Primary cilia in hard tissue development and diseases

Abstract

Bone and teeth are hard tissues. Hard tissue diseases have a serious effect on human survival and quality of life. Primary cilia are protrusions on the surfaces of cells. As antennas, they are distributed on the membrane surfaces of almost all mammalian cell types and participate in the development of organs and the maintenance of homeostasis. Mutations in cilium-related genes result in a variety of developmental and even lethal diseases. Patients with multiple ciliary gene mutations present overt changes in the skeletal system, suggesting that primary cilia are involved in hard tissue development and reconstruction. Furthermore, primary cilia act as sensors of external stimuli and regulate bone homeostasis. Specifically, substances are trafficked through primary cilia by intraflagellar transport, which affects key signaling pathways during hard tissue development. In this review, we summarize the roles of primary cilia in long bone development and remodeling from two perspectives: primary cilia signaling and sensory mechanisms. In addition, the cilium-related diseases of hard tissue and the manifestations of mutant cilia in the skeleton and teeth are described. We believe that all the findings will help with the intervention and treatment of related hard tissue genetic diseases.

Keywords

primary cilia / bone / mechanical sensing / hard tissue / cilium-related bone disease / tooth

1 Introduction

In vertebrates, bone is the architectural framework of the body structure and is critical for supporting physiologic loading and protecting internal organs. At the embryonic stage, bone is differentiated from the mesoderm and formed through intramembranous ossification or endochondral ossification [1,2]. Bone is renewed continuously throughout life to maintain its strength, maintain mineral homeostasis, and repair microdamage during bone remodeling [3]. Bone modeling and remodeling, which are both regulated by various physiological and mechanical factors, require the cooperation of osteoblasts and osteoclasts that direct the formation of new bone and the resorption of old bone, respectively [2,4].

In the skeletal system, the cell types involved in development and homeostasis regulation mainly include osteoprogenitor cells, chondrocytes, osteoblasts, osteocytes derived from mesenchymal stem cells (MSCs), and osteoclasts derived from hematopoietic stem cells [5]. At sites where long bones are formed via endochondral ossification, MSCs aggregate and differentiate into osteoprogenitor cells, which differentiate into chondroblasts and chondrocytes. Chondrocytes proliferate and form a hyaline cartilage framework and release alkaline phosphatase (ALP). This event is followed by hypertrophy and chondrocyte apoptosis. Dead chondrocytes create a barren matrix into which osteoprogenitors are recruited to derive osteoblasts [2]. Osteoblasts localize on bone matrix surfaces, synthesize and secrete bone matrix, and initiate matrix mineralization. Osteocytes are osteoblasts that are surrounded by matrix and live within lacunae. Osteoclasts absorb mineralized bone and participate in bone remodeling and healing [2,4]. In postnatal bone homeostasis regulation, a group of cells, which are defined as multipotent stem cells, act as the sources of bone healing or regeneration. These stem cells can be isolated from bone marrow, periosteum, skeletal muscle, fat, and umbilical cord blood and differentiate into specialized cells to function in remodeling, intramembranous ossification, or endochondral ossification [6].

Emerging evidence indicates the importance of primary cilia in bone development and cell function. Since primary cilia were first discovered on chondrocytes in 1967 [7], an increasing number of studies have focused on exploring their roles in chondrocyte biology, specifically endocytosis, infiltration, and apoptosis [8,9]. Further experiments found that primary cilia also exist in other cell types of the skeletal system, such as MSCs [10], MC3T3-E1 osteoblastic cells, MLO-Y4 osteocyte-like cells [11,12], and calvarial osteoblasts and osteocytes [11,13], and play important roles in bone development and remodeling. However, the existence of primary cilia on osteoclasts has yet to be proven [14].

Cilia are categorized into motile and primary cilia [15]. Primary cilia are present on the surfaces of almost all cell types in mammals, whereas motile cilia are found on respiratory epithelia and sperm flagella. Primary cilia appear like tiny antennas that protrude from the cell surface [16]. First named by Sergei Sorokin in 1968 [17], primary cilia have been considered as vestigial structures of no importance. However, increasing evidence suggests that primary cilia are functional organelles that not only can detect chemical or mechanical signals in the extracellular environment [18] but can also regulate cell mitosis [19] and signal transduction [20].

Primary cilia consist of the cilia axoneme, basal body, transition zone, and cilia membrane (Fig. 1). The axoneme is the main part of primary cilia and is composed of a ring of nine microtubule doublets. In contrast to the “9+ 2” motile cilia in respiratory epithelia or sperm flagella, primary cilia lack a central pair and other attachments, such as molecular motors and axonemal dyneins. Thus, primary cilia are nonmotile [21]. Cilia axonemes extend directly from a basal body that sits beneath the cell surface. This basal body consists of “9+ 0” microtubule triplets and is derived from the mother centriole; thus, the presence of primary cilia can inhibit the cell cycle [22]. Cilia microtubules have dynamic instability [23], and α-tubulin is acetylated to maintain microtubule stability [24]. Knocking out the mother centrosome localization protein Nde1 elongates cilia; this effect then delays the cell cycle and disrupts cell growth [25,26]. A special structure called the “transition zone” is assembled at the junction of the axoneme and basal body, which is called the “cilia gate,” and regulates the amount and speed of specific protein exchange between the cilia and the cell body, thereby controlling the strength of signal transduction [27]. For example, transmembrane protein 107 (TMEM107) is enriched in this region, and its mutation decreases the number of cilia [28]. A cilia axoneme is surrounded by a specialized membrane containing a set of specific receptors and channels, some of which are different from those contained by the cell membrane. This membrane is considered as the most important part of primary cilia because it serves as a receptor that senses external signals [29].

