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Role of K+ and Ca2+-Permeable Channels in Osteoblast Functions

Bone-forming cells or osteoblasts play an important role in bone modeling and remodeling processes. Osteoblast differentiation or osteoblastogenesis is orchestrated by multiple intracellular signaling pathways (e.g., bone morphogenetic proteins (BMP) and Wnt signaling pathways) and is modulated by the extracellular environment (e.g., parathyroid hormone (PTH), vitamin D, transforming growth factor β (TGF-β), and integrins). The regulation of bone homeostasis depends on the proper differentiation and function of osteoblast lineage cells from osteogenic precursors to osteocytes. Intracellular Ca2+ signaling relies on the control of numerous processes in osteoblast lineage cells, including cell growth, differentiation, migration, and gene expression. In addition, hyperpolarization via the activation of K+ channels indirectly promotes Ca2+ signaling in osteoblast lineage cells. An improved understanding of the fundamental physiological and pathophysiological processes in bone homeostasis requires detailed investigations of osteoblast lineage cells. This review summarizes the current knowledge on the functional impacts of K+ channels and Ca2+-permeable channels, which critically regulate Ca2+ signaling in osteoblast lineage cells to maintain bone homeostasis.


Introduction
Bone modeling initially occurs during skeletal development and comprises two modes: intramembranous ossification and endochondral ossification [1]. During intramembranous ossification, mesenchymal cells are directly converted to osteoblasts, whereas during endochondral ossification, mesenchymal cells are differentiated into cartilage cells before conversion into osteoblasts [1]. Preosteoblasts derived from multipotent mesenchymal osteoprogenitor cells further differentiate into mature osteoblasts according to osteogenic signals. Mature osteoblasts are responsible for bone formation in bone modeling and remodeling. Therefore, the proliferation and differentiation of osteoblast lineage cells play an important role in the regulation of bone homeostasis [2].
Various growth factors and signaling molecules contribute to osteoblastogenesis. For instance, bone morphogenetic proteins (BMP) belonging to the transforming growth factor β (TGF-β) superfamily have a pivotal role in controlling osteoblast differentiation [3]. BMP signaling involves both canonical and non-canonical pathways. Canonical signaling is a SMAD-dependent pathway involving three types of SMADs: receptor-regulated SMADs (R-SMADs), common-mediator SMADs (Co-SMADs), and inhibitory-SMADs. The activation of type I BMP receptors phosphorylates R-SMADs (SMAD1, 5, and 8) to form a heterotrimeric complex with Co-SMAD, SMAD4. In the nucleus, this SMAD complex acts as a transcription factor to regulate the expression of BMP target genes [4]. On the other hand, non-canonical signaling activates SMAD-independent pathways, such as mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K)/Akt [5]. Additionally, integrins, which are heterodimeric transmembrane cell adhesion complexes composed of α and β chains, are expressed in osteoblasts and contribute to bone formation [6]. Integrins promote osteoblast differentiation by interacting with major extracellular matrix mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K)/Akt [5]. Additionally, integrins, which are heterodimeric transmembrane cell adhesion complexes composed of α and β chains, are expressed in osteoblasts and contribute to bone formation [6]. Integrins promote osteoblast differentiation by interacting with major extracellular matrix components, such as fibronectin and collagen type I (Col1) [7]. The activation of integrin signaling triggers osteogenesis, indicated by the induction of osteoblast genes, such as the transcription factor Runt-related transcription factor 2 (Runx2), phosphorylation of focal adhesion kinase (FAK), and extracellular signal-regulated kinase 1/2 (ERK1/2) [8]. Runx2 forms heterodimers with the core binding factor β (CBFβ) to control the expression of osteoblast marker genes including Col1a1, Spp1, Ibsp, Bglap2, and Fn1, and regulates multiple steps during osteoblast differentiation [9].
In many cases, [Ca 2+ ] i mobilization is controlled by intracellular Ca 2+ store activation and Ca 2+ influx. Membrane hyperpolarization induced by K + channel activation increases the electromotive driving force of Ca 2+ entry through voltage-independent, store-operated, Ca 2+ -permeable channels in non-excitable cells [17][18][19]. Therefore, the membrane potential is one of the key biophysical signals in osteoblast lineage cells [20]. The present review summarizes the most recent evidence regarding K + channels and Ca 2+ -permeable channels (Orai/stromal interaction protein (STIM), transient receptor potential (TRP), and mechanosensing Piezo subfamilies) in osteoblast lineage cells, which are a group of cells comprising mesenchymal progenitors, preosteoblasts, osteoblasts (often called mature osteoblasts), bone-lining cells, and osteocytes, focusing on their role in skeletal development.

