about in part by their own metabolic activities (11). The fact that mature chondrocytes are postmitotic cells can explain why articular cartilage possesses a limited healing response to various injuries.

The cell-to-ECM ratio in articular cartilage is approximately 1 to 10 by volume, which refers to the importance of proper matrix composition in cartilage function (12). Cartilage ECM consists of four main molecule groups: collagens, proteoglycans (PGs), glycosaminoglycans (GAGs) and non-collagenous glycoproteins. The cartilage-specific collagen type II accounts for more than 80% of all collagens present in the matrix. The primary function of collagen type II fibrils is to provide tensile strength; moreover, its highly regular arrangement can be accounted for the zonal architecture of articular cartilage (13). Collagen type IX binds other matrix components to the collagen network; collagen type XI adjusts the length of ECM fibres; and collagen type VI is an essential component in binding chondrocytes to the matrix (14).

PGs that form a gel-like ground substance are also essential ECM components; they provide stability and durability during compressive load. Aggrecan is the most characteristic cartilage-specific PG. Aggrecan monomers consist of chondroitin-sulphate and keratan-sulphate side chains bound to a core protein; by connecting to hyaluronic acid, one of the fundamental cartilage GAGs, the monomers form huge PG aggregates. By having a very high negative charge, glycosaminoglycan side chains can strongly bind to water. The water content of the ECM is of crucial importance: it provides the milieu for diffusion of oxygen and nutrients. This is especially important when considering that chondrocytes can be millimetres away from the nearest blood vessel. Together with GAGs, water also provides cartilage with characteristics similar to a shock absorber: under compressive load water flows out and then it flows back in, bringing about the structural basis of elasticity (15). On account of the high negative charge of PGs, chondrocytes are exposed to an unusual ionic environment because GAG side chains attract large numbers of cations, such as Na+, K+ and Ca2+ ions, creating a high extracellular osmolarity and probably influencing the resting membrane potential of these cells (11, 16). Non-collagenous glycoproteins can either connect chondrocytes to the matrix (e.g. tenascin, fibronectin, chondronectin or anchorin CII) or provide connections between matrix molecules, including matrilin-1 and link protein (17).

Ultrastructurally, it is the high level of organisation of major ECM components (mainly that of collagen type II fibres) that clearly distinguishes mature articular cartilage from other types of hyaline cartilage. The orientation of collagen type II fibres, slight changes in ECM composition, as well as the morphology and function of chondrocytes bring about 4 distinct zones in articular cartilage (1) the superficial or tangential zone, (2) the middle (or transitional) zone, (3) the deep (or radial) zone, and (4) the zone of calcified cartilage (Figure 1) (for a review, see (18)). Obviously, the superficial zone of articular cartilage is a critical component of the mature tissue because its collagen fibrils are oriented parallel to the surface, providing resistance to shear forces in the joint. Chondrocytes in this zone were also proved to be characterized by unique gene expression profiles: they secrete lubricin, a glycoprotein-like molecule reported to provide lubrication for articular surfaces, and its role in various joint diseases (rheumatoid arthritis and osteoarthritis) has also been implicated (19). Moreover, several studies suggest that articular cartilage has a different source than epiphyseal cartilage: in the rabbit, cartilage lining the ends of bones at birth is replaced by bone and cartilage of the articular surfaces is formed anew at the beginning of post-natal life by appositional growth (13). In accordance with this concept, cells with stem cell-like properties were isolated from the tangential zone of developing articular cartilage in mice and cattle (20). Notwithstanding these preliminary results concerning the development of the superficial zone of articular cartilage, a deeper understanding of the establishment of the zonal architecture of mature articular cartilage is required in order to create bioengineered cartilage with appropriate biomechanical properties.

5. CHONDROGENESIS IS REGULATED BY INTERPLAY BETWEEN NUMEROUS INTRA- AND EXTRACELLULAR FACTORS

Cartilage appears prior to bone formation during skeletal development; it is derived from the undifferentiated embryonic connective tissue (mesenchyme) during a process that starts with the condensation and nodule formation of chondroprogenitor cells. Proper cellular density is a crucial factor at the initial step of either in vivo or in vitro cartilage formation; consequently, the majority of in vitro chondrogenesis models apply unusually high number of cells (more than 15 M chondrogenic cells/mL) to mimic condensation and these models are therefore known as micromass or high density cell cultures (HDC) (21, 22). When chondroprogenitor cells isolated from limb buds of chicken (or less frequently mouse) embryos are used to establish HDC, this system is referred to as a primary chondrogenesis cell culture model. A great advantage of this model is the inherent ability to form hyaline cartilage within one week. Data of our own laboratory discussed here were gained from experiments using chicken HDC.

Besides transient appearance of Ca2+ dependent intercellular junctions in precartilage nodules (N-CAM and N-cadherin; (23)), the differentiation of chondroprogenitor cells into chondroblasts is governed by a number of growth factors and other signal molecules, including fibroblast growth factor (FGF), transforming growth factor-beta (TGF-beta), bone morphogenic protein (BMP) and Wnt families; (24). Cells in precartilaginous aggregates change morphology, and the resultant round-shaped phenotype seems to be essential for proper chondrogenesis, as inappropriate culture conditions of isolated chondrocytes had led to loss of round cellular morphology and a consequent failure to secrete cartilage-specific ECM (25). Changes in the phenotype of chondroprogenitor cells is also associated with altered gene expression pattern; in particular, expression of the chondrocyte-specific transcription factor Sox9 markedly increases, which is regulated by members of the FGF, TGF-beta, BMP and Wnt families (26). Inevitably, the expression of Sox9 in committed osteochondroprogenitor mesenchymal cells during condensation is the first step towards chondrogenesis to actually take place. Sox9 is responsible for the expression of two other Sox transcription factors, Sox5 and Sox6, and these three Sox transcription factors (often referred to as the Sox trio) in turn control the expression of a number of ECM macromolecules, including collagen types II, VI, IX and XI, CD-RAP (cartilage-derived retinoic acid-sensitive protein) and aggrecan (for a detailed review, see (27)). As a consequence, the ECM surrounding the differentiating chondrogenic cells is also subject to profound changes: cartilage-specific matrix components, most importantly collagen type II and aggrecan are laid down soon after their differentiation (28). Noteworthy that there is a reciprocal interplay between chondrocytes and cartilage-specific ECM: synthesised by chondrocytes, the characteristic composition and organization of ECM is pivotal for maintaining the appropriate morphology and function of chondrocytes (29).

In-depth identification of major intracellular signalling pathways that govern the molecular steps controlling chondrogenesis is essential to be able to externally influence cartilage formation. Mitogen-activated protein kinases (MAPKs) are important components of chondrocyte signalling involved in the conversion of extracellular stimuli into cellular responses and thus coordinate proliferation, differentiation and gene expression. The involvement of MAPKs in chondrogenesis is the subject of several review articles (for details, see (30)) and is only briefly discussed here. The three MAPK pathways contribute to the regulation of chondrogenesis to a various extent: while JNKs seem not to be involved in this process, p38s and ERKs are key regulators of chondrogenic differentiation; in chicken HDC, an opposing role of p38 and ERK has been reported: while the p38 MAPK pathway promotes, activation of the MEK/ERK pathway represses in vitro chondrogenesis (31). The results of our laboratory also confirmed the negative role of the ERK1/2 pathway in the regulation of in vitro chondrogenesis in the same model (22, 32).

