Transcriptional, epigenetic and microRNA regulation of growth plate

Abstract

Endochondral ossification is a critical event in bone formation, particularly in long shaft bones. Many cellular differentiation processes work in concert to facilitate the generation of cartilage primordium to formation of trabecular structures, all of which occur within the growth plate. Previous studies have revealed that the growth plate is tightly regulated by various transcription factors, epigenetic systems, and microRNAs. Hence, understanding these mechanisms that regulate the growth plate is crucial to furthering the current understanding on skeletal diseases, and in formulating effective treatment strategies. In this review, we focus on describing the function and mechanisms of the transcription factors, epigenetic systems, and microRNAs known to regulate the growth plate.

1. Introduction

Chondrocyte differentiation of mesenchymal cells is an important bone formation event. Many bones are formed through the process of endochondral ossification, while others, such as the skull, part of the jawbone, and the clavicle are formed via membranous ossification [1]. Skeletal formation begins with the aggregation of mesenchymal cells, which promotes differentiation into chondrocytes and formation of cartilage primordium. The cartilage primordium then proliferates, and the central portion undergoes a cell maturation process in which it differentiates into hypertrophic chondrocytes. The continuation of this process promotes the longitudinal growth of cartilage, which gradually replaces the hypertrophic zone in the center of the bone (the primary ossification center) through angiogenesis.

As the bone continues to grow, the chondrocyte proliferation/differentiation process is confined to structures called growth plates located at both epiphyses. In addition, a secondary ossification center is formed at each epiphysis, and the zone structure of the growth plate becomes visible [2]. The growth plate can be roughly distinguished from the resting, proliferating, prehypertrophic, and calcified zones. The resting and proliferating zones primarily express ACAN and COL2A1, and the hypertrophic zone expresses COL10A1. Meanwhile, the calcified chondrocyte zone expresses VEGFA, MMP13, and SPP1 and creates a scaffold for bone formation [2] via attracting osteoblast precursors derived from perichondral cells, and osteoclasts derived from blood vessels to this zone. Furthermore, the calcified chondrocytes themselves are divided into those that differentiate into osteoblasts and those that undergo apoptosis [3,4].

This paper outlines the functions of the transcription factors, epigenetic systems, and microRNAs that have been shown by prior studies to be involved in the process of bone growth in the growth plates.

2. Transcription factors

Transcription factors are proteins involved in the initiation and regulation of gene transcription via binding to promoters and regulating the expression of downstream genes. During development specifically, transcription factors regulate the expression of multiple genes and promote differentiation of stem cells into mature cells. To date, the following transcription factors have been reported to have functions in the growth plates: SOX5, SOX6, SOX9, GLI, TRPS1, the CREB/ATF family, ZFP521, RUNX2, MEF2C, FOXA, and HIF1 (Fig. 1; Table 1).

Fig. 1.

The network of transcriptional factors active in the growth plate. Solid black arrow, positive regulation; black dashed arrow, negative regulation.

Table 1.

Summary of transcription factors related to growth plate development.

Name	Functional site	Phenotype of each KO mice	Function	Reference
SOX9	Resting zone, proliferating zone, pre-hypertrophic zone	Mesoderm condensation dysplasia	Positive regulation of Col2a1, Aggrecan, Negative regulation of Col10a1, Runx2	[5–19]
SOX5,6	Resting zone, proliferating zone, pre-hypertrophic zone	Single null mice: mild phenotype, double null mice: cartilage dysplasia	Positive regulation of Col2a1, Aggrecan	[8,17,19,20]
GLI1	Proliferating zone, pre-hypertrophic zone, hypertrophic zone	Promoting osteoblast differentiation in the early stage	Positive regulation of Ihh signaling	[42–44]
GLI2	Resting zone to pre-hypertrophic zone	Increment of proliferating chondrocytes and hypertrophic chondrocytes and decrement of bone tissue	Positive and negative regulation of Ihh signaling depending on the level of it	[45,46]
GLI3	Proliferating zone	Decrement of proliferating zone	Positive and negative regulation of Ihh signaling depending on the level of it	[38,47–50]
TRPS1	Pre-hypertrophic zone to Hypertrophic zone	Increment of proliferating zone but the number of cells are not changed	Promotion of chondrocyte proliferation and induction of chondrocyte apoptosis	[55–61]
CREB	Proliferating zone	Dysplasia of proliferating zone and hypertrophic zone	Promotion of chondrocyte proliferation via Ptch	[72–79]
ATF2	Proliferating zone	Dysplasia of proliferating zone and hypertrophic zone	Promotion of chondrocyte proliferation via Ptch	[83–87]
ATF4	Proliferating zone	Dysplasia of proliferating zone and hypertrophic zone	Promotion of chondrocyte proliferation via Ptch	[88,89]
ZFP521	Pre-hypertrophic zone	Decrement of proliferating zone, early hypertrophy transition, and shortening of growth plate	Negative regulation of Runx2	[96,97]
RUNX2	Pre-hypertrophic zone	Shortening of long bone, decrement of the number of hypertrophic chondrocyte, impaired calcification of chondrocytes	Negative regulation of Ihh and Vegfa, positive regulation of Col10a1and Mmp13	[98–101]
MEF2C	Prehypertrophic zone to Hypertrophic zone	Shortening of long bone, failure of differentiation from proliferating chondrocytes to hypertrophic chondrocytes	Positive regulation of Runx2	[117–121]
FOXA	Hypertrophic zone	Dwarfism, impaired hypertrophy and calcification of chondrocytes	Positive regulation of Col10a1	125–127
HIF1	Proliferating zone	Mesoderm condensation dysplasia	Positive regulation of Sox9 and Vegfa	[137–142]

