The role of Ca2+/Calcineurin/NFAT signalling pathway in osteoblastogenesis

Abstract The bone remodelling process is closely related to bone health. Osteoblasts and osteoclasts participate in the bone remodelling process under the regulation of various factors inside and outside. Excessive activation of osteoclasts or lack of function of osteoblasts will cause occurrence and development of multiple bone‐related diseases. Ca2+/Calcineurin/NFAT signalling pathway regulates the growth and development of many types of cells, such as cardiomyocyte differentiation, angiogenesis, chondrogenesis, myogenesis, bone development and regeneration, etc. Some evidences indicate that this signalling pathway plays an extremely important role in bone formation and bone pathophysiologic changes. This review discusses the role of Ca2+/Calcineurin/NFAT signalling pathway in the process of osteogenic differentiation, as well as the influence of regulating each component in this signalling pathway on the differentiation and function of osteoblasts, whereby the relationship between Ca2+/Calcineurin/NFAT signalling pathway and osteoblastogenesis could be deeper understood.

and development, such as regulating cardiomyocyte differentiation, chondrocyte differentiation, myocyte hypertrophy, angiogenesis, myogenesis, skeletal development and regeneration, etc. [8][9][10][11][12][13][14][15] Studies have shown that the (Down syndrome critical region 1) DSCR1 gene on chromosome 21 of patients with Down syndrome is overexpressed. DSCR1 gene is highly expressed in myocardium, striated muscle, neuronal cells and T cells, etc., the peptide expressed by this gene regulates CaN through competing with CaN to inhibit the CaN signalling pathway. 16,17 The symptom of skeletal dysplasia in Down syndrome patients is thought to be relevant to the overexpression of the DSCR1 gene. 18 Sun et al. 19 discovered that the level of bone formation of mice lacking the CaN Aα subtype was observably reduced and the mice showed osteoporosis. In addition, some studies reported that the activation of CaN was detected in the first batch of bone cells developed at the foetal stage 20 ; acute and rapid bone loss occurred after organ transplantation patients were treated with CaN inhibitors 19 ; mice and rats were treated with equivalent doses of calcineurin inhibitors, and increased bone resorption and bone loss could also be observed. 21

| OS TEOB L A S TOG ENE S I S A ND REL ATED S IG NALLING PATHWAYS
Osteoblasts are mainly derived from bone marrow mesenchymal stromal cells (BMSCs). BMSCs have the ability to differentiate into osteoblasts, adipocytes and chondrocytes, many transcription factors participate in the process of inducing BMSCs to differentiate into osteoblasts, such as runt-related transcription factor 2 (RUNX2), β-catenin and osteoblast-specific transcription factor (Osterix), etc. 24 After BMSCs are induced to differentiate into osteoblasts, they can secrete an uncalcified bone precursor composed of type I collagen, which is osteoid. 25 Subsequently, mature osteoblasts secrete vesicles, and the alkaline phosphatase (ALP) in the vesicles combines with calcium ions to form hydroxyapatite, thereby osteoid calcification is achieved. The cytoplasm of osteoblasts embedded in the osteoid reduces and the osteoblasts then transform into osteocytes. 26 In addition to the bone formation function, osteoblasts can also secrete a variety of cytokines and meet the needs of various physiological and pathological changes in autocrine and paracrine manners. 2,27 The differentiation of BMSCs into osteoblasts is regulated by a variety of signalling pathways, such as wingless-type MMTV integration site (Wnt), transforming growth factorβ/bone morphogenetic protein (TGFβ/BMP), Hedgehogs and fibroblasts growth factor (FGF) signalling pathways. 28 TGFβ/BMP increase the expression of RUNX2 by activating Smad and mitogen-activated protein kinase (MAPK) signalling pathways. [29][30][31] The active fragments of Hedgehogs can bind to the G protein-coupled receptor Smoothened (Smo), and also caused the increase of RUNX2 expression level by activating Smad. 28,32 FGF binds to its receptor to cause receptor dimerization, and promotes osteogenic differentiation by activating its downstream signalling pathways such as MAPK, JNK, PKC and PI3K. 33 In BMSCs, Wnt protein transmits signals through canonical and non-canonical pathways. 34 The canonical Wnt signalling pathway is mediated by β-catenin. Under unstimulated condition, β-catenin in the cytoplasm is phosphorylated by the complex of glycogen synthase kinase-3β (GSK-3β), adenomatous polyposis coli (APC) and Axin, and together form a degradation complex. The complex will be further ubiquitinated and degraded by the proteasome system. Wnt protein binds to Frizzled and low-density lipoprotein receptor related protein 5/6 (LRP5/6) receptors complex, causing inhibition of GSK-3β activity, allowing β-catenin to be released as a monomer and accumulate in the cytoplasm. 35,36 Then β-catenin translocates to the nucleus and induces the expression of its target genes such as RUNX2 and PPARγ. 24 The non-canonical Wnt signalling pathway also plays an important role in the recruitment, maintenance and differentiation of BMSCs, the Ca 2+ /CaN/NFAT pathway has been shown to be activated by the non-canonical Wnt signalling pathway during the differentiation of BMSCs into osteoblasts. [37][38][39] The secreted glycoprotein Wnt functions in the form of autocrine or paracrine. Frizzled on the cell membrane belongs to the G proteincoupled receptor, its N-terminal can bind to the Wnt protein, and then cause the activation of PLCγ, activated PLCγ increases inositol 1,4,5-triphosphate (IP3) level, and then promotes the release of Ca 2+ from ER into the cytoplasm by activating the IP3 receptor, and activates CaN by activating CaM to promote the nuclear translocation of NFAT. 37,40 BMSCs also express calcium-sensing receptor (CaSR), which activates PLCγ in response to increase in extracellular Ca 2+ concentration, thereby producing IP3, promoting the release of Ca 2+ from ER and causing the increase in intracellular Ca 2+ concentration. 41  other osteogenic-related genes transcription and then promote osteogenic differentiation. 46 Figure 1 exhibits the process of osteoblasts differentiation, which Ca 2+ /CaN/NFAT signalling pathway involved in.