Little is known about the upstream regulators of the MAPK pathways in chondrocytes. As far as the extracellular regulators are concerned, members of the TGF-beta and FGF families, retinoic acid, and integrins are reported to differentially activate p38 and ERK MAPK pathways (30). By considering that the MAPKs relay extracellular stimuli to cellular responses it is not surprising that these pathways are also implicated in mechanotransduction in chondrogenic cells: proliferation and differentiation, via altered MAPK activity, was also found to be influenced by mechanical stimuli during chondrogenesis (30, 33).

Transient protein phosphorylation is the most common posttranslational protein modification, which influences activity of many signalling proteins. Among the intracellular factors that regulate chondrogenesis various Ser/Thr protein kinases and Ser/Thr phosphoprotein phosphatases (PPs) were identified. In chicken HDC, expression of various protein kinase C (PKC) isoforms (PKC alpha, gamma, epsilon, zeta, lambda and iota) was reported and they were found to be mostly required at the early stages of in vitro chondrogenesis (34). Both PKC alpha (35) and PKC delta (32) were found to be positive regulators of chondrogenesis. In the same experimental model, the cAMP-dependent protein kinase A (PKA) is reported to be required for precartilage nodule formation by regulating N-cadherin expression and exerts its role via modulation of PKC alpha activity (36). Our laboratory has investigated the involvement of PP1 and PP2A in this process and found that PP2A was a negative regulator of in vitro chondrogenesis as administration of its inhibitor, okadaic acid, significantly enhanced cartilage matrix production; moreover, it also resulted in elevated PKA enzyme activity levels (37, 38). To the contrary, the Ca2+-sensitive PP2B (also referred to as calcineurin) was reported to positively control chondrogenesis and to be involved in the regulation of ERK1/2 activity (22). Signalling pathways that govern chondrogenesis in various chondrogenic models are summarised in Figure 2.

Many of the signalling components outlined above (e.g. PKC, PP2B) are at least partially calcium-sensitive; activity of the MEK/ERK pathway can also be modulated by Ca2+-calmodulin (39). Appropriate change (amplitude, frequency) in cytosolic Ca2+ concentration is often a prerequisite for proliferation or differentiation to take place. Consequently, Ca2+ sensitive signalling pathways may play an indispensable role also in chondrogenesis.

6. CALCIUM SIGNALLING: A SINGLE MESSENGER WITH DIVERSE FUNCTIONS

Calcium (Ca2+) is among the most ancient second messengers of eukaryotic cells and is involved in the regulation of various physiological and biochemical processes including secretion, muscle contraction, metabolic processes, gene expression, cell division and apoptosis (40). In the extracellular space, Ca2+ concentration is in the range of 10-3 M; in contrast, Ca2+ concentration in the cytosol is significantly lower, generally by four orders of magnitude (~10-7 M). Rise in cytosolic Ca2+ can either be the result of Ca2+ influx from the extracellular space or release from certain cell organelles, including the endoplasmic reticulum (ER) or the mitochondrium (referred to as Ca2+ stores). The large electrochemical gradient between these compartments enables the flux of Ca2+ ions into the cytosol across Ca2+ channels located in either the plasma membrane or in the membrane of the ER or mitochondria. Eventually, the increased cytosolic Ca2+ concentration, via numerous effector molecules, evokes the characteristic cellular response, which is terminated by restoring the basal cytosolic Ca2+ concentration by active Ca2+ pump or exchange mechanisms (41).

The Ca2+ signal is precisely regulated both temporally and spatially. The versatility of Ca2+ dependent signalling pathways lies in the target proteins with the help of which Ca2+ can exert its functions. All proteins that are involved in the regulation and maintenance of Ca2+ homeostasis in cells can be considered as members of a global Ca2+-signalling toolkit; all cells express and utilise the most appropriate members of the toolkit that are best required for their proper function (42). Members of the Ca2+-signalling toolkit include plasma membrane receptors, G-proteins, voltage- and ligand-gated Ca2+ channels, receptors of the endoplasmic reticulum (inositol-1,4,5-trisphosphate receptor (IP3R), ryanodine receptor (RyR)), chaperons (calsequestrin, calreticulin), Ca2+ pumps (plasma membrane Ca2+ ATPase (PMCA), sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)), and various effectors (calmodulin, classic PKC isoenzymes, NFAT and CREB transcription factors; (42)). Members of the Ca2+-signalling toolkit that are functionally expressed in MSCs and differentiating and mature chondrocytes are summarised in Table 1.

As noted above, Ca2+ signalling pathways control nearly every aspect of cellular processes, including proliferation and differentiation (41). Consequently, since these are very important functions of MSCs, extensive research concerning the Ca2+ homeostasis of these cells has been started at the beginning of the previous decade, including Ca2+ entry and elimination processes and signalling pathways that translate Ca2+ signals into cellular responses. Undifferentiated MSCs are per se non-excitable cells; however, as a result of their differentiation programme, they can give rise to many different cells types, including excitable cells (e.g., muscle and nerve cells; (43)). Despite accumulating knowledge concerning the Ca2+ homeostasis and ion channel assemblage (this latter is also referred to as the 'channelome') of non-excitable cells including mature chondrocytes and the growing evidence regarding their involvement in the regulation of metabolic processes and life cycle of these cells, the contribution of these pathways and various ion channels in the differentiation programme is not yet fully understood. Based on that, Ca2+ signalling pathways are expected to play pivotal roles in governing the differentiation of non-excitable MSCs to various excitable and non-excitable cells. Moreover, it is also anticipated that a transition in ion channel composition also accompanies (and probably regulates) differentiation of MSCs. As shown in Figures 3 and 4, mesenchymal stem cells and mature chondrocytes possess a distinct, although partially overlapping set of Ca2+ ion channels or receptors involved in the regulation of their Ca2+ homeostasis.

7. CA2+ ENTRY PROCESSES IN MSCS AND IN DIFFERENTIATING OR MATURE CHONDROCYTES

Ca2+ can enter the cytosol mainly from two sources: either from the extracellular space or from intracellular Ca2+ stores. The high number of plasma membrane Ca2+ channels are conventionally divided into four groups: voltage-operated calcium channels (e.g., L- and T-types); ligand- or receptor-operated calcium channels (e.g., N-methyl-D-aspartate or NMDA type glutamate receptors, transient receptor-potential (TRP) channels, ionotropic purinergic P2X Ca2+ channels); second messenger-operated channels (e.g., arachidonate-regulated Ca2+ channel); and Ca2+ release-activated Ca2+ channels (CRAC) that couple extracellular Ca2+ entry and intracellular Ca2+ release via the activation of store-operated Ca2+ channels (42). The other main source of cytosolic Ca2+ is release from intracellular Ca2+ stores, which is regulated by two distinct Ca2+ channels: the inositol-1,4,5-trisphosphate receptors (IP3R) and the ryanodine receptors (RyR; (44)). Ca2+ release from internal stores is agonist-dependent and voltage-independent. For the process of store-operated Ca2+ entry (SOCE), whereby Ca2+ influx from the extracellular space is activated in response to depletion of intracellular Ca2+ stores in the ER, interaction between STIM (a Ca2+ sensor in the ER) and the Orai family (plasma membrane Ca2+ channels) plays an important role (for a recent review, see (45)).