All ciliary proteins are synthesized in the cytosol and transported to cilia via intraflagellar transport (IFT), which is an evolutionarily conserved process that is required for axonemal growth and cilia maintenance [30,31]. IFT proteins form 2 complexes, complex A (IFT-A) and complex B (IFT-B). IFT-A components connect cargo to dynein-2 motors and carry it in the retrograde tip to the base direction, whereas IFT-B components bind proteins with kinesin-2 motors and carry them from the base to the tip in the anterograde direction [16,32,33]. Some ciliary proteins, including polycystin-1 (PC-1) and polycystin-2 (PC-2), smoothened (Smo), suppressor of fused (SuFu), and transcription factor glioma (Gli), are important components of signaling pathways, such as Hedgehog (Hh), Wnt, and PDGFRα [14,29,34]. The deficiency of primary cilia leads to abnormal Hh signaling and profound ectopic Wnt signaling [20]. In addition, multiple receptors and ion channels present on the primary cilia perceive signals, such as mechanical, light, chemical, and electronic field stimuli, from the surrounding environment. Cilia transduce these signals through various proteins and signaling pathways into cells [16,35]. Proteins with abnormal morphology or other defects cause reduced mechanical or biochemical susceptibility, which leads to abnormal signaling pathways [36]. However, the details of signal transduction into coordinated cellular responses by cilia remain elusive.

2 Primary cilia signaling in bone development

Cilia deficiency leads to ectopic signal transduction and skeletal system deformation [34,37]. Multiple signaling pathways associated with primary cilia have important effects on bone development and homeostasis. These pathways include Hh, Wnt, Notch, bone morphogenetic protein (BMP), PDGFR, and TGF (Fig. 2).

2.1 Hh

Hh signaling is one of the major pathways that regulates osteogenesis, embryonic bone development, and postembryonic bone homeostasis. Indian Hh (Ihh) regulates cartilage maturation during endochondral ossification, and Sonic Hh (Shh) regulates the patterning of the forming skeleton. Mice with the Ihh mutation have short statures and short, narrow ribs [38]. Day and Yang found that Hh signaling is greatly increased in all the bone tissues in mice with a single mutation in Ptch1, leading to the proliferation of cells especially in the perichondrium and articular cartilage [39]. The signaling component proteins Smo, SuFu, and Gli reside in cilia and are transported via IFT [34]. When Hh signaling is activated, Shh binds to the ligand receptor Ptc1 and releases Smo, which then moves to the cilium tip under facilitation by IFT-B and suppresses SuFu. This effect, in turn, releases the SuFu inhibition of Gli and activates Gli to form Gli2A and Gli3A, which are transported to the nucleus and activate the expression of downstream target genes [20,39,40].

The loss of the Hh signaling pathway when cilia are missing leads to various skeletal deformities [31]. Some mutations in ciliary proteins block the response of Hh ligands, and other mutations block the activation of ligand-independent pathways [34]. Ihh and Shh disturbances can be seen in the endochondral ossification of Ift88 conditional knockout (cKO) mice [41]. Prx1-cre;Ift88^fl/n mutant mice exhibit severe polydactyly, and Gli1 expression is nearly abolished at E11.5. All prx1-cre;Ift88^fl/n and prx1-cre;Kif3a^fl/n mutants show a dramatic reduction in the overall length of skeletal elements and accelerated hypertrophic differentiation [41]. Osterix-cre;Ift80 mice present osteopenia and defective osteoblast differentiation [31]. Silencing IFT80 causes cilia loss or shortens cilia length, leading to the loss of Hh signaling; a decrement in Gli2; and the suppression of the expression of osteoblastic marker genes, including runt-related transcription factor (RUNX2), OCN, BMP2, and ALP, thus resulting in the inhibition of ALP activity and cell mineralization. The expression of these genes can be rescued by the overexpression of Gli2 or the application of a Smo antagonist, indicating that IFT80 regulates bone differentiation and mineralization via Gli2 pathways, as well as osteoblastic marker genes [31].

2.2 Wnt

The canonical Wnt/β-catenin pathway promotes cell cycle and differentiation, whereas the noncanonical Wnt pathway regulates cell polarity and migration [42]. Wnt/β-catenin signaling acts in the Ihh pathway and downstream of Osx, regulating the whole osteogenic process and bone formation through intramembranous and endochondral ossification [39,43]. Primary cilia attenuate the canonical Wnt signaling pathway by sequestrating β-catenin and preventing it from entering the nucleus, thus restricting the activation of downstream Wnt target genes [44]. Ciliotherapy with lithium chloride increases the length of primary cilia on MSCs; this effect leads to an increase in β-catenin localization to the nucleus [45]. Wnt components, such as Inversin, Fat4, and glycogen synthase kinase-3β (GSK-3β), are located in primary cilia [32]. GSK, inversin, and Kif3a [46] negatively regulate canonical Wnt/β-catenin. The ciliary protein NPHP3 inhibits canonical Wnt but activates noncanonical Wnt by binding with inversin [32,47]. After treatment with Ift88 siRNA or the Rho kinase inhibitor Y27632 to inhibit the formation of actin stress fibers, cilia length and number are reduced; these effects have been proven to affect MSC response to Wnt3a. The level of nuclear active β-catenin, the activation of the canonical Wnt pathway, and the proportion of cells entering the cell cycle all increased [36,48].

2.3 Platelet-derived growth factors

Platelet-derived growth factors (PDGFs) are growth factors that promote cell proliferation and migration. PDGF molecules bind to specific cell surface receptors, namely, PDGFRs, which are receptor tyrosine kinases that control a series of cellular processes, including osteogenic proliferation, cell survival, and endoplasmic reticulum (ER)–Golgi transport regulation; the latter function is the key to collagen secretion during skull development [49–52]. PDGFRα is located on the surfaces of primary cilia in fibroblasts during growth arrest [53]. PDGFRα expression is upregulated during ciliogenesis, and the expression and ligand-mediated activation of its receptor are blocked in Ift88^−/− and Ift172^−/− mouse embryonic fibroblasts (MEFs) [53,54]. Primary cilia contain the signaling machinery required for the pathway from PDGFRαα activation to Mek1/2 through Ras–Raf, controlling cell growth and proliferation [53]. PDGFRα regulates Akt phosphorylation through PDGFRα activation. The deletion of Ift20 downregulates the transcription and translation of PDGFRα, thus suppressing PDGF–Akt signaling, resulting in decreased osteogenic proliferation and increased cell death rate [52].

