The therapeutic potential of the Wnt signaling pathway in bone disorders.

Abstract

The Wnt pathway plays a critical role in development and differentiation of many tissues, such as the gut, hair follicles, and bone. Increasing evidence indicates that Wnts may function as key regulators in osteogenic differentiation of mesenchymal stem cells and bone formation. Conversely, aberrant Wnt signaling is associated with many osteogenic pathologies. For example, genetic alterations in the Wnt signaling pathway lead to osteoporosis and osteopenia, while inactivating mutations of Wnt inhibitors result in a hyperostotic skeleton with increased bone mineral density. Hyperparathyroidism causes osteopenia via induction of the Wnt signaling pathway. Lithium, often used to treat bipolar disorder, blocks a Wnt antagonist, decreasing the patient's risk of fractures. Thus, manipulating the Wnt pathway may offer plenty therapeutic opportunities in treating bone disorders. In fact, induction of the Wnt signaling pathway or inhibition of Wnt antagonists has shown promise in treating bone metabolic disorders, including osteoporosis. For example, antibodies targeting the Wnt inhibitor Sclerostin lead to increased bone mineral density in post-menopausal women. However, such therapies targeting the Wnt pathway are not without risk, as genetic alternations may lead to over-activation of Wnt/β-catenin and its association with many tumors. It is conceivable that targeting Wnt inhibitors may predispose the individuals to tumorigenic phenotypes, at least in bone. Here, we review the roles of Wnt signaling in bone metabolic and pathologic processes, as well as the therapeutic potential for targeting Wnt pathway and its associated risks in bone diseases.

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Current Molecular Pharmacology, 2011, 4, 000-000 1

1874-4672/11 $58.00+.00 © 2011 Bentham Science Publishers Ltd.
The Therapeutic Potential of the Wnt Signaling Pathway in Bone Disorders
Eric R. Wagner1, Gaohui Zhu1,2, Bing-Qiang Zhang1,3, Qing Luo1,2, Qiong Shi1,3, Enyi Huang1,4,
Yanhong Gao1,5, Jian-Li Gao1, Stephanie H. Kim1, Farbod Rastegar1, Ke Yang1,6, Bai-Cheng He1,2,
Liang Chen1,3, Guo-Wei Zuo1,3, Yang Bi1,2, Yuxi Su1,2, Jinyong Luo1,3, Xiaoji Luo1,3, Jiayi Huang1,3,
Zhong-Liang Deng1,3, Russell R. Reid1, Hue H. Luu1, Rex C. Haydon1 and Tong-Chuan He*,1,2,3
1Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, IL,
USA; 2Stem Cell Biology and Therapy Laboratory, The Children’s Hospital of Chongqing Medical University,
Chongqing, China; 3Key Laboratory of Diagnostic Medicine Designated by Chinese Ministry of Education and the Af-
filiated Hospitals of Chongqing Medical University, Chongqing, China; 4School of Bioengineering, Chongqing Univer-
sity, Chongqing, China; 5Department of Geriatrics, Xinhua Hospital of Shanghai Jiatong University, Shanghai, China;
6Department of Cell Biology, the Third Military Medical University, Chongqing, China
Abstract: The Wnt pathway plays a critical role in Development and differentiation of many tissues, such as the gut, hair
follicles, and bone. Increasing evidence indicates that Wnts may function as key regulators in osteogenic differentiation of
mesenchymal stem cells and bone formation. Conversely, aberrant Wnt signaling is associated with many osteogenic pa-
thologies. For example, genetic alterations in the Wnt signaling pathway lead to osteoporosis and osteopenia, while inac-
tivating mutations of Wnt inhibitors result in a hyperostotic skeleton with increased bone mineral density. Hyperparathy-
roidism causes osteopenia via induction of the Wnt signaling pathway. Lithium, often used to treat bipolar disorder,
blocks a Wnt antagonist, decreasing the patient’s risk of fractures. Thus, manipulating the Wnt pathway may offer plenty
therapeutic opportunities in treating bone disorders. In fact, induction of the Wnt signaling pathway or inhibition of Wnt
antagonists has shown promise in treating bone metabolic disorders, including osteoporosis. For example, antibodies tar-
geting the Wnt inhibitor Sclerostin lead to increased bone mineral density in post-menopausal women. However, such
therapies targeting the Wnt pathway are not without risk, as genetic alternations may lead to over-activation of Wnt/?-
catenin and its association with many tumors. It is conceivable that targeting Wnt inhibitors may predispose the individu-
als to tumorigenic phenotypes, at least in bone. Here, we review the roles of Wnt signaling in bone metabolic and patho-
logic processes, as well as the therapeutic potential for targeting Wnt pathway and its associated risks in bone diseases.
Keywords: Wnt, osteoporosis, sclerostin, ?-catenin, osteogenesis, fracture, osteogenic differentiation, LRP5/6.
INTRODUCTION
Discovered in the 1980s, the Wnt (Wingless and int-1)
signaling cascade is involved in embryonic Development
and homeostasis, through regulation of cell proliferation,
differentiation, and apoptosis [1, 2]. The signaling pathway
associated with tumorigenesis was first reported when the
APC protein was found to interact with ?-catenin in patients
suffering from familial adenomatous polyposis (FAP) [1, 2].
The expression of Wnt proteins is regulated throughout De-
velopment [3]. It has been well established that Wnts play an
integral role in many physiologic and pathologic processes.
Wnt genes encode a family of approximate 20 cysteine-rich
secreted glycoproteins, which interact with cell surface re-
ceptors and trigger a cascade of intracellular events. There
are three distinct pathways through which Wnt signaling can
be transduced (Fig. 1). The canonical pathway converges on
the transcriptional regulator ?-catenin (Table 1). Wnt pro-
teins, such as Wnt 1, Wnt 3a, or Wnt 8, bind to the Frizzled
(Frz) receptors and co-receptor LRP5/6 (low-density lipopro-
tein-receptor-related protein 5/6), leading to ?-catenin stabi-
lization and translocation to the nucleus to initiate transcrip-