7.1. Voltage-operated Ca2+ entry pathways

Ca2+ influx pathways across the plasma membrane represent the main entry of Ca2+ into the cytosol of most cell types; moreover, release from intracellular Ca2+ stores also plays important roles. Since human MSCs (hMSCs) have become commercially available, Ca2+ signalling pathways (including Ca2+ entry processes) have been thoroughly investigated. In the seminal work of Kawano and co-workers, two main Ca2+ entry pathways were reported in undifferentiated hMSCs: voltage-operated Ca2+ channels (VOCCs) and store-operated Ca2+ channels (SOCs; (46)). As SOCs are responsible for Ca2+ entry in majority of non-excitable cells examined, presence of a store-operated Ca2+ current (ISOC) in hMSCs that is highly selective for Ca2+ is in accordance with previous findings (47).

VOCCs can be subdivided into several subtypes based on their characteristics: L- (CaV1.1, 1.2, 1.3, 1.4), P/Q- (CaV2.1), N- (CaV2.2), R- (CaV2.3) and T-type (CaV3.1, 3.2, 3.3) channels (44). Evidence of the functional expression of L-type Ca2+ channels was reported based on their sensitivity to dihydropyridines (although only 15% of cells measured exhibited small inward currents that could be blocked by nicardipine), but no N-type Ca2+ channels could be detected in hMSCs (46). Similar findings were reported in mouse embryonic stem cells (mESCs), where a capacitive Ca2+ entry, rather than influx via VOCCs was found to be the main basal Ca2+ entry pathway (48). In fact, no mRNA expression of L-, T- or P/Q-type Ca2+ channels could be detected in the latter study. In spite of the observations that VOCCs seem to contribute to Ca2+ entry to a lesser extent compared to store-operated Ca2+ entry processes in hMSCs, L-type Ca2+ channels were also identified in human embryonic stem cells (49) and in rat MSCs (50). Moreover, Zahanich and colleagues demonstrated that although L-type Ca2+ channels are involved in the maintenance of Ca2+ homeostasis in differentiated osteoblasts, functional expression of these channels is not required for osteogenic differentiation of hMSCs (51).

Expression of voltage-operated Ca2+ channels has also been reported during chondrogenesis. In organoid cultures of limb buds derived from early mouse embryos, VOCCs were detected to co-localise with beta-1 integrin, Na/K-ATPase and epithelial sodium channels in mechanoreceptor complexes in developing chondrocytes (52). Indeed, expressions of various CaV1.2 and 3.2 channel subunits were detected in chondrocytes in developing murine embryos by immunohistochemical staining procedures; moreover, protein expressions of the same L-type Ca2+ channels were identified in the ATDC5 chondrogenic cell line (53). Voltage-gated Ca2+ channels have long been known to be expressed by articular chondrocytes and contribute to elevation of cytosolic Ca2+ concentration by insulin-like growth factor-1 (54). Xu and co-workers recently demonstrated that Ca2+ influx via VOCCs is also necessary for signalling pathways in stimulated articular chondrocytes (55). Preliminary findings of our laboratory also implicate the involvement of various voltage-gated Ca2+ channels during in vitro chondrogenesis in chicken HDC established from distal limb buds of 4-day-old chicken embryos. In contrast to murine ESCs, where no rise in cytosolic Ca2+ concentration was observed when cells were exposed to external solution containing high concentration of K+ (48), chondrifying cells in chicken HDC exhibited large Ca2+ transients by exposure to high concentrations of K+ (56), implicating the functional expression of VOCCs. These differences may be the consequence of the more differentiated state of cells in HDC compared to embryonic stem cells. Notwithstanding these interesting results, further studies are required to clarify the precise role of voltage-dependent Ca2+ channels during chondrogenesis.

7.2. Ligand-operated Ca2+ entry pathways

7.2.1. Purinergic signalling pathways

Besides VOCCs, the other main Ca2+ entry pathway across the plasma membrane is via ligand-operated Ca2+ channels (44). A typical example of these channels is the family of purinergic receptors. Activated by extracellular nucleotides, purinergic receptors are conventionally divided into two major types: P1 receptor families are sensitive to adenosine, while ligands for P2 receptor families include ATP, ADP, and UTP. P2 receptors are further divided into two major receptor subtypes: P2Y and P2X. Members of the metabotropic P2Y receptor subtype are 7 transmembrane domain-containing G-protein coupled receptors (GPCRs) whose activation leads to the release of intracellular Ca2+ from IP3-sensitive Ca2+ stores. The ionotropic P2X receptors are ATP-gated bona fide ion channels that allow Ca2+ influx across the plasma membrane (57). In hMSCs, Kawano and colleagues found that GPCRs, mainly metabotropic purinergic (P2Y) receptors participated in the regulation of Ca2+ homeostasis, by an autocrine-paracrine ATP signalling mechanism (58). ATP was reported to be secreted from hMSCs via hemi-gap junctions to stimulate P2Y1 receptors in the plasma membrane. Moreover, in a more recent study the subtypes P2X6, P2Y4, and P2Y14 were found to be pivotal regulators in adipose tissue-derived adult MSC commitment during adipogenic and osteogenic differentiation (59). The functionality of purinergic receptors in human skin-derived MSCs was demonstrated by Orciani and colleagues when they reported that MSCs responded to administration of ATP by elevation of cytosolic concentration via P2X receptors (60). In spite of the growing evidence regarding the emerging role of purinergic signalling in stem cells (for a review, see (61)), as the majority of work was performed on neuronal or muscle precursors, current knowledge is still considerably limited concerning the possible involvement of extracellular nucleotide-gated signalling during the differentiation of MSCs towards other lineages.

The purinergic concept in the regulation of chondrogenesis was first suggested by Meyer and co-workers in 2001 (62). They reported that P2Y1, a GPCR whose activation leads to the release of Ca2+ from intracellular stores, was a negative regulator of chondrogenesis in chicken HDC as overexpression of this receptor has led to attenuated cartilage matrix production in these cell cultures. Moreover, they also showed evidence that these cells released ATP, the ligand of P2Y1 into the culture medium. In line with these experiments, results of our laboratory also support the hypothesis that purinergic signalling is involved in the chondrogenic differentiation of chicken mesenchymal cells (63). In contrast to the above findings, we concluded that ionotropic P2X receptors, rather than metabotropic P2Y receptors, were primarily involved in positively regulating in vitro chondrogenesis in chicken limb bud-derived HDC, and we confirmed that the plasma membrane expression of P2X4 receptor was at least partially responsible for a major Ca2+ influx required for differentiation of chondroprogenitor mesenchymal cells in this system. The other ionotropic (P2X1, P2X5, P2X7) and metabotropic (P2Y1, P2Y2, P2Y4) purinergic receptors whose expression was detected in the plasma membrane are probably involved in the maintenance of basal cytosolic Ca2+ concentration (63).