Regulatory Mechanism of Ca 2+ Signaling by Membrane Potential in Non-Excitable Cell
[Ca 2+ ] i is a ubiquitous second messenger regulating various cellular functions, such as proliferation, secretion, differentiation, migration, and apoptosis [21]. In quiescent cells, [Ca 2+ ] i is maintained at very low levels (50-100 nM). The stimulation of cells with agonists, such as cytokines, hormones, or growth factors, results in [Ca 2+ ] i rises via Ca 2+ influx and/or Ca 2+ release from the ER and mitochondria. In electrically non-excitable cells, including osteoblast lineage cells, membrane hyperpolarization increases the driving force for Ca 2+ signaling. A direct link has been demonstrated between membrane hyperpolarization and Ca 2+ signaling in epithelial cells [22], endothelial cells [23], cancer cells [19], microglia [24], and many types of immune cells [25]. The main Ca 2+ entry route in these cells is not through voltage-gated Ca 2+ channels but through Orai or TRP channels with high Ca 2+ permeability. Membrane hyperpolarization promotes osteogenic differentiation in human mesenchymal stem cells (MSCs) [20], and the K + channels play a pivotal role in the regulation of the membrane potential hyperpolarization. Therefore, a functional analysis of K + channels may provide crucial insights into the functions of osteoblast lineage cells ( Figure 2).
In many cases, [Ca 2+ ]i mobilization is controlled by intracellular Ca 2+ store activation and Ca 2+ influx. Membrane hyperpolarization induced by K + channel activation increases the electromotive driving force of Ca 2+ entry through voltage-independent, store-operated, Ca 2+ -permeable channels in non-excitable cells [17][18][19]. Therefore, the membrane potential is one of the key biophysical signals in osteoblast lineage cells [20]. The present review summarizes the most recent evidence regarding K + channels and Ca 2+ -permeable channels (Orai/stromal interaction protein (STIM), transient receptor potential (TRP), and mechano-sensing Piezo subfamilies) in osteoblast lineage cells, which are a group of cells comprising mesenchymal progenitors, preosteoblasts, osteoblasts (often called mature osteoblasts), bone-lining cells, and osteocytes, focusing on their role in skeletal development.

Regulatory Mechanism of Ca 2+ Signaling by Membrane Potential in Non-Excitable Cell
[Ca 2+ ]i is a ubiquitous second messenger regulating various cellular functions, such as proliferation, secretion, differentiation, migration, and apoptosis [21]. In quiescent cells, [Ca 2+ ]i is maintained at very low levels (50-100 nM). The stimulation of cells with agonists, such as cytokines, hormones, or growth factors, results in [Ca 2+ ]i rises via Ca 2+ influx and/or Ca 2+ release from the ER and mitochondria. In electrically non-excitable cells, including osteoblast lineage cells, membrane hyperpolarization increases the driving force for Ca 2+ signaling. A direct link has been demonstrated between membrane hyperpolarization and Ca 2+ signaling in epithelial cells [22], endothelial cells [23], cancer cells [19], microglia [24], and many types of immune cells [25]. The main Ca 2+ entry route in these cells is not through voltage-gated Ca 2+ channels but through Orai or TRP channels with high Ca 2+ permeability. Membrane hyperpolarization promotes osteogenic differentiation in human mesenchymal stem cells (MSCs) [20], and the K + channels play a pivotal role in the regulation of the membrane potential hyperpolarization. Therefore, a functional analysis of K + channels may provide crucial insights into the functions of osteoblast lineage cells ( Figure 2).

Figure 2.
In non-excitable cells, voltage-independent Ca 2+ channels are the main Ca 2+ -permeable channels. Membrane hyperpolarization followed by K + channel activation increases the driving Figure 2. In non-excitable cells, voltage-independent Ca 2+ channels are the main Ca 2+ -permeable channels. Membrane hyperpolarization followed by K + channel activation increases the driving force for Ca 2+ influx. Consequently, K + channels indirectly regulate osteoblast functions, including proliferation, differentiation, and mineralization, by controlling intracellular Ca 2+ signaling in osteoblast lineage cells.