*Address correspondence to this author at the Molecular Oncology Labora-
tory, Department of Surgery, The University of Chicago Medical Center,
5841 South Maryland Avenue, MC3079, Chicago, IL 60637, USA; Tel:
(773) 702-6216; Fax: (773) 834-4765;
E-mail: tche@surgery.bsd.uchicago.edu
tion [4-7]. Wnt-Frz interactions are often described as pro-
miscuous, since a single Wnt protein may bind to multiple
Frz proteins [8]. The Wnt-Frz/LRP5/6 interaction activates
the associated kinases, which in turn phosporylates the intra-
cellular protein disheveled (Dvl). Activation of Dvl inhibits
glycogen synthase kinase 3 ? (GSK3?), resulting in subse-
quent stabilization of ?-catenin (Fig. 1) [9]. Stabilized ?-
catenin then translocates to nucleus and forms a transcrip-
tional complex with lymphoid-enhancer binding factor
(Lef)/T-cell specific transcription factors (Tcfs) to regulate
target gene expression [10]. Examples of the ?-catenin/Tcf
target genes include c-Myc, cyclin D1 and PPAR? [11-14].
In the absence of Wnt ligands, a complex consisting of
GSK3?, Axin and adenomatous polyposis coli (APC), phos-
phorylates the N-terminal of ?-catenin and initiates its pro-
teosomal degradation [4, 5].
The non-canonical Wnt pathways, which occur inde-
pendently of ?-catenin, include the calcium dependent and
cell polarity pathways (Fig. 1). Well characterized non-
canonical Wnts include Wnt 5a and Wnt 11 [7]. The cal-
cium-dependent Wnt pathway plays important roles in em-
bryonic Development, cell migration and motility, and pos-
sibly tumor suppression [5, 15]. In the calcium-dependent
Wnt pathway, the Wnt ligands bind to Frz receptor and trig-
ger intracellular calcium release by heterotrimeric G protein
stimulation. The released intracellular calcium activates pro-
tein kinase C (PKC) or calcineurin (CaCN), which then turns
on transcription factors Elk-1 and NF-AT [16]. The cell

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Wagner et al.

