| Ca 2+ /CaN/NFAT signalling pathway
Increased intracellular concentration of Ca 2+ can activate CaN by interacting with calmodulin (CaM). 47 CaM is dumbbell-shaped, its C-terminus and N-terminus each contain a globular domain, and the two globular domains are connected by a flexible helical joint region. Each globular domain of CaM has a pair of Ca 2+ binding sequences, and after binding Ca 2+ , a hydrophobic surface is exposed. 15 This hydrophobic surface can bind to a variety of CaM target proteins, including CaN. CaN is widely expressed in brain, lung, skeletal muscle, heart valve, myocardium, kidney, spleen, bone and other tissues, [48][49][50] it is a type of serine/threonine phosphatase, and is a heterodimer, which is structurally composed of catalytic subunits (CnA) and regulatory subunit (CnB). 51 CaN to be activated. 53 Activated CaN can dephosphorylate multiple substrates, including NFAT. 44 NFAT contains a few domains, the regulatory domain of which are highly phosphorylated under the inactive state, which covers the nuclear localization sequence and makes the NFAT protein to remain in the cytoplasm. 51  F I G U R E 1 Concise schematic diagram of Ca 2+ /CaN/NFAT signalling pathway involved in the process of osteoblast differentiation. The combination of Wnt and the N-terminal of Frizzled causes the activation of PLCγ and increases the level of IP3, which in turn activates the IP3 receptor to promote the release of Ca 2+ from ER into the cytoplasm, and activates CaN by CaM to promote the nuclear translocation of NFAT. CaSR activates PLCγ in response to an increase in the extracellular Ca 2+ concentration to cause an elevation in the intracellular Ca 2+ concentration. In addition, the storage depletion of Ca 2+ in ER can also lead to Ca 2+ influx through Orai1/STIM1. L-VGCC and NCX all regulate Ca 2+ influx, which further results in the activation of the Ca 2+ /CaN/NFAT signalling pathway. NFAT and Osterix form transcription complexes, which in turn, trigger the transcription of osteoblast-related genes The Na + /Ca 2+ exchanger (NCX) mediates Ca 2+ flowing into the cells mainly through reverse exchange, 66 and plays its role on the surface of osteoblasts, regulates the concentration of Ca 2+ in osteoblasts and promote bone matrix mineralization. 57,67 The CaSR belonging to the G protein-coupled receptors can sense the extracellular Ca 2+ concentration and instantaneously mobilize intracellular Ca 2+ flux. 68 Previous studies have shown that CaSR signals mediate the osteogenic differentiation of BMSCs in vitro and bone formation in vivo, and CaSR agonists can promote the proliferation, differentiation and matrix mineralization of osteoblasts. 41,69,70 In addition, Ca 2+ influx caused by mechanically sensitive channels also promotes osteogenic differentiation 71 ; Orai1 gene knockout leads to impaired osteoblast differentiation and mineralization. 43 Overexpression of Pannexin 3, which is the Ca 2+ channel of ER, can increase the intracellular Ca 2+ concentration to promote osteogenic differentiation, 72 and the depletion of Ca 2+ in the ER induced by IP3 causes STIM1 to accumulate at the junction of the ER and cell membrane, STIM1 interacts with Orai1 protein and activates the SOC channel, causing Ca 2+ influx, 73 this process has also been demonstrated to take a part in dental pulp cells (DPCs) osteogenic differentiation and mineralization. 57,74 In addition, in the process of Ca 2+ regulating osteogenic differentiation, there are also crosstalks between various Ca 2+ channels. inhibited. 78 When the extracellular Ca 2+ concentration increases to 50 mM, it will hinder the normal adhesion of cells. 79 Therefore, proper concentration of Ca 2+ treatment can enhance osteogenic differentiation and mineralization, but excessively high concentrations of Ca 2+ may disrupt Ca 2+ homeostasis and cause abnormal cell function.