K + Channel Superfamilies in Osteoblast Lineages
K + channels are particularly important in maintaining the resting membrane potential and determining the shape and duration of the action potential. K + channels may also regulate the cell volume, proliferation, differentiation, and migration of a wide range of cell types including osteoblast lineage cells (Table 1). Approximately 80 members are classified into voltage-gated K + (K V ) channel superfamily, inward-rectifier K + (Kir) channel superfamily, Ca 2+ -activated K + (K Ca ) channel superfamily, and two-pore domain K + (K 2P ) channel superfamily [26] (Figure 3). MC3T3-E1 cells prolifeataion [40] two-pore K + channel K 2P 2.1 human osteoblasts and MG-63 cells functional expression [41] K 2P 3.1, K 2P 5.1, K 2P 9.1 MG-63 cells prolifeartion [42] K V channel is a tetramer composed of four identical subunits consisting of six transmembrane (TM) (S1-S6) domains with a pore region (S5-P-S6), in which seven amino acidic residues, TTVGYGD that forms the structure of the ion-selectivity filter. K V channels are also characterized by containing a voltage-sensor domain in which the S4 contains positively charged amino acids that constitute the voltage-sensing elements. Kir channel is also a tetramer composed of four identical subunits consisting of two transmembrane domains connected by a pore domain, in which the ion-selectivity filter with the characteristic TXGYG signature sequence. K Ca 1.1 channels have seven transmembranes (S0-S6). K Ca 2 and K Ca 3.1 channels have six transmembranes (S1-S6) like K V channels but also contain an intracellular domain to bind CaM. The pore domain of the K Ca 1.1 channel is assigned to the region contained between S5 and S6 segments, which include the signature sequence of TVGYG. K Ca 2 and K Ca 3.1 channels have six TM segments and a pore loop region (between S5 and S6) containing the characteristic signature sequence GYGD. Then, K 2P channel subunits are composed of four TM segments and two pore domains with the characteristic TxGy/FG motif; the 4TM/2P structure defines the membership in the K 2P channel family [26]. 5   KV channel is a tetramer composed of four identical subunits consisting of six transmembrane (TM) (S1-S6) domains with a pore region (S5-P-S6), in which seven amino acidic residues, TTVGYGD that forms the structure of the ion-selectivity filter. KV channels are also characterized by containing a voltage-sensor domain in which the S4 contains positively charged amino acids that constitute the voltage-sensing elements. Kir channel is also a tetramer composed of four identical subunits consisting of two transmembrane domains connected by a pore domain, in which the ion-selectivity filter with the characteristic TXGYG signature sequence. KCa1.1 channels have seven transmembranes (S0-S6). KCa2 and KCa3.1 channels have six transmembranes (S1-S6) like KV channels but also contain an intracellular domain to bind CaM. The pore domain of the KCa1.1 channel is assigned to the region contained between S5 and S6 segments, which include the signature Figure 3. Phylogenetic tree topology of K + channels, voltage-gated K + (K V ) channels (A), Ca 2+ -activated K + channels (B), inward-rectifier K + (Kir) channels (C), and two-pore K + (K 2P ) channels (D).

Voltage-Gated K + (K V ) Channel Superfamily
The K V channel superfamily is the largest group of K + channel superfamilies. K V channels represent a diverse group of membrane proteins with 12 distinct subfamilies (K V 1-K V 12). K V channels are well-known for regulating electrophysiological processes such as action potential generation and excitation-contraction coupling in excitable cells, such as neurons and myocytes [43]. In excitable cells, hyperpolarization followed by K V channel activation reduces [Ca 2+ ] i levels by voltage-gated Ca 2+ channel blockades, thereby inhibiting neurotransmission and muscle contraction. In contrast, in non-excitable cells, hyperpolarization followed by K V channel activation increases [Ca 2+ ] i levels by facilitating Ca 2+ influx through voltage-independent, store-operated Ca 2+ -permeable channels, thereby promoting cell proliferation, migration, and differentiation [43]. K V channels also influence cell cycle progression through cell volume regulation [43].
Yang et al. [27] showed that treatment with tetraethyl ammonium, a K V channel inhibitor, increased the matrix mineralization of human bone marrow-derived mesenchymal stem cells (BMMSCs) during osteogenic differentiation. The pharmacological blockade of K V 7.3 with linopirdine augmented the mineralization during osteoblast differentiation of osteoblast-like MG-63 and Saos-2 cells through the release of glutamate [27]. Furthermore, several studies have shown that K V 10.1 (also referred to as ether-à-go-go) enhanced the proliferation of MG-63 and Saos-2 cells [28,29]. The functional expression of the K V 2.1 channel substantially contributed to 17β-estradiol-sensitive K + currents in MG-63 cells [30].

Inward-Rectifier K + (K ir ) Channel Superfamily
The K ir channel superfamily is best known for its role in maintaining resting membrane potential in cardiac/vascular myocytes, neurons, and pancreatic β cells, regulating muscle contraction/relaxation, action potential firing, and insulin release [44]. K ir channels also regulate bone and cartilage development, as reported by Ozekin et al. [45].
Andersen-Tawil syndrome (ATS) is caused by mutations in the KCNJ2 gene, which encodes K ir 2.1, in patients exhibiting cardiac arrhythmia, periodic paralysis, dental defects, cleft lip/palate, micrognathia, hypertelorism, low-set ears, and limb patterning defects. The disruption of K ir 2.1 also leads to limb defects, hypoplastic craniofacial structures, and cleft palate in mice, craniofacial defects in Xenopus laevis, and morphological defects in Drosophila [46][47][48]. Recent studies showed that K ir 2.1 disruption inhibited mammalian facial development by inhibiting BMP signaling [31]. Sacco et al. [32] demonstrated the role of K ir 2.1 in osteoblast during osteogenesis. Osteoblasts derived from healthy control participants were able to produce bone matrix; however, osteoblasts from ATS patients, as well as in K ir 2.1 knockout mice, were unable to synthesize bone matrix [32]. On the other hand, the loss of K ir 2.1 function induced the dephosphorylation of SMAD and inhibited the BMP signaling pathway, resulting in impaired osteoblastogenesis [49].
ATP-sensitive K + (K ATP ) channels, which are heteromultimers (1:1) of K ir 6.x, and sulfonylurea receptors, regulate a variety of cellular functions by coupling cell metabolism with membrane potential [44]. Osteogenic differentiation of MSCs strongly upregulated Kir6.2, whereas Kir6.1 and SUR2A showed no significant change. The elevated Kir6.2 expression may contribute to in vitro differentiation of MSC according to the metabolic state of the developing tissue [33]. K ATP channels are related to alkaline phosphatase activity and bone mineralization in rat primary osteoblast cells [34]. The activation of K ATP channels protects osteoblasts from high-glucose-induced injury by promoting osteoblast proliferation and preventing apoptosis [34].

