Research progress on the hedgehog signalling pathway in regulating bone formation and homeostasis

Abstract Bone formation is a complex regeneration process that was regulated by many signalling pathways, such as Wnt, Notch, BMP and Hedgehog (Hh). All of these signalling have been demonstrated to participate in the bone repair process. In particular, one promising signalling pathway involved in bone formation and homeostasis is the Hh pathway. According to present knowledge, Hh signalling plays a vital role in the development of various tissues and organs in the embryo. In adults, the dysregulation of Hh signalling has been verified to be involved in bone‐related diseases in terms of osteoarthritis, osteoporosis and bone fracture; and during the repair processes, Hh signalling could be reactivated and further modulate bone formation. In this chapter, we summarize our current understanding on the function of Hh signalling in bone formation and homeostasis. Additionally, the current therapeutic strategies targeting this cascade to coordinate and mediate the osteogenesis process have been reviewed.

ligands, including two homologous genes, Ptch1 and Ptch2. Smo is a seven transmembrane protein that functions as a signal sensor.
Generally, vertebrates contain three Glis proteins, namely, Gli1, Gli2 and Gli3, which are transcription factors with zinc finger structures.
Usually, Gli1 and Gli2 function primarily as transcriptional activators, while Gli3 acts as a repressor of Hh signalling. Sufu is a negative regulator of the Hh pathway.
Generally, the Hh pathway is triggered by binding of the Hh protein to its receptor Ptch. In the absence of the Hh ligands, Ptch is usually located around the primary cilia and suppresses the activity and key functions both individually as well as in coordination with other signalling cascades in terms of Wnt, BMP and parathyroid hormone-related protein (PTHrP) during skeletal development and bone repair. [5][6][7] More importantly, the dysregulation of Hh signalling could lead to bone-related diseases in terms of osteoarthritis, osteoporosis and bone defects. In these settings, this review aims to organize and review the functions of Hh signalling in bone repair and regeneration.

