Wnt signaling: Essential roles in osteoblast differentiation, bone metabolism and therapeutic implications for bone and skeletal disorders

Abstract

Wnt signaling executes an indispensable performance in osteoblast differentiation, bone development, homeostasis, and remodeling. Wnt signals trigger the intracellular Wnt signaling cascade to initiate regulating the implication of β-catenin in the bone environment. Going through the novel discoveries done via high-throughput sequencing technologies on genetic mouse models, we highlighted the significant contribution of Wnt ligands, co-receptors, inhibitors, their related skeletal phenotypes in mouse models and the similar bone disorders clinically observed in human beings. Moreover, the crosstalk between Wnt signaling pathway and BMP, TGF-β, FGF, Hippo, Hedgehog, Notch and PDGF signaling pathways is thoroughly demonstrated to be the underlying gene regulatory network that orchestrates osteoblast differentiation and bone development. We also introspected the significance of Wnt signaling transduction in the reorganization of cellular metabolism by stimulating glycolysis, glutamine catabolism, and fatty acid oxidation in osteoblast-lineage cells that display an important regulatory arbor in the cellular bioenergetics of the bone. Throughout this evaluation, most to date therapeutical approaches towards osteoporosis and other bone maladies found in human beings, are formulated with an aspiration to holistically revamp the present clinical applications involving various monoclonal antibodies therapies that lack specificity, efficacy, and safety into more requisite advanced therapeutics that satisfy these three requirements for further clinical considerations. Conclusively, our review provides comprehensive scientific findings related to the fundamental significance of Wnt signaling cascades in skeletal system and the underlying gene regulatory network with other signaling pathways enlightening researchers with the possibility to further integrate the identified target molecules into therapeutic strategies for skeletal disorders treatment in the clinic.

Introduction

Bone is an exceptionally vigorous organ that goes through frequent resorption by osteoclasts and a brand-new development by osteoblasts during the remodeling process. The four types of cells that make up bone, the stiff tissue that constitutes the skeleton in vertebrates, are osteoblasts, bone lining cells, osteocytes, and osteoclasts.¹ It has significant roles in the body, including motility, resistance, and soft tissue protection, as well as Ca²⁺ and PO₄ storage and bone marrow nurturing.2, 3, 4 Evidence also shows that osteocytes can sense mechanical stimuli and orchestrate the bone remodeling process by modifying their environment.5, 6, 7 The old bone is replenished through a composite process of bone remodeling that includes three phases¹: the resorption of bone tissue by osteoclasts,² the growth progression to new bone creation, and³ the completed bone production by osteoblasts.^8,9 This process occurs as a result of the coordinated actions of osteoblasts, osteoclasts, osteocytes, and bone lining cells, within the bone cavities, which form the fundamental multicellular unit (BMU).^9,10 Besides its self-mending abilities, there are some deficiencies that bone is unable to provide regeneration for, therefore various signaling pathways actively respond to these deficiencies and one of them is the Wnt signaling pathway.¹¹

The Wnt pathway is a developmentally conserved route that contains a series of signaling pathways that regulate a variety of biological activities ranging from embryonic growth to tissue regeneration.12, 13, 14, 15 In both human beings and mice mammals, there are 5 genes in C. elegans and 7 genes in Drosophila that encode for the large family of Wnt secreted protein growth factors and regulate the behavior, adhesion, and polarity of the cells. Wnt is an abbreviation for wingless, the Drosophila segment polarity gene, and integrated (int-1), the vertebrate analog. This signaling pathway controls a plethora of functions with the help of Wnt ligands, R-spondin proteins, norrin and other receptors found on the cell surface that trigger downstream gene expression and determine the specificity of Wnt pathway.^12,16 In addition, the Wnt signaling pathway may be separated into the canonical mechanism that depends on β-catenin's activity and the non-canonical mechanism that doesn't depend on β-catenin's activity. These two major divisions are subdivided into the Wnt/planar cell polarity (PCP) and the Wnt/Ca²⁺ pathways and are required for osteocyte cell migration and patterning throughout embryonic maturation, for tissue homeostasis and regeneration and most notably during bone remodeling and homeostasis in bone.^17,18 Consequently, dysregulations of Wnt signaling have been associated with a variety of human skeletal diseases. Further studies are required to understand the underlying issues that lead to a cavity in targeted therapy of Wnt-linked diseases.^12,19

Canonical and non-canonical Wnt signaling pathways

Wnt/β-catenin signaling: canonical Wnt pathway

Canonical Wnt signaling pathway promotes the production of β-catenin protein in the cytoplasm and is a requisite process for fetal maturity and metabolic equilibrium.¹² When the canonical Wnt ligand is missing in this specific pathway, it is a destructive complex of proteins, that includes serine–threonine kinases glycogen synthase kinase 3β (GSK-3β) and casein kinase 1α (CK1α), the protein Axin, the cytoplasmic phosphoproteins Dishevelled (DVL) and Adenomatous Polyposis Coli (APC), that calls for β-catenin's phosphorylation and eventually its destruction via the degrading actions of ubiquitin-proteasome system (UPS).^20,21 On the other side, when the canonical Wnt ligand is not missing, its cell surface receptor frizzled (FZD) and co-receptor low-density lipoprotein receptor-related protein 5/6 (LRP5/6) attach themselves to it causing DVL and Axin proteins to assemble near the plasma membrane, thus deconstructing the destructive complex of proteins. This process results into the accumulation of β-catenin in the cytoplasm and eventually its entrance into the nucleus thus triggering gene expression upon its molecular connection with T-cell factor/lymphoid enhancer factor (TCF/LEF)^16,22, 23, 24, 25 (Fig. 1).

Figure 1.

Canonical and non-canonical Wnt signaling pathways. Canonical Signaling- Wnt-Off: At the plasma membrane, both the Frizzled receptor (FZD) and LRP5/6 receptor stand inoperative, when in lack of Wnt ligand. In the cytoplasm, the destruction complex (consisted of GSK-3β, APC, CK-1 and Axin) bind and phosphorylate β-catenin, followed by its proteasomal degradation. In the nucleus, Wnt target gene expression remains off. Canonical signaling- Wnt-On: When Wnt ligand binds to the Frizzled receptor, an active receptor complex is formed by sequestering LRP5/6 receptor. This brings the inactivation destruction complex and signalosome formation mediated by Dishevelled (DVL). Thus β-catenin, after translocating into the nucleus, binds to T cell factor gene region and forms a transcriptional protein-activator complex in order for Wnt target genes to become expressable. Non-canonical Wnt/PCP pathway: Wnt ligand binds to the Frizzled receptor thus recruiting Dishevelled. They form a complex, with co-receptors such as ROR and RYK included, in order for RhoA/Rock pathway to be activated and actin to become polymerized. This particular pathway can instigate JNK pathway's operation via Frizzled-Dishevelled-ROR/RYK system or VANGL planar cell polarity protein-ROR/RYK system. Non-canonical Wnt/Ca²⁺pathway: Frizzled receptor sequesters ROR&RYK receptors when Wnt ligand binds and trigger the membrane proteins Dishevelled and Guanine nucleotide-binding proteins to operate and consequently activate Plc enzyme to release Ca²⁺ from within the cell. High amounts of Ca²⁺ trigger other pathways to initiate their activity assisted by activating receptor molecules like CaN (calcium-dependent serine–threonine phosphatase), CaM-kinase II (Ca²⁺/calmodulin-dependent protein kinase II) and PKC (protein kinase C).

Wnt/PCP and Wnt/Ca²⁺ signaling: non-canonical Wnt pathways

The non-canonical Wnt/PCP pathway operates separately from β-catenin's activity. It combines cell mobility with tissue polarity via GTPase RhoA and/or c-Jun N-terminal kinase catalysis (JNK).²⁶ PCP pathway initiates functioning only when ligands like WNT5A, WNT5B, and/or WNT11, bind to their receptors Frizzled 3 or Frizzled 6 and their co-receptors ROR1/2 (Receptor tyrosine kinase-like orphan receptor 1 and 2), or PTK7 (Protein tyrosine kinase 7).²⁷ Other proteins such as VANG (11/12), Prickle (1/2), Cadherin EGF LAG (CELSR 1/2/3), DVL (1/2/3), and Ankyrin repeat domain 6 (ANKRD6) are major participants in this signaling catalyzation²⁸ (Fig. 1). Since this pathway is dependent on the Dishevelled protein's activity, it is exactly these group of proteins DAAM1 and DAAM2 (Dishevelled-associated activator of morphogenesis proteins) as well as MAP kinases (Mitogen activated protein) that trigger the Rho-associated protein kinase to initiate the RhoA-ROCK pathway or the RAC-JNK signaling pathway, sequentially. Contrastingly, in the situation of non-canonical Wnt pathway's independence from the activities of the Dishevelled protein, it is RTKs (Receptor tyrosine kinases), G proteins and PLC (Phospholipase C) that trigger the serine/threonine protein phosphatase calcineurin and cause the NFAT (Nuclear factor of activated T cells), via calcium release, to assemble inside the nucleus.^26,29,30 Other pathways, such as NLK (Nemo-like kinase) signaling can initiate functioning in the presence of high level of calcium released in the cytoplasm, via CaMKII (Calcium/calmodulin dependent kinase II)'s activity.