2.1. SOX9, SOX5, and SOX6

SOX9, which encodes a transcription factor with a high mobility group box (HMG box), is a causative gene for congenital osteochondrodysplasias, including campomelic dysplasia (CD) and acampomelic campomelic dysplasia (ACD). SOX9 is known to be a master gene that plays an essential role in chondrocyte differentiation [5–7]. In fact, undifferentiated mesenchymal cells neither aggregate nor initiate chondrogenesis in Sox9-knockout cells [5,8]. Hence, SOX9 is involved in undifferentiated mesenchymal cell aggregation and differentiation into cartilage progenitor cells. As part of a SOX-trio, SOX9 then induces Sox5 and Sox6 activity and expression of the cartilage-specific genes, Col2a1 and Acan (encodes aggrecan) [9–12], supporting the regulation of cartilage precursor cell differentiation into proliferating chondrocytes [8,13]. SOX9 also activates and maintains Col10a1 expression in prehypertrophic chondrocytes [14]; meanwhile prehypertrophic chondrocytes deficient in SOX9 either die immediately or differentiate into immature osteoblasts [15]. The primary roles of SOX9 include suppressing differentiation into hypertrophic chondrocytes, blocking RUNX2, which plays an important role in chondrocyte maturation [16–18], and suppressing COL10A1 expression in hypertrophic chondrocytes [18,19]. Further, overexpression of Sox9 in hypertrophic chondrocytes delays chondrocyte hypertrophy [14]. In summary, SOX9 plays an essential role in maintaining the growth plate through its involvement in the aggregation of mesenchymal cells and their differentiation into precursor, proliferating, and prehypertrophic chondrocytes.

It has been reported that SOX5 and SOX6 are not necessarily required at the aggregation stage of undifferentiated mesenchymal cells before they differentiate into prochondrocytes [20]. Mice lacking either Sox5 or Sox6 develop a mild skeletal defect, whereas mice deficient in both exhibit more severe cartilage damage [20]. Furthermore, in chondrocytes deficient in both SOX5 and SOX6, the expression of cartilage-specific genes, such as Col2a1 and Acan, is reduced despite normal expression of Sox9. Hence, there appears to be a cooperative function of the trio of SOX5, 6, and 9 that is essential for early chondrocyte differentiation and growth plate formation [8,17,19,21].

Genetic analysis studies of CD and ACD have identified SOX9 enhancers within an approximately 2 Mb non-coding region located between SOX9 and KCNJ2, a nearby coding gene upstream of SOX9. These enhancers serve to activate transcription in several distinct organs including cartilage [22–27]. Additionally, a recent ChIP-seq analysis using the CRISPR/Cas system, which enables genome editing and analysis of transgenic and knockout mice, revealed the existence of Rib-Cage Specific Enhancer (RCSE) located approximately 1 Mb upstream of Sox9 [28]. Multiple additional analyses with CRISPR-ChIP-mass spectrometry (CRISPR-ChIP-MS) demonstrated that the transcription factor STAT3 regulates the expression of Sox9 via this RCSE region [28]. Furthermore, Hall et al. reported that STAT3 regulates Sox9 expression as a key modulator of chondrocyte generation and differentiation [29].

SOX9 has, therefore, been established as an essential factor in chondrocyte generation and differentiation, which form and maintain the growth plate. Future research should focus on elucidating how SOX9 forms a network with other transcription factors, including STAT3, involved in growth plate generation and maintenance.

2.2. GLI

GLI (a GLI-Kruppel family member), a transcription factor that contains a Zn finger domain, is involved in targeted gene expression of the hedgehog signal pathway and Indian Hedgehog (IHH) signal mediation. In the growth plate, IHH, among the hedgehog ligands, is present in both the prehypertrophic and hypertrophic zones and plays an essential role in chondrocyte proliferation, maturation, and late differentiation [30,31]. Sonic hedgehog (SHH) mediates cartilage progenitor cell generation. In fact, in the absence of both IHH and SHH, SOX9 is prevented from properly regulating chondrocyte differentiation [32,33].

The GLI family of proteins is primary composed of three homologs in mammals, which are expressed in all chondrocytes and act as activators or repressors depending on the IHH signal level [34,35]. Gli1 is expressed in the proliferating zone, prehypertrophic zone and hypertrophic zone. Gli2 is expressed in each chondrocyte zone except for hypertrophic zone. Gli3 is strongly expressed in the proliferating zone [36–38]. GLI2 and GLI3 act as repressors when the Hh signal is absent and as activators when the signal is present [39–41].

An analysis of Gli1−/− mice showed that GLI1 is important for promoting osteoblast differentiation in the early stage, however, no clear change in the growth plate is observed during chondrogenesis [2,42]. Meanwhile, other reports showed that in Gli1−/− mice, the expression region of the osteoblast marker ALP is reduced and disappears in the perichondrium, and ectopic expression of chondrocyte markers, such as Col2a1 and Col10a1, can be observed [43]. Further, overexpression of Gli1 in C3H10T1/2 cells suppresses the expression of the Sox9-trio, and GLI1 inhibits the promotion of SOX9-dependent Col2a1 expression [43]. Similarly, a ChIP-assay showed that Gli1 overexpression inhibits SOX9 recruitment near the enhancer region of the Col2a1 first intron [43]. It has also been reported that Gli1 may regulate Sox9 expression via a far-upstream enhancer [44]. However, the effect induced by GLI1 on chondrogenesis has not yet been fully elucidated and, therefore, requires further analysis.