Ca 2+ -Activated K + (K Ca ) Channel Superfamily
The K Ca channel superfamily consists of eight members: K Ca 1.1/BK, K Ca 2.1/SK1, K Ca 2.2/SK2, K Ca 2.3/SK3, K Ca 3.1/SK4/IK, K Ca 4.1/K Na 1.1, K Ca 4.2/K Na 1.2, and K Ca 5.1. Due to sequence similarity in the pore region and C-terminus CaM-binding domain, the small-conductance (K Ca 2.1, K Ca 2.2, K Ca 2.3) and intermediate-conductance K Ca (K Ca 3.1) channels belong to the same genetic lineage (KCNN genes) [50]. Distinct from K Ca 2.x and K Ca 3.1, the large-conductance K Ca (K Ca 1.1) has voltage-dependent gating characteristics with sequence similarities to K V channels. K Ca 1.1 has unique Ca 2+ -binding domains at the C-terminus, RCK1/2 domains (referred to as "Ca 2+ bowls"), required for Ca 2+ -dependent channel activation [50].
K Ca 1.1 is activated by depolarization and [Ca 2+ ] i elevation. Due to these properties, the K Ca 1.1 activation results in repolarization and the closing of voltage-dependent Ca 2+ channels in neurons and smooth muscle cells [51,52]. Therefore, K Ca 1.1 serves as a negative feedback regulator of membrane potential and [Ca 2+ ] i in excitable cells. In contrast, K Ca 2.2 activation hyperpolarizes and thereby promotes Ca 2+ influx through TRPC in brain capillary endothelial cells [53]. K Ca 3.1 activation results in elevated [Ca 2+ ] i in human cancer cells [19,54] and immune cells [25]. Taken together, K Ca channels have an important role in the positive feedback mechanism in Ca 2+ signaling in non-excitable cells. K Ca 1.1 especially plays a critical role in osteoclast activation. For instance, K Ca 1.1 deficiency in mice induced osteopenia due to enhanced osteoclast resorption subsequent to the autonomous release of cathepsin K [55]. K Ca 1.1 is also functionally expressed in human malignant osteoblast-like osteosarcoma cells (SaM-1, MG-63, and SaOS-2) and osteogenic precursor cells (C1) [35].
Hei et al. [36] showed that K Ca 1.1-null mice osteoblasts significantly decreased the ability for proliferation and mineralization, thereby decreasing bone mineral density and trabecular bone volume of the tibia and lumbar vertebrae. Integrin-linked kinases regulate bone formation and repair. K Ca 1.1 proteins interact with integrin β1 in osteoblasts, and the integrin-mediated activation of MAPK-ERK and FAK signaling pathways promotes osteogenesis-related expression of genes, such as Runx2. The blockade of integrin signaling by K Ca 1.1 knockdown in BMMSCs caused impaired bone formation and osteoblast differentiation [37].
Vascular calcification is a hallmark of cardiovascular disease with chronic kidney disease. One of the key events is the transition of contractile vascular smooth muscle cells (VSMCs) into a non-contractile osteoblast-like phenotype. The activation of K Ca 1.1 channels ameliorated vascular calcification via Akt/FoxO1 signaling pathways in calcified VSMCs [38]. On the other hand, K Ca 3.1 channels are expressed in proliferative undifferentiated VSMCs [56]. The pharmacological blockade of K Ca 3.1 inhibited the calcification medium (Ca 2+ /PO 4 3− )-induced upregulation of osterix and osteocalcin in VSMCs and suppressed mineralization during VSMC calcification by simultaneously enhancing osteopontin expression [39] and by interfering with TGF-β and nuclear factor-kappa B (NF-κB) signaling [39].
Many studies have firmly established the important roles of vitamin D (VD) in calcium metabolism and bone development [57]. The VD endocrine system has a beneficial effect on bone homeostasis, but VD receptor (VDR) stimulation has direct positive or negative effects on bone mass depending on the osteoblast development stage. Yamamoto et al. [58] showed that VDR in osteoblasts served as a negative regulator of bone homeostasis due to osteoclastogenesis induced by the receptor activator of NF-κB ligand, RANKL. On the other hand, Sooy et al. [59] reported that osteoblast differentiation was promoted in calvarial osteoblasts obtained from VDR-deficient mice. Our recent study showed that the functional activity of K Ca 3.1 positively regulated the proliferation of mouse preosteoblast MC3T3-E1 cells by enhancing Ca 2+ signaling [40]. K Ca 3.1 activity was suppressed in cells treated with VDR agonists following the downregulation of transcriptional/epigenetic modulators of K Ca 3.1, such as Fra-1 and HDAC2. Conclusively, a VDR agonist-induced decrease in K Ca 3.1 activity suppressed cell proliferation in mouse preosteoblasts. Therefore, the combined treatment of K Ca 3.1 activators and vitamin D preparations may enhance therapeutic effects on bone disorder involving bone mass reduction.