Mutations in Wnt components generate deficient skeletal phenotypes in mouse models and humans

Wnt ligands mutation in osteoblasts

WNT1. WNT1 plays a crucial function during osteoblastogenesis. Given that WNT1 is found in mesenchymal stem cells (MSCs), osteoblasts, and osteocytes, it activates the canonical pathway and suppresses MSCs differentiation into adipocytes causing the production of osteoblasts to increase.^31,32 Studies have shown that WNT1 plays a principal role in the early stages of fetal growth. For instance, purebred Wnt 1-KO mice emerged with infant mortality whilst hybrid Wnt1-KO mice underwent lenient low bone density without a weakened bone development and ossification.³¹ At a later stage, WNT1 activates mTORC1 pathway promoting osteoblasts' differentiation and mineralization as studies have shown that the inhibition of mTORC1 in the Wnt1 gain-of-function mouse model was associated with reduced mineralization and bone formation.³³ Due to WNT1's strong linkage with bone growth and formation, loss-of-function mutations associated with WNT1 are responsible for diseases like premature osteoporosis and osteogenesis imperfecta in humans.34, 35, 36

WNT3A. WNT3A ligand has a major contribution to differentiating mesenchymal stem cells into osteoblastic cells via the canonical Wnt pathway as well as the non-canonical Wnt/Protein kinase C pathway.^37,38 It may also prevent the process of programmed cell death in both mature and immature osteoblastic cells39, 40, 41 as well as mesenchymal stem cells’ further progression to transform into chondrocytic cells.⁴² Reported experiments have resulted in defective fetal development to non-surviving Wnt3a-KO mouse models.⁴³ Moreover, a patient diagnosed with skeletal dysplasia was found with a wild-type alteration on WNT3A gene.⁴⁴ Other findings have shown that the treatment of ESCs (embryonic stem cells) in mouse with recombinant Wnt3a, which positively affects the activity of β-catenin, downregulated the osteogenic expression.⁴⁵

WNT4. WNT4 contributes to osteoblastic differentiation via activating the non-canonical Wnt pathway and hinders both the inflammation of bone as well as bone remodeling.^46,47 Wnt 4-KO mice have displayed a delay in chondrocyte maturation and an early postnatal death while Wnt4-cKO female mice showed decreased femoral BMD.^48,49 WNT4 can hinder RANK to attach to the TRAF6 in osteoclastic cells (TNF receptor-associated factor 6) suppressing the RANKL-induced osteoclast differentiation.⁴⁷ Experimenting with mouse models has shown that WNT4 ligand functions as a protective element against bone resorption as well as against bone destruction. Via in vitro reports, it was demonstrated that the main alterations of Wnt4 gene as well as the knockdown of a protein coding gene named Zinc Finger and BTB/POZ Domain Containing 40 (ZBTB40) occurring in osteoblastic cells, resulted in a suspended osteoblastic development and ossification.49, 50, 51

WNT5A. WNT5A has a distinct role in stimulating the differentiation of both osteoblastic and osteoclastic cells by enhancing or hindering the canonical Wnt pathway in bone.52, 53, 54^,55 This ligand is responsible for independent-GSK-phosphorylation-β-catenin-deterioration.⁵⁸ It can also hinder the genes for β-catenin's adjustment to transcript via the non-canonical Wnt signaling pathway and T-cell factor (TCF) intervention.⁵⁴ Moreover, the WNT5A expression in osteoblasts is promoted by sphingosine-1-phosphate (S1p).⁵⁹ Experiments have shown Wnt5a knockout mouse models to display a diversity of developmental anomalies, a delay in chondrocyte hypertrophy and skeletal ossification, and perinatal death.^57,60 Meanwhile, the Wnt5a conditional knockout mouse models have displayed under-expressed RANK levels in osteoclast progenitors that led to breakdown of old bone, and a damaged osteoblast development.⁵⁷ In addition to that, the excessive signaling activity of WNT5A ligand and neurotrophic tyrosine kinase, receptor-related 2 (ROR2) has shown to cause bone depletion in rheumatoid arthritis.^56,57 Opposite to Wnt3a, the treatment of ESCs in mouse models with a recombinant Wnt5a upregulated various kinases' activity such as PKC, CaMKII and JNK while antagonizing the activity of β-catenin thus increasing the osteogenic proliferation and displaying a potential application in tissue engineering.⁴⁵ Loss of function alterations occurring in WNT5A gene have clinically shown to be a cause for Acral dysostosis with facial and genital abnormalities (Robinow syndrome).^53,61

WNT5B. WNT5B is associated with MSC differentiation and chondrocyte proliferation during bone development.^60,62 Furthermore, WNT5B gene has been found to be overexpressed in female osteoarthritis (OA) patients.^63,64 Wnt5b-KO mice displayed a delayed bone ossification due to the overexpressed Wnt5b in chondrocytes.⁶⁰

WNT7A. WNT7A is necessary in the development of cranium, face, and limbs during the formation process of the embryo.^65,66 Upregulation of WNT7A reduced RUNX2 expression thus reducing the osteogenic cell proliferation, while the downregulation of WNT7A in MSCs increased RUNX2 expression thus promoting osteogenic cell proliferation.⁶⁷ Wnt7a-KO mice models display impaired limb development.⁶⁸ Alterations like loss of functions for the WNT7A gene was shown to be a great cause for the Fuhrmann malady, the Al-Awadi-Raas-Rothschild disease (AARRS) and lastly the Santos affliction in human.^65,69,70 These diseases have the ectodermal dysplasia physical composition accompanied by acute limb deficiencies.^65,69

WNT7B. WNT7B ligand functions as an enhancing factor for osteoblast differentiation in mice, prenatally and postnatally⁴¹ through the non-canonical activation of mTORC1 and PKC δ signaling.^37,41 Wnt7b knockout mouse models were shown perinatally dead.⁷¹ In occasions where particular parts of Wnt7b gene in osteogenic cells were missing, a delayed chrondocytic development and a deficient osteoblastic differentiation mediated by the osterix transcription factor were displayed.^37,41 Overexpression of Wnt7b gene, in osteoblasts and chondrocytes of mice, resulted in accelerated bone formation and denser bones.^33,41 In humans, increased WNT7B expression has been linked to osteoarthritis (OA) patients and rheumatoid arthritis (RA) patients.^72,73

WNT9A. WNT9A arranges appropriately chondrocyte development prior to ossification and maintains the joint stability.^48,74 During osteoblastic differentiation, Wnt9a expression decreases.⁴⁰ Wnt9a knockout mouse models have displayed bone malformations like, incomplete arthrodesis, low levels of bone ossification, indications of skeletal dysplasia, metaplastic cartilage and lastly, neonatal death.⁴⁸

WNT10A. WNT10A favors ostosis by hindering the formation of adipocytes during osteoblastogenic process, via the Wnt-canonical signaling pathway.^56,75 Wnt 10a knockout mouse models have displayed reduced bone ossification, bone marrow adipose tissue development and tooth development.76, 77, 78 Due to this ligand's significant role in dentinoblast differentiation, WNT10A gene alterations have displayed abnormal development of ectodermally-derived organs (also known as ectodermal dysplasia) in humans, causing absence of teeth (also known as anodontia) as well as the schöpf-schulz-passarge syndrome (also known as the odontoonycho-dermal dysplasia, OODD).79, 80, 81, 82

WNT10B. WNT10B ligand functions as a regulator in the process of osteoblastic cells formation via the Wnt canonical pathway. It hinders PPARγ and C/EBPα (also known as the family of transcription factors during adipogenesis) from expressing accurately and enhances RUNX2 to express properly.⁸³ WNT10B maintains and renews osteoblastic mesenchymal stem cells in the bone marrow⁸⁴ due to its highly expressed level in numerous progenitor cellular compartments.⁸⁴ In Wnt 10b knockout mouse models, the mineralized bone volume (BV/TV), bone density level (BMD) and the number of trabeculae (Tb.N) were all shown to be substantially reduced, when contrasted with homozygous mice.³² WNT10B has been implied in various human disorders.⁸⁵ In humans, homozygous missense mutations in WNT10B cause split limb malformation with autosomal recessive inheritance, dental anomalies including here oligodontia and osteosarcoma.^53,86, 87, 88

WNT16. WNT16 is involved in the maintenance of postnatal bone homeostasis by regulating both canonical and non-canonical Wnt targets in osteoblasts.⁸⁹ Wnt 16 is found to be more highly expressed in cortical bone than in trabecular bone⁹⁰ as both Wnt16 knockout and the conditional knockout mouse models displayed reduced bone density level, low density in cortex, weakened bones and increased impermeability in cortex, while the trabeculated bone tissue's volume remained unaltered.^90,91 Alternatively, mouse models with a high expression of Wnt16 genes, in both their osteoblastic and osteocytic cells of trabeculae and cortex compartments, displayed an increased bone density level as well as a more resistant bone.92, 93, 94 WNT16 gene, has long been linked to with various bone physical compositions such as bone density level (BMD), the resistance of bone, bone linear framework, the density of cortex and lastly bone rupture possibility.95, 96, 97, 98 Researchers assume for WNT16 ligand to contribute to hindering the process of bone formation in two ways, by increasing the osteoprotegerin (also known as the osteoclastogenesis inhibitory factor, OCIF) or activating the osteoclastic hematopoietic progenitors via ostoblastic mediation.^90,99