Fig. (1). Schematic representation of Wnt signaling pathways. Wnt ligands can activate either the canonical pathway (through ?-catenin) or
the non-canonical pathway (through Ca++ signaling or Rac/Rho pathway) to achieve distinct biological functions. In the canonical pathway,
Wnt ligand binding of LRP 5/6 receptors induces them to interact with Frz proteins, leading to the phosphorylation of Disheveld (Dvl). Acti-
vating Dvl, then inhibits the Wnt antagonist complex, consisting of Axin, Glycogen Synthase Kinase 3 ? (GSK3?), and Adenomatous Poly-
posis Coli (APC), which then stabilizes ?-catenin and enables its translocation to the nucleus and interaction with the Tcf/Lef transcription
factors. The non-canonical pathways include the calcium dependent and the cell polarity pathways. The calcium dependent pathway utilizes a
heterotrimeric G protein to stimulate a cascade including phospholipase c, IP3, and DAG which concludes in intracellular calcium release and
calcineurin activation. In the cell polarity pathway, Frz activates Rho and Rac and proceeds through JNK pathway to activate the transcription
factor c-Jun.
Table 1.

Potential Therapeutic Strategies by Targeting Wnt Signaling at Different Levels
Mechanism Intervention Therapeutic Effect Reference
Agonize Wnt Signaling Molecules
?-Catenin
Transmits Wnt signal to nucleus, binds
TCF-Lef-1
DCA (activates ?-catenin)
Enhanced Osteogenic Signaling and Differ-
entiation
[96]
Antagonize Wnt Signaling Molecules
Sclerostin Inhibits Wnt coreceptors LRP 5/6 Anti-Sclerostin Abs Decreased BMD in Spine and Hip [4, 5]
Dkk-1 Bind, Internalize LRP Receptors Anti-Dkk-1 Abs Increased Bone Mass and Osteogenesis [90]
SFRPs
Bind, competitively inhibit Frz Recep-
tors
Small-molecule inhibitors of
SFRPs
Increased Bone Formation and resultant
Bone Density
[80, 92]
Tcf/Lef-1 Binds ?-catenin, activates transcription Quercetin (blocks the association
of ?-catenin to TCF/Lef-1)
Decreases Wnt Signaling and Wnt associ-
ated Osteogenesis
[97]
GSK-3
Phosphorylates ?-catenin to lead to its
degradation
Lithium and BIO (blocks GSK-3
to inhibit ?-catenin degradation)
Increased BMD in ovariectomized rats,
decreased fracture incidence
[87, 98, 99]
Targeting Wnt Cross Talk
Gleevec Tyrosine Kinase Inhibitor Anti-Cancer Therapies Downregulates ?-catenin signaling pathway [100]
PPAR? and Reti-
noic Acid
Stimulate Adipogenesis Synthetic Agonists Ligands
Downregulates Wnt/?-catenin signaling
pathway
[89, 101]
For therapeutic purpose, Wnt signaling can be targeted at the multiple levels of the signaling cascade, such as at the extracellular, cell membrane, and intracellular levels. Exemplary
interventions targeting each signaling nodal point are given and discussed in the text.

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The Therapeutic Potential of the Wnt Signaling Pathway Current Molecular Pharmacology, 2011, Vol. 4, No. 1 3
polarity pathway functions through Frz receptors, activating
Rho and Rac, which lead to c-jun NH2 terminal kinase
(JNK) activation [17]. This pathway may modify the actin
cytoskeleton, while also plays a role in cell proliferation,
differentiation, and apoptosis [9, 18]. However, many as-
pects in the non-canonical Wnt signaling pathways are
poorly understood.
In this review, we primarily focus on the canonical Wnt
pathway. The Wnt pathway plays an important role in many
physiologic and pathologic processes throughout the body.
Abnormal Wnt signaling has been associated with many dis-
eases, ranging from cancer to bone disorders. Investigations
into the Wnt pathway have shed light on its role in os-
teogenesis, highlighting its role in associated pathologies.
These lines of studies have also offered opportunities for
therapeutic manipulation of the Wnt signaling components,
from the receptors, signal transducers, and downstream tar-
gets to the antagonists.
WNT ANTAGONISTS
The negative regulation of the Wnt signaling pathway
can occur both outside and within the cell (Fig. 2). Extracel-
lular inhibitors of the Wnt proteins include Wnt Inhibitory
Factors (WIFs), Dickkopfs (Dkks), secreted frizzled-related
proteins (SFRPs), Kremen1 and 2 (Krm 1/2), and Sclerostin
(Sost) (Fig. 2 and Table 1). These regulatory molecules act
by either binding the Frz (SFRPs) or LRP 5/6 (Sclerostin,
Dkks, Krm) receptors to prevent Wnt association, or by di-
rectly binding the Wnt proteins (WIFs) [5]. Dkk 1 and Dkk 2
are secreted proteins that bind to the LRPs, leading to a
cross-linking and internalization of receptor [11]. The SFRPs
inhibit Wnt signaling through sharing a similar ligand bind-
ing domain with Frz receptors [19]. The transmembrane pro-
teins kremens are involved in promoting LRP inactivation
[11]. Sclerostin is a secreted protein that binds and blocks
the Wnt co-receptors LRP 5/6. Intracellular inhibitors in-
clude the GSK3?-Axin-APC complex and Chibby (Cby)
(Fig. 2). Cby competes with Tcf/Lef-1 to block ?-catenin
signaling [20]. This nuclear control involves modifying the
Tcf and Lef transcription factors in the Wnt signaling cas-
cade [21, 22]. The antagonist regulation of the Wnt cascade
is critical in a variety of disease processes, and therefore, has
tremendous potential in therapeutic regimens.
WNT SIGNALING IN NON-OSTEOGENIC DIFFER-
ENTIATION
The Wnt signaling cascade plays a crucial role in many
aspects of Development, and tissue renewal. Abnormalities
in the Wnt signaling pathway may disrupt homeostasis by
altering normal physiologic processes. Gut epithelium is the
most dynamic example of self-renewal, as the new epithelial
cells form in a matter of 3-5 days. In this process, stem cells


