| REG UL ATI ON OF MSC OS TEOG ENI C D IFFERENTIATI ON BY HH S I G NALLING
The osteogenic lineage commitment of MSCs is regulated by mechanical signals, paracrine factors, cytokines, chemokines and growth factors within their niche, which then activate a variety of signalling cascades, including Hh ( Figure 2). 7 A previously conducted study identified that recombinant N-terminal Shh (ShhN) promoted the proliferation and osteogenic differentiation of rat bone marrow MSCs (BMMSCs) in vitro, as evidenced by enhanced ALP activity, increased osteogenesisrelated gene expression and matrix mineralization. 8 Additionally, the implantation of MSCs overexpressing ShhN significantly accelerated bone formation in vivo; specifically, a 4-mm segmental bone allograft model in immunodeficient mice was established, and the modified MSC administration significantly promoted bone defect reconstruction via improving donor cell survival and differentiation, along with scaffold revascularization at the bone defect site. 9 Considering that Shh and Nel-like 1 protein (Nell-1) both possess osteoinductive potential, a combination therapy using ShhN with Nell-1 was established, and this particular combination strategy was demonstrated to markedly facilitate the osteogenic differentiation of adipose-derived MSCs (hASCs) when compared with either cytokine alone. Additionally, the pro-osteogenic function delivered by Nell-1 alone could be abolished in response to the Hh signalling inhibition with a Smo antagonist (cyclopamine). This particular combination cytokine strategy may be of potential therapeutic benefit for bone regeneration. 10 Additionally, Hh signalling was found to be involved in the osteoblastic differentiation deficiency of BMMSCs under high glucose (HG) conditions. In specific, HG delivered an inhibitory effect on BMMSC osteogenic differentiation; however, the addition of recombinant Shh alleviated the inhibitory function induced by HG, where cells transfected with Shh lentivirus demonstrated increased matrix mineralization nodules, higher ALP activity and expression levels of bone sialoprotein(BSP), osteopontin (OPN) and bone morphogenetic protein 4 (BMP-4). Additionally, a tooth extraction model in diabetes mellitus rats was established to verify the in vitro results. As expected, Shh administration promoted bone formation within the extraction socket. 11,12 In a recent study, Hh signalling was reported to be involved in the osteogenesis process, and the Hh gene was required for the loading-mediated osteogenic differentiation of the murine MSC line C3H10T1/2. 13 Dexamethasone, a well-known promoter for osteoblast differentiation, was found to enhance ALP activity and type I collagen expression and up-regulate Shh expression levels during the F I G U R E 1 A simplified display of the Hh signalling pathway. In the presence of the Hh protein, the combination of Hh and Ptch abolishes the suppressive effects on Smo delivered by Ptch; then, Hh signalling is activated and the Hh signals are transducted into cells. After that, transcription factors Glis family are dissociated from a suppressive complex containing Sufu and further activated. Subsequently, the Hh signalling downstream target genes that contribute to certain cellular activities are modulated. Hh, hedgehog; Ptch, patched receptor; Smo, smoothened receptor; Gli, glioma-associated oncogene; Sufu, suppressor of fused osteoblast differentiation process. Interestingly, both the mRNA and protein expression of Ihh and Gli1 were down-regulated. 14 The Hh pathway also participated in the BMMSC osteogenic differentiation process induced by simvastatin. Specifically, simvastatin administration resulted in an enhanced osteogenic differentiation capacity, indicated by up-regulated expression of COL1, ALP and osteocalcin (OCN), and increased ALP activity. More importantly, BMMSCs treated with simvastatin expressed higher levels of Ihh and Gli1, and more nuclear translocation of Gli1 was observed.
Contrasting effects were observed after the BMMSCs were exposed to cyclopamine (an Hh signalling inhibitor), indicating that simvastatin promotes BMMSC osteogenic differentiation, at least in part, via the Hh pathway. 15 During mandibular development, ciliary protein Ift88 was found to participate in chondrogenesis and osteogenesis at least partially via the Shh pathway. 16 Additionally, low-level laser irradiation was also found to promote osteoblast proliferation via Hh signalling. 17  and further studies demonstrated that the underlying mechanisms may lie in that Gli1 + cells promoted angiogenesis via the Gli-HIF-1a signalling. 19 Additionally, a variety of microRNAs (miRNAs) have been reported to get involved in the osteogenesis process mediated by Hh signalling. For example, miR-342-3p was identified as a therapeutic agent that can accelerate the osteogenic differentiation of human umbilical mesenchymal stem cells (UCMSCs) by down-regulating Sufu to activate Shh signalling. 20 Another study also verified that miR-342-3p was highly expressed in hUCMSCs during osteogenic differentiation, and miR-342-3p overexpression markedly upregulated the expression levels of osteogenic-related genes (ALP, Cbfa1 and OPG) by activating Hh signalling. 21 miR-196a could reverse the MSC osteogenic differentiation obtained in osteoporosis mice by targeting GNAS to further activate Hh signalling, 22 whereas miR-467g was found to be an inhibitor for osteoblast differentiation, and could negatively regulate the osteogenesis process via Ihh/runtrelated transcription factor 2(RUNX2) signalling. 23

| HH S I G NALLING AND BONE-REL ATED DISE A SE
The Hh pathway plays a crucial role in skeletal development and bone repair, and the dysregulation of Hh signalling could lead to bone-related diseases, including osteoarthritis, osteoporosis and bone fracture. For example, osteoporosis results from decreased bone formation by osteoblasts in parallel with increased bone resorption by osteoclasts ( Figure 2). Under this context, modulating Hh signalling to manipulate osteoprogenitor cells to augment the osteogenic differentiation potential and enhance bone formation properties is of great significance.

| Osteoporosis
Osteoporosis is a metabolic bone disease represented by continuous destruction of bone mass and microstructure due to the imbalance of bone formation and resorption. Based on the current F I G U R E 2 Hedgehog signalling plays an important role in regulating MSC differentiation and maintaining bone homeostasis. Hh signalling inhibits MSC differentiation into adipocytes, whereas it promotes their differentiation into chondrocytes and osteoblasts, further getting involved in maintaining bone homeostasis where osteoblasts mediate bone formation, and osteoclasts dominate the bone resorption. Additionally, the dysregulation of Hh signalling could lead to bone-related diseases in terms of osteoarthritis, osteoporosis and bone fracture. MSC, mesenchymal stem cell understanding, dysfunction of MSCs with impaired osteogenic potential contributes to osteogenesis disorders, especially the osteoporosis development. For example, oxidative stress was verified to deliver its suppressive effect on MSC osteogenic differentiation and facilitate age-related osteoporosis by inhibiting Hh signalling. 18 A previous study reported that miR-196a was poorly expressed when its direct downstream target GNAS was overexpressed in osteoporosis mice. After transfection with miR-196a mimic, the MSCs (isolated from osteoporosis mice) dis- indicating that Ptch1 may represent a promising modulatory target for osteoporosis treatment. 25