2.4 TGF-β/BMP

The TGF-β superfamily is a large regulatory peptide that includes the model transforming growth factor-β family, BMP, and other families. TGF-β and BMP receptors are located in primary cilia. Primary cilia mediate cell proliferation, differentiation, and migration by adjusting the output of canonical and noncanonical TGF-β signals and balancing the output of TGF-β/BMP signals [55,56]. In addition, the disruption of BMPs leads to embryonic lethality, limb patterning abnormalities, and cartilage condensation loss [57]. In the fracture model constructed with Col2α1^creIFT80^f/f mice, the activation of the TGF-β signaling pathway is impaired, which leads to the interruption of cartilage callus formation and reduction in vascularization [58]. In smokers, the disruption of TGF-β signaling in MSCs under the mediation of cigarette smoke extract impairs endochondral ossification and delays fracture healing [59]. The elimination of primary cilia via transfection with IFT88 siRNA can block the activation of the BMP–Smad1/5/8 signal induced by pulsed electronic fields (PEMFs) and inhibit the increase in osteoblast differentiation and maturation [60]. The TGF-β1-mediated activation of SMAD3 induces human MSC recruitment, which is regulated by primary cilia [61].

3 Primary cilia perception in bone

3.1 Mechanosensing roles of primary cilia

Mechanical stimulation is one of the most important factors in bone modeling and remodeling [3]. Bone cells need mechanical stimulation to maintain their survival and vitality. The lack of mechanical loading results in the apoptosis of bone cells and may lead to the initiation of bone resorption [62]. Bone lacunae and canaliculi are fluid-filled channels that house osteocytes; the latter obtain nourishment and communicate with neighboring osteocytes and osteoblasts from and through fluid [63,64]. Daily walking, running, or jumping creates normal cycles of tension and compression in long bones. Microdeformation generates strain gradients in the medullary cavity, resulting in the development of hydrostatic pressure and fluid flow through lacunae and canaliculi [65]. The fluid in the osteocyte network sloshes back and forth, thus generating membrane-deforming shear forces on the osteocyte network [66,67]. Bone cells detect loading or fluid flow through the cell body and dendritic process and cilial bending [68] and react accordingly to initiate downstream signals. In vitro experiments have indicated that oscillating fluid shear (OFS) can promote MSC proliferation, differentiation, and early osteogenesis [69–72]. Mechanical stimulation induces the osteogenesis of periosteal progenitor cells with an increase in osteogenic markers [66]. When human MSCs (hMSCs) are exposed to fluid shear (FS), the expression of bone formation markers increases [73,74]. By contrast, simulated microgravity inhibits the proliferation, osteoblastic, differentiation, and mineralization of rat calvarial osteoblasts (ROBs) [75].

In 2001, Praetorius first proved that primary cilia in the kidney are mechanically sensitive and respond to bending with calcium entry [76]. In 2003, Whitfield proposed that in osteocytes, cilia act as mechanical sensors and bend in response to pulses of extracellular fluid that are generated during walking and running cycles [66]. Malone et al. also suggested in 2007 that cilia on bone cells possess physical properties that are consistent with a flow-sensing function [12]. Primary cilia have been proven to perceive hydrostatic pressure applied on the cell body [77] and mediate mechanosensing in bone-like cells [33]. The loading-induced activation of the periosteal bone surface necessitates primary cilia, thereby initiating the recruitment of progenitor cells and osteogenic differentiation [78]. Mechanical stimulation also affects the formation of primary cilia. Simulated microgravity abrogates the formation of primary cilia and decreases the proportion of ciliated ROBs. Residual cilia visibly shorten and appear dotted [75].

With intact primary cilia, periosteal osteochondroprogenitors (OCPs) show an immediate osteogenic response to fluid shear [79]. Primary cilia regulate the oscillatory fluid flow (OFF)-induced expression of osteogenic markers. The levels of markers, such as osteopontin (OPN), cytokine prostaglandin E2 (PGE2), and COX2, significantly increase in MC3T3-E1 osteoblasts and MLO-Y4 osteocytes after exposure to OFF [12]. Osteoblasts, osteoclasts, and OPCs with intact primary cilia increase the levels of PGE2, COX2, OPN, and RUNX2 in response to physical stimulation, but these changes are absent from cilium-blocked cells [14,79]. PGE2 is considered as the first component of the activation cascade in osteocytes during the initiation of bone mechanosensing, and primary cilia induce the release of PGE2 in response to fluid flow [12,62,80]. The damage or removal of cilia by chloral hydrate suppresses the release of PGE2 and the deposition of calcium [81]. In addition, primary cilia regulate the mechanical stimulation-induced expression and secretion of RANKL and OPG from osteocytes, which regulate the differentiation of osteoclasts [82]. A brief summary is shown in Fig. 3.

The length of primary cilia is also sensitive in response to overloading or detecting subtle forces. Delainesmith et al. showed that primary cilia on MLO-A5 cells subjected to OFF shorten and become stub-like [81]. Liu et al. proved that drag force and torque in a cilium with a length of 8 mm are significantly greater than those in a cilium with a length of 2.5 mm [83]. Overloading decreases the length of primacy cilia or even disassembles cilia on chondrocytes [84]. Nonloaded tendon cells show increased cilia length, which decreases with loading [85].

KIF3a is one of the subunits of the anterograde directed kinesin-II motor protein, and the disruption of KIF3a leads to inappropriately assembled and truncated primary cilia [86]. The disruption of KIF3a in osteoblasts leads to an attenuated response to fluid FS stress in vitro and impaired cilium-related signaling pathways, such as Hh and Wnt [87]. The loss of KIF3a decreases the number and length of primary cilia [31] and results in the loss of cilia on MSCs. The conditional silencing of Kif3a results in severe patterning defects in the craniofacial area, a split sternum, and polydactyly [88]. The specific deletion of cilia or KIF3a in osteoblasts and osteocytes results in a decrease in loading-induced bone formation. After exposure to cyclic ulnar loading, new bone formation in 2.3-Cre;Kif3a^fl/fl mutants is significantly reduced compared with that in the control group [89]. Col1-Cre;Kif3a^fl/fl mice lacking primary cilia show impaired cellular mechanical sensing and osteogenesis in the area around the implant. Loading-induced bone repair is also impaired in the peri-implant zone with the attenuation of cell proliferation and the deposition and synthesis of collagen fibers along the strain field. This research also suggested that primary cilia influence the strain-induced orientation of cells and the matrix [90].

