1. Wnt -4 Stimulation
of Human Bone
Marrow Stromal
Stem Cells and
Mesenchymal
Stem Cells of the
Maxilla
Gayathri Vijayakumar
Poly ID# 0408513
Guided Studies- Fall 2010
Nicola C. Partridge, Ph.D.
Professor, Chair
Basic Science & Craniofacial
Biology
E-mail: ncp234@nyu.edu
Michael D Turner, D.D.S., M.D.
Assistant Professor
Oral and Maxillofacial Surgery
E-mail: mdt4@nyu.edu

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TABLE OF CONTENTS
INTTRODUCTION ....................................................................................................................... 3
OVERVIEW ................................................................................................................................... 4
WNT PROTEINS ...................................................................................................................................... 4
WNT SECRETION & EXTRACELLULAR TRANSPORT ..................................................................... 7
WNT RECEPTION.................................................................................................................................... 8
WNT SIGNAL TRANSDUCTION PATHWAYS .................................................................................... 9
REGULATION OF WNT SIGNALING.................................................................................................. 14
WNT SIGNALING IN BONE FORMATION ......................................................................................... 15
WNT-4 ..................................................................................................................................................... 17
PTH & WNT PROTEINS (WNT-4) ........................................................................................................ 20
WNT PROTEINS & STEM CELLS................................................................................................................. 24
ROLE OF WNT SIGNALING IN MESENCHYMAL STEM CELLS ....................................................................... 26
CONCLUSION ............................................................................................................................ 30
REFERENCE ............................................................................................................................... 31

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1. INTRODUCTION
Wnt proteins are palmitoylated and glycosylated ligands that play a well established role in
embryonic patterning, cell proliferation and cell determination [1][2], besides playing a
significant role in cell cycle arrest, differentiation, apoptosis and tissue homeostasis. As a result,
aberrations in the Wnt signaling pathway are associated with birth defects as well as a multitude
of diseases, most notably cancer [3]. Wnt proteins can be categorized into two types: the
canonical and non-canonical.
Wnts have important roles in regulating many aspects of skeletal development,
from limb formation to chondrogenesis and osteoblast maturation [4]. In addition to
promoting osteoblast maturation, Wnts may play a role in lineage commitment of mesenchymal
precursor cells by preventing adipogenesis is the hypothesized default pathway for mesenchymal
stem cells that do not receive proper inductive signals to
become osteoblasts, chondrocytes, myocytes, or other mesodermal cells [5].
Parathyroid hormone (PTH) regulates calcium homeostasis in addition to stimulating bone
formation [6]. Human and experimental animal studies have shown that PTH
increases osteoblast activity and number via increased differentiation and survival, increases
bone mass and bone strength, and decreases fracture rate [7]. Due to its anabolic actions, PTH is
currently the only clinically available anabolic agent used to effectively treat osteoporosis. [8][9].
In vivo microarray analysis has shown that PTH regulates molecules of the Wnt signaling
pathway in bone [10]. PTH has been demonstrated to stimulate bone lining osteoblasts to
produce non-canonical Wnt-4 in vivo, and exogenous Wnt-4 has been shown to regulate
osteoblast differentiation through non-canonical Wnt signaling pathway [11].

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Wnt signaling has been implicated in the control over various types of stem cells and may act as
a niche factor to maintain stem cells in a self-renewing state [12]. Recent studies have shown that
Wnt-3a stimulates proliferation while inhibiting osteogenic differentiation of hMSCs [17], while
non-canonical Wnt-5a enhanced osteogenic differentiation but had no effect on hMSC
proliferation [18]. Most significantly, Wnt-4 has been shown to stimulate osteogenesis in hMSCs
isolated from craniofacial tissue through a novel p38 non-canonical Wnt signaling pathway that
is a known pathway associated with osteogenic differentiation [19][20].
Preliminary findings from Dr. Nicola C. Partridge’s lab at the Department of Basic Science and
Craniofacial Biology at NYU-CD have shown that Wnt-4 is stimulated by PTH in vivo [11], and
that Wnt-4 enhances proliferation and osteogenesis of mouse bone marrow stromal stem cells
(BMSSC) in vitro. From this it is hypothesized that Wnt-4 has an important function in
proliferation and differentiation of BMSSC.
The specific aims of this study are to:
1) Assess the Wnt-4 effect on proliferation on human bone marrow stromal stem cells
2) Assess the Wnt-4 effect on differentiation of human bone marrow stromal stem cells
3) Assess the Wnt-4 effect on the proliferation and differentiation of human dental pulp
stem cells.
2. Overview
2.1 Wnt Protiens
Together with the other families of secreted factors such as FGF, TGF-beta, and Hedgehog
proteins,Wnt proteins are implicated in a wide variety of biological processes. The first Wnt gene