Regarding GLI2, Gli2-null mice die shortly after birth due to numerous organ failures including skeletal failure. Analysis of the fetuses of these mice revealed that the number of proliferating and hypertrophic chondrocytes increases compared to that in wild type mice, resulting in an increase in cartilage in the growth plate, decrease in bone tissue and delayed endochondral ossification [45,46]. These results indicate that GLI2 plays an essential role in normal endochondral ossification, and the deletion of Gli2 increases the number of immature chondrocytes. A subsequent report showed that mice of Col2-Cre;R26ΔNGli2/+, with one ΔNGli2 and one wild-type allele at the ROSA26 locus, at E14.5 decreases in the proliferating zone, and increases in the hypertrophic zone. The premature onset of hypertrophy may not be due to a decrease in parathyroid hormone like hormone (Pthlh), as Pthlh expression is unchanged.

Ptch1, a direct transcriptional target of Hh signaling, is normally expressed from the proliferating zone to the prehypertrophic zone, while being absent from the hypertrophic zone. However, in the case of Col2-Cre;R26ΔNGli2/+, Ptch1 is expressed in all chondrocyte zones, including the hypertrophic zone. That is, GLI2 acts as a repressor of the Hh signal during chondrocyte differentiation [45]. It has also been reported that GLI2 acts positively on chondrocyte hypertrophy and controls vascular invasion of hypertrophic cartilage during endochondral ossification [34,45], suggesting that it promotes the differentiation of proliferating chondrocytes into hypertrophic chondrocytes.

Regarding GLI3, the proliferating zone is more reduced in Gli3−/− mice than in wild mice, suggesting that hypertrophic differentiation may be accelerated in Gli3−/− mice [38]. The expression level of PTHLH does not differ significantly between wild and Gli3−/− mice [38], suggesting that the acceleration of hypertrophic differentiation may occur independently of PTHLH. As described above, GLI3 exists as a full-length activator in the presence of Hh signaling; however, in the absence of Hh signaling, GLI3 undergoes phosphorylation by a plurality of kinases and takes on a repressor form [47–49]. In C3H101/2 cells, the number of GLI3 transcription factors in the repressor form is reduced by stimulation of the Hh ligand and increased by stimulation of Pthlh [34]. Ihh−/−; Gli3−/− mice have been analyzed to determine how GLI3 operates downstream of the IHH signal and results demonstrate that in Ihh−/− mice, chondrocyte proliferation is strongly impaired, and the initiation of differentiation into hypertrophic chondrocytes is accelerated as evidenced by cells with a phenotype lacking a distinct columnar chondrocyte region. Conversely, the morphology and arrangement of the proliferating zone are recovered in the Ihh−/−;Gli3−/− mice compared to that in the Ihh−/− mice [38,50]. Moreover, chondrocyte proliferation is clearly reduced in Ihh−/− mice, whereas the phenotype is significantly recovered in Ihh−/−;Gli3−/− mice. In Ihh−/− mice, the expression of Col10a1 spreads throughout the cartilage, while in Ihh−/−;Gli3−/− mice, it is restricted to the center of the cartilage [38,50]. Finally, the expression of Pthlh is eliminated in Ihh−/− mice, whereas the expression of Pthlh is reactivated in Ihh−/−; Gli3−/− mice. Cumulatively, these results indicate that GLI3 acts as a strong repressor of the Ihh target gene [38,50].

Mice overexpressing Ihh under control of the Col2a1 promoter (Col2a1-Ihh mice) were analyzed to investigate the function of the active and repressor forms of Gli3 in chondrocytes. Whereas Col2a1-Ihh mice show accelerated differentiation of distal chondrocytes into columnar chondrocytes and a delay in the onset of hypertrophic differentiation, Col2a1-Ihh;Gli3−/− double mutants rescued the delayed hypertrophic differentiation. Furthermore, the zone of proliferating chondrocytes in Col2a1-Ihh;Gli3−/− mice decreased compared to that in Col2a1-Ihh mice. The size of distal chondrocytes is slightly reduced in Col2a1-Ihh mice and strongly reduced in Col2a1-Ihh;Gli3−/− mice, compared to wild type [38].

In summary, GLI1 is thought to be involved in the regulation of SOX9 expression and in cartilage differentiation, however, the precise mechanism remains unclear. What is known is that GLI2 promotes cartilage differentiation of proliferating chondrocytes into prehypertrophic and hypertrophic chondrocytes. Further, GLI3 functions differently in its activator and repressor forms with the activator form promoting differentiation of distal chondrocytes into columnar chondrocytes and maintaining proliferating chondrocytes, and the repressor eliciting the opposite effect. The Hh signal suppresses the processing from the activator to the repressor form, while PTHLH acts positively on the processing and is independent of the Hh signal in cartilage differentiation.