Two-Pore Domain K + (K 2P ) Channel Superfamily
The background leak K 2P channel subfamily is involved in diverse physiological functions such as ion homeostasis, hormone secretion, cell development, and excitability and is modulated by mechanical stretch, heat, intracellular/extracellular pH, lipids, and temperature [60]. The human K 2P channel subfamily consists of 15 members possessing four transmembrane segments and two-pore-forming domains and assembling as dimers [61]. K 2P 2.1 (tandem of P-domains in a weakly inward rectifying K + (TWIK)related K + channel 1, TREK1), a mechanosensitive K 2P channel member, has been highly expressed in excitable cells [62]. TREK1 is also expressed in human primary osteoblasts and MG-63 cells and is involved in the maintenance of resting membrane potential in human osteoblasts [41]. Of the15 K 2P members, five (K 2P 3.1 (TWIK-related acid-sensitive K + channel 1, TASK1), K 2P 5.1 (TASK2), K 2P 9.1 (TASK3), K 2P 16.1 (TWIK-related alkaline pH-activated K + channel 1, TALK1), and K 2P 17.1 (TALK2)) are known to be influenced by extra-and intracellular pH changes [63]. Acid-sensitive K 2P channels (K 2P 3.1, K 2P 5.1, and K 2P 9.1), especially, are functionally expressed in MG-63 cells. The extracellular acidosis-induced inhibition of acid-sensitive K 2P channel activity reduced MG-63 cell proliferation, indicating that these channels are associated with osteoblast proliferation [42]. The impacts of K 2P channels on significant roles in native osteoblast lineages have not been established. However, Cid et al. [64] showed that K 2P 5.1 was expressed in hyaline cartilage from the trachea and the articular surface of the knee. Further studies are expected to provide evidence that K 2P channels have physiological and pathophysiological roles in bone/cartilage homeostasis.