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mouse Wnt-1, was discovered in 1982 as a proto-oncogene activated by integration of mouse
mammary tumor virus in mammary tumors [21]. With the molecular identification of the
Drosophila segment polarity gene wingless (wg) as the orthologue of Wnt-1 [22][23] and the
phenotypic analysis of Wnt-1 mutations in the mouse [24], it became clear that Wnt genes are
important regulators of many developmental decisions [25][26].
Table 1 [21] Phenotypes of Wnt mutations in mouse, Drosophila, and C. elegans
Gene Organism Phenotype Organism Phenotype
Wnt-1 Mouse Loss of midbrain and
cerebellum
Wnt-2 Mouse Placental defects
Wnt-3A Mouse Lack of caudal somites and
tailbud
Wnt-4 Mouse Kidney defects
Wnt-7A Mouse Ventralization of limbs
Wingless Drosophila Segment polarity, limb
development, many others
Dwnt-2 Drosophila Muscle defects, testis
development
lin-44 C.elegans Defects in asymmetric cell
divisions
mom-2 C. elegans Defects in endoderm induction
and spindle orientation
The Wnts comprise a large family of protein ligands that affect diverse processes such as
embryonic induction, generation of cell polarity and the specification of cell fate. In addition to
influencing developmental processes, recent studies point to a key role for Wnt signaling during
adult homeostasis in the maintenance of stem cell pluripotency [27]. Wnts are defined by amino
acid sequence rather than by functional properties [28-29]. As many as 19 mammalian Wnt
homologues are known and are expressed in temporal spatial patterns. Shared features of all
Wnts include a signal sequence for secretion, several highly charged amino acid residues, and

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many glycosylation sites. Wnt proteins also display a characteristic distribution of 22 cysteine
residues. overexpression in tissue culture cells, several different N-linked glycosylated
intermediate Wnt protein products are observed in cell lysates [30-32], suggesting that Wnt
protein processing and secretion are highly regulated processes.
Even though the primary amino acid sequence of Wnts suggests that they should be soluble,
secreted Wnt proteins are hydrophobic and are mostly found associated with cell membranes and
extracellular matrix (ECM), the reason being that Wnt proteins are lipid modified by the
attachment of a palmitate on the first conserved cysteine residue within the protein family and on
a serine in the middle of the protein which may be necessary for:
 Wnt signaling as well as secretion
 May be necessary for their glycosylation.
 Might also aid in Wnt transport between cells as glycosylation might increase Wnt
interactions with heparin sulfate proteoglycans (HSPGs) present on the surface of Wnt
responding cells.
 Potentially anchoring Wnt proteins into the membrane for sustained signaling. [33-34].
There are a total of 7 related Wnt genes in the Drosophila genome and 19 in the human genome
(some with multiple isoforms) that generally have close orthologues in mice. Orthologous Wnt
gene products (proteins with the same function in different species) are often very highly
conserved. [35] Based on their ability to induce secondary body axis in Xenopus embryos, the
Wnt family of proteins is grouped into two functional classes. The Wnt-1 class (Wnts-1, 2, 3, 3a,
7b, 8, 8b, 10b) is able and Wnt-5a (Wnts-4, 5a, 5b, 6, 7a, 11) is unable to induce a secondary
body axis [36].

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2.2 Wnt Secretion and Extracellular Transport
Two recent genetic screens have identified the multipass transmembrane protein Wntless
(Wls)/Evenness interrupted (Evi)/Mom-3 as acting in the secretory pathway to promote the
release of Wnts from producing cells [37]. A model of Wnt secretion and realease is shown in
figure 1.Exogenously derived lipoproteins termed ―argosomes‖ are implicated in moving Wnts
and other lipid-modified proteins such as Hedgehogs [38]. A model is proposed wherein
palmitoylated proteins associate with lipoprotein particles on the extracellular face of cells.
Traffic of Wnt proteins from one cell to the next requires this association [38]. In addition,
transcytosis may regulate Wnt movement. It has been proposed that the retromer complex
promotes the association of secreted Wnts with other proteins required for ligand transport, such
as lipoprotein particles. [39]
Figure 1 [40]: Model of Wnt secretion and release. Wnt is lipid modified in the ER and is transported to the Golgi,
where it binds to Wls. Next, the Wnt–Wls complex is transported to the plasma membrane and Wnt is released (1).