Wnt-co-receptors in osteoblasts

LRP. LRP stands for low-density-lipoprotein-receptor-related-protein and belongs to the low density lipoprotein receptor family, recognized in the cell surface,¹⁰⁰ with endocytic and signaling functions.¹⁰¹ These LRP integral polypeptides are composed of an outer-cell compartment which binds itself to proteins that are present in the extra-cellular matrix, and an inner-cell compartment that functions as an interceder in signaling mechanism transductions.¹⁰² In experimental phases, both Lrp5-KOs and cKOs mice in skeletal cells display a low bone density level postnatally, caused by a decreased skeletal development processing.103, 104, 105, 106, 107, 108 Point-mutations occurring in Lrp6 gene are linked to anodontia, the disorder related to congenital absence of more than six teeth (also known as ogliodontia) and lastly high bone mass phenotype.109, 110, 111 The Lrp6 knockout mice, during the development of embryo, display lost non-proximal limb appendage as well as shortened transverse bones.¹⁰⁵ Moreover, cKO mouse models have also confirmed the involvement of Lrp6 in postnatal bone metabolism.^106,108,112 Various researchers tend to acknowledge the Lrp5-mediated regulation of Wnt-signaling pathway in mature osteoblast cells found in the cortex as well as the Lrp5-mediated regulation of Wnt-signaling pathway in premature osteoblast cells found in trabeculae, to enhance the bone mass density's well-development.^{105,106,108,113} Lrp4 is necessary to enhance the inhibitory function of sclerostin inhibitor in the canonical Wnt pathway114, 115, 116, 117 amongst other inhibitors such as DKK1 and SOSTdc1.^116,118 LRP4 ligand is also known to promote DKK1 and SOSTdc1's inhibiting activity in the Wnt-signaling pathway.^116,118 In addition, mutations in Lrp4 cause Cenani–Lenz syndrome (CLS), sclerosteosis-2 and bilateral syndactyly.^114,119, 120, 121, 122 Lrp4-KO mutant mouse models absent neuromuscular functions and the inability to breathe¹¹⁵ whilst, Lrp4-cKO mouse models do not.^115,117 Lrp4 is also shown to apply its functions in hindering the canonical pathway and eventually affect the osteoblastic development process, during in-vitro and in-vivo animal experimental phases.

In humans, alterations related to LRP5 gene appear linked to two types of skeletal physical compositions such as, osteogenesis perfecta (OPPG) and increased bone mineral density (HBM). OPPG is highly linked to loss of function mutations in LRP5 gene that cause the Wnt-canonical pathway to become inoperative, while gain of function mutations are linked to the reduction of propinquity for other inhibiting elements found in the cell's surface such as SOSTdc1 and DKK1, an event that eventually generated the increased bone mineral density phenotype to display.123, 124, 125, 126, 127, 128

ROR. ROR1/2 are type I transmembrane protein tyrosine kinases that belong to the ROR family of kinases. Ror1/2 genes are highly expressed in most main skeletal compartments during the formation of embryo but significantly reduced in mature tissue compartments.¹²⁹ Ror2 gene, especially, is distinctly expressed in fully-developed osteoclastic cells¹³⁰ and has been outlined to modulate bone length, via interacting with two other Wnt ligands such as WNT5A and WNT9A.^129,131 The Ror2 knockout mouse prototypes have shown traits of nanism characterized by shortened or misshaped bones, anomalous face typology, congenital defective ventricle, and lastly blue skin disorder (also known as cyanosis).^57,132, 133, 134 ROR2-WNT5A moderate signaling controls the osteoclastic development and ossification therewith preserving proper amounts of BMD while being initiated by the constitutively active form of RhoA.^57,130,134 Inactive loss of function ROR2 gene modification is clinically shown to be linked to the less common type of robinow syndrome (RRS). On the other hand, gain of function alterations are associated with BDB (also known as brachydactyly type B), a condition characterized hypoplasia or aplasia, also in human beings.^130,135, 136, 137, 138

KRM. KRM1/2 are single-spanning trans-membrane receptors that display excessive binding interaction for Wnt inhibitors like Dickkorpf 1 and 2 (DKK1 and DKK2).^139,140 However, Kremen 1 is shown to have a more extensive expression habit while Kremen 2 is mainly highly expressed in bone cells [109]. Therefore, when the inhibitor DKK1 is not present, KRM 1/2 attaches to receptor LRP6 thus activating the Wnt signaling pathway.^141,142 Krm1 knockout mouse models and premature Krm2 knockout mouse models appear alive with no malformations and a normalized BMD in general,¹⁴³ while mature Krm2 knockout mouse models show an increased BMD.¹⁴¹ In humans, KRM1 alterations have been associated with ectrodactyly and the congenital absence of more than six teeth (also known as oligodontia).¹⁴⁴ Putting these data altogether, KRM 1 and KRM 2 appear superfluously present in amount during skeletal development with especially, KRM 2, playing a significant role during bone remodeling process.

LGR. G-protein-coupled receptors rich in leucine (LGRs) subfamily consists of three specific types 4, 5 and 6.¹⁴⁵ Specifically, LGR4 is broadly expressed in various body compartments, like bone cells, adipocytic cells and myocytic cells since premature stages of embryo's formation and development until the maturity stages.146, 147, 148 LGR4 has also demonstrated to inhibit the development of osteoclasts mediated by a type II membrane known as RANKL.¹⁴⁹ Lgr4 fully knockout mouse models are shown at risk of dying in their early stages of life exhibiting an abnormal light and small body as well as shortened bones.147, 148, 149, 150 Lgr4 knockout mouse models appear to have a reduced bone mineral density caused by a diminished process of ossification and bone mineralization thus obstructing the regular process of osteoblastogenesis in the early stages of embryogenesis and reducing the uncalcified bone matrix (also known as the osteoid) development.^147,150 Lgr5 is expressed in osteoblastic progenitors during bone development and formation. Lgr5 knockout mouse models display traits of tongue-tie disease (also known as ankyloglossia) a congenital oral anomaly that reduces the mobility of the tongue tip accompanied by acute respiratory distress syndrome (ARDS) and lastly death in their newly days of being born.¹⁵¹ LGR6 enhances Wnt pathway downstream of the BMP signaling pathway.¹⁵² Although Lgr6 knockout mouse models have displayed a normal pattern of development, LGR6 is compulsory for multi-tissue restoration (also known as the digit tip regeneration) of bone in mouse stereotypes.¹⁵³

Wnt inhibitors in osteoblasts

DKK. Dickkopf (DKK) is comprised of four genes that code for the Dickkopf-related proteins.1, 2, 3, 4 Members DKK1, DKK2 and DKK4 have a similar structure and act through the canonical Wnt pathway, while DKK3 has no effect through the canonical Wnt pathway¹⁵⁴ as DKK1, DKK2 and DKK4 can interact with LRP5/6 while DKK3 does not.155, 156, 157 DKK1 binds to LRP5/6 co-receptors and inhibits the canonical Wnt pathway and is mostly found in osteoprogenitors, osteoblasts, osteocytes, and adipocytes.^158,159 DKK1 is known to hinder osteoblastic and chondrocytic differentiation, promote adipocytic development and prompt osteoclastic development thereby enhancing the osteoprotegerin-RANKL binding.158, 159, 160, 161, 162 Dkk 1 knockout mouse models manifest a deficiency in bone development, like head and limb abnormalities and ending up dying in a short time, while the hybrid Dkk1 knockout mice and those characterized by a specialized lineage of ostoblastic cells appeared to have a high bone mineral density as well as bone composition.^159,160,163, 164, 165 Deregulated expression levels of Dkk1 are linked to different skeletal afflictions, characterized by the death of osteocytes and bone marrow (also known as femoral head avascular necrosis/osteonecrosis (ONFH)),¹⁶⁶ bone deformity (also known as osteitis deformans/Paget disease) as well as rheumatoid arthritis, osteoarthritis, and multiple myeloma.^162,167,168 DKK2 attaches strongly to Kremen 1/2 and LRP5/6 to prevent Wnt to attach to the respective cell-surface molecules. However, when Kremen 2 is not present, LRP 6 gets activated without any DVL-mediation activity. Consequently, the Dkk 2 knockout mice display a reduced level of osteoblastogenesis and a high level of osteclastogenesis mediated by the osteoprotegerin-RANKL activity, causing low bone mass (osteopenia).^140,169

SOST. SOST is a glycoprotein rich in cysteine that acts as an inhibitor of the Wnt pathway, by blocking LRP5/6 co-receptors’ activity.^170,171 SOST transcripts are found in high levels in osteocytes as well as in bone marrow and cartilage.¹⁷² High BMD and bone strength were seen in Sost-KO mice, as well as an increase in the number of osteoblasts that recapitulate the phenotypic of sclerosteosis or van buchem disease.^173,174 In addition, the mice with specific overexpression in osteocytes have shown increased levels of RANKL.^175,176 In humans, alterations of SOST gene are linked to diseases such as sclerosteosis, van buchem disease and craniodiaphyseal dysplasia, an extremely rare sclerosing bone dysplasia, that is caused by missense mutations in SOST gene and decreases its extracellular secretion.¹⁷⁷ SOST inhibits osteoblast function and stimulates osteocyte function by detaching bone formation and resorption. In addition, explorations done in vitro and in vivo have shown SOST to increase and promote adipogenesis by inhibiting the Wnt pathway.178, 179, 180 As a result, the findings show that SOST inhibits bone growth while also stimulating bone resorption.