| Calmodulin in osteoblastogenesis
Calmodulin is regulated by intracellular Ca 2+ and activates a variety of downstream target proteins after binding Ca 2+ . It is precisely because there are many types of CaM downstream target proteins, the cell functions that CaM participates in are also diverse, such as inflammation, metabolism, apoptosis and so on. 80 Conversely, CaM can also affect intracellular Ca 2+ flux by regulating Ca 2+ channels, such as through IP3R and P/Q type calcium channel. 15 CaM participates in the process of parathyroid hormone (PTH) and vitamin D3 in regulating osteoblast differentiation through Ca 2+ signals, 81 and Smad1 in the BMP signalling pathway can directly bind to CaM, so that the activity of Smad1 is increased, thereby promoting osteogenic differentiation. 82 Trifluoperazine, a CaM inhibitor, is demonstrated to inhibit the osteogenic differentiation of MC3T3-E1 cells and bone formation in mice, and has a dose-dependent inhibitory effect on the activity of ALP in rat skull. 83,84

| Calcineurin in osteoblastogenesis
The calcium-sensitive protein CaM can activate calcineurin under the condition of low and continuously increasing intracellular Ca 2+ concentration. 85 The activation of CaN can affect a variety of physi-  89 and it is reported that Endothelin-1 (ET-1) activated CaN signalling pathway when acting as an anti-apoptotic factor for osteoblasts. 90 Therefore, the exact conclusions and specific mechanisms of CaN inhibitors regulating osteoblast differentiation need to be further studied. It is worth noting that many studies have also mentioned the influence of osteoblast function when they reported that osteoblast differentiation is regulated by CaN signalling pathway, and they all claimed that the impacts on osteoblast function are the same as those on osteoblast differentiation, but they did not first culture mature osteoblasts and then regulate CaN signalling pathway, instead, they directly analysed the changes in osteoblast function through the mineralization level of osteoblasts whose differentiation degree has been altered. Therefore, the conclusions about the regulation of osteoblast function by CaN signalling pathway is not precise.

| Nuclear factor of activated T cell in osteoblastogenesis
In the inactive state, NFAT protein localizes in the cytoplasm due to the hyperphosphorylation of its N-terminal regulatory domain. After Ca 2+ activates CaN through CaM, CaN dephosphorylates NFAT and exposes the nuclear localization sequence to cause its nuclear translocation. 91 In the nucleus, NFAT acts as a transcription factor to promote the transcription of target genes and NFATc1 itself. It can be inferred that in the Ca 2+ /CaN/NFAT pathway, CaN not only regulates the dephosphorylation and nuclear translocation of NFAT but also enhances its expression; therefore, NFAT as a transcription factor can cause its own self-amplification effect. 18 Some previous studies reported that CaN/NFAT had a positive regulatory effect on osteogenic differentiation, and confirmed that the overexpression of NFAT in vivo and in vitro could promote osteogenic differentiation, 45 after expressing the constitutively active variant of NFATc1 in osteoblasts in mice, the mice showed increased bone mass. 92 Similarly, mice lacking NFAT had reduced bone formation and low bone mass, 45 the inactivation of NFATc1 and NFATc2 markedly inhibited the differentiation and function of osteoblasts. 93 Besides, the promoter of the Fra-2 gene contains three potential NFAT consensus sequences, and the combination of NFAT with Fra-2 will cause the negative regulation of Fra-2, 86