2.3. TRPS1

TRPS1 (Trichorhinophalangeal syndrome type 1) is a gene that causes trichorhinophalangeal syndrome, resulting in abnormalities in the hair, nose, phalanges, and skeleton [51–53]. TRPS1 is known to act as either a transcriptional activator or repressor [54]. Trps1-null mice develop chondrodysplasia and die shortly after birth, primarily due to respiratory failure [55,56]. In growth plates, TRPS1 is mainly expressed in proliferating chondrocytes at E14.5, however, is restricted to prehypertrophic chondrocytes at E18.5 [57,58]. Moreover, in E18.5 Trps1−/− mice, the growth plate shows no significant changes in the hypertrophic zone, while the proliferating and prehypertrophic zones are enlarged [55,57]. According to bromodeoxyuridine (BrdU) analyses, the cell proliferating capacity of chondrocytes decreases throughout the growth plate in Trps1−/− mice. The hypertrophic chondrocytes of TUNEL-positive mice clearly decrease in the hypertrophic zone of Trps1−/− mice, with decreased caspase-3 expression and increased Bcl-2 expression throughout the whole growth plate [55]. These results suggest that TRPS1 promotes chondrocyte proliferation and also induces chondrocyte apoptosis.

The expression of phosphorylated STAT3 increases in Trps1−/− chondrocytes, and the number of phosphorylated STAT3 present in the nucleus has also been shown by immunostaining to increase in the growth plate of Trps1−/− mice, suggesting that TRPS1 regulates both cell proliferation and apoptosis by suppressing the STAT3 signal [55]. Further, in Trps1−/− mice, expanded PTHLH expression in the proliferating zone has been observed, supporting the model that states TRPS1 represses PTHLH to maintain a normal chondrocyte structure [58].

Trps1−/− mice exhibit abnormal perichondrium calcification, which may be caused by an increase in Ihh signaling and a suppression of chondrocyte differentiation via disrupted coordination of Trps1 by GLI3 and RUNX2 [57,59]. Furthermore, during differentiation into osteoblasts, TRPS1 suppresses osteocalcin expression and negatively regulates bone matrix formation [60]. Trps1 and Gli3 are expressed together with Wnt5a in the prehypertrophic zone, and Wnt5a is regulated by TRPS1 and GLI3 [61]. More information is needed regarding the potential cooperative action of TPRS1 with other signals, including those of the IHH/GLI family and WNT.

2.4. CREB/ATF family

The CREB/ATF family is a group of transcription factors that bind to CRE and have a symmetric common DNA sequence (TGACGTCA). Among them, CREB was the first 43kD transcription factor to be identified and is located at the end of the following typical signal cascade in cells: G protein-coupled receptor, AC (adenylyl cyclase) activation, increased cAMP concentration, and phosphorylation by PKA [62]. In addition, CREB is notable because it was the first reported example of a transcription factor that regulates gene expression by self-phosphorylation [63,64]; self-phosphorylation (at Ser133) promotes binding to CREB-binding protein (CBP)/p300 and enhances transcription of downstream genes.

CBP/p300, a histone acetyl-transferase, plays a role in increasing the expression of the CREB target gene by acetylating histones [65]. CREB is under the control of several other regulatory factors and has been reported to be regulated by co-activator cAMP-regulated transcriptional co-activators (CRTCs) [66,67]. There are three types of CRTCs (CRTC1, CRTC2, and CRTC3), which have a common domain structure. CRTCs are usually phosphorylated in the cytoplasm by SIK2 (salt-inducible kinase 2) and remain inactive due to the 14-3-3 protein interaction. However, when calcineurin (Ser/Thr phosphatase) is activated by increased intracellular calcium concentration, phosphorylation by SIK2 is suppressed, and dephosphorylation by calcineurin, translocation into the nucleus, and binding to CREB result in increased activity [68,69].

In addition to CREB, many other homologs have been identified in the CREB/ATF family, such as CREB-1 (CREB), CREB-2 (ATF-4), CREB-3, CREB-5, CREM, ATF-1 (TREB36), ATF-2 (CRE-BP1), ATF-3, ATF-5 (ATFX), ATF-6, ATF-7, and B-ATF. Each of these transcription factor families has a basic leucine zipper (b-ZIP) structure consisting of a basic amino acid cluster and a leucine zipper region at the C-terminal, and the same CREB/ATF family or b-ZIP domain forms a homo-heterodimer with a transcription cofactor (such as AP-1) and binds to CRE [70,71].

The expression of CREB is ubiquitous in the growth plate, however, phosphorylated CREB is specifically found in the proliferating cartilage zone [72], where the gene downstream of CREB is activated. In Creb-knockout mice, lung dysfunction and suppression of T-cell differentiation were reported, however, no skeletal abnormalities were observed [73]. This may result from compensation by other CREB/ATF families. To examine the essential role of CREB family in the growth plate, transgenic mice of a dominant negative Creb (A-Creb) [74] were generated and examined using a chondrocyte-specific collagen type 2 promoter [72]; it was determined that shortening of the limbs occurs in A-Creb mice [72]. The growth and hypertrophy of chondrocytes in the growth plate are suppressed, as is the formation of the growth plate [72]. Furthermore, in these mice, the expression of Ptch is reduced independently of IHH, indicating that CREB promotes chondrocyte proliferation via PTCH and plays an important role in the formation of a proliferative zone [72].

PTHLH and the BMP family have been reported as factors that activate CREB in chondrocytes of the growth plate. In Pthlh knockout mice, as in A-Creb mice, developmental deficiencies in the limbs and lungs have been observed, and the proliferation zone in the growth plate is shortened [75]. PTHLH promotes CREB phosphorylation and AP-1 formation [76]. The activation of CREB and AP-1 enhances the expression of downstream genes such as cell cycle-related genes and proliferation-related genes, thereby promoting cell proliferation and suppressing hypertrophy.