The functional characterisation of purinergic signalling pathways in articular chondrocytes have been performed almost two decades ago (64, 65). Mature chondrocytes have also been reported to express both P1 and P2 purinoreceptor genes (66) and that detailed analyses revealed that members of both p2x (67) and p2y (68) purinoreceptors were expressed and functional in chondrocytes. Although their role played in Ca2+ homeostasis of adult cartilage cells is not fully characterised, their involvement in chondrocyte mechanotransduction pathways is implicated (see below).

7.2.2. N-methyl-D-aspartate receptor mediated pathways

L-glutamate is the major excitatory neurotransmitter that can activate ionotropic (iGluR) and metabotropic (mGluR) receptors. Ionotropic glutamate receptors are bona fide ligand-gated non-selective cation channels, and based on their selective pharmacological agonists they are subdivided into alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate receptors (NMDAR). While AMPA and kainate receptors are mostly permeable to K+ and Na+, NMDARs are primarily permeable to Ca2+ (44). NMDARs are heterotetrameric complexes that are comprised of two obligatory NR1 subunits and two others of four distinct NR2 (NR2A, NR2B, NR2C or NR2D) and/or two NR3 (NR3A or NR3B) subunits (for a recent review, see (69)).

Conventionally, glutamate signalling has been considered as the primary excitatory pathway in the central nervous system; therefore, the involvement of NMDARs in the regulation of differentiation, survival and electrophysiological properties has been extensively investigated in neurons (70). However, glutamate receptor expression and function in non-neuronal tissues is also reported (for a review, see (71)). Interestingly, functional characterisation of glutamaterg signalling pathways, including NMDAR expression and function remains elusive in undifferentiated mesenchymal and embryonic stem cells; these pathways have only been investigated during differentiation into neural precursors and nerve cells (70). Nonetheless, owing to the emerging role of NMDARs in non-excitable cells, the importance of glutamaterg signalling in MSCs, at least partially via NMDARs, can be implicated.

Recent findings demonstrated the importance of glutamaterg signalling in skeletal tissues. NMDARs have proved to play a complex role in bone remodeling (72) and have been reported to be involved in differentiation of osteoblasts and osteoclasts (73). In ex vivo organotypic cultures of fetal mouse tibias, Takahata and colleagues demonstrated that mRNAs for the NR1, NR2D and NR3A NMDAR subunits were constitutively expressed in developing chondrocytes and that glycine probably acts on these NMDARs to induce terminal differentiation of these cells (74). Mature human articular chondrocytes have also been reported to express NMDARs (75) and that they are active components of mechanotransduction pathways (76). In accordance with these results, we were also able to confirm the mRNA and protein expressions of the NR1 NMDAR subunit in chicken HDC. Furthermore, NR1 subunit expression was found to be affected by mechanical stimulation in chondrogenic cells (33). Nonetheless, the importance of glutamate signalling in the regulation of cartilage development is yet to be clarified.

7.2.3. Transient Receptor Potential (TRP) pathways

Accumulating evidence suggests that transient receptor potential (TRP) channels play prominent roles in regulating the intracellular Ca2+ concentration in non-excitable cells. TRPs are clearly remarkable proteins: they constitute a large and functionally versatile family of cation-conducting channels, and at the same time, they have also been considered as unique cell sensors. Of the more than 50 TRP channels identified thus far in many species, the 28 mammalian TRP channels are conventionally divided into 6 subfamilies: canonical (TRPC), vanilloid (TRPV), melastatin (TRPM), ankyrin (TRPA), mucolipin (TRPML), polycystin (TRPP), and TRPN ("no mechanoreceptor potential C"); most of them comprises numerous members (77). Members of the TRP subfamilies are reported to be expressed in majority of cells and tissues examined and found to regulate many basic physiological processes, including nociception, temperature and osmosensation or muscle contraction. Functioning as non-selective cation channels, the molecular structure of TRPs highly resembles that of voltage-operated ion channels; upon activation, TRPs can cause cell depolarisation. TRPs may not only act as bona fide cation channels that can enable a rise in cytosolic free Ca2+ concentration, but (1) their activity may influence gating of VOCCs mainly in excitable cells; (2) TRP channels can regulate the driving forces for Ca2+ entry, mainly in non-excitable cells, by de- or hyperpolarizing the plasma membrane via Ca2+ -dependent activation of other ion channels; and (3) TRPs themselves are influenced (activated or inhibited) by changes of intracellular Ca2+ concentration (78). While the majority of TRPs are located in the plasma membrane and allow for Ca2+ influx (all the TRPCs, all TRPVs, TRPM1, 2, 3, 6, 7, and 8, TRPA1, TRPP2, 3, and 5 and TRPML1, 2, and 3), some of the TRP channels are reported to be mainly localised in intracellular membranes (such as TRPML1 and TRPP2) (77).

Kawano and colleagues have detected the mRNA expression of TRPC4 (and not that of TRPC3, 5 and 7) in hMSCs and implicated its involvement in the store-operated Ca2+ currents characteristic to these cells (46). As the function of TRP channels has primarily been investigated in the nervous system especially concerning nociception, currently available data regarding the expression and role of various TRPs in mesenchymal stem cells is mostly limited to neurogenesis. Nonetheless, the importance of TRPM7 in the survival of bone marrow-derived MSCs has been recently reported (79). Weick and co-workers demonstrated that TRPC1 and TRPC4 act as molecular targets for controlling neurite elongation in human embryonic stem cell-derived neurons (80). That MSCs express canonical TRPC1, 2, 4 and 6 mRNA and that TRPC1 may be involved in stem cell proliferation has been recently shown (81).

Data available concerning the putative role of TRPs during chondrogenesis is also sparse. Muramatsu and co-workers revealed that TRPV4 is a positive regulator of Sox9: besides the fact that the mRNA expression profile of TRPV4 highly resembled those of chondrogenic marker genes in the chondrogenic cell lines ATDC5 and C3H10T1/2, pharmacological activation of TRPV4 induced Sox9 reporter activity (82). Furthermore, they also showed that the Ca2+-calmodulin pathway was required for this process. As far as TRP channel expression in mature chondrocytes is concerned, Gavenis and colleagues have reported that articular chondrocytes isolated from knee joints of osteoarthritic patients express various TRP ion channels (83). That TRPV4 is an osmotically active ion channel in mature chondrocytes and that it is also an important component of chondrocyte mechanotransduction pathways has been reported (84, 85). Furthermore, resting membrane potential in chondrocytes was found to be at least partially regulated by TRPV5 (86).