Fig. (2). Functions of some naturally occurring Wnt antagonists at cell surface. Wnt signaling is tightly regulated at cell surface level by
several antagonists that can target receptors Frizzled (Frz), co-receptors (LRP5/6), or Wnt ligands themselves. Extracellular antagonists in-
clude Sclerostin, which binds and inhibits LRP 5/6, Wnt Inhibitory Factors (WIFs) that bind Wnt ligands, and secreted frizzled-related pro-
teins (SFRPs) that bind Frz and competively inhibit the LRP interaction with Frz. Kremens (Krm) and Dickkopfs (Dkk) facilitate LRP inac-
tivation through internalization of the receptor. The intracellular complex Glycogen Synthase Kinase 3 ? (GSK3?), Axin, and Adenomatous
Polyposis Coli (APC) inhibit ?-catenin translocation to the nucleus. The recently discovered Chibby (Cby) blocks ?-catenin association with
its transcription factors in the nucleus.

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4 Current Molecular Pharmacology, 2011, Vol. 4, No. 1
Wagner et al.
form differentiated crypt precursors lining the base of the
villi, enabling regeneration of the overlying epithelial layers
[23]. Wnt signaling is an integral part in this process. Lack
of Tcf4 or over-expression of the antagonist Dkk-1 leads to
the absence of these progenitor cells, while Wnt stimulation
results in cell proliferation and paneth cell terminal differen-
tiation [23-25].
Another rapidly renewing cell regeneration cycle involv-
ing stem cells occurs within the hair follicles. Bulge stem
cells migrate to the base of the follicle to undergo terminal
differentiation and form the precortex [11]. Wnt signaling is
critical in this differentiation process, activating bulge stem
cells to initiate the cascade and hair follicle formation [11].
Mutants in the transcription factor Lef1 have few hair folli-
cles, while the overexpression of ?-catenin leads to excessive
hair [26, 27]. Wnt signaling also functions as important regu-
lators of the hematopoietic microenvironment. Wnt proteins
and ?-catenin overexpression promote proliferation and self-
renewal of hematopoietic progenitor cells [28]. Furthermore,
Tcf1 knockout leads to a decrease in thymocyte progenitors,
impacting the production of T-lymphoid lineages [29].
WNT SIGNALING AND CROSS-TALK WITH OTHER
PATHWAYS DURING OSTEOGENIC DIFFEREN-
TIATION
The Wnt pathway plays an integral role in bone Devel-
opment and osteogenic homeostasis [6, 9, 10]. Wnt ligands
have been identified as critical mediators for limb bud initia-
tion and Development, joint formation, and limb morpho-
genesis[9]. Experimental deficiencies of non-canonical Wnt
signal transducers lead to abnormal bone formation through-
out Development [30, 31]. Recent studies indicate that Wnts
are important regulators of osteogenic differentiation of
mesenchymal stem cells (MSCs). MSCs are pluripotent bone
marrow stromal progenitor cells that retain the ability to dif-
ferentiate into a variety of tissues, including bone, adipose,
muscle, cartilage, and tendons (Fig. 3). This differentiation
process is tightly regulated by complex signaling events [32,
33]. Wnt signaling is crucial in regulating both osteogenic
and adipogenic lineage-specific differentiation [6, 34], as
Wnts can promote osteoblastic precursors into more differ-
entiated osteoblasts and serve as negative regulators of adi-
pogenesis (Fig. 3) [35, 36]. Canonical Wnt/?-catenin, acts
synergistically with the osteogenic regulator Runx2 and
promotes the differentiation pathway toward osteogenic pre-
cursors [37, 38]. The non-canonical Wnt pathway has also
been shown to suppress the adipogenic regulator PPAR?,
and enhances Runx2, inducing osteogenesis [34, 39], as Wnt