In the growth plate, PTHLH in the resting cell zone and IHH in the hypertrophic zone regulate chondrocyte differentiation through mutual feedback [30,31]. In recent years, it has been reported that skeletal stem cells are present in the PTHLH-positive cells of resting chondrocytes and play a role in determining the fate of the chondrocytes [77,78]. CREB is one of the major downstream targets of the PTHLH signal, suggesting the potential role in these skeletal stem cells. In addition, SIK2/SIK3, which are located downstream of PTHLH receptor gene PTH1R, regulate the proliferation and differentiation of chondrocytes via phosphorylation of class 2 HDACs [79]. Although SIK2/3 also regulates CRTCs, which are CREB co-activators, the function of CRTCs in chondrocytes has not yet been fully illustrated.

Among the other CREB/ATF families, ATF-2 and ATF-4 have the most obvious functions in the growth plate. ATF-2 forms a heterodimer with Jun and consists of transcriptional activation domains that are phosphorylated by stress-activated protein kinases (SAPKs) such as JNK (Jun amino-terminal kinase) and p38 [80–82]. Most Atf-2-deficient mice die within one month of birth. As for bone formation, the growth plate is shortened and cartilage hypoplasia is observed [83,84]. ATF-2 gene expression is found in the quiescent and proliferative zones and is involved in the distribution of CREB phosphorylation. ATF-2 binds to the CRE region of cyclin A, cyclin D1, and BCL2 to promote gene expression and chondrocyte proliferation [85–87].

ATF-4 is expressed in the proliferating and prehypertrophic cell zones. RSK2, the causative gene of Coffin-Lowry syndrome, which results in shortening of the femur and suppression of membrane ossification (delay of fusion), phosphorylates ATF-4. In Atf-4 knockout mice, delayed osteogenesis and decreased bone density are observed during development [88]. The lack of ATF-4 disrupts and shortens the columnar structure of proliferating chondrocytes in the growth plate. Conversely, the hypertrophic cartilage zone is enlarged, suggesting a different effect from those of other CREB families. ATF-4 has been reported to bind directly to the promoter region of IHH and activate its transcription [89].

BBH2H7 (box B-binding factor 2 human homolog on chromosome 7) is a trans-membrane transcription factor newly identified as belonging to the CREB/ATF family [90] that is usually present in the ER membrane. In Bbh2h7 knockout mice, proliferating chondrocytes in the growth plate are decreased, and apoptosis has been shown to be enhanced [91]. The expression of Atf-5 and Mcl1 decreases, indicating that BBH2H7 binds to the CRE region of the Atf-5 promoter region and enhances its expression in the chondrocytes of these mice. As ATF-5 has been reported to act as an anti-apoptotic agent in various other cells such as blood cells, the ER-BBH2H7-ATF-5 axis may also be involved in suppressing apoptosis of chondrocytes [90].

2.5. ZFP521

ZFP521 (Evi3, EHZF) produces a Krüppel-like zinc finger protein and is a zinc finger gene with 30 C2H2 domains [92,93]. Like the homologous ZFP423 (OLF, EBF-related ZFP (OAZ)), it has been reported to suppress hematopoietic cell differentiation [93–95]. ZFP521 is expressed in prehypertrophic chondrocytes in the growth plate. In vitro studies have shown that its expression increases in osteoblasts and that it regulates the expression of BMP and PTHLH. The ZFP521 protein binds to RUNX2, whose protein is among the master transcription factors for osteoblast differentiation and suppresses its transcriptional activity and the subsequent expression of osteoblast marker genes such as osterix and osteopontin, resulting in the inhibition of osteoblast differentiation in vitro [96]. It has also been reported that PTHLH increases the expression of ZFP521 in prehypertrophic cells [78]. Zfp521 chondrocyte-specific knockout mice showed a phenotype similar to that of Pthlh knockout mice and Pth1r chondrocyte-specific knockout mice, showing a decrease in the proliferating zone, an early hypertrophy transition, and shortening of the growth plate [97]. In the Zfp521 knockout mice, expression of RUNX2 and the target genes of RUNX2, such as osteoblast differentiation markers, is increased. The expression of genes involved in the cell cycle, such as cyclin D1, is reduced, and apoptosis is increased in growth plate chondrocytes [96]. Thus, ZFP521 plays a central role in the control of chondrocyte proliferation and differentiation downstream of PTHLH/PTH1R, in cooperation with RUNX2.

2.6. RUNX2

RUNX2 (CBFA1) and RUNX3 (CBFA3) play a role in differentiating proliferating chondrocytes into hypertrophic chondrocytes during growth plate development. RUNX2 is expressed in the late condensation stage of chondrogenesis, and after being reduced in proliferating chondrocytes, its expression is increased again in prehypertrophic chondrocytes. It is also known to be strongly expressed in osteoblast and perichondral cells [98,99]. In Runx2 knockout mice, osteoblasts are not formed, the systemic long stem bone is shortened, the hypertrophic chondrocyte count is reduced, hypertrophic chondrocyte calcification is inhibited, and expression of the Runx2 downstream genes Opn and Mmp13 is decreased [100,101].