Although the exact role of TRPs in chondrocyte differentiation and maturation is still not well understood, given that TRPV4 is a stretch-activated channel that allows for stretch-activated Ca2+ entry (78), it may be hypothesised that TRPV4 ion channels (and possibly other stretch-activated TRPs) may be key regulators of chondrogenesis by translating mechanical forces into Ca2+ mobilization.

7.3. Ca2+ Release from internal stores and store-operated Ca2+ entry

Ca2+ release from intracellular Ca2+ stores (primarily from the ER) represents the other main source of cytosolic Ca2+ besides influx from the extracellular space across the plasma membrane. Investigation of Ca2+ release from internal stores in MSCs has led to the conclusion that IP3Rs are the primary factors that allow for Ca2+ release from the ER (although to a variable extent, all three IP3R subtypes were found to be expressed at the mRNA level in hMSCs), while RyR, the other main Ca2+ release channel was not found to be involved in this process as its functional expression (either at the mRNA level or by local application of the RyR-agonist caffeine during Ca2+ measurements) could not be proved in hMSCs (46). Murine ESCs have proved to utilise the same Ca2+ release pathway: mRNA expressions of IP3R subtypes 1, 2 and 3 were confirmed, but no mRNAs for ryanodine receptors could be detected in these cells (48). Mature chondrocytes were also reported to possess internal Ca2+ stores that are releasable via the activation of the PLC-IP3R pathway (87). The involvement of RyR in impact-induced chondrocyte death in mature equine chondrocytes was reported by Huser and Davies (88). Our findings are in accordance with the above results in that internal Ca2+ stores in developing chondrocytes of chicken HDC also contained IP3Rs, rather than RyRs, and the PLC-IP3R pathway was found to be functioning in these cells. Nonetheless, internal Ca2+ stores seemed to contain relatively low amounts of releasable Ca2+ in differentiating cells of chicken HDC (63, 89).

In electrically non-excitable cells, Ca2+ entry pathways are dominated by store-operated Ca2+ entry (SOC) channels; by definition, their activation is dependent on the depletion of internal Ca2+ stores (90). Given that human MSCs are essentially non-excitable cells, Kawano and co-workers reported that hMSCs primarily utilise SOC pathways (ISOC) rather than influx via VOCCs, although they refrained from characterising these molecules in detail (46). The store-operated Ca2+ channels are activated by store depletion, which has been confirmed by local administration of cyclopiazonic acid (CPA) or thapsigargin (SERCA inhibitors) to MSCs during single cell Ca2+ measurements. Attempts have been made towards identifying the molecules for ISOC, and the role of TRP family proteins (most importantly, TRPC4) was implicated. The same group reported that ISOC is the main Ca2+ entry pathway also in murine ESCs and found that TRPC1 and TRPC2 might be responsible for Ca2+ entry in these cells (48). Signs of SOC pathways have been recorded in differentiating chicken chondroprogenitor mesenchymal cells in HDC during single cell Ca2+ measurements (89); however, our results contradict observations reported in human and murine stem cells. Cells in chicken HDC were found to primarily depend on the availability of free extracellular Ca2+: chelation of extracellular Ca2+ by EGTA almost completely abolished cartilage matrix production by preventing differentiation and proliferation of these cells, and at the same time significantly reduced cytosolic free Ca2+ concentration. Also, removal of available extracellular Ca2+ during single cell fluorescent Ca2+ measurements immediately resulted in slightly lower Ca2+ levels (approx. 10 nM), reflecting on the sensitivity and dependence of Ca2+ homeostasis of these cells on extracellular Ca2+. Furthermore, long-term treatments with CPA did not have any effect on cartilage matrix production, nor on proliferation rate of cells, also supporting the hypothesis that the contribution of internal Ca2+ stores to the Ca2+ homeostasis of differentiating chicken chondrocytes is insignificant (89). To the contrary, in mature porcine articular chondrocytes, the importance of internal Ca2+ stores in the regulation of Ca2+ homeostasis was implicated, without a significant contribution from Ca2+ influx (91); these findings also support the concept that a profound change in Ca2+ homeostasis, as well as in the Ca2+-signalling toolkit complement may characterise each step of differentiation processes from undifferentiated pluripotent cells to committed progenitors and mature cells.

Although the existence of SOC pathways that represent the major Ca2+ entry to non-excitable cells (via Ca2+ release-activated Ca2+ channels or CRAC) has long been known, the characterisation of the molecular machinery that orchestrates SOCE took almost 20 years to accomplish. Two major SOCE components have been identified; the stromal interaction molecules (STIM) and the Orai ion channel family (for a review, see (92)). STIM1 is a Ca2+ sensor protein located in the ER membrane possessing an EF-hand domain that activates SOCE upon Ca2+ store depletion, whereas its homologue STIM2 is mainly involved in the maintenance of basal cytosolic and ER Ca2+ levels. Upon store depletion, STIM1 redistributes to sub-plasma membrane puncta, where it co-localizes with Orai1 to form the basic subunit required for SOCE. The close apposition of the ER and plasma membrane at the STIM1-Orai1 clusters enables SERCA pumps to rapidly and efficiently refill the ER Ca2+ stores (92). In spite of previous results providing evidence that SOCE pathways are responsible for the main Ca2+ processes in MSCs, the characterisation of these molecules in undifferentiated mesenchymal stem cells has not been performed yet. However, Darbellay and colleagues reported that STIM1 and Orai1-dependent SOCE is involved in myoblast differentiation in myogenic stem cells (93). In more committed endothelial progenitor cells, mRNA expressions of molecules that mediate SOCE, including TRPC1, TRPC4, Orai1, and STIM1, were identified (94). As yet, no data are available concerning the expression and role of these proteins either in developing or in mature chondrocytes.

Over a decade ago, a novel way of non-capacitive Ca2+ entry pathway has been described in many cell types including both electrically excitable and non-excitable cells that is entirely independent of store depletion and is rather dependent on receptor-mediated generation of arachidonic acid (95). While arachidonate-regulated Ca2+-sensitive (ARC) channels are implicated to be involved in the modulation of the frequency of agonist-induced Ca2+ oscillations (see below), SOC channels determine the amplitude of sustained Ca2+ signals. Calcineurin was found to be the key downstream target that differentially relays signals from these two Ca2+ channels: oscillatory Ca2+ signals generated by the activation of ARC channels fail to increase calcineurin activity, while a long-term sustained rise in cytosolic Ca2+ concentration is required for calcineurin activation (96). In spite of the growing body of evidence regarding the activation and function of ARC channels, the molecular characterisation of this novel kind of channels has only recently been revealed and proved to be a pentameric structure primarily made up of Orai1 and Orai3 subunits (97). Owing to the fact that these ARC channels have originally been described only a decade ago (95), their characterisation in MSCs has not been performed yet. In fact, based on experiments by using La3+, a blocker of non-selective cation channels, Kawano and colleagues concluded that an unknown, novel Ca2+ entry pathway that is irrespective of store depletion was functioning in hMSCs, which contributed to Ca2+ oscillations (98) - this channel (among other candidates) could have well been an ARC channel. Although no data are available regarding the presence of ARC channels in chondrocytes, arachidonate metabolism has long been known to regulate in vitro chondrogenesis in chicken limb bud-derived HDC (99); therefore, the involvement of these channels during chondrocyte differentiation may be hypothesised.