| Osteoarthritis
The dysregulation of Hh signalling can lead to osteoarthritis, which is characterized by progressive degeneration of articular cartilage, and in Ihh-depleted mice, the expression of osteoarthritis-related markers, including MMP-13 and collagen type X, was significantly downregulated. Thus, the inhibition of Ihh signalling could be regarded as a promising strategy to prevent or treat osteoarthritis. 26 Additionally, a study conducted by Ruiz-Heiland G concluded that Hh signalling blockade could be protective in that this particular inhibition treatment could block the formation of collagen type X and hypertrophic chondrocytes and inhibit osteophyte formation. 27 Recently, a Smospecific inhibitor named taladegib was verified to be a promising agent for osteoarthritis treatment, since it controlled chondrocyte hypertrophy by down-regulating the expression of MMP13, collagen type X and RUNX2 via Smo/Gli1 signalling. 28  Hh target gene expression, which suggests promising therapeutic alternatives for fracture treatment in smokers, especially for delayed fracture healing. 32 Additionally, Hh signalling was observed to be involved in impaired bone healing in the setting of diabetes mellitus.
A previous study found that inhibition of Hh signalling suppressed the expansion of injury-induced mouse skeletal stem cells (mSSCs), further impairing bone healing in diabetic mice. Then, a slow release hydrogel was utilized to precisely deliver recombinant Ihh/Shh to the local fracture site, which led to an accelerated fracture repair effect because the impaired expansion and osteogenic potential of mSSCs in response to injury were restored. 33 A previously conducted study demonstrated that Shh-positive and Gli1-positive cells were localized along the surface of the newly formed bone; further study identified that Shh and Gli1 were co-localized with RUNX2 and OSX at the fracture site, implying that Shh-Gli1 signalling regulates intramembranous and endochondral ossification processes within bone fracture healing. 34

| S MALL MOLECULE S/B IOLOG IC AL MATERIAL S IN HEDG EHOG S I G NALLING AND OS TEOG ENE S IS REG UL ATI ON
In recent years, much progress has been made in discovering and applying small molecules or bioactive materials to regulate stem cell commitment, and target-based manipulation provides substantial insights into therapeutic strategies for committing MSCs to tissue regeneration. 35 In this context, employing small molecule agents/ bioactive materials to modulate Hh signals represents a promising therapeutic approach for the treatment of bone-related diseases, and these natural or synthetic agents are promising for promoting osteogenesis for bone repair/regeneration (Table 1).

| Purmorphamine
Purmorphamine, a Smo receptor agonist, was demonstrated to induce the osteogenic differentiation of human endometrial stem cells seeded on a collagen/HA scaffold, where the ALP level and RUNX2 expression were both up-regulated. 36 Besides, the pro-osteogenic effect of purmorphamine was demonstrated, to an extent, similar to that delivered by BMP-4. Purmorphamine administration increased cellular proliferation and up-regulated osteogenic gene expression by activating Hh signalling, resulting in bone formation. 37  utilizing purmorphamine for the treatment of bone-related disease have been widely studied due to its bone regenerative properties. 38,39

| Smoothened agonist (SAG)
Smoothened agonist has been identified as an activator of Hh signalling, which facilitated the translocation of Smo from the cytoplasm to the primary cilium and stabilized it in its active form. 49 Lee and colleagues found that SAG administration led to accelerated osteoblast differentiation in vitro and promoted calvarial bone healing in vivo, which may be applied for bone defect treatment. 50 Further studies found that the administration of SAG combined with Nell-1 significantly accelerated calvarial bone defect healing, as demonstrated by increased bone volume and bone thickness as well as increased defect vascularization. 51 A previous study found that Kruppel-like factor 4 (KLF4) inhibited osteoblast differentiation by repressing basal Hh activity. After SAG treatment, the decreased expression of osteoblastic genes and mineralization delivered by KLF-4 was significantly up-regulated. 52 Also, the administration of SAG combined with helioxanthin derivative markedly promoted bone formation and finally achieved bone healing (in a rat femur bone defect model) without cell transplantation. 53 Under this context, the combination therapy involved with SAG and other molecules, like BMP-2, may represent a promising strategy for bone repair. 54 and Patch1, was significantly down-regulated. 56 Astragaloside IV is an effective agent isolated form Astragali Radix (Chinese medicine).