Runx3-knockout mice show only a slight delay in hypertrophic chondrocyte formation and angiogenesis into cartilage tissue, whereas Runx2/Runx3 double-knockout mice do not exhibit any cartilage maturation. This phenotype of Runx2/Runx3 double-knockout mice is more marked than that of Runx2 single-knockout mice, suggesting Runx2/3 cooperativity [102]. In Runx2 overexpressing mice, chondrocytes are promoted to hypertrophic chondrocytes, causing bone formation in cartilage [103–105]. In dominant negative Runx2 transgenic mice, hypertrophic chondrocyte formation is inhibited [99,104,106].

ATF3, NKX3–2, and SOX9 are reported to be negative regulators of RUNX2. ATF3 suppresses cyclin D1 and cyclin D4 [107], whereas the cyclin D1/CDK4 complex promotes RUNX2 proteolysis and enlargement of proliferating chondrocytes [108]. SOX9 primarily increases the expression of NKX3-2 in proliferating chondrocytes, and NKX3–2 acts as a negative regulator of RUNX2, preventing proliferating chondrocytes from undergoing hypertrophic chondrogenesis [8,109]. RUNX2 binds to the regulatory region of its downstream genes, which include IHH [102], VEGFA [110], COL10A1 [111,112], and MMP13 [113]. RUNX2 is also known to interact with SMADs and promotes hypertrophic cartilage via BMP signals [114–116].

2.7. MEF2C

MEF2Cand MEF2D members of the myocyte enhancer factor 2 (MEF2) family, are expressed in the growth plates of prehypertrophic chondrocytes and hypertrophic chondrocytes [117]. Chondrocyte-specific Mef2c-knockout mice have shortened long bones, delayed chondrocyte hypertrophy, and reduced Runx2 expression [117]. Conversely, mice overexpressing constitutive active Mef2c-Vp16 have immature and excessive endochondral ossification [117]. MEF2C is believed to be an upstream effector of RUNX2, and its protein binds to the enhancer region of RUNX2 together with DLX5/6, SOX5/6, SP7, SMAD1, TCF7, and CTNNB1; and is responsible for inducing and maintaining RUNX2 expression in hypertrophic chondrocytes [118]. Along with RUNX2 activity, MEF2C activity is also regulated by HDAC4 [117,119,120]; meanwhile, PTHLH positively regulates HDAC4 by inhibiting the activity of SIK3. Hence, the PTHLH – SIK - class II HDACs - RUNX2/MEF2C cascade has been comprehensively characterized [121].

2.8. FOXA

FOXA belongs to the hepatocyte nuclear factor-3 (HNF-3)/forkhead family and is known to be widely expressed throughout vertebrate development. FOXA2 and FOXA3 were first reported to be expressed throughout the skeletal system during development [122–124], especially in hypertrophic chondrocytes [125–127]. Foxa3 knockout mice do not have a prominent phenotype, but mice with a carboxyl-specific Foxa2/Foxa3 double-knockout exhibit marked disorders of dwarfism and chondrocyte hypertrophy and calcification. They also show reduced expression of hypertrophic chondrocyte markers such as Mmp13, collagen 10, and alkaline phosphatase [125–127]. The micro-mass culture of chicken limb bud-derived mesenchymal stem cells for cartilage induction results in increased expression of Foxa1 and Foxa2. FOXA has been shown to bind to the enhancer region of collagen 10 [127].

2.9. HIF-1

Hypoxia-inducible factor (HIF) plays a role in enabling the survival and differentiation of hypoxic cells, reducing oxygen consumption, and simultaneously increasing oxygen supply to tissues [128,129]. HIF-1 is composed of two helix-loop-helix proteins, HIF-1α and HIF-1β, in the PER-ARNT-SIM subfamily [130,131], which are ubiquitously expressed [132]. HIF-1α becomes activated when the oxygen concentration is below 5%, while the tissue half-life is extremely short in tissues of 5% or more. On the other hand, HIF1-β is activated independently of oxygen concentration [133–135]. Under hypoxic conditions, HIF1-α and HIF1-β form a dimer, which functions by binding to the promoter of the hypoxia-responsive gene [136]. Hif1-α limb-specific knockout mice show growth impairment with delayed formation of cartilage primordium [137]. In growth plates, HIF1-α promotes SOX9 expression and enhances glycolytic enzymes and glucose transporters to promote cartilage differentiation and adapt to hypoxia [138,139]. HIF-1α also enhances angiogenesis by increasing the expression of VEGFA and promotes the supply of oxygen to chondrocytes [140,141]. In addition, it promotes the hydroxylation of collagen so that chondrocytes in hypoxic conditions can also secrete collagen [142]. Lastly, it inhibits mitochondrial respiration and enhances survival of hypoxic chondrocytes [143]. As for HIF-2a, it has little effect on COL10A1 regulation in endochondral ossification and is believed to contribute to the regulation of COL10A1, MMP13, and VEGFA expression [139], as well as in the osteoarthritic changes in joints [144–146].

2.10. HDACs

Histone deacetylases (HDACs) are enzymes that deacetylate histones, which are major components of chromatin structure, and play an important role in promoting chromatin assembly and repression of transcription. There are two major classes of histone deacetylase (HDAC) genes [147]. Class I (HDACs 1, 2, 3, and 8) are widely expressed and mainly consist of catalytic domains. Class II (HDACs 4, 5, 7, and 9) show an expression pattern depending on the cell type, have an N terminal extension, and act on specific transcription factors to respond to various signaling pathways [148]. The effects of HDACs 3, 4, and 7 on epigenetic control during growth plate development have been reported as described below (Fig. 2) (Table 2).