8. CA2+ ELIMINATION PROCESSES IN MSCS, DIFFERENTIATING AND MATURE CHONDROCYTES

In general, basal cytosolic Ca2+ concentration is in the range of 100 nM. In order to eliminate the cellular signal and attenuate Ca2+ dependent signalling pathways, restoration of the resting cytosolic Ca2+ concentration is required. The plasma membrane Ca2+-ATPase (PMCA) and the Na+/Ca2+-exchanger (NCX) are the primary factors that eliminate cytosolic Ca2+ ions towards the extracellular space, while the sarco(endo)plasmatic reticulum-Ca2+-ATPase (SERCA) located within the membrane of ER is involved in the accumulation of cytosolic Ca2+ in the intracellular stores. The fourth pump mechanism is the mitochondrial uniporter that pumps Ca2+ ions into mitochondria (42).

In hMSCs, Kawano and co-workers were the first to describe the functional expression of PMCA and NCX (98). In this work, they also demonstrated that these Ca2+ extrusion mechanisms are important contributors to the maintenance of Ca2+ homeostasis in these cells by sustaining oscillatory changes in cytosolic Ca2+. The same group has also reported the functional expression of both PMCA and NCX in mouse ESCs (48). Of the PMCA isoforms, PMCA-1 and PMCA-4 mRNA expression was detected in stem cells, while all three NCX isoforms (NCX-1, -2, and-3) were found to be expressed in these models. In comparison with PMCA and NCX, SERCA and the mitochondrial uniporter only play minor roles in Ca2+ elimination processes. Nevertheless, numerous data gained by the application of well-known SERCA inhibitors (thapsigargin, CPA) have indirectly provided evidence that SERCA was active and functioning both in MSCs (46) and in developing chondrocytes (89). In mature articular chondrocytes, only the presence and function of NCX has been reported (100).

9. TEMPORAL CHARACTERISTICS OF CA2+ DEPENDENT SIGNALS

Besides the wide variety of the Ca2+-signalling toolkit mentioned above, the precise temporal regulation of cytosolic Ca2+ concentration also significantly contributes to the versatility of Ca2+ dependent signalling pathways. In certain cell types (e.g. cardiac muscle cells or neurons), the effectors respond to changes of cytosolic Ca2+ concentration within milliseconds, whereas in other cells the same kinds of changes require seconds or even minutes. Cellular proliferation and differentiation are key processes that both require long-term, sometimes sustained changes in cytosolic Ca2+ concentration.

9.1. Day-by-day variation of cytosolic Ca2+ concentration

The dependence of in vitro chondrogenesis on extracellular Ca2+ has long been known (101) as high concentrations of this cation promoted chondrogenic differentiation in chicken HDC as early as the condensation or aggregation phase. Extracellular Ca2+ was also reported to modulate cell differentiation during skeletogenesis in chicken embryonic calvaria, where low concentrations enabled chondrogenesis (102). Treatment with the L-type channel-specific blockers nifedipine and verapamil during chondrogenesis in mouse limb bud-derived HDC attenuated differentiation, as well as mineralisation (103). Based on the above results, and given that chondrogenesis is regulated by signalling pathways that are Ca2+ sensitive; moreover, as cellular differentiation processes are per se dependent on Ca2+, the concept of Ca2+ dependent regulation of chondrogenesis has emerged.

In chicken HDC established from limb bud-derived mesenchymal cells, we have reported a day-by-day variation in cytosolic Ca2+ concentration measured by single cell fluorescent Ca2+ imaging (89). Spontaneous final commitment and differentiation of majority of chondroprogenitor mesenchymal cells occurs on culturing day 3 in this chondrogenesis model and a significant amount of hyaline cartilage is formed by day 6 of culturing. Interestingly, a characteristic peak in cytosolic free Ca2+ concentration was found on day 3 (approximately 140 nM, as compared to 100 nM prior to or after this day). Furthermore, the occurrence of this peak in cytosolic Ca2+ concentration was found to be indispensable to proper differentiation of cells in HDC: chelation of extracellular Ca2+ on culturing days 2 or 3 resulted in significantly lower cytosolic Ca2+ concentration, and chondrogenesis, as well as cartilage matrix production was almost completely abolished. Moreover, uncontrolled influx of Ca2+ via A23187 Ca2+ ionophore had dual effects: a 1.25-fold increase in cytosolic Ca2+ concentration significantly promoted chondrocyte differentiation and matrix production, while a 1.5-fold increase was already detrimental and negatively affected in vitro chondrogenesis (89). As these interventions were only effective when applied prior to or during day 3; furthermore, as rate of cellular proliferation has proved to be the most sensitive parameter, Ca2+ dependent signalling may primarily exert its chondrogenesis-promoting or inhibitory effects via modulation of proliferation. All these findings support the hypothesis that a sustained rise in cytosolic Ca2+ concentration (but only to a certain extent) at the time of final commitment of chondrogenic cells is a prerequisite to chondrogenic differentiation in these cultures.

9.2. Ca2+ oscillations in mesenchymal stem cells and chondrocytes

Various cell types (importantly, many types of non-excitable cells) were described to exhibit Ca2+ oscillations in response to sustained stimuli. During these oscillations, the increase of cytoplasmic Ca2+ levels is in the range of 50-600 nM. It is obvious that the mere existence of Ca2+ oscillations adds another dimension besides the amplitude of Ca2+ signals: the Ca2+ signal now has a frequency (104). The exact physiological significance of Ca2+ oscillations in non-excitable cells has not been fully characterised yet; however, these periodic changes have been reported in case of almost every cell type examined and implicated in having a role in controlling many processes, such as fertilisation, proliferation, secretion, muscle contraction, etc. A possible role of oscillatory elevation of cytosolic Ca2+ concentration may lie in activation of Ca2+-sensitive transcription factors. During Ca2+ oscillations, Ca2+ concentration can periodically exceed the activation threshold for transcription factors, whereas a small, constant increase in cytosolic Ca2+ concentration cannot accomplish this (105). Thus, by appropriately decoding information embedded in the frequency and/or amplitude of oscillatory Ca2+ signals, the differential activation of different genes may drive cells towards specific developmental pathways (106). This is particularly important for MSCs, and it can be hypothesised that oscillatory increases of cytosolic Ca2+ concentration are involved in more complex functions during differentiation towards various lineages.