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The hydrophobic Wnt protein either remains associated with the plasma membrane or binds directly to lipoprotein
particles or HSPGs to facilitate spreading and gradient formation (2). After the release of Wnt, Wls is internalized
through AP-2/clathrin-mediated endocytosis (3) and is transported back to the TGN through a retromer-dependent
trafficking step (4). An alternative possibility is that Wnt is not released at the plasma membrane, but is
reinternalized together with Wls (5). Dissociation of Wnt from Wls may take place in endosomes, after which Wnt
is released (possibly in association with lipoprotein particles) through a recycling endosomal pathway (6). Also in
this scenario, Wls is recycled back to the TGN through a retromer-dependent transport step (7). Wnt may also be
reinternalized through a Reggie-1/Flotillin-2 dependent pathway (8), which may lead to the release of a more
mobile, micelle-like form of Wnt or to the association of Wnt with lipoprotein particles (6).
2.3 Wnt Reception
The seven-pass transmembrane protein
Frizzled (Fz) protein was first receptor found
to transduce a Wnt signal [41]. . Fz proteins
contain a large extracellular domain
containing a conserved motif comprised of
10 cysteine residues called the cysteine-rich
domain (CRD) that has been shown to bind
Figure 2 [21]: Structure of Frizzleds and FRPs. Frizzled
to multiple Wnts with high affinity. At the receptors are characterized by an N-terminal signal peptide,
a cysteine-rich ligand-binding domain (CRD) followed by a
cytoplasmic side, Fzs may interact directly hydrophilic linker,seven transmembrane regions, and a
cytoplasmic tail. FRPs are secreted proteins with a CRD
similar to Frizzleds. In addition, they contain a region with
with the Dishevelled protein, a known
similarity to netrins, secreted proteins involved in axon
guidance.
mediator of Wnt signaling [42-44].
Following Wnt binding, it is thought that Fzs form a co-receptor complex with single-pass
transmembrane proteins of the low-density lipoprotein (LDL) family called Lrp5 and -6 to
transduce the canonical Wnt signal. Lrp5 and -6 proteins have a relatively small intracellular
domain and a large extracellular domain containing several potential protein interaction
domains [45].

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There are other proteins with known Wnt-binding
domains that can serve as receptors for Wnt ligands.
The single-pass tyrosine kinase Ror2, although
structurally distinct from Fz receptors, is involved
in other forms of Wnt signaling. Another well-
characterized Wnt-binding domain is the Wnt
inhibitory factor (WIF) module, which is also found
in the cell surface atypical receptor tyrosine kinase Figure 3: Different receptors that Wnt proteins
can bind to.
Ryk [46][47]. http://www.stanford.edu/group/nusselab/cgi-
bin/wnt/receptors
2.4 Wnt Signal Transduction Pathways
Wnt proteins signal through the canonical and the non-canonical pathways which are composed
of three independent signal transduction pathways (the Wnt/β-catenin pathway, the
Wnt/Ca+2 pathways, or the Wnt/planar polarity pathway) that are used to regulate the expression
of different genes. The Wnt/β-catenin pathway is commonly referred to as the canonical
pathway. It promotes cell fate determination, proliferation, and survival by increasing β-
catenin levels and altering gene expression through Lef/Tcf transcription factors [48]. The non-
canonical Wnt/Ca+2 pathway stimulates heterotrimeric G proteins, increases intracellular
calcium levels, decreases cyclic GMP levels, and activates protein kinase C to induce NF-
AT and other transcription factors [49]. The non-canonical Wnt/planar polarity pathway activates
Rho/Rac GTPases and Jun N-terminal kinase to modulate cytoskeletal organization and gene
expression. Distinct Wnt ligands probably act through specific Frizzled (Fzd) receptors to initiate
each [49].

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Figure 4: Wnt Signaling Pathways and their Implications
The canonical Wnt signaling pathway (see Figure 5) is activated when Wnts interact with
Lrp/Fzd receptor complexes as shown in the middle portion of figure 4. Receptor engagement
activates an unknown kinase(s) (K) that phosphorylates the cytoplasmic tail of Lrp5/6. These
phosphorylated residues (P) serve as docking sites for Axin and the APC, Dsh, β-catenin
complex. A GSK3β binding protein (GBP) is also mobilized after receptor ligation and excludes
GSK3β from the proximal receptor complex. β-Catenin thereby escapes phosphorylation events
that normally promote its ubiquitination (U) by E2 ligases and degradation by the proteosome
(right side of this figure). As β-catenin levels rise above those needed to bridge cadherins to the

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actin cytoskeleton (lower left of this figure), some β-catenin molecules travel to the nucleus
where they interact with Lef1/Tcf transcription factors to either increase or decrease the
expression of specific target genes. β-Catenin displaces nuclear co-repressors (CoR) from
Lef1/Tcf to facilitate the expression of genes involved in cell cycle progression (e.g. cyclin D1)
and survival (e.g. c-myc). β-Catenin and Lef1/Tcf suppress other genes, such as osteocalcin
(OCN) and E-cadherin, through unknown mechanisms, but that may involve interactions with
other transcription factors (TF). Soluble antagonists block the canonical Wnt signaling pathway
and promote β-catenin degradation via two mechanisms.
Figure 5 [49]: The Canonical Wnt Signaling Pathway
Soluble frizzled related proteins (Sfrp) bind free Wnt molecules and compete with surface
receptors (top right of figure 5). In contrast, Dkks interact with extracellular domains in Lrp5/6

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and recruit them to complexes containing Krm (top left of figure 5). This trimolecular complex is
internalized to lysosomes where Lrp5/6 are either degraded or recycled to the surface. Dkk
therefore decreases Lrp5/6 cell surface expression to regulate Wnt signaling [49].
Figure 6 [55]: Nuclear activity of ß-catenin
Accumulated ß-catenin then translocates to the nucleus, replaces Groucho from TCF, and
activates target genes. ß-catenin forms a complex with TCF and the transcription factors Brg1
and CBP. Lgs and Pygo also bind to -catenin, possibly driving its nuclear localization in addition
to playing a direct role in transcriptional activation. Negative regulation of signaling is provided
by NLK (Nemo-like kinase) which phosphorylates TCF, and ICAT (inhibitor of catenin) and
Chibby, which are antagonists of ß-catenin. In addition to TCF, two other DNA-binding proteins
have been shown to associate with ß-catenin: Pitx2 and Prop1. In the case of Prop1, ß-catenin
can act as a transcriptional activator or repressor of specific genes, depending on the co-factors