3.2 Ca²⁺ signaling in primary cilium-related sensing

Calcium is one of the most important cations in the human body and plays an important role as a second messenger in signal transduction. Intracellular Ca²⁺ ion concentrations are regulated by a variety of ion pathways, including mechanically sensitive Ca²⁺ channels and transient receptor potential (TRP) channels, that are activated at the cell membrane. TRP channels are categorized into 7 families: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPP (polycystin), TRPML (mucolipin), and TRPN (Drosophila NOMPC) [91]. Most TRP channel proteins have selective permeability for calcium ions and respond to the osmotic pressure and pH of the extracellular environment.

TRPV4 regulates the response of renal epithelial cells to osmotic pressure [92]. TRPV4 acts as a mechanical sensor in osteocytes and chondrocytes and mediates cellular response to loading and MSC response to mechanical stimuli [93]. TRPV4 is also expressed on the surfaces of osteoclasts [94]. TRPV4 regulates osteogenesis and osteoclast activity by mediating the localization of NFATc1 to the nucleus [72,95], activating a key signaling pathway in osteogenesis, and promoting osteogenic differentiation [96]. NFATc1 can also act downstream of RANKL, thus promoting the terminal differentiation of osteoclasts [97]. The osteogenic potential of MSCs isolated from Trpv4-knockout mice is inhibited [98]. TRPV4 is required in the mechanical transduction of MSCs, Ca²⁺ signaling induced by OFS, and the expression of early osteogenic genes [93]. TRPV4 affects the terminal differentiation of osteoclasts, and Trpv4^−/− mice exhibit an increase in bone mass compared with the control group [95]. TRPV4 deficiency prevents nonloading-induced decreases in bone mass and bone formation rate and inhibits osteoclast proliferation [94]. Masuyama et al. showed that the activation of TRPV4 by drugs enhances osteoclast activity and decreases bone mass [99]. However, they did not subject mice to loading in this experiment. TRPV4 is expressed in highly stressed areas of ​​the cell membrane surface, especially primary cilia [93,100,101]. TRPV4 function requires the existence of an intact cilia structure [101]. Some TRPV4 agonists or drugs, such as lithium chloride, hydrogen sulfide, and interleukin-1, can be used to extend primary cilia on the surfaces of bone cells [102,103]; enhance the level of mechanical stimulation; and possibly amplify the osteogenic response to prevent bone loss from disuse.

PC1 is a cell-surface-expressed G protein-coupled receptor that is encoded by the PKD1 gene [104], and PC2 is a TRP-type Ca²⁺-permeable nonselective cation channel that is encoded by PKD2. PC2 interacts with PC1 in a functional complex that is often implicated in a common signaling pathway [105]. PC1 is part of the mechanosensing complex in renal epithelial cells. When the extracellular domain of PC1 is activated in response to fluid flow, the PC2 ion channel opens, allowing Ca²⁺ influx. This intraciliary Ca²⁺ influx is thought to lead to the release in intracellular calcium stores from the ER [106]. Homozygous mutant Pkd1^m1Bei/m1Bei mice show delays in intramembranous and endochondral bone formation, and heterozygous Pkd^m1Bei mice demonstrate osteopenia with decreases in Runx2-II and osterix expression, indicating that this phenotype is caused by osteoblast dysfunction [11]. PC1 is also expressed in osteocytes and participates in mechanical transduction, playing important roles in bone development and homeostasis [11]. Mice with PC1 deficiency display reduced loading-induced bone formation in response to mechanical loading [107]. Knocking down Pkd1 in osteoblasts leads to enhanced adipogenesis, which is similar to the effect of kif3a knockdown [87,108]. Interestingly, the further deletion of kif3a in pkd1-deficient mice reverses skeletal abnormalities by restoring the balance between osteogenesis and adipogenesis [108]. However, the mechanism of this reversal is elusive.

Studies on PC2 in different tissues have provided different results. Jin et al. suggested that Ca²⁺ signaling in mouse kidney LLCPK cells induced by mechanical loading requires the presence of primary cilia and PC2 [109], whereas Malone et al. demonstrated the opposite results: Ca²⁺ signaling in MC3T3-E1 osteoblasts and MLO-Y4 osteocytes induced by fluid flow is independent of cilia and PC2 [12]. These results are identical to those of Lee et al. [100]. Other experiments found that Ca²⁺ influx is absent from MC3T3-E1 cells after stimulation by fluid flow [110]. These results suggest that the role of primary cilia in mechanically induced Ca²⁺ influx is tissue-specific, which may be related to the different types of Ca²⁺ channels in the cell membrane and primary cilia.

3.3 Induction of primary cilia by electromagnetic field signals

The fluid in the osteocyte network sloshes back and forth, thus generating streaming ion potentials and piezoelectric currents on the osteocyte network [66]. Electromagnetic fields (EMFs) have been shown to prevent bone loss because of disuse or ovariectomy in mice and can replace medicine for osteoporosis treatment [111–113]. The Food and Drug Administration reported that PEMFs are a safe and effective way to treat osteoporosis [114]. However, the optimal parameters, such as frequency and intensity, and internal mechanism of this treatment need to be further clarified. PEMFs can promote the proliferation, mineralization, and maturation of ROBs [115], and PEMF or electrical field stimulation (EFS) can increase the expression of the osteogenic markers BMP2, COL1, and RUNX2 [35,115]. These processes require the participation of primary cilia. PEMFs promote osteogenesis through the activation of the soluble adenylyl cyclase (sAC)/cAMP/PKA/cAMP response [116], and sAC and p-PKA also localize to primary cilia [117]. After inhibiting primary cilia with iRNA, the promoting effect of 50-Hz PEMFs on osteogenesis differentiation and maturation is abrogated [115]. Yan et al. used IFT88 siRNA to inhibit primary cilium synthesis, leading to significantly decreased expression levels of osteogenic and mineralization marker genes in ROBs [115]. Cai et al. showed that primary cilia act as sensors for EFS, which can enhance the osteogenic response in osteoprogenitor cells by simultaneously upregulating the expression of mRNA in ciliary structure proteins and PKD1 [35]. Wnt10b is localized to the base of primary cilia and disappears or is released after sinusoidal EMF (SEMF) treatment. Zhou et al. found that 50 Hz 1.8 mT SEMFs increase peak bone mass in vivo, enhance the expression of Wnt10b, and activate Wnt10b/β-catenin signaling, thus promoting the expression of osteogenic-related proteins. This process is abolished when ciliary synthesis is inhibited [118].