By using Fluo-3 imaging techniques, Kawano and co-workers were the first to describe that undifferentiated hMSCs exhibited spontaneous Ca2+ oscillations with an average frequency of 1 oscillation/120 sec (46). These oscillations were sustained for a short period even in the absence of free extracellular Ca2+, although with lower amplitudes, which implicates that intracellular Ca2+ stores play important roles in the generation of these oscillatory changes besides Ca2+ influx via plasma membrane ion channels. Consistent with this hypothesis, administration of the SERCA blockers thapsigargin or CPA completely abolished these oscillations; moreover, the fact that the non-selective IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) also inhibited oscillations further supported the theory that these oscillations were primarily dependent on the internal Ca2+ stores (46). Nonetheless, Ca2+ influx is also required for long-term sustainment of these oscillations as Ca2+ influx is necessary to refill the intracellular Ca2+ stores upon depletion. As far as the contribution of Ca2+ extrusion pathways to the Ca2+ oscillations is concerned, both PMCAs and Na+/Ca2+ exchangers (NCX) were found to be involved in sustaining Ca2+ oscillations as pharmacological inhibition of PMCAs and NCXs with carboxyesin or caloxin 2A1 and Ni2+ or KBR7943, respectively, completely blocked these periodic Ca2+ transients (98). The same group also reported that an autocrine/paracrine purinergic loop via the metabotropic purinergic receptor P2Y1 is a crucial regulator of oscillations in MSCs (58). In hMSCs, ATP was demonstrated to be secreted from cells via hemi-gap junction channels, thereby triggering and maintaining Ca2+ oscillations by an autocrine mechanism.

Ca2+ oscillations were also identified and partially characterised in cells of chicken limb bud-derived chondrifying mesenchymal cell cultures by our group (56, 89). However, several features of these oscillations were found to be different from those reported by Kawano and co-workers. First, the periodicity of these oscillations was significantly (approx. 10 times) shorter, with approximately 4-5 transients/minute (Figure 5). Second, the oscillations in chondrifying mesenchymal cells were found to be mainly dependent on the availability of extracellular Ca2+ as removal of free Ca2+ from the bath solution immediately abolished oscillations in most of the cells examined. Third, the involvement of VOCCs in the generation and sustainment of these Ca2+ oscillations in differentiating chondrocytes has been implicated (56).

As described earlier, Ca2+ oscillations can trigger the activation of Ca2+-dependent transcription factors depending on the frequency (NFAT, CREB, etc.) that regulate important cellular functions such as proliferation and differentiation (107). Kawano and co-workers demonstrated that Ca2+ oscillations were required for nuclear translocation of NFAT, as blockade of Ca2+ oscillations by interfering with the ATP autocrine/paracrine signalling pathway prevented this process. Furthermore, when MSCs differentiated to adipocytes, Ca2+ oscillations were not detectable any more, and the nuclear signal for NFAT has also decreased (58). Dephosphorylation by calcineurin is a prerequisite to nuclear translocation of NFAT (108). As calcineurin enzyme activity was found to be differentially regulated by elevated or lower cytosolic Ca2+ concentration in differentiating chondrocytes (89), it implies that this phosphatase may be one of the crucial factors by which Ca2+ oscillations govern cellular proliferation or differentiation processes. Similar to the above-mentioned results, the frequency of Ca2+ oscillations in HDC was found to be decreased at a differentiation state-dependent manner and could not be detected in mature HDC, which implies that Ca2+ oscillations are probably involved in the activation of signalling pathways that control differentiation in chondrifying HDC (56). Moreover, the master transcription factor of chondrogenesis, Sox9 has been reported to be transported into the nucleus by calmodulin in a cytosolic Ca2+ concentration-dependent manner (109).

Mature chondrocytes have also been reported to exhibit spontaneous, as well as mechanically induced Ca2+ oscillations in sliced pieces of cartilage and these two types of oscillations were found to be different from one another (68). Consistent with data mentioned above regarding the purinergic concept of the regulation of Ca2+ oscillations in hMSCs, these oscillatory changes in cytosolic Ca2+ concentration in chondrocytes were also mediated by a purinergic signalling pathway. Mature chondrocytes were found to exhibit agonist-induced Ca2+ oscillations regulated by mechanisms similar to those in hMSCs; although the rhythmic activity was most importantly triggered by the intracellular stores via activation by ATP, Ca2+ influx was required to sustain these oscillations (110).

Although we are far from the detailed characterisation of Ca2+ oscillations in non-excitable cells including undifferentiated MSCs, it is clear that different patterns of Ca2+ oscillations may differentially regulate signalling pathways that eventually lead to diverse and various responses dependent on the type of the cell and probably on their differentiation stage.

10. CA2+ SIGNALLING DURING MECHANOTRANSDUCTION

Mechanical forces have long been known to be key external stimuli that keep musculoskeletal tissues, in particular bone and cartilage in a healthy condition. These tissues are equipped with a not yet fully characterised set of signalling molecules including mechanosensitive ion channels that convert the effects of external stimuli to appropriate biochemical signals during a process called mechanotransduction. The effects of mechanical load have been extensively studied in articular cartilage, in which the unique biomechanical characteristics of the ECM provide a special milieu for chondrocytes that withstand extraordinarily high tensile forces especially in the hip and knee (111). Numerous research groups carried out experiments with bioreactors that were able to apply various loading regimes, including compressive forces (both constant and intermittent), hydrostatic pressure, tensile strain and shear stress (112, 113). Mechanical stimulation has been widely applied on both differentiating and mature musculoskeletal tissues; however, the nature of cellular response to physical stimuli-besides being cell- and tissue-specific-seems to vary according to the type of load. Nevertheless, cyclic compressive loading seems to be the most relevant as it best mimics physiological stimuli on articular cartilage during normal physical activity. That articular cartilage in vivo undergoes intermittent compression within the range of 3 to 10 MPa during normal activities has been recently reported (114). Compressive forces have been described to enhance collagen and proteoglycan synthesis in articular chondrocytes; moreover, the involvement of integrin signalling and the MAPK/ERK pathway was also implicated (115). Although both integrins and the ERK pathway are important mediators of chondrocyte mechanotransduction, Ca2+ signalling seems to be the major regulator. Various components of the Ca2+-signalling toolkit have been implicated as mediators of mechanotransduction pathways in chondrocytes (Table 2). When exposed to short-term cyclic compression, mature articular chondrocytes responded by the activation of voltage-gated L-type calcium channels and stretch-activated cationic channels, which allowed Ca2+ influx resulting in the elevation of cytosolic Ca2+ concentration (115). Besides VOCCs, involvement of ligand-gated Ca2+ channels in mechanotransduction pathways of chondrocytes has been thoroughly investigated. Of these, Millward-Sadler and co-workers were the first to describe that ATP has a role in the response of adult human chondrocytes to mechanical stimulation, via P2Y2 metabotropic purinergic receptors; moreover, they also found that osteoarthritic cartilage-derived chondrocytes responded differentially to ATP and therefore, the importance of the purinergic pathway in the progression of this disease was implicated (116). More recently, a mechanosensitive purinergic Ca2+ signalling pathway via P2Y receptors was also identified in isolated chondrocytes embedded in agarose (117). Moreover, Garcia and Knight provided the first evidence that mechanical loading triggers ATP-mediated purinergic signalling pathways via ATP release through hemichannels in agarose-embedded chondrocytes (118). As outlined above, TRPV4 cation channels are expressed by articular chondrocytes and can be gated by osmotic and mechanical stimuli; indeed, this receptor was found to play a critical role in the maintenance of joint health in a study performed by Clark and co-workers on knee joints of TRPV4-deficient mice (119). Besides TRPV4, other members of the TRPV ion channel family are also involved in mechanotransduction (for a review, see (120)); however, their role in chondrocytes has not been thoroughly investigated yet. As far as the involvement of NMDA receptors is concerned, role of this ion channel in chondrocyte mechanotransduction pathways has also been implicated (76). Results of our research group also suggest that cyclic compressive force applied during in vitro chondrogenesis of chicken HDC influences Ca2+ homeostasis via NMDA receptors (33).