SFRPS. SFRPS are secreted glycoproteins that share similarity with FZD receptors. They can bind to Wnt ligands and FZD receptors and eventually block both canonical and non-canonical Wnt pathways.¹⁸¹ SFRP1 and 2, amongst others, attach themselves to WNT5A whilst SFRP3 only prevents WNT1 and 8.¹⁸² In bone, during ossification process the Sfrp1 gene is overexpressed thus deactivating the canonical Wnt pathway and boosting both the osteoblast and osteocyte cell death.^183,184 Sfrp1-KO mice have resulted with an increased bone trabecular area, a decrease in osteoblast apoptosis and enhancement of osteogenesis and mineralization.^183,184 On the other side, when Sfrp2 gene was overly expressed, the mice resulted with an augmented production of mesenchymal stem cells and an abatement in apoptosis, thus inhibiting the accurate procession of osteogenesis and chondrogenesis. SFRP3 is also linked to mature osteoblastogenesis and programmed cell death (also known as apoptosis) promotion in mesenchymal stem cells.^182,185 Both osteoblasts and osteoclasts hold an over-expressed level of SFRP4 in embryogenesis and postpartum periods, displaying the significance of this glycoprotein in the growth of bone and the loss of bone linked to stress oxidation and age.^186,187 Gene alterations in SFRP4 are known to cause bone disorders such as metaphyseal chondrodysplasia (also known as the Pyle's disease), characterized flaring of the ends of long bones amongst other abnormalities.¹⁸⁸ Upraised SFRP4 levels are linked to osteomalacia, characterized by a soft and more flexible bone.¹⁸⁹

WIF-1. Wnt inhibitory factor-1 is an excreted Wnt inhibitor that acts as a negative feedback regulator of the Wnt canonical pathway. WIF-1 gene expression is increased during osteoblast differentiation as well as in the treatment of MSCs with Bmp 2.^185,190,191 Alterations of WIF-1 gene are closely related to its methylation and this fact has been used to propose, Gossypol, for treatment against osteoporosis as it promotes bone proliferation through methylation and inhibition of the WIF-1 promoter.¹⁹² However, WIF-1 methylation is associated with bone tumors although it is epigenetically silenced in human osteosarcoma cell lines.¹⁹⁰

Ultimately, a documentation of the roles of most Wnt signaling pathway constituents during osteoblastogenesis, osteclastogenesis and osteocytogenesis are outlined in Table 1. Gene variations of Wnt elements are linked to their respective physical compositions in mouse stereotypes. Their related human disorder is also presented in detail. These encompassing interpretations provided from both past and current research highlight the importance of Wnt signaling in assisting the differentiation of MSCs precursors into osteoblastogenesis as well as in impeding the bone development process.

Table 1.

Genetic alterations of Wnt signaling molecular components in mouse phenotype and their related human disease.

Gene	Method	Cre line (Tissue)	Phenotype(s)	Reference	Related human skeletal disorder
Wnt 1	KO	Germline	Homozygous resulted in perinatal death. Heterozygous resulted with mild osteopenia.	31	LOF mutations can cause early-onset of osteoporosis (OMIM: 166710) or osteogenesis imperfecta (OI) (OMIM: 166200)
Wnt 3	cKO	RARβ-Cre (Forelimb Ectoderm) or MSX2-Cre (Hindlimb Ectoderm)	Resulted in the loss of forelimbs (RARβ-Cre) or hindlimbs (Msx2-Cre).	193	N/A
Wnt3a	KO	Germline	Heterozygous resulted in low amounts of BMD, BV/TV, Tb N, and a high amount of Tb Sp.	43,44	May be linked to Skeletal Dysplasia (OMIM: 618870)
Wnt 4	KO	Germline	Homozygotes resulted in delayed chondrocyte maturation and early postnatal death.	48,49	N/A
Wnt 4	cKO	Germline	Female mice resulted in a decreased femoral BMD.	48,49	N/A
Wnt5a	KO	Germline	Homozygotes resulted in delay in chondrocyte hypertrophy and skeletal ossification as well as perinatal death. Heterozygotes resulted in low BMD, BV/TV, Tb N, and a high Tb Sp.	57,61	Robinow Syndrome (OMIM: 180700)
Wnt5a	cKO	Germline	Osteoclast and bone formation as well as bone resorption.	57	N/A
Wnt5b	OE	Chondro-osteo progenitors	Resulted in a delayed bone ossification due to the overexpressed Wnt5b in chondrocytes.	60,63	Overexpressed Osteoarthritis in female patients (OMIM: 165720).
Wnt7a	KO	Germline	Frequent impaired limb development.	65,69,70	Al-Awadi/Raas-Rothschild/Schinzel phocomelia syndrome (OMIM:276820); Santos syndrome (OMIM: 613005).
Wnt7b	cKO	Dermo1-Cre (Mesoderm)	Homozygotes resulted alive with a low ossification and anomalous chondrocytes and osteoblasts.	37	N/A
Wnt7b	KO	Germline	Resulted in delay in chondrocyte maturation, accelerated bone formation and perinatal death.	41,71,73	Linked to osteoarthritis (OMIM: 165720); Rheumatoid arthritis patients (OMIM: 180300).
Wnt9a	KO	Germline	Resulted in skeletal abnormalities, reduced mineralization and chondroid metaplasia and probable prenatal death.	48	N/A
Wnt 10a	KO	Germline	Resulted in decrease in bone mineralization, adipose tissue, bone marrow and dental formation.	77,80	Linked to odontoonychodermal dysplasia OODD (OMIM: 257980)
Wnt 10b	KO	Germline	Resulted in decrease in bone volume fraction, BMD, and tb.N.	32,86,88	Homozygous missense mutations are linked to split limb malformation, oligodontia (OMIM: 106600) as well as osteosarcoma (OMIM: 165660).
Wnt 16	KO & cKO	Germline	Resulted in a decreased BMD level, cortex density, skeletal strength and an increased porous cortex.	93,96	N/A
Wls	cKO	OC-Cre (Osteoblasts)	Resulted in low BMD, trabeculae, calvaria and BV/TV.	194	N/A
Fzd2	KO-LacZ	Germline	Homozygotes resulted less viable with smaller sized skeletal elements.	195	N/A
Fzd9	KO	Germline	Resulted in low vertebral Tb N, osteoblastic surface, osteoid apposition, and mechanical strength in long bones.	196	LoF mutations may be linked to Williams-Beuren syndrome (OMIM: 194050)
Lrp4	KO	Germline	Homozygotes resulted in perinatal death.	197	Cenani-Lenz Syndactyly Syndrome (OMIM: 212780)
Lrp5	KO-Genetrap	Germline	Decreased BMC, BMD, as well as cortical and trabecular bone mass.	103	Osteoporosis-Pseudoglioma Syndrome (OMIM: 259770)
CGOF		DMP1-Cre (Osteocytes), PRX1-Cre (Limb mesenchyme)	Resulted in high bone mass with DMP1-Cre and high bone mass with PRX1-Cre.	107	Osteosclerosis, autosomal dominant (OMIM: 144750)
GOF		Germline	Resulted in high in cortical bone mass, BV/TV, Tb N and very thick trabeculae with a low Tb Sp.	107,198	Osteopetrosis, autosomal dominant 1 (OMIM: 607634)
CGOF		DMP1-Cre (Osteocytes), PRX1-Cre (Limb mesenchyme), COL1A1-Cre (Mature osteoblasts)	Resulted in high bone mass with DMP1-Cre, high bone mass with Prx1-Cre and no enhancement of bone mass with COL1A1-Cre.	107,199	High bone mass (OMIM: 601884)
Lrp6	KO-LacZ	Germline	Homozygotes resulted in close to perinatal death, shortening of the axial skeleton and limb defects. Heterozygotes resulted in a low BV/TV.	113,200	Associated with low bone mass (OMIM: 610947) in CAD.
Dkk 1	KO	Germline	Homozygotes resulted in perinatal death, severe craniofacial malformation, and ectopic digits. Heterozygotes resulted in high BV/TV, mineral apposition rate, osteoblast surface, and mechanical strength.	159,163	Associated with rheumatoid arthritis (OMIM: 180300).
Dkk 2	KO	Germline	Homozygotes were viable. Resulted in osteopenia with defective mineralization level and high osteoclast numbers.	201	N/A
Sost	KO-LacZ	Germline	Resulted in high bone mass.	173,198,202	Sclerosteosis 1 (OMIM: 269500)
	cKO-Hypomorphic	COL1A1-Cre (Mature osteoblasts)	Resulted in increased BV/TV and osteoblast surface.	203	Van Buchem disease (OMIM: 239100) and Craniodiaphyseal dysplasia (OMIM: 112860)
	OE	Osteoblasts and osteocytes	Resulted in osteopenia with disarranged bone construction, thin cortex, low Tb.N and osteochondrodysplasia.	204	N/A
Sfrp1	KO-LacZ	Germline	Resulted in reduced age-related trabecular bone loss mostly in female mice.	183	N/A
Sfrp2	KO	Germline	Resulted in truncated metacarpus and phalanges with a retarded ossification and a low rate of chondrocytic cells.	205	N/A
Sfrp3	cKO	E2A-Cre (Germline)	Resulted in high BMD level, and cortex density.	206	N/A
Sfrp4	OE	Various	Resulted in decreased rate of bone acquisition, BV/TV, and Tb size.	207	N/A
Dvl2	KO	Germline	Resulted in viable mice with twisted tails, anomalous vertebras, and fusion in ribs.	208	N/A
Axin 1	KO (Fu-TG1)	Germline	Homozygotes died at days 9.5. Heterozygotes had tail bisections and fused ribs.	209	Caudal duplication anomaly (OMIM: 607864)
Axin 2	KO-LacZ	Germline	Resulted in skull bone fusion from high mineralization rate and bone ossification, and low chondrocyte growth zone.	210, 211, 212	Oligodontia-colorectal cancer syndrome (OMIM: 608615)
Apc	cKO	COL2A1-Cre (Chondro-osteo progenitors)	Homozygotes resulted in PNM, truncated limbs, abnormal craniofacial construction, highly abnormal vertebras, and immature chondrocyte and osteoblast cells.	213	N/A
Gsk3β	Germline		Resulted in nanism with truncated limbs and vertebras, and a COL2A1 under-expression.	214	N/A
Gsk3β	Germline		Homozygotes resulted in neonatal mortality. Resulted in low skull ossification and craniofacial features. Heterozygotes resulted in high density of cortex and trabeculae bones.	215, 216, 217	N/A
β-catenin	COL2A1-Cre (Chondro-osteo progenitors)		Homozygotes resulted in neonatal mortality with bone deficiencies with truncated limbs, loss of transverse tarsal joint leading to a fused bone and a dome-like skull.	218	N/A
Tcfl (Tcf 7)	Germline		Homozygotes displayed viability. Resulted in low bone mass.	219	N/A