5a is able to promote osteoblastogenesis, while acting as a
co-repressor of PPAR? and subsequent adipogenesis [39].
These counter-regulatory functions are integral to terminal
osteogenic differentiation (Fig. 3).
Wnt proteins play an important role in regulating adipo-
genic, chondrogenic and myogenic Development [40, 41].
Canonical Wnt ligands are able to suppress both adipogene-
sis and chondrogenesis [37, 38]. Inhibition of Wnt signaling
leads to transdifferentiation of myoblasts to adipocytes [42].
Both Wnt antagonists Dkk1 and Dkk2 are required for ter-
minal differentiation of osteoblasts, suggesting a complex
regulatory loop in the differentiation cascade [43, 44]. None-
theless, Wnt proteins may play a key role in both promoting
and regulating the complex cascade associated with MSC
differentiation into mature osteocyte during regenerative
processes.
Wnts cross-talk with bone morphogenetic proteins
(BMPs) in regulating MSC differentiation. In particular,
BMPs 2, 6, and 9 act as major osteogenic inducers [33].
Wnt3a acts synergistically to induce osteogenic differentia-
tion with BMP9, while ?-catenin knockdown or Frz antago-
nist overexpression blocks BMP9 induced osteogenic differ-
entiation and bone formation, inducing chondrogenic matrix
production [45]. Addition of BMPs to ?-catenin has been
proven to be critical for ectopic bone formation, although ?-
catenin alone is able to induce osteogenic differentiation, not
bone formation [33, 46]. Both Wnt antagonist Dkk-1 and ?-
catenin null inhibit the BMP-2 induced bone formation, indi-
cating that these pathways may cross-talk at the interaction
of Smad-4 and ?-catenin [47]. Thus, as two important media-
tors of osteogenesis, the cross-talk between Wnts and BMPs
represents a critical step towards understanding and targeting
this differentiation process.
During osteogenic regulation, Wnt signaling may cross-
talk with the Hedgehog (Hh) and Notch signaling pathways.
Hh is crucial for embryonic skeletal Development and en-
dochondral bone formation [48, 49]. Wnt signaling regulates
key mediators in the Hh signal transduction, Gli2 and Gli3
[50]. Notch signaling is also important for skeletal Devel-
opment. A deficiency in the Notch signal mediator preseni-
lin-1 leads to defects in the axial skeleton [51]. There ap-
pears to be a conserved domain between Notch signaling
molecules and the transcription factors Tcf/Lef-1, enabling
the Notch signaling mediators to inhibit the canonical Wnt
pathway [52]. Other mechanisms associated with osteogenic
induction through Wnt signaling involve activation of PKC?,
as well as, Src/ERK and PI3K/Akt signaling pathways [53].
Further investigations into the cross-talks between these sig-
naling pathways with Wnt signaling are imperative to elicit
the mechanisms underlying MSC differentiation and bone
Development.
WNT SIGNALING IN OSTEOGENESIS AND OS-
TEOGENIC PATHOLOGIES
The canonical and non-canonical Wnt signaling path-
ways play an important role in bone metabolism and os-
teogenesis, and contribute to osteogenic pathologies, as
shown in both animal studies and genetic disorders in hu-
mans. Many signaling components of the Wnt pathways thus
may represent potential drug targets for treating pathologies
of the skeletal system.
Wnt Co-Receptors as Regulators of Osteogenesis and
Bone Mineral Density
Abnormalities in LRP5 have significant impacts on
skeletal homeostasis. Osteoporosis pseudoglioma syndrome
(OPPG) is caused by inactivating mutations of the LRP5
gene [54]. The OPPG patients are characterized with pheno-
types of osteoporosis and decreased bone mineral density
[55]. Similar results have been seen in LRP5 knockout mice
[56, 57]. However, LRP5 activating mutations lead to in-