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present. The participation of any particular ß-catenin complex in transcriptional regulation is
highly cell type-dependent.[55]
The non-canonical pathway can be broadly classified into 2 branches based upon phenotypic
response; the Planar Cell Polarity (PCP) pathway and the Wnt/ Ca2+ pathway [50]. However
some authors have classified the pathways as Wnt/calcium signaling, Wnt/PCP signaling,
Wnt/JNK signaling and Wnt/Rho signaling [56]. The PCP pathway is involved in cellular
asymmetry, and it is this cellular asymmetry that controls the rigid architectural orientation of
epithelial tissues and sensory organs (e.g. inner ear cochlea), as well as the morphology and the
migratory processes of mesodermal cells undergoing gastrulation. Activation of PCP signaling
occurs basically through the binding of Wnts to Frizzled (Fz) receptors alone, without LRP co-
receptor involvement. These signals activate Dishevelled (Dvl), which in turn leads to the
activation of the GTPases Rho and Rac. Activated Rac subsequently stimulates JNK activation
[50].
The second branch of non-canonical signaling – the Wnt/Ca2+ pathway – is characterized by
Wnt-Fzd-induced PLC (phospholipase C) activation and the resultant increase of cytoplasmic
Ca2+ levels. These Ca2+ fluxes activate several Ca2+-responsive proteins, such as PKC (protein
kinase C) and CaMKII(calcium/calmodulin-dependent kinase II). CaMKII has been shown to
activate the transcription factor NFAT, TAK1 (TGF-beta activated kinase), and NLK (Nemo-like
kinase, all of which have the net effect of decreasing intracellular cGMP and consequently,
antagonizing Wnt/beta-catenin/TCF signaling [51-52]. The Wnt/Ca2+ signaling pathway is
involved in regulating cellular adhesion, cytoskeletal rearrangements, and other developmental
processes, such as dorsoventral patterning and tissue separation in embryos [53].

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Figure 7 [54]: Non- canonical pathways
2.5 Regulation of Wnt Signaling:
Wnt signaling is tightly regulated by members of several families of secreted antagonists
1. Wnt signaling requires interaction with frizzled receptors (Fz) and the presence of a
single-pass transmembrane molecule of the Lrp family (Lrp5 or 6) which can be inhibited
by members of the secreted frizzled-related protein (Sfrp) family and Wnt inhibitory
factor 1 (WIF-1).

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2. Dkk1 binds to Lrp with high affinity and to another class of transmembrane molecules,
the Kremens. Dkk1 promotes the internalization of Lrp and makes it unavailable for Wnt
reception by forming a complex with Lrp and Kremen [57].
3. Lrp coreceptor activity is also inhibited by members of sclerostin (SOST gene product).
4. Chibby is a nuclear antagonist that binds to the C terminus of β-catenin [58].
5. Another β-catenin binding protein, ICAT, can block the binding of β-catenin to TCF and
also can lead to the dissociation of complexes between β-catenin, LEF,
and CBP/p300 [56].
6. TCF can be phosphorylated by the mitogen-activated protein (MAP) kinase-related
protein kinase NLK/Nemo.
7. The phosphorylation of TCF/LEF by activated Nemo is thought to diminish the DNA-
binding affinity of the β-catenin/TCF/LEF complex, thereby affecting transcriptional
regulation of Wnt target genes [56].
2.6 Wnt Signaling in Bone formation:
The Wnt signal transduction pathway has been implicated in bone formation: patients suffering
from osteoporosis–pseudoglioma syndrome have an inactivating mutation in the Wnt co-
receptor LRP5 [13], whereas an activating LRP5 mutation is associated with high bone mass
syndrome [14] [15]. Analysis of LRP5-deficient mice revealed a decreased number
of osteoblasts suggesting that Wnt signaling stimulates bone formation at the level of
osteoprogenitor proliferation [16].