Notes: BMD, Bone mineral density; CAD, Coronary artery disease; CGOF, Conditional gain of function; COL1A1/COL2A1, Collagen, type I/type II, alpha 1; DMP1, Dentin matrix acidic phosphoprotein 1; E2a/Tcf 3, Transcription factor 3/E2A immunoglobulin enhancer-binding factors E12/E47; GOF, Gain of function; LOF, Loss of function; MSX2, Homeobox protein MSX-2; NA, Not applicable; OC, Osteocalcin; OE, Overexpression; OMIM, Online Mendelian Inheritance in Man; PNM, Perinatal mortality; PRX1, Peroxiredoxin 1; RARβ, Retinoic acid receptor beta; Tb.N, Trabecular number; Tb.Sp, Trabecular separation; TMD, Tissue mineral density.

Crosstalk between Wnt pathways and other signaling pathways in osteoblasts differentiation and bone metabolism

Multiple former and recent publications have investigated the integration of Wnt signaling pathway with other signaling pathways such as BMP, TGF-β, FGF, Notch, Hedgehog, and PDGF in osteoblast differentiation. This cross–functional interaction serves as a gene regulatory network that coordinates bone orientation, development, and flexibility, which are an attribute to the skeletal well-development (Fig. 2 and Table 2).

Figure 2.

Cross-functional signaling between Wnt and other signaling pathways in osteoblasts. Wnt and Bmp: Wnt/β-catenin signaling is stimulated by Bmp signaling activity by creating β-catenin/TCF/LEF/RUNX2 complex thus increasing the Wnt expression, inhibiting DVL and decreasing BTRC expression, oppositely Wnt signaling is inhibited when Bmp activates Wnt antagonist Dkk 1 and Sost expression thus preventing β-catenin translocation in the nucleus. Wnt and TGF-β: TGF-β stimulates Wnt ligands gene expression to increase and Axin protein gene expression to decrease via Wnt signaling activation. Wnt, BMP and FGF: FGF expression is increased by Bmp signaling and vice versa, thus indirectly increasing and decreasing DKK levels, respectively. The Fgf-mediated activation of P13K-AKT pathway or MAP kinase pathway causes the GSK-3β to phosphorylate and further translocate β-catenin into the nuclei while the inhibition of SNAIL phosphorylation hinders the E-cadherin-mediated translocation of β-catenin into the nuclei. Wnt and Hippo: In the nucleus, YAP forms transcriptional complexes with TEADs (Transcriptional enhanced associate domains), inducing downstream gene expression. Wnt and Hh: Smoothened (SMO) catalyzes a series of reactions in order to release glioma-associated oncogene (GLI) from the deactivated compound and translocate it to the nucleus to operate target gene expression. During the post-translational modification (PTM) phase, GLI and Suppressor of fused homolog (SUFU) negatively regulate the Hh signaling and hinder β-catenin to translocate to the nuclei, while Smoothened positively promotes β-catenin production. Wnt and Notch: Notch proteins initiate the expression of Hes and Hey genes thus hindering the well-functioning of Wnt signaling and restricting β-catenin-mediated transcription. Wnt and PDGF: PDGF signaling pathway can stimulate the SMAD1/5/8/RUNX2 complex to induce gene production and guide the differentiation of bone.

Table 2.

Cross-functional signaling between Wnt and other signaling pathways in osteoblasts.

Molecules of respective pathway	Crosstalk signaling target molecules	Molecular mechanism's results	Reference
BMP and WNT	SMAD complex with TCF/LEF/β-catenin	Increased osteoblast differentiation	256,257
TGFβ1	WNTs increased, Axin 1/2 decreased	Increased chondrocyte differentiation, Decreased adipocyte differentiation	258,259
TGF-β1 stimulation	Increased BMP2	Stimulates ectopic bone formation	260
Decreased level of SMAD4	β-catenin activity increased	Increased bone formation	261,262
BMPR1A stimulation	SOST increased, increased DKK1 stimulates β-catenin to decrease	Decreased bone mass	263
BMP2	Decreased β-TRCP stimulates β-catenin to increase.	Increased osteoblast differentiation, Increased chondrocyte hypertrophy.	264,265
FGF-FGFR3	TGF-β	Mediates embryonic bone formation	266
FGFR stimulation	Increased BMP2 and TGF-β1	Increased osteogenic expression	267
FGR2 stimulation	Increased Bmp-induced osteogenesis	Increased osteoblast differentiation	268,269
BMP2 stimulation	Increased Fgf-induced osteogenesis	Increased osteoblast differentiation	270
Notch	Increased Bmp-induced ALP	Stimulated SMAD and Notch	271
Ihh stimulation	Increased Bmp-induced osteogenesis	Bone formation	272
Ihh and Bmp stimulation	Increased ALP; Increased Ihh	Long bone development	272
YAP	β-catenin	Increased β-catenin transcriptional activity and downstream genes	273,274
TAZ	β-catenin, DVL	Knockdown of TAZ significantly increased DVL phosphorylation and β-catenin nuclear levels	275
MST1/2	β-catenin	Deletion of MST1/2 led to activation of β-catenin	276
GLI stimulation	Increased BMP2	Normal osteoblast differentiation	277
PDGF	Increased MSCs	Highly mitogenic factor and chemotactic for MSCs, osteoblasts and perivascular cells	278

Notes: BMP, bone morphogenetic protein; FGF, fibroblast growth factor; GLI, glioma-associated oncogene; Ihh, Indian hedgehog; MSCs, meristematic stem cells; MST1/2, macrophage-stimulating protein; TGF-β, transforming growth factor-β; PDGF, platelet-derived growth factor; TAZ, transcription adaptor putative zinc finger; YAP, Yes-associated protein.

Wnt and Bmp signaling in osteoblasts

Wnt and BMP pathway interact in mesenchymal cells during chondrocyte or osteoblast differentiation. For instance, BMP2 can promote differentiation of chondrogenic cells while β-catenin has no effect or even inhibits the mechanism.^220,221 Moreover, BMP2 blocks ALP (Alkaline phosphatase) induction while, the Wnt/β-catenin pathway plays a direct role in upregulating ALP induction in mesenchymal cells. β-Catenin interacts with SOX9, that mediates the transcription of collagen, and causes ubiquitination-mediated degradation in chondrocytes.222, 223, 224 However, BMP2 inhibits β-catenin–SOX9 interaction by activating p38 MAPK factor suggesting that BMP signaling indirectly promotes chondrogenesis by blocking Wnt/β-catenin signaling. Wnt signaling can repress BMP-induced activation by over-expressing Dkk1 gene that automatically enhance BMP4-triggered apoptosis in vertebrate limb development thus indicating that Wnt signaling inhibits apoptosis induced by BMP signaling.²²⁵ Other results confirm the cooperative interaction between SMAD1/4 and β-catenin in gene expression during mesenchymal cell differentiation.²²⁶ Also, the loss of bone morphogenetic protein receptor type 1a (Bmpr1a) gene in both osteoblastic and osteocytic cells has shown to block the proper expression of various metabolic-promoting Wnt genes in the cortex and bring about an overly-expressed Wnt7b gene that displayed an effective operation in completely rescuing the deficiency in periosteum's growth.^227,228 These data illustrate the functional role of Wnt signaling in BMP2-regulated mesenchymal cell differentiation.

Wnt and TGF-β signaling in osteoblasts

β-Catenin signaling pathway is a process through which TGF-β regulates osteoblastogenesis in hMSCs (human mesenchymal stem cells). Through RNAi approaches, was shown that TGF-β1 uplifted the amounts of β-catenin and augmented β-catenin- T-cell factor/lymphoid enhancer factor transcription rates in human mesenchymal stem cells through both SMADand non-SMAD pathways. Moreover, it was shown that after knockdown of β-catenin with siRNA, transforming growth factor beta 1 (TGF-β1), activin receptor-like kinase 5 (ALK-5), protein kinase A (PKA) and Jun N-terminal Kinase (JNK), hinder osteoblastogenesis in hMSCs in vitro. In recent findings it was demonstrated that when mesenchymal progenitor cells are treated with TGF-β, a β-catenin-responsive firefly luciferase reporter plasmid (also known as TOPflash), is activated and eventually β-catenin is translocated into the nucleus without the activation of Wnt signaling pathway.²²⁹ Also, cooperation between TGF-β and WNT3A led to high amounts of nucleic β-catenin. Transcriptionally, SMADs and β-catenin can form a complex that is imported into the nucleus to trigger osteoblast-dependent gene expressions however, further investigation is needed to better-understand this interaction.