While the above results were gained on chondrocytes cultured in vitro, data concerning the effects of mechanical stimuli in vivo are sparse. Nonetheless, mechanically induced calcium signalling has been confirmed in articular chondrocytes in situ (121); moreover, the requirement of a well-defined movement pattern during in vivo embryonic joint and articular cartilage formation has also been published (122, 123).

Our current understanding is even more limited regarding mechanotransduction in MSCs. Proper mechanical loading does not only improve the characteristics of developing or mature cartilage but it is implicated to be involved in determining the fate of differentiating mesenchymal cells (124, 125). Mechanical stimulation induces MAPK activation and controls MSC proliferation (126). Application of various mechanical stimuli including cyclic mechanical stress, shear stress or low intensity ultrasound to manipulate MSC differentiation towards different lineages have been recently reviewed by Titushkin and colleagues (127). They envision that mechanical factors are key regulators of MSC differentiation, and that a delicate interplay and tight coupling between Ca2+ homeostasis, MAPK pathways and biomechanical remodelling is required for lineage-specific commitment and differentiation of MSCs.

11. CONCLUSIONS

Mesenchymal stem cells represent a new and exciting horizon for cell-based tissue regeneration techniques. To achieve this, recent attempts have been focused on the manipulation of stem cell differentiation towards specific lineages, including chondrocytes. To this end, a better understanding of the molecular machinery governing lineage-specific differentiation is required. Ca2+ is generally accepted as a critical player in cellular differentiation processes. As outlined in this review, MSCs are characterised by the functional expression of numerous members of the Ca2+-signalling toolkit. Non-excitable MSCs possess the capability of differentiating to various lineages, including both excitable and non-excitable cells. There is evidence that an adaptation of Ca2+-signalling toolkit to function of mature cells derived from ESCs and MSCs is indispensable. This also supports the concept that a profound change in Ca2+ homeostasis, as well as in the assemblage of Ca2+-signalling toolkit characterises differentiation from undifferentiated pluripotent cells to committed progenitors and mature cells. Key targets of general or local changes in cytosolic Ca2+ concentration are the extraordinarily wide range of Ca2+-sensitive signalling pathways including-among others-various protein kinase/phosphoprotein phosphatase systems, that may ultimately enable distinct gene expression profiles via differential activation of key transcription factors (NFAT, CREB, etc.), giving rise to lineage-specific differentiation. Therefore, a better understanding of these pathways may ultimately allow us to control MSC differentiation to required cell types. Although appropriate methodologies are readily available to characterise changes in the calcium dynamics and associated signalling pathways, we still do not fully understand whether the observed (and yet-to-be discovered) changes are simply consequences or rather critical prerequisites to MSC differentiation. An in-depth understanding of these mechanisms will undoubtedly lead to a more effective development of MSC-based regenerative tissue engineering techniques for prevention and treatment of articular cartilage disorders and aid strategies for the optimization of such approaches for cartilage repair.

12. ACKNOWLEDGEMENTS

This work was supported by grants from the Hungarian Science Research Fund (OTKA CNK80709) and TÁMOP-4.2.1/B-09/KONV-2010-0007 project implemented through the New Hungary Development Plan, co-financed by the European Social Fund. C.M. is supported by a Mecenatura grant (DEOEC Mec-9/2011) from the Medical and Health Science Centre, University of Debrecen, Hungary. The authors acknowledge Prof. Pál Gergely, Prof. László Csernoch and Prof. László Kovács for their valuable help and comments in writing the manuscript.

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Abbreviations: 2-APB: 2-aminoethoxydiphenyl borate; ACI: autologous chondrocyte implantation; ADP: adenosine diphosphate; AMPA: alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; ARC: arachidonate-regulated Ca2+ channel; ATP: adenosine triphosphate; BMP: bone morphogenic protein; cAMP: cyclic adenosine monophosphate; CD-RAP: cartilage-derived retinoic acid-sensitive protein; CPA: cyclopiazonic acid; CRAC: Ca2+ release-activated Ca2+ channel; CREB: cAMP responsive element binding protein; ECM: extracellular matrix; ER: endoplasmic reticulum; ERK: extracellular signal-regulated kinase; ESC: embryonic stem cell; FGF: fibroblast growth factor; GAG: glycosaminoglycan; GPCR: G-protein coupled receptor; HDC: high density cell culture; iGluR: ionotropic glutatame receptor; IP3R: inositol-1,4,5-trisphosphate receptor; JNK: jun N-terminal kinase; MAPK: mitogen-activated protein kinase; MEK: mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; mGluR: metabotropic glutatame receptor; MSC: mesenchymal stem cell; N-CAM: neural cell adhesion molecule; NCX: Na+/Ca2+-exchanger; NFAT: nuclear factor of activated T-lymphocytes; NMDAR: N-methyl-D-aspartate type glutamate receptors; OA: osteoarthritis; PG: proteoglycan; PKA: protein kinase A; PKC: protein kinase C; PMCA: plasma membrane Ca2+ ATPase; PP1, PP2A and PP2B: phosphoprotein phosphatase 1, 2A and 2B; RyR: ryanodine receptor; SERCA: sarco/endoplasmic reticulum Ca2+ ATPase; SOCE: store-operated Ca2+ entry; STIM: stromal interaction molecule; TGF-beta: transforming growth factor-beta; TRP: transient receptor-potential channel; TRPA: ankyrin TRP; TRPC: canonical TRP; TRPM: melastatin TRP; TRPM: mucolipin TRP; TRPN: "no mechanoreceptor potential C" TRP; TRPP: polycystin TRP; TRPV: vanilloid TRP; UTP: uridine triphosphate; VOCC: voltage-operated Ca2+ channel

Key Words: Mesenchymal Stem Cells, Chondrocytes, High Density Cultures, Ca2+ toolkit, Oscillations, Ion Channels, Review

Send correspondence to: Roza Zakany, Department of Anatomy, Histology and Embryology, University f Debrecen MHSC, Nagyerdei krt 98, H-4032 Debrecen, Hungary, Tel: 36-52-255-567, Fax: 36-52-255-115, E-mail: roza@anat.med.unideb.hu