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Figure 8 [56]: Role of canonical Wnt signaling in skeletal development. Mesenchymal stem cells have the ability
to differentiate into chondrocytes or osteoblasts, depending on the environmental cues. Canonical Wnt signaling
is regulated to control the lineage progression between chondrocyte and osteoblast. If there is inadequate
canonical Wnt signaling, differentiation towards chondrocyte lineage is encouraged. However, the maturation of
chondrocytes requires the presence of canonical Wnt signaling. Canonical Wnt signaling is also required for the
progression of osteoblast progenitor cells toward osterix positive osteoblasts and then osteocalcin-positive
osteoblasts. Canonical Wnt signaling represses osteoclastogenesis by increasing the osteoprotegrin (OPG)
expression, therefore the OPG/RANKL ratio. Green plus signs indicate positive effects of Wnt; red circle minus
signs indicate inhibitory effects of physiological canonical Wnt signaling
The effects of Wnt signaling on chondrogenesis are complex and have been implicated in the
regulation of chondrogenic differentiation and hypertrophy [56]. Chondrocyte-specific
inactivationof β-catenin using Col2a1-Cre transgene leads to decreased chondrocyte proliferation
and delayed hypertrophic chondrocyte differentiation [59]. In another loss-of-function
model, Dermo 1-Cre transgene was used to delete the β-catenin gene in the mesenchymal
precursors of both chondrocytes and osteoblasts. It was shown that there is a significant delay
in chondrocyte maturation in conditional knockout embryos [60]. At the same time Activation
of β-cateninin limb and head mesenchyme repressed the expression of Sox9, a factor essential
for chondrogenesis, thereby preventing mesenchymal cells from differentiating into skeletal
precursors [59]. In the absence of β-catenin, the expression of early osteoblast markers, such
as collagen I, osterix, and osteocalcin was greatly diminished [60].

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Figure 9 [49]: Effects of Wnt signaling on osseous cells. The canonical Wnt signaling pathway
promotes the proliferation, expansion and survival of pre- and immature osteoblasts. Dkks,
Sfrps, and Wif-1 antagonize Wnt signaling in osteoblasts to facilitate death of immature cells,
but they may also downregulate the pathway in mature cells to induce terminal differentiation.
Wnt-4:
Wnt-4 is a signaling factor with multiple roles in organogenesis, a deficiency that leads to
abnormal development of the kidney, pituitary gland, female reproductive system, and mammary
gland. Wnt4 is conserved throughout vertebrates and was originally studied in non-mammalian
vertebrates such as zebrafish, chicken and xenopus [61]. Several studies have tried to gauge the
varied functions of this protein, giving the impression that this is indeed a highly conserved
protein essential for proper organ formation and development. Some of the findings are as
follows:
1. It is essential for nephrogenesis. The Wnt-4 gene is expressed in the
assembling nephrons and the medullary stromal cells during kidney development The

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role of Wnt-4 signaling in controlling mesenchyme to epithelium transformation
and kidney tubule induction is well established [63].
2. Wnt-4 signaling plays also a role in the determination of the fate of smooth muscle
cells in the medullary stroma of the developing kidney. Smooth muscle α-actin (α-SMA)
is markedly reduced in the absence of its signaling [62].
3. Wnt-4 plays an important role in female sexual development and ovarian function. Wnt-4
deficiency leads disturbed development of the internal genitalia in mouse and human, and
to a dramatic reduction of mouse oocytes. The expression of Wnt-4 protein
in human fetal ovaries was high during mid-pregnancy, when new follicles are also
rapidly being formed. This implies that Wnt-4 may have a substantial role in regulation
of the follicle formation in human ovaries [64].
4. In the reproductive system, Wnt-4 is specifically involved in Müllerian duct formation,
sex-specific blood vessel formation, oocyte maintenance and repression of
steroidogenesis. Partial XY male-to-female sex-reversal is observed in mice lacking Wnt-
4 as well as in one human patient carrying a Wnt-4 point mutation [61].
5. During mammalian embryogenesis, Wnt-4 is expressed in the gonads of both sexes
before sex determination events take place and is subsequently down-regulated in the
male gonad [61].
6. Wnt4 regulates mammary gland development in response to hormonal changes that take
place during pregnancy and may also play a part in mammary gland tumorigenesis.
Expression of Wnt4 is inversely correlated with cell proliferation in the mouse mammary
C57 mg cell line, suggesting that Wnt4 participates in restricting cell proliferation. On the
other hand, the elevated expression of theWNT4 gene in fibroadenomas, and occasionally

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also in malignant tumor tissue, implies that changes in WNT4 signaling are associated
with abnormal cell proliferation in human breast cells [68].
7. Screening of the multiple adult human tissues Figure 10 [65]. WNT4 transcripts in human adult
(A) and embryonic (B) tissues. Three mRNA bands,
revealed three human WNT4 mRNA bands of 1.5, 1.5, 2.4 and 4.3 kb in size, are seen in the adult
tissues and one, 2.4 kb, in the embryonic liver and
2.4, and 4.3 kb in size (Figure 8). The most kidney tissues. The ages of the embryos from 6 to
12 weeks are indicated below the sample rows. The
common 1.5 kb transcript was detected in a arrows show the positions of ribosomal
RNA.
number of tissues: the adrenal
gland, placenta, liver, mammary
gland, prostate, spinalcord, stomach, thyroid, trach
ea, skeletal muscle and small intestine, the signal
being strongest in the adrenal gland and placental
samples.
8. The involvement of Wnt-4 in normal mammary
gland and ovary development suggests that Wnt-
4 germline mutations may be associated with
the human cancer predisposition [65].
9. Wnt-4 is critical for development of the adrenal gland, the zona glomerulosa of which is
incomplete at birth in Wnt-4-deficient mice, probably as a result of significantly reduced
aldosterone production. In addition, Wnt-4 represses the migration of steroidogenic
adrenal precursors into the gonad [66]. In mouse spleen, Wnt1and Wnt-4 signaling
regulates differentiation of the thymocytes, the number of which is decreased in
compound mutants [67].