Orai/STIM
Orai1 is a pore-forming subunit of the Ca 2+ release-activated Ca 2+ (CRAC) channel that mediates Ca 2+ influx in most non-excitable cells via a store-operated Ca 2+ entry (SOCE) mechanism [95]. [Ca 2+ ] i changes are important signaling pathways in the regulation of osteoblast linage function [10], but causal roles remain highly controversial.
Studies on Orai1-deficient mice provided the first evidence of the importance of Orai1 for osteoblastic bone formation. The genetic deletion of Orai1 impaired bone development in the mice and resulted in osteopenia due to defective osteoblastic bone formation [65,66]. In addition, Choi et al. [67] showed that Orai1 contributed to the proliferation and differentiation of osteoblast lineage cells at various stages of maturation and that Orai1 deficiency attenuated the proliferation, differentiation, and Col1 secretion of mesenchymal progenitors and primary calvarial osteoblasts in Orai1-deficient mice. Orai1 deficiency also decreased the expression of an osteoid osteocyte marker gene, phosphate-regulating gene with homologies to endopeptidases on the X chromosome (Phex), a mineralizing osteocyte marker gene, dentin matrix protein 1 (DMP1), and a mature osteocyte marker gene, fibroblast growth factor 23 (FGF23) in mouse cortical long bone [67]. Taken together, Orai1 plays a crucial role in the differentiation of osteoblast lineage cells from mesenchymal progenitors into osteocytes.
Additionally, Liu et al. [68] showed that overexpression of Orai1 promoted human cartilage-derived MSC differentiation to osteoblasts. Lee et al. [69] reported on the Orai1 contribution to osteogenic effects of BMP2. Differentiation of BMMSCs into bone-forming osteoblasts requires the orchestrated regulation of signaling pathways, such as BMP signaling [96]. BMP signaling induces SMAD1/5/8-dependent signal transduction via BMP receptor types I and II, which activates the expression of Runx2, osterix, and osteoblast differentiation markers [97]. BMP-induced osteogenic conditions failed to induce increased osteogenic differentiation markers in BMMSCs of Orai1-deficient mice. These results suggest that Orai1 regulates osteogenic differentiation through BMP signaling. Interestingly, constitutively activated BMP signaling reversed the inhibition of osteogenic differentiation in Orai1-deficient mice. Collectively, BMP signaling is the downstream pathway of Orai1-mediated osteogenic differentiation. FGF23, mainly produced by osteocytes and osteoblasts, is a pivotal regulator of renal functions such as phosphate/calcium handling and vitamin D 3 homeostasis [98][99][100]. SOCE through Orai1 induces FGF23 production in rat osteoblast-like UMR106 cells [70,71]. Glosse et al. [101] showed that the blockade of SOCE decreased upregulated FGF23 expression induced by AMP-dependent kinase (AMPK) inhibition. Therefore, inhibitory AMPK-mediated FGF23 production may be regulated by Orai1-mediated SOCE activity in osteoblasts [101].
Peroxisome proliferator-activated receptors (PPARs) are nuclear transcriptional factors and are composed of PPARα, PPARβ/δ, and PPARγ [102]. PPARα is expressed in many organs, including bone [103]. Stimulation with a PPARα agonist induces AMPK activation in osteoblast lineage cells, resulting in a decrease in SOCE activity, whereas the pharmacological and genetic blockade of PPARα promotes FGF23 expression [104]. Thus, PPARα activation downregulated FGF23 expression through the AMPK-mediated suppression of SOCE in osteoblasts.
The ER Ca 2+ sensor STIM1 is an essential component of the SOCE process. Overexpression of STIM1 in MC3T3-E1 cells promotes osteoblast differentiation and matrix mineralization with the upregulation of osteogenic markers, such as Runx2, Col1, and BMP4 [72]. Therefore, the loss of STIM1 function leading to SOCE dysregulation may be associated with impaired ER Ca 2+ signaling in several skeletal-and tooth-related disorders [105]. Furthermore, gain-of-function mutations of Orai1 and STIM1 gave rise to tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK), forming a clinical spectrum encompassing muscle weakness, myalgia, and cramps, and additional multi-systemic signs, including short stature [106]. Silva-Rojas et al. [107] generated a mouse model carrying the most recurrent STIM1 gain-of-function mutation, STIM1 R304W, found in patients with TAM/STRMK. Moreover, microcomputed tomography analyses revealed a severe skeletal phenotype with thinner and more compact bone in STIM1 R304W mice [107,108]. Increased osteoclastogenesis was observed in bone marrow cells derived from patients with TAM/STRMK [109]. Bone formation and resorption are intricately balanced processes driven by bone-forming osteoblasts and bone-resorbing osteoclasts. Therefore, to gain further in vivo information on the contributions of SOCE activity to osteoblastogenesis, the role of SOCE activity in osteoclastogenesis needs to be comprehensively examined.

TRPM Channels
Shear stress is physiologically generated by blood flow and interstitial fluid [112]. Fluid flow is a potent stimulator of osteoblasts and generally can be of three types: (1) steady (fixed in magnitude and direction); (2) pulsating (oscillating in magnitude but fixed in direction); and (3) oscillatory (oscillating in magnitude and periodically reversing direction). Roy et al. [73] demonstrated that oscillatory fluid flow, but not steady and pulsating flow, induced high Ca 2+ flicker activity at Ca 2+ microdomains with a localized high [Ca 2+ ] i in MG-63 cells. The localization of TRPM7 in lipid rafts as Ca 2+ microdomains was critical for Ca 2+ flicker activity [73]. In human MSCs, TRPM7 stimulated by fluid flow promoted [Ca 2+ ] i , resulting in osteogenic differentiation [74,75].
Clinical pulsed electromagnetic field stimulation is widely applied to promote bone regeneration in both the United States and Europe as a therapeutic approach for patients with musculoskeletal disorders [113]. Although the underlying mechanisms of electromagnetically induced osteogenesis are not yet completely elucidated, several studies have indicated the benefit of applying electromagnetic stimulation in bone-healing processes. Magnetically induced electrostimulation is an attractive approach to osteogenic differentiation [114] and osteoporosis prevention [115]. Indeed, the stimulation by electric fields upregulated TRPM7 expression, leading to the migration of human osteoblasts from femoral heads of patients undergoing total hip replacement [76].
Mechanical stresses regulate bone formation by upregulating RANKL expression in osteoblasts. Acute mechanical stress induced by hypoosmotic shock promoted TRPM3 and TRPV4 activation, resulting in increased Ca 2+ -mediated RANKL and NFATc1 expressions in primary mouse calvarial osteoblasts [77]. NFATc1 is required for parathyroid hormone (PTH)-related peptide-induced RANKL expression in C2C12 and primary-cultured mouse calvarial cells [116]. The [Ca 2+ ] i rise through TRPM3 and TRPV4 as a mechanosensor are critical to NFATc1 activity and the subsequent physiological activity of osteoblasts.