Fig. 2.

The histone deacetylase network in the growth plate. Solid black arrow, positive regulation; black dashed arrow, negative regulation.

Table 2.

Summary of epigenetic factors related to growth plate development.

Name	Functional site	Phenotype of each KO mice	Function	Reference
HDAC3	Hypertropihc zone	Shortening of long bone	Deacetylation of Histon3,4. negative regulation of Il6, Mmp13, Saa3	[149–152]
HDAC4	Proliferating zone	Early chondrocyte hepertorophy and calcification	Negative regulation of Runx2, Mef2c	[79,153–155]
HDAC5	Proliferating zone	No phenotype	Negative regulation of Runx2, Mef2c (function as an assistant of HDAC4)	[79]
HDAC7	Proliferating zone	The ratio of proliferating zone increases, whereas the ratio of hypertrophic zone decreases. Bone mineral density of cancellous bone decreases.	Negative regulation of B catenin	[156]
ARID5b	Proliferating zone	Early chondorogenic disorders	Demethylation of H3K9me2 → promotion of Col2 expression	[157,158]
SIRT6	Proliferating zone	The ratio of resting zone increases, whereas the ratio of proliferating zone and hypertrophic zone decreases.	Promotion of binding of ATF4 to the promoter region of Ihh	[161]

2.10.1. HDAC3

HDAC3 is widely expressed throughout the growth plate and is particularly prominent in hypertrophic chondrocytes. Hypertrophic chondrocytes of Prx-Cre:Hdac3 knockout mice are more prone to apoptosis than wild type mice, and upregulation of Mmp3, Mmp10, and Fgf21 causes the shortening of long bones [149]. The target gene for HDAC3 in the growth plate is not known, but it is known that HDAC3 regulates transcription of RUNX2, ZFP521, and HDACs 4, 5, and 7 in osteoblasts [148,150,151]. In an analysis using Col2CreERT:Hdac3 knockout mice, CRE induction using tamoxifen was performed in P5 mice, and the results indicated that the shortening of long bones can be confirmed at eight weeks of age. In these mice, acetylation of histones such as H3 and H4, cytokines such as IL6, MMP13, and SAA3, and genes involved in matrix remodeling increased [152].

2.10.2. HDAC4

HDAC4 plays a major role in nascent endochondral ossification. Hdac4 knockout mice display an early chondrocyte hypertrophy and calcification phenotype similar to that of Runx2 overexpressing mice and die early after birth, suggesting that HDAC4 suppresses Runx2 [119,153]. Hdac4 knockout mice and Col2-Cre mice show marked shortening of the growth plate, and conversely, phenotypes of Osx-Cre mice show increases in proliferating chondrocytes during endochondral ossification [121].

HDAC4 is located in the nucleus in proliferating chondrocytes and in the cytoplasm by the calmodulin-dependent kinase IV in proliferative chondrocytes, suppressing RUNX2 [154]. Furthermore, during differentiation of prehypertrophic chondrocytes into hypertrophic chondrocytes, the degradation of HDAC4 is promoted by mitogen-activated protein kinase (MAPK) p38, and as a result, cell differentiation during endochondral ossification is promoted [155]. PTHLH, like HDAC4, is known as a suppressor of chondrocyte hypertrophy, and the PTHLH signal promotes HDAC4 dephosphorylation and nuclear translocation. Recently, it has been shown that HDAC5 functions as an adjunct when HDAC4 becomes dysfunctional in the pathway, and that HDAC4/HDAC5 suppress the transcriptional activity of MEF2 [79].

2.10.3. HDAC7

HDAC7 is highly expressed in the resting and proliferative zones. In an analysis of Hadc7-knockout mice after birth using Col2-CreERT mice, the proportion of the proliferating zone in the growth plate was shown to increase while that of the hypertrophic zone decreased, resulting in a phenotype of reduced bone mineral density in the cancellous bone [156]. It has further been reported that HDAC7 functions in the proliferating zone by negatively regulating the activity of beta-catenin, which is involved in chondrocyte proliferation [156].

3. Other epigenetic regulators of growth plate development

3.1. ARID5

ARID5 has been identified as a transcriptional cofactor of SOX9, one of the important transcription factors involved in endochondral ossification [157,158]. Arid5b knockout mice show early chondrogenic disorders [158]. Col2a1 is known as a target gene of SOX9 (9–12), and ARID5b regulates Col2a1 gene expression along with SOX9. ARID5b recruits PHM2, a jmjC histone lysine demethylase, to demethylate H3K9me2, thereby promoting transcription of Col2a1 [158].

3.2. SIRT6

Sirtuin 6 (SIRT6) is a NAD+-dependent histone deacetylase that targets acetylated H3K9 and acetylated H3K56 [159,160]. SIRT6 is expressed in prehypertrophic chondrocytes and proliferating chondrocyte in the growth plate [161]. Sirt6 knockout mice have an increased resting zone thickness, whereas the proliferating and hypertrophic zones exhibit a decreased thickness phenotype. As a mechanism, it is believed that SIRT6 regulates the proliferation of chondrocytes by promoting the binding of ATF4 to the promoter region of IHH [161].