Furthermore, EFS changes the oscillation patterns of cytoplasmic Ca²⁺ [119–121], and the defects of primary cilia do not decrease EFS-induced Ca²⁺ influx [35]. The transduction of EFS signaling through primary cilia in hASCs is independent of stretch-activated channels but may depend on a voltage-gated calcium channel [35]. After blocking voltage-activated calcium channels, the electrical stimulation-induced osteogenic response is completely eliminated [122–124]. These results may provide a theoretical basis for the treatment of bone loss by EFS-assisted drugs that act on ion channels.

4 Primary cilium-related bone diseases

Ciliopathy is a hereditary disease that affects almost every important organ and tissue [125,126], including the brain, kidney, retina, liver, and bone, manifesting as intracranial cysts and microcephaly [22], autosomal dominant polycystic kidney disease [16], and retinal dystrophy and degeneration [127], suggesting the importance of primary cilia in development and body function. Cilium deficiency in the skeletal system is classified as primary cilium-related bone disease [128]. The main clinical features of these diseases include shortened long bones, polydactyly or brachydactyly, short or horizontal ribs, reduced rib cage volume, conical epiphyses, and facial deformities.

4.1 Bone defects in primary cilium-related syndromes

Generally, cilium-related bone diseases develop as syndromes. Among the best-studied ciliopathies are Mainzer–Saldino syndrome (MZSDS [OMIM 266920]), oral–facial–digital syndrome (OFD), Bardet–Biedl syndrome (BBS [MIM 209900, MIM 613610, MIM 614099]), Sensenbrenner syndrome or cranioectodermal dysplasia (CED [MIM 218330]), and short rib–polydactyly syndromes (SRPs). SRPs are divided into six subgroups: Saldino–Noonan syndrome (SRP type I [OMIM 263530]), Majewski syndrome (SRP type II [OMIM 263520]), Verma–Naumoff syndrome (SRP type III [OMIM 263510]), and Beemer–Langer syndrome (SRP type IV [OMIM 26986]). The other two are Jeune syndrome (also known as Jeune asphyxiating thoracic dysplasia (JATD) [OMIM 611263, OMIM 613091, OMIM 613819, OMIM 614376]) and Ellis–van Creveld (EvC [OMIM 225500]) [16,38,129–131]. Some diseases with known phenotypes and pathogenic genes are shown in Tables 1 and 2.

The typical skeletal phenotypes of SRPs are short ribs, a constricted thoracic cage, metaphyseal dysplasia, and a trident aspect of the acetabular roof. Some cases manifest with defective ossification in the calvaria, vertebrae, and the bones of the hands and feet [38,132]. Polydactyly is consistently observed in SRP type II and EvC, common in types I and III, and rare in type IV and JATD [133]. SRPs differ by variable visceral malformations and metaphyseal appearance [134]. SRP type I is characterized by severely shortened and flipper-like limbs and the transposition of the great vessels and atretic lesions of the gastrointestinal and genitourinary systems [132], and type II presents with a malformed epiglottis and larynx [135]. SRP type III is accompanied by multiple anomalies of major organs, including the heart, intestines, genitalia, kidneys, liver, and pancreas [136,137], and type IV shows brain defects; absent internal genitalia; and renal, bile, and pancreatic cysts [138].

Compared with that of other SRPs with high mortality caused by cardiopulmonary failure, the survival rate of JATD is 40%–80% [139,140]. JATD and SRP types I–IV have typical phenotypes of the skeletal system, such as shortened ribs and bell-shaped thorax, but patients with JATD have a lower probability of having respiratory insufficiency [141]. Other manifestations are short bones, polydactyly, irregular spurs on the acetabular bone and metaphysis, and cone-shaped epiphyses [141–144]. Surviving patients occasionally present retinal degeneration, polycystic kidneys, and hepatopathy [145]. Similar to patients with JATD, individuals affected with MZSDS present cone-shaped epiphyses and retinal dystrophy but also have occasional deformities in other organs, such as cerebellar ataxia, abnormal proximal femur, and liver fibrosis [141,146].

CED is characterized by abnormal ectodermal manifestations, including short fingers, sparse hair, and malformed teeth [147]. In addition, patients with CED have similar clinical phenotypes as patients with Juene syndrome, namely, short stature, short limbs, short ribs, asphyxiate thorax, polydactyly, renal failure, and liver fibrosis. The potential pathogenic genes of CED are still being explored [148]. Walczak-Sztulpa et al. stained cultured fibroblasts from patients with CED and found significantly reduced cilium frequency and length in fibroblasts [148]. Gene scanning showed that hypomorphic mutations in IFT122, whose production is a component of the primary ciliary IFT-A complex, cause an abnormal ciliary phenotype in homozygous carriers. Furthermore, an abnormal phenotype similar to that in patients with CED is observed in zebrafish embryos with ift122 knockout [148]. Other cases show the overlap of these cilium-related bone disease phenotypes, indicating interplay among ciliary genes that affect multiple downstream signaling pathways [129,141].

4.2 Pathogenic genes in cilium-related bone diseases

Case reports and gene testing data show that common pathogenic genes for cilium-related bone diseases include the IFT protein, dynein, and the basal body protein of primary cilia. Typical mutant genes include components of the transport complex, such as IFT122, IFT43, WDR35, WDR19/IFT144, TTC21B/IFT139, IFT140, and WDR56/IFT80; the dynein component genes DYNC2H1 and NEK1; and the basal body protein genes EVC and EVC2 (Fig. 4).