Wnt, Fgf and Bmp signaling in osteoblasts

During limb development, a regulatory system displayed by FGF, Wnt and BMP signaling interaction is observed to manage the cell proliferation and development.²³⁰ This regulatory system induces cell death in the anterior margin of the limb by either inhibiting Wnt or FGF signaling or by activating Bmp signaling through failing in regulating Dkk, Fgf 8 or Bmp 4 expression.²³¹ Through experiments, the repression of FGF signaling is considered crucial in inducing DKK gene expression, by hindering its expression. Similarly, DKK together with BMP4 hinder Fgf8 gene expression, playing a dominative role in apoptosis.²³¹ Glycogen synthase kinase 3 (GSK-3β) also functions as an intermediator in assisting the Wnt-FGF signaling amalgamation.²³² Moreover, osteoblast differentiation during bone development can be stimulated by Fgf 2 via Wnt pathway where Fgf2 deletion reduced the expression of WNT10B, LRP5 and β-catenin genes.²³³ During exogenous Fgf2 administration in marrow stromal cells of Fgf2-KO mice, β-catenin was accumulated in nucleus thus increasing the effect of mineralization.²³³ In the absence of endogenous Fgf2, SOST mRNA and SOST protein expression were increased thus reducing osteoblast differentiation and bone development in Fgf2-KO mice.²³⁴

Wnt and Hippo signaling in osteoblasts

Wnt and Hippo signaling cooperate to regulate each other's activity for precise regulation of biological phenomenon. YAP/TAZ (Yes-associated protein 1/Transcription adaptor putative zinc finger) interact with β-catenin and DVL and regulate their levels and activities.²³⁵ Apparently, there is a combinational interaction of YAP/TAZ co-activators in promoting skeletal formation. The absence of genes coding for the transcriptional YAP/TAZ proteins has shown to decrease bone size therefore causing a condition similar to osteogenesis imperfecta in Osx-Cre mouse line.²³⁶ Yap and/or Taz gene deletion in Osx-Cre mouse line has also shown to produce a damaged thickness in periosteum by negatively affecting the process of differentiation in osteo-chondro-progenitor cells.²³⁷ Other transgenic mouse lines with a Yap/Taz deletion, such as Prx-Cre and Dmp-Cre, have resulted in fetal death and a disbalance in the osteoblast/osteoclast cells ratio with a low number of osteoblastic cells and a high number of osteoclastic cells, respectively.²³⁸ This phenomenon occurs due to the suppressive effect of YAP/TAZ co-activators in osteo-chondro-progenitor cells that cause the osteoblasts number to decrease and a promotive effect in adult osteoblastic cells that cause the osteoblasts number to increase and positively enhance the skeletal development and fracture repair.^238,239 Altogether, the given data identify YAP/TAZ as key transcriptional regulators in osteoblast, osteoclast, and osteocyte mediated bone remodeling.^236,240

Wnt and hedgehog signaling in osteoblasts

Wnt and Hh signaling integration is pivotal for the development and differentiation of embryogenesis. During the limb regeneration process, Hh pathway inhibits the activation of Wnt signaling²⁴¹ and hinders the expression of β-catenin in order to cause cartilaginous disorders.²⁴² The Ihh signaling activation during bone repair is another important evidence that demonstrates the synergetic interaction of these two pathways.²⁴³ While Wnt signaling pathway's activity is enhanced in mature osteoblast cells, the Ihh signaling pathway's activity is enhanced in early phases of osteoblast cells differentiation in order to promote bone repair and formation.²⁴³ The Ihh signaling is also highly operative in skeletal cancerous cells and the deletion of PTCH1 gene (protein patched homolog 1) causing the benign bone tumor of enchondroma and the malignant neoplasm known as osteosarcoma, demonstrates that. This phenomenon occurs while PTCH1 deletion enhances the WNT5A and WNT6 ligands expression causing the β-catenin's activity to increase and originate bone abnormalities.²⁴⁴

Wnt and Notch signaling in osteoblasts

Wnt and Notch signaling pathways are two highly-cooperative pathways that well-manage cell fate proliferation especially the bone cell formation process.²⁴⁵ In human cells, both inactive and active form of Notch signaling pathway when limited to the membrane fraction and nucleus can inhibit Wnt/β-catenin signaling by seizing β-catenin through endocytosis to prevent its interactivity with T cell factor/lymphoid enhancer factor family (TCF/LEF). The nuclear-limited Notch binds to β-catenin forming a composite that interrupts Wnt/β-catenin signaling and restricts β-catenin-induced transcription through the expression of downstream target genes HES (Hairy and enhancer of split-1) and HEY (Hairy/enhancer-of-split related with YRPW motif protein) repressors.²⁴⁶ When the activation of HES and HEY repressors is blocked, there is no displayed modification in the function of Notch intracellular domain (NICD), in order to inhibit Wnt signaling.²⁴⁶ In bone cells, the link between these two pathways is represented by JAG1, one of the five surface-interactive ligands of Notch pathway,²⁴⁷ and osteocytes with JAG1 deletion containing a higher amount of other Notch ligands and receptors, demonstrates this cooperation. Also, Notch stimulates β-catenin's activity by hindering the GSK-3β activity in order to stabilize the β-catenin-activator SNAIL (Zinc finger protein SNAI1).^248,249 Commonly, Wnt and Notch have opposite functions and whenever one of them is obstructed, the other pathway reacts to the Wnt–Notch interaction.²⁵⁰ Among other factors, HIF-1α (hypoxia-inducible factor-1α) is one that upregulates the NICD domain levels and promotes Notch pathway but, on the other side, it obstructs the activity of Wnt pathway therefore modifying the growth of osteoblast cells.²⁵¹

Wnt and PDGF signaling in osteoblasts

Platelet-derived growth factor receptor alpha (PDGF-Rα) is necessary for a normal development of skull and face bones.²⁵² In general, PDGF-Rβ can potentially regulate the role of MSCs however and reduce mitogenesis and enhances osteogenesis.²⁵³ PDGF-R is involved in osteoblatogenesis process mediated by WNT3A in MC3T3-E1 cells (subclone 4 pre-osteoblasts) with PDGF-R reducing osteoblast proliferation induced by the WNT3A ligand.²⁵⁴ PDGF-R is transactivated by WNT3A ligand via SRC (non-receptor tyrosine kinase) anchoring to DVL2 and stimulating the activity of its tyrosine kinases.²⁵⁵ Moreover, WNT3A binds to LRP5/6-FZD receptors and activates DVL, which stimulates β-catenin-independent pathways via the transactivation of PDGF-R in osteoprogenitors and osteoblastic cells and therefore result in bone formation increment.²⁵⁴

Osteoblastic utilization of glucose, glutamine and fatty acids via Wnt-stimulation

Wnt stimulates β-oxidation in osteoblasts

Wnt-mediated mechanisms of metabolism in osteoblastic cells were observed to maintain LRP5 and LRP6 effectors' intersecting activities.²⁷⁹ LRP5 variants developed modifications in body configuration not seen in the LRP6 variants such as white adipose tissue depots increased in size and whole-body energy expenditure reduced. The genetic fingerprinting of osteoblastic cells containing defective LRP5 genes displayed physical traits of varied triglyceride catabolic reaction caused by an under-expressed gene activity taking place in mitochondria in deviant osteoblasts during the saturated fats oxidation.²⁷⁹ Frey et al²⁸⁰ explored if the operation of this mitochondrial type of oxidation executed by LRP5 gene was associated with β-catenin's stimulation. Treatments of primary osteoblasts in vitro demonstrated that only WNT3A, WNT10B and WNT16 ligands were shown to be linked to β-catenin activation, suggesting the canonical mechanism to be the cause of alteration induction. On the other side, mature osteoblastic cells that were missing the activation of β-catenin, encountered a hindering of osteoblastic cells' further development as well as a significant depletion in oxidative reactions and adenosine triphosphate's accumulation, in spite of glycolysis activity being enhanced. Anyhow, the manipulated β-catenin in vivo, resulted in loss of function for LRP5 as well as an increase in adipose tissue mass and serum fatty acids (Fig. 3).²⁸⁰

Figure 3.

Glucose, glutamine and fatty acids utilization in osteoblasts via Wnt signaling regulation. Wnt ligand binds to FZD and LRP5/6 receptors which leads to degradation compound's deactivation, which is composed of Dishevelled, Axin, GSK-3β, APC's and CKlα (casein kinase 1). Through this mechanism, in mature osteoblastic cells, β-catenin's amount increases in the cytoplasm then translocates to the nuclei and assisted by the TF (also known as sequence-specific DNA-binding factor), the mitochondrial fatty-acid β-oxidation gene expression initiates. In osteogenic cells, mTORC1 signaling pathway is stimulated by Wnt pathway and causes the amount of glutamine amidohydrolase, the initiative enzyme in the glutaminolytic pathway, to increase. Wnt pathway also stimulates mTORC2 signaling pathway in order to adjust the proper number of proteins engaged in the glycolytic pathway. Notes: MLST8, mTOR associated protein, LST8 homolog; RAP, RAS/RAP GTP-binding protein; RIC, resistance to inhibitors of cholinesterase; SIN1, stress activated protein kinase interacting protein 1; TSC1/2, tuberous sclerosis proteins 1 and 2.