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Figure 11 [65]. Mapping of the human WNT4 locus to chromosome region 1p36.12 by prometaphase FISH. (A)
Hybridization signals for WNT4 (green signals) and BAC RP11-285H13 (red signals). (B) Chromosomal
location of BAC RP11-285H13 to the region 1p36.11a-1p36.11b and BAC RP11-145C4 to the region 1p36.13c-
1p36.13d (C) Hybridization signals for WNT4 (green signals) and BAC RP11-145C4 (red signals).
2.7 PTH and Wnt Proteins (WNT-4)
PTH is a single chain polypeptide with 84 amino acids and is the principle regulator of
calcium homeostasis in vertebrates. However, although continuous infusion of PTH induces
bone loss, intermittent administration of PTH results in bone formation [69]. Binding
of PTH and PTHrP to PTH1R activates two signaling pathways in osteoblasts: the PKA pathway
which is responsible for the majority of the calciotropic and skeletal actions of PTH, the PKC
pathway leading to accumulation of 1, 4, 5-inositol triphosphate and increased intracellular
calcium. This pathway has been found to regulate IGF-binding protein-5 [70].
A number of studies suggest that the PTH and Wnt signaling pathways do indeed overlap.
Following treatment of osteoblastic cells with N-terminal PTH, an increase in β-catenin that was
measured in whole cell lysates by Western blot was seen [71].

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Figure 12: PTH regulation of Wnt-4
Furthermore, PTH increased the level of β-catenin expression in mouse osteoblastic cells
(MC3T3-E1) via both PKA and PKC signaling pathways [74]. More recently, PTH was shown to
activate β-catenin signaling in osteoblasts in vitro and in vivo by direct recruitment of LRP6 to
PTH/PTH1R complex. In vivo studies confirmed that intermittent PTH treatment led to an
increase in amount of β catenin in osteoblasts (immunohistochemical analysis with antibody
to β-catenin) with a concurrent increase in bone formation in rat [75].
Partridge et al. also demonstrated a link between PTH and Wnt4 expression in bone [72]. In
vivo microarray analysis of intermittent and continuous PTH 1–34 showed

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that PTH regulated Wnt4 in bone. PTH was shown to stimulate Wnt4 primarily through
the PKA pathway and Wnt4 treatment in osteoblasts induced early expression of bone marker
genes and stimulated key canonical Wnt pathway genes. This finding suggested cross-talk
between the Wnt signaling cascades [73].
Previous studies at Dr. Partridge’s lab at the Department of Basic Science & Craniofacial
Biology at NYU-CD has showed that Wnt-4 is significantly regulated by PTH in vivo after
anabolic and catabolic protocols in bone lining osteoblasts. As pointed out earlier Partridge et
al. demonstrated that PTH stimulated Wnt-4 expression in cultured osteoblastic cells and that
this stimulation is PKA dependent and is a primary response to PTH. It has also been shown
that exogenous Wnt-4 enhances bone marker gene expression during osteoblast
differentiation by activating the non-canonical Wnt/Ca2+ and Wnt/PCP signaling pathways
but does not significantly stimulate the canonical Wnt/ß-catenin pathway. Most significantly,
Wnt-4 has been shown to stimulate osteogenesis in hMSCs isolated from craniofacial tissue
through a novel p38 non-canonical Wnt signaling pathway that is a known pathway
associated with osteogenic differentiation [19][20]. Therefore it can be hypothesized that non-
canonical Wnt signaling plays a significant role in bone and that Wnt-4 may be an important
molecule in PTH’s anabolic effect.[72]
Real time TR-PCR results have shown that PTH stimulates Wnt-4 mRNA expression in all
phases of osteoblastic differentiation but is greatest in the mineralization phase and maximal
stimulation occurs 8h after PTH treatment. Partridge et al. has demonstrated that non-
canonical Wnt-4 does not increase pre-osteoblastic proliferation or the expression of bone
marker genes at proliferation day 7, but enhances bone marker gene expression (runx2,
osterix, osteocalcin, alkaline phosphatase, MMP-13) significantly in the absence of

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differentiation-promoting factors such as ascorbate. Continuous treatment of rat primary
osteobalsts with Wnt-4 in an osteogenic environment showed that there was increased
osteocalcin mRNA and alkaline phosphatase expression in late stage differentiation with
increased mineralized nodules. In addition there was increased relative expression of runx2
and osterix mRNA during osteoblast differentiation. It was also found that Wnt-4 acute
treatment in the early stages of primary cell differentiation was much more effective at
stimulating the relative gene expression of runx2, osterix, osteocalcin, alkaline phosphatase,
and collagen-1a mRNA expression. Together these suggest that Wnt-4 may promote the
differentiation of osteoblasts as well as uncommitted cells in the bone environment as part of
PTH’s anabolic effect.[72]
Figure 13: Effect of WNT-4 on osteoblast proliferation & differentiation