Abnormalities in different ciliary components lead to different phenotypes. The alteration of the IFT-A component disorganizes the assembly and/or maintenance of the ciliary structure by impairing retrograde IFT through the redistribution of ciliary proteins. The staining of the axonemes of fibroblasts from IFT144-deficient individuals revealed a significant reduction in the number of ciliated cells and shortened cilia length [149]. The mutation of IFT140, a core protein of the IFT-A complex, results in shortened primary cilia and the accumulation of IFT-B proteins at the bases of cilia [150]. The mutation of IFT-B genes often leads to loss of cilia [29]. For example, the silencing of IFT80 causes the shortening or loss of cilia [31]. Chondrocytes from affected individuals with mutant DYNC2H1, a cytoplasmic dynein involved in retrograde transport, have shortened primary cilia with bulbous distal ends or no discernible cilia projecting from cell surfaces. In DYNC2H1 mutants, the early stage of cilial formation remains intact, and the basal body is normally anchored at the plasma membrane, whereas the elongation and/or maintenance of the axoneme by IFT-A required to form the mature primary cilia is abnormal [151]. DYNC2H1 encodes a subunit of the cytoplasmic dynein complex that is associated with the light intermediate chain (DYNC2LI1) and directly participates in the contact and translocation of the dynein complex along microtubules through its large motor domain [152]. In mammalian tissue, the colocalization of DYNC2H1, DYNC2LI1, and IFT pathway homologs supports the generation and maintenance of cilia [131].

Mutations in genes that encode components of IFT, especially the IFT-A complex, are major causes of skeletal ciliopathies [129,144]. IFT140 is one of the most studied IFT-A genes among disease-related genes. Schmidts et al. found that all biallelic IFT140 patients present with renal disease in early childhood with notable retinal involvement but have a nonlethal thorax-related clinical course and no polydactyly. The IFT140 mutation has also been detected in patients with JATD [129,153]. At least 6 other genes are related to JATD, namely, DYNC2H1, IFT80, IFT139, IFT140, WDR19, and WDR34 [129–131,139–141,144, 149,154,155], indicating a complex correspondence between genotype and phenotype. Schmidts et al. concluded that the IFT140 mutation is a rare cause of JATD in general but is a frequent cause of JATD with renal involvement [141]. In 2012, Perrault et al. found that the IFT140 mutation is the only cause of MZSDS [129]. In the following year, Halbritter et al. added IFT172 (an IFT-B component) to the pathogenic gene list [156]. IFT140 and IFT172 are the only two pathogenic genes found in MZSDS to date. Studies on mouse models of gene mutations and knockouts have found that Ift140 mutant or Ift140-knockout mouse embryo development is slower than wild-type embryo development with the highest fatalities in midpregnancy and that survivors present with exencephaly with polydactyly. Other skeletal deformations include severe rib defects, failed calvarial development, and cervical vertebral fusion [153]. The conditional knockdown of Ift140 in different cells leads to different phenotypes. For example, the deletion of Ift140 in renal collecting ducts results in severe early cystic kidney disease [143]. After the knockout of Ift140 in osteoblast precursor cells, mice are smaller in size and weight than wild-type mice and have reduced expression levels of genes involved in bone formation and osteogenic processes [157].

IFT80 and DYNC2H1 have been verified to be associated with JATD and SRP type III [131,151,154]. Rix et al. established a model of Ift80^gt/gt mice and found that the percentage of survival to birth is less than 3%. Postnatal survivors present marked growth retardation, constricted thoracic cages, ribs with irregular margins between bone and cartilage, shortened long bones, and a characteristic bilateral preaxial polydactyly of the hind limbs wherein a single extra digit is formed and is markedly longer than the other digits [130]. Defects in cytoskeletal microtubule architecture are also observed in mutant SRP chondrocytes with an approximately 2-fold increase in acetylated α-tubulin in the SRP samples relative to that in the control. The abnormalities identified in SRP raise the possibility that the loss of DYNC2H1 function may affect the movement of organelles and the transport of critical cargo necessary for signal transduction and skeletal development [151]. Merrill et al. speculated that homozygosity with two null alleles results in early embryonic lethality, whereas a combination of a null mutation and a variety of missense mutations may lead to a range of phenotypes [151].

WDR34 concentrates mostly around the centrioles and basal body and is considered as a linker between cytoplasmic dynein-1 and IFT dynein-2 motors [144]. Cases with mutations in WDR34 may account for up to 10% of all JATD cases [144]. A previous report demonstrated that WDR34 inhibits the NF-kB pathway component TAK1 in cell culture [158]. The mutation of the WDR34 homolog dync2i1 in zebrafish leads to a typical ciliopathic phenotype [159]. However, whether the axonemal localization of WDR34 may be affected in these deficient cells remains unclear [144]. The proportion of ciliated cells and cilial length are not significantly affected in any of the Wdr34-knockdown ATDC5 cell lines (mouse chondrocyte progenitor cells) [144]. This finding is similar to the results reporting the absence of loss or malformation of cilia in the Ift80-deficient hypomorphic JATD mouse model, in which a low level of Ift80 expression allows ciliogenesis but does not rescue Hh signaling defects [130]. The achieved level of WDR34 knockdown may not be sufficient to result in a ciliogenetic defect [144]. Altogether, the function of WDR34 in the IFT dynein-2 complex remains to be further investigated.

EVC, the basal body protein, is unnecessary for ciliary synthesis. Evc^−/− mice show growth plate dysplasia of the long bones but retain normal Ihh expression in the growth plate; however, the expression of the downstream genes Ptch1 and Gli1 is significantly reduced [160]. Evc/Evc2 defects lead to the failure of Sufu/Gli3FL dissociation, and Gli3R increases after treatment with the Smo agonist SAG [161]. The functions of primary ciliary genes are summarized in Table 3.

5 Roles of primary cilia in craniofacial development

Craniofacial bones, as components of the skeletal system, have similar structural characteristics as bones in other parts of the body but undergo unique and complex developmental processes. As the embryo differentiates, neural crest (NC) cells in the ectodermal germ layer undergo epithelial-to-mesenchymal transition and migration. NC cells then differentiate into facial bones, cartilage, and teeth (except enamel). The entire process involves many different signaling pathways, such as BMP, FGF, and Wnt, which together establish a regulatory network and play essential roles in the evolution of NC [162]. The connection between primary cilia and many developmental signaling pathways is critical for craniofacial development [163]. More than 30% of ciliopathies are characterized by craniofacial phenotypes, including midfacial hypoplasia, cleft lip and palate, retrognathia, and craniosynostosis. The frequency and severity of craniofacial phenotypes underscore the importance of cilia during the development of craniofacial complexes [128]. Several important cilium-related craniofacial syndromes exist.