Kim et al²⁸¹ examined the skeletal and metabolic phenotypes of a mouse model to investigate the requirements and contributions of lipid homeostasis in bone development and vice versa and eventually, the study demonstrated the disruption of the expression of carnitine palmitoyl transferase 2 (CPT2), a highly required enzyme for mitochondrial fatty-acid β-oxidation in osteoblasts and osteocytes being caused by CPT2 deficiency. However, the seriousness of the matter was determined by the sex of the mutant. Cpt2-male-mouse-variants demonstrated an impermanent low mechanical competence in trabeculae or Tbvf (also known as the trabecular bone volume fraction). On the other side, Cpt2-female-mouse-variants showed a low mechanical competence in trabeculae in thighbones (distal femur) and the fifth lumbar spine vertebrae (L5) all the time. It is important to point out that Cpt2-female-mouse-variants cumulated osteoid with an enhanced adjustment period (lag period) during the mineralizing process. The sex dimorphic phenotype is linked to whether the estrogen hormone adjusts fuel selection according to both in vivo and in vitro studies. In male mutants' CPT2 deficient osteoblasts, cultured in vitro, the glucose uptake was increased, which was not shown in female mutants’ CPT2 deficient osteoblast cultures treated with exogenic estrogen.²⁸¹ Similarly important is the fact that the abnormal amount of lipids (dyslipidemia) has shown significant alterations in bone metabolism equilibrium and caused trabeculae to decline in hyperlipidemic and high cholesterol diet mice.²⁸² Altogether, these studies illustrate the importance of fatty acid catabolism in normal osteoblast function and bone mass development which is highly influenced by both sex and food diet.

Wnt stimulates glycolysis in osteoblasts

Glucose is the vitality origin for most mammalian cell types and here it is required to normalize osteoblast function. Wnt signaling pathway is intrinsically linked to carbohydrate metabolism, therefore, Esen and colleagues²⁸³ illustrated the increase of carbohydrate attainment via the stimulation of various gene expressions, such as glucose transporter 1 (GLUT1), HK2 (known as hexokinase II), PDC kinase (known as pyruvate dehydrogenase complex kinase) and lactic acid dehydrogenase (LDH) via WNT3A and WNT10B ligands. This was shown to be more efficacious than the usage of insulin-cellosaurus cell line (ST2) as a stimulant. Interestingly enough, it was shown that the Wnt3a stimulated lactate production suggesting that Wnt signaling activated aerobic glycolysis instead of oxidative phosphorylation.²⁸⁴

Esen et al²⁸³ also demonstrated via gene knockdown studies, that in both lrp5^−/− mice and those in which Lrp5 expression was eradicated by Osterix-Cre, displayed a reduction in glycolytic enzyme expression in bone which did not continue via alterations in the activity of either GSK3β or β-catenin activity because these effectors were altered as inhibitors and did not impact glucose utilization. Alternatively, the mammalian target of rapamycin complex 2 (mTORC2) was promoted by the activity of WNT3A ligand assisted by the RAC Family Small GTPase 1 (RAC1) and consequently enhancing the carbohydrate utilization via gene overexpression (Fig. 3).

Through RNA-Seq technique, it was demonstrated that the increased number of genes caused by Wnt3a-stimulated cellosaurus cells (ST2) was exceeded by the number of genes with a decreasing effect in gene expression.²⁸⁵ Gene downregulation is linked to chondrocytic and adipocytic differentiation identified via CCAAT and CEBPA detectors. To conclude, Wnt signaling controls the genomic landscape and fate-specification of osteoprogenitors by hindering carbohydrate to enter the Krebs or citric acid cycle whereby both citric acid and acetyl coenzyme A catalyzation are decreased.

Wnt stimulates glutaminolysis in osteoblasts

Glutamine is the most abundant free amino acid in circulation and is not only an important oxidative fuel, but also a precursor for the synthesis of non-essential amino acids, nucleotides, and the antioxidant. Experimental research has put forward the necessity for GLN (glutamine) addition in order to assist the process of osteoblast-mediated ossification.²⁸⁶

The stimulation of the ST2 cell is caused by Wnt factors during osteoblast differentiation and bone associated with glutamine metabolism.²⁸⁷ This stimulation increased glutamine content. Glutamine catabolism, resulted from toxicological hindrance of glutaminase catalyzation, was considered necessary for stimulating osteoblastogenesis in an artificial environment and enhancing BMD via the Hemoglobin subunit mu (Hbm) gene well-expression found in the LRP5 cell surface receptor in the living body of an Hbm-variant. Simultaneously, the “fight or flight” reaction and the over-expression of particular genes encoding for protein utilization and their three-dimensional structure acquisition were initiated by glutaminolysis. Furthermore, research on this mechanism, stipulate that the mTORC1 (mechanistic target of rapamycin complex 1) pathway stimulates glutamine usage, therefore, glutamine attainment and consumption in response to Wnt signaling constitutes a molecular resistance mechanism that sustains metabolism, ER (endoplasmic reticulum) status²⁸⁷ and oxidoreduction equilibrium.²⁸⁸

The association of Wnt signals with various human skeletal disorders and their forms of treatment

Taking into consideration to date therapeutical advancements related to Wnt signaling pathway, we elaborate the association of numerous muscular-skeletal maladies with Wnt ligands, recent and innovative prospective forms of treatment, pre-symptomatic observations acquired by both human and animal samples, potential novel therapies, and lastly the aspects at issue that should be taken into consideration when performing clinical interventions (Table 3).

Table 3.

The association of Wnt signals with various human skeletal disorders and their forms of treatment.

Human skeletal disease	Wnt-related molecules involved	Results of mutations	Existing therapies	Potential novel therapies	Clinical issues	Reference
Osteoporosis (OR)	LRP5, WNT3Aa upregulation, SOST overexpression.	High bone resorption and low bone formation caused by agedness and reduced amount of estrogen.	Drugs like Raloxifene (also known as SERM), diphosphonates, and denosumab (monoclonal antibody therapy) hinder bone resorption.	Anti-Sost monoclonal antibody (Romosuzab, BPS804, etc) stimulating bone formation and suppressing bone resorption by enhancing osteoblast differentiation and OCIF/OPG production, treating HPP and OI.	Romosozumab treatment is linked to cardiovascular incidents.	289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302
			Teriparatide (parathyroid hormone PTH) an effective anabolic agent promotes bone remodeling.	Caveolin-3 serving as a biological indicator by enhancing ossification and inhibiting OR.	A mixture of antibodies that attack both DKK1 and SOST has shown great possibilities to be effective in the enhancement of bone-rupture healing.
Osteoarthritis (OA)	β-catenin activation, WNT7B overexpression and SOST expression.	Joint-related (Articular) cartilaginous tissue impairment, Development of bone spur (osteophyte), and solidification of the under-cartilaginous-bone (subchondral).	Treatment with P.O NSAIDs and HA joint shot (injection).	Sirolimus (Rapamycin) decreasing MMP13 gene expression and reducing cartilaginous demolition.	The systematic hindering of Wnt signaling pathway has shown possibilities of affecting the bone remodeling process non-positively.	73,303, 304, 305, 306, 307, 308, 309
				Small Wnt pathway inhibitor molecules (like SM04690) protecting cartilaginous tissues from demolition.
				Mianserin, an anti-R-spondin 2 agent, inhibiting the abnormal functioning of Wnt pathway in OA.
Rheumatoid Arthritis (RA)	TNF-α induction of DKK1 expression and Sost expression; Increased WNT5A expression;	Bone resorption stimulation; Erosive Arthritis (Bone erosion); Pro-inflammatory cytokine expression enhancement; Cartilaginous demolition via MMPs production stimulation by Wnt5a ligand.	MTX treatment (a traditionally synthesized DMARD) and other biological DMARDs functioning as anti-inflammatory cytokines factors.	Possible treatment of OCD (easing the pain caused by cartilaginous demolition and inflammation) by hindering Wnt5A gene expression via the WNT5A/ROR2 signaling pathway.	The monoclonal antibody treatment with Denosumab have no apparent premises on giving positive results in cartilaginous demolition.	310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323
	Decreased SFRP5 expression.
Osteosarcoma/OS (Primary Bone Tumors)	WIF-1; DKK3; SFRP2.	Metastatic cell proliferation facilitation by both Wnt ligands and inhibitors.	Standardized treatment using MMT, combination CTx, RTx, and surgery.	GSK-3β treatment has shown great premises in suppressing the further-production of osteogenic sarcoma cells by activating the Wnt signaling pathway.	The inhibitory function of GSK-3Β is done through a distinct Wnt-independent mechanism that calls for additional research in order for actual application to take place.	324, 325, 326, 327, 328, 329, 330, 331
					Virus-like-particle-based anti-HER2/neu vaccine as well as CAR-T cells also show great premises for the future.
Multiple Myeloma (MM)	SOST overexpression.	Osteolytic lesions from the high concentration of SOST.	Treatment of osteolytic lesions with Zoledronic acid (also known as Zometa) and Denosumab (monoclonal antibody treatment).	Antibodies targeting Sost and suppressers targeting a protein complex (also known as proteasome inhibitor) integrated has displayed an acceptable method for treating osteolytic lesions and decrease the proliferation of metastatic cells.	There are concerns whether the usage of antibodies targeting SOST in MM patients aggravate the MM disease by increasing the proliferation of metastatic cells.	332, 333, 334, 335
Bone Metastasis	DKK1 overexpression	Osseous metastatic disease (bone metastasis) stimulation caused by osteoclastic cells high-differentiation.	Usage of Zoledronic acid and Denosumab to treat bone lesions.	The expression of DKK1 gene serving as biological indicator to predict and prevent the osseous metastatic disease in the upcoming years.	Further research is needed when considering if bone metastasis and other tumors are biologically affined or not.	336,337

Notes: CAR-T, chimeric antigen receptor T cells; CTx, chemotherapy; DMARDs, disease-modifying antirheumatic drugs; HA, hyaluronic acid; HPP, hypophosphatasia; MMP13, matrix metallopeptidase 13; MMPs, matrix metalloproteinases; MMT, multi-modal therapy; MTX, methotrexate; NSAIDs, non-steroidal anti-inflammatory drugs; OCD, osteochondritis dissecans; OCIF, osteoclastogenesis inhibitory factor; OI, osteogenesis imperfecta, P.O, per Os (oral administration); OPG: osteoprotegerin; RA, rheumatoid arthritis; RANKL, receptor activator of nuclear factor kappa-Β ligand; SERM, selective estrogen receptor modulators; RTx, radiotherapy; HER2/neu, human epidermal growth factor receptor 2.