| DIVERSE C A 2+ /C AN/NFAT S IG NALLING PATHWAY MODUL ATING COMP OUNDS , WHI CH REG UL ATE OS TEOG ENI C D IFFERENTIATI ON
Decreased differentiation or dysfunction of osteoblasts will lead to a variety of skeletal diseases. The Ca 2+ /CaN/NFAT signalling pathway has been shown to be closely related with the physiological activities of osteoblasts. We have summarized compounds that have a regulatory effect on this signalling pathway and at the same time modulate osteoblastogenesis, aiming to provide new ideas for the exploration of treatment options for osteogenesis-related diseases. Table 1

| Zinc
Zinc is essential in the process of skeletal development, 1-50 μM zinc has been shown to inhibit osteoblast apoptosis and promote the proliferation and differentiation of osteoblasts, 98   However, in colon carcinoma cells, cyanidin inhibited the increase in intracellular Ca 2+ level caused by neurotensin, 108

| Apocynin
The inhibitor of NADPH oxidase, apocynin, is a kind of methoxysubstituted catechol. When MC3T3-E1 cells are exposed to antimycin A and resulting in excessive ROS production, 0.01-1 μM apocynin can scavenge ROS, protect MC3T3-E1 cells and promote their osteogenic differentiation. 118 In BMMs, apocynin reduces Ca 2+ influx by blocking Ca 2+ channels except the two pore channel 2 (TPC2) and

| Trifluoperazine
Trifluoperazine (TFP) can inhibit the activity of CaM and further restrain the activation of CaMKII. 10 μM TFP inhibits osteogenic differentiation, and also shows the ability to reduce the formation and mineralization of osteoblasts in the calvarial model of mouse pups. 83 Komoda et al. 84 also confirmed the inhibitory effect of TFP on ALP activity in rat calvaria and its inhibitory effect on the proliferation and osteogenic differentiation of MC3T3-E1 cells in vitro.

| Cyclosporin A and FK506
Cyclosporin A and FK506 are CaN inhibitors and are widely used to reduce rejection reaction after organ transplantation. Low concentrations of CsA (less than 1 μM in vitro and 35.5 nM in vivo) have been shown to increase the expression of Fra-2 to promote the transcription of osteogenic genes, thereby promoting osteogenic differentiation. 86,87 High concentrations of CsA (more than 1 μM in vitro and in vivo) inhibit osteogenic differentiation and bone formation. 86 Similarly, low concentrations of FK506 (less than 1 μM in vitro) promote osteogenic differentiation, 88 whilst high concentrations of FK506 can also reduce BMP-2 induced osteogenic differentiation both in vivo and in vitro. 45 The osteoinhibitory effect of high concentration of CsA and FK506 is believed to be exerted by inhibiting the formation of NFAT-Osterix-DNA complex. Over the years, the relationship between Ca 2+ /CaN/NFAT signalling pathway and bone metabolism has been explored in many ways, this signalling pathway has a wide range of effects on cell fate, and the mechanisms involved are far-reaching. There are still many unknown or unexplained relationships between Ca 2+ /CaN/NFAT signalling pathway and osteoblastogenesis, further exploration in this field is needed to broaden the way for the study of bone formation regulation and bone-related diseases development.

ACK N OWLED G EM ENTS
This work was funded by National Natural Science Foundation of China (Nos. 82072405 and Nos. 81571816).

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no conflict of interest.

AUTH O R CO NTR I B UTI O N S
W.X. and R.R. conceived the aims and structure of the review. R.R. and J.G. collected the articles and wrote the original draft of the manuscript. Y.C., Y.Z. and L.C. reviewed and edited the manuscript.
W.X. acquired the funding. All authors have read and agreed to the published version of the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All the data are available from the corresponding author by request.