TRPV Channels
TRPV1 deficiency results in the reduced osteogenic differentiation potential of BMM-SCs [78]. On the other hand, bone marrow cells derived from TRPV1-and TRPV4-deficient mice showed enhanced osteoblast differentiation activity [79]. Gain-of-function mutations of TRPV4 induced congenital skeletal dysplasias, including metatropic dysplasia (MD) [117]. Patients with MD exhibit short limbs at birth due to a defect in long bone development and progressive kyphoscoliosis. Nonaka et al. [118] reported that the novel gain-of-function mutation TRPV4 L619F was involved in the chondrogenic differentiation of MSCs and accelerated the osteogenic differentiation of MSCs obtained from patients with MD [80]. Osteoblasts expressing TRPV4 L619F exhibited increased [Ca 2+ ] i, levels; thus, calcification was enhanced with upregulated Runx2 and osteocalcin expression. Indeed, the expression and nuclear translocation of NFATc1 were upregulated in osteoblasts expressing TRPV4 L619F. These findings indicate that MD-associated disorganized endochondral ossification is induced by disordered osteogenic differentiation through the TRPV4-Ca 2+ /NFATc1 signaling axis [80].
A biologically active N-terminal fragment of human PTH, PTH , is used clinically to increase bone volume in osteoporosis. Pozo et al. [81] showed that PTH (1-34) promoted Ca 2+ influx through TRPV4 in MG-63 cells via a PKA-dependent pathway. Interestingly, the PTH-induced Ca 2+ influx significantly inhibited MG-63 cell migration. When PTH (1-34) is administered to humans or rodents, bone mass was increased due to an increased number of osteoblasts and bone formation rate [119]. PTH  also suppressed osteoblast apoptosis [120] and converted quiescent bone-lining cells into active osteoblasts [121]. Further studies will be needed to clarify the relationship between cytosolic Ca 2+ dynamics and osteoblast functions via PTH stimulation.

TRPP Channels
Autosomal dominant polycystic kidney disease is caused by inactivating mutations in the gene that encode either TRPP1 or TRPP2 [122]. TRPP1 binds to TRPP2 through their respective C-terminal coiled-coil domains to form the functional TRPP complex in the cell membrane [123]. This complex between TRPP1 and TRPP2 functions as a mechanosensor in bone and kidney [124].
Recent studies indicated that the TRPP complex played a pivotal role in osteoblastogenesis [82,83,85,86,125]. TRPP1 has been shown as a mechanosensing molecule involved in osteoblastogenesis and bone remodeling. Dalagiorgou et al. [125] reported that mechanical stress upregulated Runx2 expression by activating the TRPP1-JAK2/STAT3 signaling pathway, resulting in osteoblastic differentiation in human osteoblastic cells and, ultimately, bone formation. In addition, TRPP1 and the transcriptional coactivator TAZ formed a mechanosensing complex and contributed to osteoblastic differentiation in MSCs [82,83].
Osteoporosis is a disease characterized by low bone volume, skeletal fragility, and an increased risk of fracture. Bicaudal C homolog 1 (Bicc1) is one of the genetic determinants of patient bone mineral density, and Bicc1-deficient mice had low bone mineral density. Mesner et al. [85] indicated that TRPP2 was a downstream target of Bicc1 involved in osteoblast differentiation. In addition, a human genome-wide association study (GWAS) meta-analysis showed that single nucleotide polymorphisms (SNPs) in TRPP2 are associated with decreased bone mineral density. These findings suggest that Bicc1 is a critical determinant of osteoblastogenesis and bone mineral density by regulating TRPP2 transcription.
The osteoblast-specific depletion of TRPP2 resulted in reduced bone volume associated with decreased bone mineral density, mineral apposition rate, trabecular bone volume, and cortical thickness, and impaired biomechanical properties of bone. TRPP2-deficient osteoblasts exhibited lower basal [Ca 2+ ] i and impaired response to flow-induced Ca 2+ influx [86]. Additionally, the short hairpin RNA (shRNA)-mediated blockade of TRPP1 suppressed Ca 2+ signaling to shear fluid stress in MG-63 cells [84]. These studies suggest that Ca 2+ -permeable channel TRPP2 is coupled to mechanosensing TRPP1 in response to mechanical stress-loaded osteoblasts during osteoblast differentiation.