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2.8 Wnt Proteins and Stem Cells
The Wnt Signaling pathway has been found to play a very important role in maintaining the
potency of stem cells and stem cell fate determination. Stem cells from different locations
interpret Wnt in different ways which reflects an activation of distinct genetic programs in
response to the same signal. In addition, the time point during development at which a stem cell
is challenged by Wnt signals determines whether the cell responds by self-renewal or
differentiation. Besides the cell-intrinsic cues that influence the biological activity of Wnt in
distinct stem and progenitor cell types, the same type of stem cell might respond in different
ways to Wnts, depending on its extracellular microenvironment (illustrated in Figure 14 and 15)
[88].
Figure 14. [88] Cell-intrinsic differences among stem cells
Figure 15. [88] The effect of canonical Wnt on a particular
influence the biological function of Wnts. (a) Different stem cell
type of stem cell is context-dependent. (a) In a
types differentially respond to canonical Wnt signaling and
microenvironment X, Wnt activity is modulated by the
undergo either self-renewal or lineage commitment. The
factor(s) X. In this context, Wnt signaling elicits self-
differential responsiveness of stem cells is presumably due to
renewal. (b) However, the very same stem cell in
distinct cell-intrinsic determinants (indicated in the figure by
microenvironment Y, in which Wnts are modulated by the
differently colored cells). (b) A stem cell type of a given cell
factor(s) Y, responds to Wnt signaling by adopting a specific
lineage (indicated in various grades of green) can integrate
cell fate rather than by self-renewing. Thus, the biological
canonical Wnt signaling in different ways, depending on its cell-
activity of Wnt in a particular microenvironment is influenced
intrinsic properties which change over time. At different stages of
by the convergence of Wnt signaling with other signal
development, Wnt promotes stem cell self-renewal or lineage
transduction pathways.
commitment, or the cell loses its ability to respond to Wnt.

25. 25 | P a g e
Some of the findings regarding the role of Wnt signaling in stem cells are as follows:
1. In embryonic stem cells, over expression of Wnt1 or of stabilized β-catenin results in the
inhibition of neural differentiation [77].
2. Treatment of haematopoietic stem cells with Wnt proteins and sustained expression of β-
catenin promotes self-renewal in long-term cultures and increases the reconstitution of
haematopoietic lineages in vivo, which could have been mediated by Notch1 and
the transcription factor HoxB4. However, conditional ablation of β-catenin in
haematopoietic stem cells did not impair haematopoiesis and lymphopoiesis, suggesting
that β-catenin is not required for self-renewal and development of haematopoietic stem
cells under physiological conditions [78].
3. Wnts play major roles during central nervous system (CNS) development. Ablation
of wnt1 results in severe defects of the midbrain, the cerebellum and the
developing spinal cord, while ablation of wnt3A results in a total loss of
the hippocampus. The defects observed in Wnt mutants are possibly explained by
perturbed proliferation of stem or progenitor cells in the ventricular zone [78] [79] [80].
4. Wnt/β-catenin signaling is not only essential for the homeostasis of the intestinal
epithelium; sustained β-catenin activity has also been implicated in the formation
of colon carcinoma [78] [81].
5. In vivo manipulation of genes encoding Wnt signaling components indicates that ß–
catenin deficient stem cells fail to differentiate into follicular keratinocytes and instead
adopt an epidermal fate and thus play a very important role in the fate determination
process of epidermal stem cells [82].

26. 26 | P a g e
6. Wnt signaling has been implicated in the early stages of neural crest development, such
as neural crest induction and melanocyte formation. In neural crest stem cells, the genetic
ablation of β-catenin results in lack of melanocytes and sensory neural cells in dorsal root
ganglia. In fact, NCSCs lacking β-catenin emigrate and proliferate normally but are
unable to acquire a sensory neuronal fate. Constitutive expression of β-catenin in neural
stem/progenitor cells results in the expansion of the entire neural tube, supporting a role
of β-catenin in progenitor proliferation [83].
7. In a recent study from Nusse's laboratory, Axin2 gain-of-function mice were used to
demonstrate that Wnt3A functions as a rate-limiting, self-renewal factor to clonally
expand mammary stem cells (MaSC) [84].
2.9 Role of Wnt Signaling in Mesenchymal Stem Cells:
Mesenchymal stem cells (MSCs), otherwise termed as mesenchymal progenitor cells or marrow
stromal cells are adherent, fibroblast-like population with the potential for extensive self-renewal
and multilineage differentiation. Under appropriate culture conditions, MSCs are capable of
giving rise to osteoblasts, adipocytes, chondrocytes and myoblasts. Their multipotency, ease of
isolation and ready availability make MSCs particularly suited for tissue engineering and gene
therapy applications [76].
MSCs express a number of Wnt ligands such as Wnt-2, Wnt-4, Wnt-5a, Wnt-11 and several Wnt
receptors as well as various co-receptors and Wnt inhibitors [85]. Exogenous application of Wnt-
3a to cell cultures expands the multi-potential population of MSCs and this proliferative is
presumably achieved by the up-regulation of cyclin D1 and c-myc both of which drive cell cycle
progression to promote growth [17] [86].