5.1 Primary cilium-related craniofacial syndromes

JS and JS-related disease (JSRD) are a group of stunted growth/multiple congenital abnormality syndromes that are characterized by molar tooth sign in the brain. JS is a complex midbrain–hindbrain deformity. JS manifests with unique features in the facial area, including a large head and frontal prominence, arched eyebrows, drooping upper eyelids, wide eyes, low ears, and a triangular mouth. Currently, mutations in approximately 34 genes have been identified to be related to JS [164]. These mutations include INPP5E, ARL13B, CC2D2A, RPGRIP1L, TMEM67, NPHP1, AHI1, CEP290, CXORF5, and TMEM216 [128], with CEP290 (also known as JBTS5) being the most common. The protein encoded by CEP290 is located in the cilial transition zone. CEP290 mutant cells have the typical characteristics of elongated cilia, impaired ciliogenesis, and cilia composition defects. Some studies have proposed that targeted exon skipping provides new directions for JS treatment because it is capable of rescuing the expression of CEP290 protein, the composition defects of ciliary proteins, and the function of the transition zone in CEP290 mutant cells [165].

The OFD syndrome lineage includes 13 different genetic diseases. OFD type I ([OMIM311200]) is an X-linked dominant disorder with an incidence of approximately 1/50 000, with affected males dying during pregnancy. Its symptoms are mainly manifested in the oral cavity (lobulated tongue, tongue nodules, clefts of the hard or soft palate, accessory gingival frenulae, hypodontia, and other dental abnormalities) and the face (widely spaced eyes or telecanthus, hypoplasia of the alae nasi, median cleft or pseudocleft upper lip, and retrognathia) [166]. Genetic linkage analysis and familial analysis of OFD type I cases have revealed that this syndrome is caused by a mutation in the CXORF5 gene located in the Xp22.2 chromosome. The product of this gene is OFD1, one of the fundamental proteins of the primary cilia, and is located in the basal bodies at the base of the ciliary axoneme. OFD1 is necessary for maintaining the microtubule length stability of centrioles and the recruitment of IFT88 but can be prematurely truncated by mutations in the CXORF5 gene [167,168]. The clinical characteristics of the pathogenesis of OFD type I are related to abnormal Hh signal transduction, a depressed planar cell polarity pathway, and errors in cell cycle control that result from the dysfunctions of primary cilia [169]. The knockout of Ofd1 in mice to establish an animal model recapitulates the main symptoms of OFD1, but severity is enhanced [170].

OFD type VI is a rare phenotype of JS and is associated with a C5ORF42 mutation [171]. The craniofacial features include tongue fissure, tongue hamartoma, retrognathia, additional frenulae, and other mouth–face deformities (such as cleft lip and palate) [172]. In chicken embryos, C5orf42 silencing results in the widening of the facial midline, the appearance of abnormal primary cilia on the cell surface, and the weakening of cell response to Shh agonists [173].

BBS is an autosomal recessive genetic heterozygous disorder that affects multiple parts of the body. In addition to the common symptoms of progressive vision loss, polycystic kidneys, and postaxial hand polydactyly, patients with BBS may also present with special craniofacial appearances: brachycephaly, macrocephaly, bitemporal narrowing, male frontal balding, large ears, short and narrow palpebral fissures, a long shallow philtrum, nasal anomalies, dental crowding, midfacial hypoplasia, and mild retrognathia [174]. The mutation of BBS genes leads to abnormal tooth development, such as crowded dentition, protrusions, and enamel hypoplasia. The clinical examination of patients with BBS revealed tooth dysplasia, microdontia, short roots, or taurodontism [175–177]. To date, 21 pathogenic genes have been discovered (BBSome1–21), but the BBS protein network remains to be determined [178]. The 8 subunits of the BBSome assemble into a functional complex and play vital roles in ciliary transport. In contrast to IFT-A or IFT-B mutants, BBS mutants usually do not show significant abnormalities in cilial structure but exhibit abnormal signal transduction in cilia [179]. Zebrafish model studies have shown that the knockout of different BBS genes leads to significant overlapping phenotypes, and the consumption of the BBSome increases the Hh signal in the zebrafish fin bud, leading to skeletal abnormalities [180]. The deletion of the BBSome gene in mice results in the accumulation of Smo and Ptch1 in cilia and the reduction in the Shh response [181].

EvC and Weyers acrofacial dysostosis (MIM 193530) have been proven to be associated with EVC and EVC2 [182,183]. EvC is a rare autosomal recessive disorder that affects bone development due to chondroectodermal dysplasia [128]. Clinical features include short statures, short ribs, polydactyly, nail and tooth dysplasia, and labiogingival adhesions. Affected children have congenital cardiac defects, most frequently a defect in primary atrial septation, which is the common cause of death [184]. EvC shows a wide spectrum of abnormal oral manifestations, including serrated incisal margins, dental transposition, diastema, conical teeth, enamel hypoplasia, and hypodontia. Teeth may be erupted at birth or exfoliate prematurely [185]. Incisor hypoplasia [186], fused first and second molars, molar microdontia, and abnormal root development can be seen in mice lacking Evc [187]. Weyers acrofacial dysostosis is an autosomal dominant disorder that is clinically characterized by mild short stature, postaxial polydactyly, and nail dystrophy. Craniofacial phenotypes include ocular hypotelorism; median cleft; enamel hypoplasia; conical teeth; single central incisor or hypoplasia; and, less frequently, other organ abnormalities [188,189]. The differences between EvC and Weyers are the different genetic patterns and the severity of the phenotype [190].