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The Therapeutic Potential of the Wnt Signaling Pathway Current Molecular Pharmacology, 2011, Vol. 4, No. 1 5
creased bone mass, while knockout mutations reduce bone
mineral density from a defect in osteoblast proliferation [55,
57, 58].
The role of LRP6 is analogous to that of LRP5 in initiat-
ing the Wnt signaling pathway. As a Wnt co-receptor, muta-
tions in the LRP6 gene results in a variety of phenotypes in
adult bone homeostasis [59]. A mutation in the LRP6 gene in
humans leads to osteoporosis and other metabolic abnormali-
ties [60]. In mice, a spontaneous missense mutation in the
LRP6 gene leads to defects in somitogenesis and reduced
bone mass in adults [61]. Null mutations in the LRP6 gene
lead to prenatal lethality secondary to truncated distal limb
and axial skeletal structures [59, 62]. The critical role of
LRP6 in osteogenic Development is demonstrated when mu-
tations of both LRP5 and LRP6 synergistically delay and
prevent skeletal Development [63].
Although a few attentions have been paid to the Wnt
ligands, there appears to be some associations of the ex-
tracellular Wnt agonists with metabolic abnormalities. Wnt
10b missense mutations in humans may be linked to obesity,
while similar mutations in animals lead to a decrease in post-
natal bone density [58, 64]. Furthermore, in mice overex-
pression of Wnt 10b results in increased bone mass [65].
Conditional Wnt 7b knockout causes defects in osteoblasto-
genesis and chondrogenesis [66]. Thus, therapeutic manipu-
lations of extracellular Wnt receptor, either through mimick-
ing Wnt ligands or using blocking antibodies against their
receptors, hold tremendous potential for treating skeletal
pathologies
Wnt Intracellular Signaling During Osteogenesis
As an important mediator of the canonical Wnt signaling
pathway, ?-catenin is crucial in bone formation and regen-
eration [36]. Conditional deletion of ?-catenin in mice leads
to impaired mineralization secondary to a deficiency of ter-
minally differentiated osteblasts [67, 68]. This also impaired
endochondral ossification and ectopic chondrocyte formation
[4]. The osteopenia, or mildly decreased bone mineral den-
sity, resulting from ?-catenin knockout is associated with
increased osteoclasts [69]. These results point to the critical
role for ?-catenin in regulating the relationship between os-
teoblasts and osteoclasts.
The Tcf/Lef transcription factors are critical mediators in
the Wnt pathway’s regulation of osteogenesis through ?-
catenin. Tcf1 null mice demonstrate low bone density with
an increased number of osteoclasts [35]. This is similar to
the impaired mineralization seen in ?-catenin deficient mice,
as would be expected from the interaction of these transcrip-
tion factors with ?-catenin. Further investigation into ma-
nipulating these intracellular mediators of Wnt signaling
may enable us to target these nuclear mediators in the path-
way using biological and/or chemical agents.
Wnt Extracellular Antagonists in Osteogenesis and Bone
Pathologies
Sclerostin (Sost) is a protein secreted by osteocytes that
binds to and blocks Wnt binding to co-receptors LRP5/6,
leading to inhibiting osteogenic differentiation. It has been
shown that sclerostin is able to indirectly inhibit BMP in-

















Fig. (3). The roles of Wnt signaling in regulating mesenchymal stem cell differentiation. Canonical Wnts can inhibit adipogenic differentia-
tion, and promote osteogenic differentiation, which can be inhibited by sclerostin. Wnt signaling proteins are thought to regulate the late
stages of ostegenic differentiation, while inhibiting adipogenesis in part through its interaction with PPAR?. Sclerostin is secreted by osteo-
cytes in a negative feedback cycle to inhibit the Wny cascade. PPAR? and retinoic acid are critical regulators of adipogenesis, while Runx2,
osterix, osteocalcin, and osteopontin are involved in the regulation of the osteogenic differentiation cascade.