27. 27 | P a g e
Figure 16: Wnt-4 signaling in the bone as part of PTH’s anabolic effect

28. 28 | P a g e
In previous studies done in Dr. Partridge’s lab by Bergenstock et al., it was found that Wnt-4
increased cell proliferation and CFU-F numbers by inhibiting mouse BMSSC apoptosis and
stimulating their rate of growth. mBMSSCs treated with Wnt-4 in osteogenic and non-
osteogenic cultures showed a significant increase in osteocalcin bone marker gene expression
and a significant reduction of adipocyte marker genes (ap2, pparϒ and C/EBPα). It was found
that Wnt-4 stimulated the Wnt/ß-catenin pathway in proliferating mBMSSCs after Wnt-4
treatment. Wnt-4 did not alter ß-catenin expression but stimulated the phosphorylation of
CamKII (non-canonical Wnt/Ca2+) in differentiating mBMSSCs while inhibiting JNK
phophorylation (non-canonical Wnt/PCP) in proliferating mBMSSCs. This suggests the
divergent effect of Wnt-4 depending on the developmental stage of the cell. Since Wnt-4 is
regulated by PTH in bone, this suggests two functions for Wnt-4 in the stem cell environment
as part of PTH’s anabolic effects. The first being to act on stem cells and still uncommitted
osteoprogenitor cells to expand cell numbers which would eventually be released into
osteogenic differentiation.
The mechanism of signaling switch, that determines whether canonical or non-canonical Wnts
increase stem cell proliferation or stimulate differentiation in certain tissues is not well
understood. Now the question arises how Wnt-4 stimulates the canonical and non-canonical
pathways. One paper suggests that the balance and coordination between
nuclear/transcriptionally active beta-catenin and cytoplasmic/cytoskeletal beta-catenin couples
canonical and non-canonical Wnt signaling. Another suggestion is that the coactivators CBP and
p300, part of the Wnt signaling network of proteins, plays the integrator role [87].

29. 29 | P a g e
CREB binding protein (CBP) and p300 are
key regulators of RNA polymeraseII-mediated
transcription that encode highly related protein
accetlytransferases that bind a variety of
transcriptional regulators and other proteins.
As pointed out earlier, research done in this
lab has pointed out that continuous treatment
of mBMSSCs with Wnt-4 activates Wnt/beta-
catenin pathway in the proliferation stage in a
significant manner and then the Wnt/Ca2+
pathway is activated in differentiation, but
Figure 17 [87]: A. model of coactivator usage. Antagonizing the
CBP/beta-catenin interaction leads to the downregulation of genes
beta-catenin continues to be produced albeit that are critical for stem cell/progenitor cell maintenance and
proliferation (left arm of pathway). This also pushes the cell to
not in a significant manner as in the utilize p300 as its coactivator. The switch to p300/beta-catenin-
mediated transcription is the first critical step to initiate a
proliferation stage. Perhaps the co-activators differentiative program. B. a subset of the gene expression cassette
that is regulated by the CBP/beta-catenin arm is critical for the
CBP and p300 along with nuclear beta-catenin maintenance of potency and proliferation (e.g. Oct4, survivin, etc.).
Other genes that are similarly regulated by CBP/beta-catenin (e.g.
plays a role in making the switch from hNkd and axin2) are involved in the negative feedback of this arm of
the pathway, and initiate differentiation via a switch to the
proliferation to differentiation. p300/beta-catenin arm.
A recent paper has described that that CBP/beta-catenin mediated transcription is critical for
stem cell/ progenitor cell maintenance and proliferation, whereas a switch to p300/beta-
catenin mediated transcription is the initial critical step to initiate differentiation and a decrease
in cellular potency. A subset of the gene expression cassette (e.g. Oct4, surviving, etc.) is critical
for the maintenance of potency and proliferation, other genes such as hNkd and axin2 that are
regulated in this manner are negative regulators that stop proliferation, exit cell cycle and initiate

30. 30 | P a g e
the process of differentiation [87]. The shift from the canonical to the non-canonical pathway
relies on the activation of the PKC pathway. PKC phosphorylation of Ser89 of p300 increases
the affinity of p300 for beta-catenin both in vivo an in vitro and thus the switch occurs and the
differentiation pathway is initiated (shown in Figure 17) [87]. If this is true in the case of Wnt-4
is still to be investigated.
3. Conclusion:
Wnt-4 is significantly regulated by PTH as part of its anabolic effect in the bone environment.
Preliminary studies have revealed that Wnt-4 expression is stimulated in osteoblasts in a PKA
dependent manner and it is a primary response to PTH. It has been found to promote osteoblast
differentiation by enhancing bone marker gene expression by activating the non-canonical
pathways.
In mBMSSCs, Wnt-4 has been found to stimulate proliferation by activating the canonical
pathway and then induce differentiation along the osteoblast lineage by stimulating the non-
canonical pathway.
Next we are going to assess if the Wnt-4 can induce proliferation and differentiation along the
osteoblast lineage in human BMSSCs and human dental pulp stem cells (hDPSCs).

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