| Other small molecules
A previous study found that astragaloside IV promoted the proliferation and migration of osteoblasts to facilitate osseointegration by Hh pathway. 57 Also, resveratrol treatment could improve the osteogenic differentiation potential of MSCs in vitro via alternating the expressions of Hh target genes, which provides promising therapeutic alternatives for the treatment of bone diseases. 32  60 Nano-scaled hydroxyapatite-blasted titanium (nHA) was also utilized for osteoblast differentiation due to its anti-inflammatory potential, and Shh signalling was verified to be involved in the osteoblast process mediated by nHA, which could be regulated to guarantee osteoblast activity towards osteogenesis. 61

| HH S I G NALLING INTER AC TS WITH OTHER S I G NALLING PATHWAYS TO REG UL ATE BONE FORMATI ON
A variety of signalling cascades are involved in osteogenesis-related cell fate decisions, and osteogenesis is well orchestrated by various signalling pathways such as Wnt, BMP PTHrP and Hh ( Figure 3). In this setting, gaining a better understanding of the interaction of the "osteogenic signalling network" for tissue engineering is of great significance, and the distinct window for each signalling in terms of timing and the threshold level of its activation is pivotal. 62

| Hh-Wnt axis
Regarding the interaction of Hh and Wnt signalling, a variety of studies have reported that Hh and Wnt signalling is functionally antagonistic through common regulators, such as secreted frizzled-related

| Hh-BMP axis
In addition to Wnt signalling, other pathways also contribute to Hhinduced bone formation. Of note, BMP signalling has been validated to be required or has a positive impact on Hh-mediated osteogenesis. 79 It was reported that BMP-dependent Hh signalling was required for calvarial bone defect repair, and it regulated the interplay between suture MSCs and osteoclasts, which are both crucial for calvarial bone homeostasis and injury repair. 80 In addition, Ihh and BMP-2 were verified to deliver a synergistically effect on the osteogenic differentiation of human MSCs. 81  Further study demonstrated that Shh signalling was repressed by BMP signalling; more importantly, the decreased osteo/dentinogenic differentiation of SCAPs (mediated by Shh signalling) was enhanced. 84 These contradictory results regarding the interaction and function of Shh and BMP signalling in MSC differentiation might be related to cell types and conditions, which remain to be explored with more in-depth and detailed research.

| Ihh-PTHrP axis
Previous studies have demonstrated that the Ihh-PTHrP feedback pathway was critical to the endochondral ossification process. 85 Additionally, Ihh and PTHrP could work together to commit MSCs towards the osteoblastic lineage by inducing RUNX2. 86  as cell-secreted lipid bilayer structures, can be manipulated as therapeutic tools for any molecule of interest including Shh, to promote osteogenesis. 90 As naturally occurring secreted vesicles, EVs possess certain advantages over other carrying agents (like liposomes), including low propensity to trigger immune rejection, no toxicity concern and high stability. 91 Additionally, attention needs to be paid to fundamental problems of fine-tuning the duration and strength of the Hh axis at appropriate timing, together with other signalling cascades in terms of the Wnt, BMP and PTHrP axes, because the regulatory mechanisms involved in the Hh signalling are complicated and cell type-specific. Additionally, novel mechanisms are continuously being identified; and each new finding usually triggers another question. 92 These challenges need to be addressed before the potential of these approaches can be fully realized to facilitate bone formation and maintain bone homeostasis 93 .

ACK N OWLED G EM ENTS
This study was supported by the National Natural Science

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

AUTH O R CO NTR I B UTI O N
HZ, ZL and YC conceptualized the study; HZ, ZL and CH-Z wrote original draft; F-MC and AL contributed to writing-review and editing. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article, as no new data were created or analysed in this paper.