Therapeutic targeting of Wnt signaling components in clinical trials

Wnt molecules are implicated to well-maintain bone mass, repair bone fractures, and provide remission for tumor treatments during separate phases.^30,338 ETC1922159 and WNT-974 (selective molecule inhibitors of O-acyltransferase porcupine) reach the first phase of clinical trial and palmitoleate Wnt ligands to moderate the β-catenin productivity and maintain the uncontrolled cell-growth,339, 340, 341 Ipafricept (IPA) (transgenic integrating protein containing an extracellular human frizzled 8 receptor) engages with a Cr-domain (Cysteine-rich) of Wnt ligand and eventually hinders the activation of the Wnt signaling,342, 343, 344 Tabituximab barzuxetan (monoclonal immunoglobulin G1 targeting frizzled 10 receptor), reaching the first phase of clinical trial, experiments have shown a provision of potent remission for synovial sarcoma in mice stereotype,^345,346 Vantictumab (monoclonal immunoglobulin G2, anti-frizzled receptor) having completed the first phase of clinical trial, hinders the Wnt signaling by blocking the binding of WNT3A ligand to frizzled receptors (FZD1, FZD2, FZD5, FZD7, FZD8),347, 348, 349 Rosmantuzumab (monoclonal immunoglobulin G1 anti-human R-spondin 3) being on its clinical trial, targets Rspo3 to hinder its binding to the leucine-rich repeat-containing G-coupled receptors (LGRs) and prevent the activation of RSPO-LGR pathway thus inhibiting the proliferation of tumor cells in acute myeloid leukemia.^30,350 Foscenvivint/PRI724 (inhibiting receptor that targets β-catenin and hinders the transcriptive activity of cAMP response element-binding protein) effectively inhibits the Wnt signaling pathway by decreasing the level of β-catenin in pluripotent human embryonal carcinoma cisplatin-resistant germ cell tumor line,351, 352, 353 and Zilovertamab/Cirmtuzumab (monoclonal immunoglobulin G1, anti-receptor tyrosine kinase like orphan receptor Ror-1) blocks the activity of WNT5A ligand and hinders cell-proliferation in chronic lymphocytic leukemia^354,355 amongst others, are overly suggested to be utilized in the clinic.

In skeletal disorders treatment, targeting the Wnt signaling pathway is validated to be clinically sufficient. Gene alterations encoding for porcupine inhibitors such as WNT974 and WNT-C59 (inhibitor for Wnt3a-mediated activation of T-cell factor binding site) are documented for their detrimental outcomes in decreasing bone ossification and increasing bone remodeling activity thereby causing various skeletal disorders³⁵⁶ but with an efficacy of alendronate used in patients reported.

It is reported, through clinical observations, on the development of sclerostin moAbs (monoclonal antibodies) targeting and counterpoising sclerostin as an approving pharmacological method due to its abilities to enhance BMD, reduce bone-rupture risks and promote metabolic activity in mouse models.^293,357 It is intriguing that antisclerostin compounds increase bone formation while decreasing bone resorption in contrast to teriparatide, the only osteoanabolic agent currently approved in the USA for treating osteoporosis. Antisclerostin therapy has led to improved clinical outcomes with a favorable balance of benefits and risks, therefore this approach to osteoporosis treatment is now a welcomed addition to current options.

Utilizing other agents in targeting the Wnt signaling cascade has also shown effectiveness.

A flavonol found aplenty in herbs, named Quercetin (nonspecific protein kinase enzyme inhibitor), was shown in a recent study, functioning as an antioxidant agent enhancing the production of osteocytes and hindering the death of osteoblast cells amongst other functions. However, there are a few underlying negative results in bone coming from its utilization which require further investigation.³⁵⁸ Recent report has demonstrated, Polydatin, also known as Piecid, a stilbenoid glucoside derived found in grape juice, to enhance the production of human bone marrow mesenchymal stem cells in osteogenic cells via the crosstalk between Wnt pathway and BMP2 pathway.³³⁰ Another new report has shown the significance of a WIOTM (Wnt-induced osteogenic tissue model) “band-aid” capable of being transplanted in maintaining human skeletal stem cells and repairing bone defects in mice thus demonstrating the therapeutical significance of Wnt signaling manipulation in clinic.³⁵⁹

Summary and perspective

Bone well-development and homeostasis is vital for health maintenance, and it is highly regulated by the Wnt signaling pathway known to orchestrate the osteoblast differentiation, bone growth and repair. This review coordinates the studies on the critical role of Wnt signaling pathway for the attainment of normal bone mass and structure and their significance in generating the energy in osteoblasts-lineage cells. The identification of specific and well-conserved regulatory proteins and different signaling pathways involved at various steps of this pathway designate that the Wnt-related processes are as tightly regulated as they are highly predisposed to gain alterations and be linked to pathological processes and development of numerous abnormal skeletal conditions. Supplementary research is highly necessary to further inquest the contributions of Wnt signaling pathway on osteoblast metabolism, bone homeostasis and the energy balance of the body. The compelling evidence emerging from newly reported studies will be highly embraced and utilized for novel therapeutics design to treat or alleviate skeletal disorders or bone diseases in the clinic. In the upcoming years, researchers dedicated to carcinogenic therapies and bone disorder operation should gain a better understanding on bone-related-affairs and further supply with relevant and applicable pre-emptive inventions. In spite of all that, aspiration, and expectation for the expansion of accurate understanding and execution in this discipline and especially on Wnt signaling pathway, will always remain increasingly serviceable to human beings.

Author contributions

RV: data analysis and literature formation. GC, MW, and XZ: supervision and grant holder.

Conflict of interests

The authors declare no conflict of interest.

Funding

This work was supported by grants by Zhejiang Qianjiang Talent Program (No. 21040040-E), the Department of Sci-Tech of Zhejiang Province (No. LGF19H140002), a startup grant from Zhejiang Sci-Tech University (No. 18042290-Y, and 2021Q031), funds from National Natural Science Foundation of China (No. 81900806, and 81400489), the basic Public Welfare Planning Project of Zhejiang Province (No. LGD20C040001) and Jiaxing Science Technology Foundation (No. 2020AY10001) and Jiaxing Key Laboratory of Animal Model Generation and Precise Synthesis of New Drug Leads.

Abbreviations

BMD

Bone mineral density

BMP

Bone morphogenetic protein

CAD

Coronary artery disease

CAR-T

Chimeric antigen receptor T cells

COL1A1/COL2A1

Collagen, type I/type II, alpha 1

CGOF

Conditional gain of function

CTx

Chemotherapy

DMARDs

Disease-modifying antirheumatic drugs

DMP1

Dentin matrix acidic phosphoprotein 1

E2A/TCF3

Transcription factor 3/ E2A immunoglobulin enhancer-binding factors E12/E47

FGF

Fibroblast growth factor

GLI

Glioma-associated oncogene

GOF

Gain of function

HA

Hyaluronic acid

HER2/neu

Human epidermal growth factor receptor 2

HPP

Hypophosphatasia

Ihh

Indian hedgehog

LOF

Loss of function

MLST8

mTOR associated protein, LST8 homolog

MMPs

Matrix metalloproteinases

MMP13

Matrix metallopeptidase 13

MMT

Multi-modal therapy

MSCs

Meristematic stem cells

MST1/2

Macrophage-stimulating protein

MSX2

Homeobox protein MSX-2

MTX

Methotrexate

NSAIDs

Non-steroidal anti-inflammatory drugs

OC

Osteocalcin

OCD

Osteochondritis dissecans

OCIF

Osteoclastogenesis inhibitory factor

OE

Overexpression

OI

Osteogenesis imperfecta

OMIM

Online Mendelian inheritance in man

OPG

Osteoprotegerin

PDGF

Platelet-derived growth factor

PNM

Perinatal mortality

P.O

Per Os (oral administration)

PRX1

Peroxiredoxin 1

RA

Rheumatoid arthritis

RANKL

Receptor activator of nuclear factor kappa-Β ligand

RAP

RAS/RAP GTP-binding protein

RIC

Resistance to inhibitors of cholinesterase

RARβ

Retinoic acid receptor beta

RTx

Radiotherapy

SERM

Selective estrogen receptor modulators

SIN1

Stress activated protein kinase interacting protein 1

TAZ

Transcription adaptor putative zinc finger

Tb.N

Trabecular number

Tb.Sp

Trabecular separation

TGF-β

Transforming growth factor-β

TMD

Tissue mineral density

TSC1/2

Tuberous sclerosis proteins 1 and 2

YAP

Yes-associated protein