5.2 Primary cilia in tooth development

Teeth and bone undergo similar cell maturation processes and mineralized matrix formation [191]. Several studies have observed that primary cilia participate in every stage of tooth development [186,192,193]. At the embryo stage, the thickened epithelium differentiates into dental lamina and extends toward deep connective tissue. Terminal cells proliferate and form the tooth germ. During this period, primary cilia can be observed in epithelial thickening and the mesenchyme below the thickening [186]. The tooth germ is composed of three parts: the enamel organ, which forms the enamel; the dental papilla, which forms the dentin and dental pulp; and the dental sac, which develops into periodontal tissue. Primary cilia are expressed during the three differentiation stages of the enamel organ, dental papilla, and dental sac [186,192]. During the bud and cap stage, cilia are mostly distributed in the epithelial dental tissues and mesenchymal cells surrounding the tooth germ. Interestingly, cilia gather on the apical surface of the basal epithelial layer, whereas few cilia localize in the central area and are short in the bud stage [186]. In the bell stage, the expression of primary cilia is higher in the developing cervical loop [193] and the enamel knot area than in the stellate reticulum [186]. During this stage, the concave surface of the enamel organ displays the shape of the teeth that will form in the future. The enamel knot, the local epithelial thickening in the inner enamel epithelium, may be the signal center and may have important effects on the tooth, especially cusp morphogenesis [194,195]. Primary cilia in enamel knots are longer than in other organs [186]. Considering that cilia are involved in many signaling pathways that regulate growth and development, cilia may regulate tooth morphology through the expression of signaling pathways in enamel knots.

After the differentiation and maturation of ameloblasts during the late bell stage, undifferentiated mesenchymal cells near the inner enamel epithelium of the dental papilla are induced to grow and differentiate into odontoblasts. The primary cilia on the odontoblasts are aligned parallel to the dentin wall with the axoneme oriented toward the pulp core, and the cilia on the ameloblasts project toward the outer enamel epithelium such that the cilia on the surface of two cells are oriented in the opposite direction distal from the area where hard tissue is produced [192,196]. However, the cilia in the dental follicle or the periodontal ligament (PDL) show no obvious pattern after birth [192], suggesting that primary cilia have an important influence on regulating cell polarity and mineral deposition direction. In early postnatal and adult mice, primary cilia are detected on the edges of the PDL adjacent to the teeth and alveolar bone, and few ciliated cells are observed in the medial region of the PDL [192]. In odontoblasts and ameloblasts, cilia gradually elongate during differentiation, whereas cilia length and frequency decrease on dental stalk cells and rudimental successional dental lamina with keratinization and aging, respectively [186]. A previous in vitro study on dental pulp stem cells suggested that primary cilia elongate during odontogenesis and that the ciliated cell population also increases significantly [197].

Single variations in the key components of primary cilia also cause different types of dental dysplasia. Variations in IFT88, the primary cilial component involved in Shh signaling, lead to significant abnormal dental phenotypes. Shh, as the major ciliary signaling pathway, is involved in tooth germ development, growth, formation, and differentiation. Abnormal primary cilia have a pleiotropic effect on tooth development [198]. Tg737^orpk is a hypomorphic allele of IFT88/Polaris in which mutants have enhanced HH signals and form ectopic molars. The knockout of Ift88 in mesenchymal cells results in hyperdontia, whereas the knockout of Ift88 in epithelial cells does not result in the manifestation of the phenotype [199]. The focal domains of WNT and Shh define the sites where teeth will develop; therefore, any changes in expression can cause defects in incisor initiation. In Wnt1^Cre+Kif3a^fl/fl mice, molar germs are enlarged, and enamel organs are misshapen [186]. Furthermore, HH signaling is lost in the tooth germ, whereas WNT/β-catenin signaling is enhanced in the mesenchyme of Wnt1-cre;Kif3a^f/f mice, resulting in multiple invaginations of the epithelium instead of a single secondary enamel knot [200].

Ift140^cauli/cauli embryos exhibit severe craniofacial defects, including absent or rudimentary alisphenoid and palatine and hypoplastic and malpositioned maxilla and premaxilla [153]. Our laboratory found that the cKO of IFT140 in odontoblasts results in a reduced number of primary cilia. Dentin thickness is thinner and dentin formation is slower in Osx-Cre;Ift140^f/f mice than in the control. Ift140-cKO mice have short molar roots and hypoplastic interradicular dentin with the decreased expression of the Shh signaling molecules Ptch1, Smo, and Gli1-3. Osx-Cre;Ift140^f/f mice have impaired reparative dentin formation, whereas the number of primary cilia in the wild-type mice increases after cavity preparation, suggesting that primary cilia act in reparative dentin formation and pulp protection [201].

Tooth regeneration has always been an important topic in the field of dental tissue research. Research on the regulatory factors of dental development during different stages is helpful for researchers to explore key cellular structures and signaling pathways and to find possible methods for tooth regeneration in vitro.

6 Future perspectives

In the last two decades, a complete reversal of the opinion that primary cilia are of no great importance has been gradually accepted. Current studies have concluded that many primary ciliary genes can regulate complex signaling during bone development. Primary cilia also act as mechanical and EMF sensors that detect loading or current and voltage changes in the skeletal system and regulate bone remodeling. However, the biological response and activity mechanism of cilia and cells after external stimuli remain unclear. In the craniofacial area, primary cilia also function in the development of mandibular and intervertebral discs (IVDs). The deletion of IFT80 disrupts Hh signaling and decreases IVD marker gene expression, causing the early onset of the IVD disorder phenotype [202]. The slightly shorter and thicker mandibles of Ift88 mice than those of wild-type mice may be related to altered Hh signaling [203]. With the deepening of the understanding of auxology and the increase in research on genetic diseases, additional secrets regarding ciliary function and mechanisms in craniofacial development will be revealed. These tiny organelles are anticipated to play important roles in metabolic diseases and aging diseases of hard tissue. Research on the regulatory functions of ciliary genes may provide novel information for use in proposing potential therapeutic targets. In conclusion, further comprehensive studies are needed to reveal the biological function of primary cilia.

In addition, the etiology of ciliary hereditary osteopathies is complex, and their pathogenic genes are diverse. Embryonic lethality caused by severe skeletal diseases has introduced difficulties into population screening and animal research. Current screening studies have revealed that most cilium-related bone diseases may have multiple pathogenic genes. An intricate relationship may exist between genes and different genotypes, and multiple areas of genotypes and disease phenotypes overlap. The underlying mechanism cannot be explained by a single pathway, thus complicating the identification of therapeutic targets and the appropriate timing of treatment and interventions. The pathogenesis and treatment of primary cilium-related bone diseases require extensive long-term genetic screening and animal experiments for further clarification.

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