3.3. MicroRNAs

MicroRNAs are short, non-coding RNAs of approximately 22 bases that are very well conserved among many species. MicroRNAs suppress gene expression by binding mainly to the 3′UTR of mRNA [162,163]. In fact, more than half of human genes may be regulated by microRNAs [164] as it has been reported that they are deeply involved in various physiological phenomena and disease states [164], including chondrocyte differentiation in the growth plates. MicroRNAs are primarily transcribed as primary microRNAs, cleaved in the nucleus and cytoplasm, and ultimately biosynthesized into mature microRNAs [165]. Kobayashi et al. created a mouse model in which the Dicer gene, which has a central role in cytoplasmic cleavage, is specifically knocked down in chondrocytes, and the expression of mature microRNAs in chondrocytes is reduced [166]. In this model, skeletal formation is markedly suppressed, as is chondrocyte proliferation in the growth plate [166], suggesting that microRNAs play important roles in the growth plate. In particular, the effects of miRNA-140, 322, and 21 have been well described in growth plate development (Fig. 3; Table 3).

Fig. 3.

The microRNA network in the growth plate. Solid black arrow, positive regulation; black dashed arrow, negative regulation.

Table 3.

Summary of microRNAs related to growth plate development.

Name	Functional site	Phenotype of each KO mice	Target genes	Reference
miRNA-140	Proliferating zone	Hyposkeletal formation, Inhibition of chondrocyte proliferation	Negative regulation of Dnpep, Rala and HDAC4	[168–171]
miRNA-322	Pre-hypertrophic zone to hypertrophic zone	Slight reduction of hepertrophic zone	Unknown	[176]
miRNA-21	Proliferating zone?	In Gas5 overexpressing mice, Gas 5 suppress miRNA-21 and suppression of cell proliferation	Unknown	[181]

3.3.1. miRNA-140

Among microRNAs, the function in growth plates of miRNA-140 has been extensively examined. miRNA-140 is specifically and highly expressed in proliferating chondrocytes [167], and its knockout results in hyposkeletal formation [167,168]. In the growth plate, although there are differences among reports, chondrocyte proliferation is inhibited [168] and chondrocyte differentiation into hypertrophic chondrocytes is promoted [169].

ADAMTS5 is targeted in joints and is involved in the development of knee osteoarthritis [167]. miRNA-140 also targets mRNAs in chondrocytes, such as DNPEP [168], RALA [169], and HDAC4 [170]. It has also been shown that miRNA-140 suppresses transcription of MEF2C by suppressing P38, and consequently controls differentiation into hypertrophic cartilage [171]. miRNA-140 functions by controlling different target genes in the growth plate and joints.

miRNA-140 is present in the intron region of WWP2, and the expression of both WWP2 and miRNA-140 is regulated by SOX9 [172,173]. To dissect the exact function of WWP2 and miRNA-140 in bone formation individually, Wwp2-specific and miRNA-140-specific knockout mice have been generated. Skull formation is dysregulated in miRNA-140 knockout mice, but not in Wwp2 knockout mice [174]. Although the phenotype of the growth plate has not yet been well examined, this is an example in which the host gene and intron microRNA have different functions.

More recently, a human family was reported to have an autosomal dominant inheritance due to a gain-of-function mutation in miRNA-140 [175] that resulted in skeletal dysplasia. A mouse phenotype carrying the same mutation was consistent with the human phenotype. This is the first report of a microRNA gain-of-function mutation whose pathological condition is well-understood [175].

3.3.2. miRNA-322

miRNA-322 is strongly expressed from the prehypertrophic zone to the hypertrophic zone [176]. miRNA-322 has been shown to regulate the RAF/MEK/ERK pathway [177], which is an important pathway in many cells that translates extracellular signals in cells. Impairment of this pathway in cartilage tissue causes chondrodysplasia [178]. Analysis of miRNA-322-deletion mouse cartilage tissue using the CRE-loxP system revealed that hemizygous mutants suffer neonatal death due to respiratory failure resulting from tracheal cartilage damage [176]. In the growth plate, miRNA-322-deficient mice exhibit a phenotype of a slightly reduced hypertrophic zone [176]. The difference between the tracheal cartilage and the phenotype of the growth plate is thought to be because the ERK1/2 phosphorylation signal controlled by miRNA-322 plays a more important role in the development of tracheal cartilage [176].

3.3.3. miRNA-21

Growth-arrest-specific 5 (GAS5), an lncRNA, has an important role in mammalian growth and differentiation [179]. GAS5 acts as a miRNA-21 negative regulator to cause osteoarthritis [180]. In chondrosarcoma-derived chondrocytic cells (HCS-2/8 cells) with GAS5 overexpression, miRNA-21 is down-regulated. On the contrary, knockdown of miRNA-21 causes GAS5 up-regulation [181]. In rat models that overexpress GAS5, GAS5 suppresses miRNA-21 in chondrocytes of the growth plate. This results in suppression of cell proliferation and promotion of apoptosis [181], suggesting that miRNA-21 may play an important role in the maintenance and differentiation of growth plate chondrocytes.

4. Conclusion

In this review, we focused on transcription factors, epigenetic regulators, and microRNA involved in growth plate development and outlined their expression regions, effects, and interactions with other factors based on previous reports. From the 1990s to the present, many factors have been identified; however, our understanding of the interactions among these factors and of the network is still incomplete. We hope that the powerful analysis tools that have emerged recently will be used to increase our knowledge of musculoskeletal congenital disease mechanisms and ultimately lead to treatments.