Piezo Channels
Piezo channels are emerging as important regulators of various aspects of mechanosensing [126][127][128]. Piezo2 is predominantly expressed in sensory neurons, such as dorsal root ganglia neurons, whereas Piezo1 is primarily expressed in non-sensory tissues and nonneuronal cells and senses various mechanical stimuli, including shear stress, static pressure, and membrane stretch. Piezo channels are permeable to divalent ions such as Ca 2+ , as well as to monovalent ions [129]. Recent GWAS reports showed that human Piezo1 SNPs were associated with shorter adult height [130] and reduced bone mineral density [131]. Sugimoto et al. [87] showed that hydrostatic pressure induced the differentiation of BMMSCs into osteoblasts with enhanced BMP2 expression via the ERK1/2 and p38 MAPK signaling through Piezo1. In MC3T3-E1 cells, the combined action of both Piezo1 and TRPV4 is involved in the sensing of mechanical fluid shear stress [88], and Piezo1 is essential for Runx2 expression through the Akt/GSK-3β/β-catenin signaling pathway [89]. Indeed, Piezo1 is highly expressed in various bone tissues of mice and plays a critical role in bone formation [90] (Figure 4).
sensing [126][127][128]. Piezo2 is predominantly expressed in sensory neurons, such as dorsal root ganglia neurons, whereas Piezo1 is primarily expressed in non-sensory tissues and non-neuronal cells and senses various mechanical stimuli, including shear stress, static pressure, and membrane stretch. Piezo channels are permeable to divalent ions such as Ca 2+ , as well as to monovalent ions [129]. Recent GWAS reports showed that human Pi-ezo1 SNPs were associated with shorter adult height [130] and reduced bone mineral density [131]. Sugimoto et al. [87] showed that hydrostatic pressure induced the differentiation of BMMSCs into osteoblasts with enhanced BMP2 expression via the ERK1/2 and p38 MAPK signaling through Piezo1. In MC3T3-E1 cells, the combined action of both Piezo1 and TRPV4 is involved in the sensing of mechanical fluid shear stress [88], and Piezo1 is essential for Runx2 expression through the Akt/GSK-3β/β-catenin signaling pathway [89]. Indeed, Piezo1 is highly expressed in various bone tissues of mice and plays a critical role in bone formation [90] (Figure 4).  Osteoblast lineage-specific, Piezo1-deficient mice (Piezo1 OcnCre ) showed severely impaired bone formation and blunt, mechanical unloading-induced bone loss [90]. Moreover, Piezo1-conditional-deficient mice, Piezo1 Prrx1Cre , exhibited multiple skeletal fractures in long bones, the radius, and ulna; however, Piezo2 conditional-deficient mice (Piezo2 Prrx1Cre ) had normal bone development with no fractures. Piezo1/2 double conditional knockout neonatal mice exhibited more severe skeletal defects in the appendicular skeleton [94]. Piezo1 and Piezo1/2 knockout mice showed decreased activity in the Hippo-Yap1/TAZ and Wnt/β-catenin signaling pathway in osteoblasts, thus promoting osteoblastogenic differentiation [94]. In addition, Li et al. [91] reported that osteoblast/osteocyte-specific deletion of Piezo1 decreased bone formation and bone mass in other Piezo1 knockout mice, Piezo1 Dmp1Cre . The shRNA-mediated blockade of Piezo1 in MLO-Y4 osteocyte-like cells reduced the expression of Ptgs2 and Tnfrsf11b [89], well-known targets of fluid shear stress in osteocytes [132,133]. Furthermore, Sasaki et al. [92] reported that mechanical stimulation suppressed an osteocyte-derived negative regulator of bone formation, sclerostin mRNA expression, in murine osteocyte IDG-SW3 cells through the Piezo1-Akt pathway.
Piezo1 knockout mice, Piezo1 Runx2Cre , demonstrated a more severe osteoporotic phenotype than Piezo1 Dmp1Cre [93]. Because Runx2 was expressed in chondrocytes, as well as in osteoblasts, Piezo1 activity in chondrocytes was involved in this aggravation of osteoporosis in Piezo1 Runx2Cre mice [132]. Lee et al. [134] have described the presence of mechanosensory Piezo channels in chondrocytes, which function synergistically in response to injurious mechanical loading.
Wang et al. [135] reported that depletion of osteoblastic Piezo1 promoted bone resorption in Piezo1 Prx1Cre mice. Piezo1 in osteoblasts regulated the YAP-dependent expression of Col types II and IV, which control osteoclast differentiation, suggesting that Piezo1 can coordinate the crosstalk between osteoblasts and osteoclasts by directly sensing mechanical stress in osteoblast lineage cells.

Conclusions/Future Perspectives
Osteoporosis is a major skeletal disorder that influences bone structure and composition. Gradual bone loss results in bone fragility and significantly increases the risk of bone fracture. An estimated 1.5 million fractures occur per year in the United States [136]. Bone homeostasis depends on intracellular Ca 2+ signaling, as well as on the external calcium balance that regulates the function and differentiation of chondrocytes, osteoclasts, and osteoblast lineage cells. A number of ion channels critically contribute to the regulation of these processes, including intestinal calcium absorption and renal calcium reabsorption [137].
This review has highlighted the critical role of K + channels and Ca 2+ -permeable channels in osteoblast functions. However, the lack of understanding of their specific functions in bone formation poses a definite limit. Elucidating their involvement in bone signaling pathways could help identify molecules targeting bone biology. Emphasis should be given on data describing the cooperative coupling of K + and Ca 2+ signaling in regulating the physiological and pathophysiological frameworks of bone homeostasis.