A Review on the role of Wnt-4 in Stem Cell Biology
Wnt -4 Stimulationof Human BoneMarrow StromalStem Cells andMesenchymalStem Cells of theMaxillaGayathri VijayakumarPoly ID# 0408513Guided Studies- Fall 2010Nicola C. Partridge, Ph.D.Professor, ChairBasic Science & CraniofacialBiologyE-mail: firstname.lastname@example.orgMichael D Turner, D.D.S., M.D.Assistant ProfessorOral and Maxillofacial SurgeryE-mail: email@example.com
2|PageTABLE OF CONTENTSINTTRODUCTION ....................................................................................................................... 3OVERVIEW ................................................................................................................................... 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 ....................................................................... 26CONCLUSION ............................................................................................................................ 30REFERENCE ............................................................................................................................... 31
3|Page 1. INTRODUCTIONWnt proteins are palmitoylated and glycosylated ligands that play a well established role inembryonic patterning, cell proliferation and cell determination , besides playing asignificant 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 multitudeof diseases, most notably cancer . Wnt proteins can be categorized into two types: thecanonical and non-canonical.Wnts have important roles in regulating many aspects of skeletal development,from limb formation to chondrogenesis and osteoblast maturation . In addition topromoting osteoblast maturation, Wnts may play a role in lineage commitment of mesenchymalprecursor cells by preventing adipogenesis is the hypothesized default pathway for mesenchymalstem cells that do not receive proper inductive signals tobecome osteoblasts, chondrocytes, myocytes, or other mesodermal cells .Parathyroid hormone (PTH) regulates calcium homeostasis in addition to stimulating boneformation . Human and experimental animal studies have shown that PTHincreases osteoblast activity and number via increased differentiation and survival, increasesbone mass and bone strength, and decreases fracture rate . Due to its anabolic actions, PTH iscurrently the only clinically available anabolic agent used to effectively treat osteoporosis. .In vivo microarray analysis has shown that PTH regulates molecules of the Wnt signalingpathway in bone . PTH has been demonstrated to stimulate bone lining osteoblasts toproduce non-canonical Wnt-4 in vivo, and exogenous Wnt-4 has been shown to regulateosteoblast differentiation through non-canonical Wnt signaling pathway .
4|PageWnt signaling has been implicated in the control over various types of stem cells and may act asa niche factor to maintain stem cells in a self-renewing state . Recent studies have shown thatWnt-3a stimulates proliferation while inhibiting osteogenic differentiation of hMSCs , whilenon-canonical Wnt-5a enhanced osteogenic differentiation but had no effect on hMSCproliferation . Most significantly, Wnt-4 has been shown to stimulate osteogenesis in hMSCsisolated from craniofacial tissue through a novel p38 non-canonical Wnt signaling pathway thatis a known pathway associated with osteogenic differentiation .Preliminary findings from Dr. Nicola C. Partridge’s lab at the Department of Basic Science andCraniofacial Biology at NYU-CD have shown that Wnt-4 is stimulated by PTH in vivo , andthat 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 inproliferation 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. Overview2.1 Wnt ProtiensTogether with the other families of secreted factors such as FGF, TGF-beta, and Hedgehogproteins,Wnt proteins are implicated in a wide variety of biological processes. The first Wnt gene
5|Pagemouse Wnt-1, was discovered in 1982 as a proto-oncogene activated by integration of mousemammary tumor virus in mammary tumors . With the molecular identiﬁcation of theDrosophila segment polarity gene wingless (wg) as the orthologue of Wnt-1  and thephenotypic analysis of Wnt-1 mutations in the mouse , it became clear that Wnt genes areimportant regulators of many developmental decisions .Table 1  Phenotypes of Wnt mutations in mouse, Drosophila, and C. elegansGene Organism Phenotype Organism PhenotypeWnt-1 Mouse Loss of midbrain and cerebellumWnt-2 Mouse Placental defectsWnt-3A Mouse Lack of caudal somites and tailbudWnt-4 Mouse Kidney defectsWnt-7A Mouse Ventralization of limbsWingless Drosophila Segment polarity, limb development, many othersDwnt-2 Drosophila Muscle defects, testis developmentlin-44 C.elegans Defects in asymmetric cell divisionsmom-2 C. elegans Defects in endoderm induction and spindle orientationThe Wnts comprise a large family of protein ligands that affect diverse processes such asembryonic induction, generation of cell polarity and the specification of cell fate. In addition toinﬂuencing developmental processes, recent studies point to a key role for Wnt signaling duringadult homeostasis in the maintenance of stem cell pluripotency . Wnts are defined by aminoacid sequence rather than by functional properties [28-29]. As many as 19 mammalian Wnthomologues are known and are expressed in temporal spatial patterns. Shared features of allWnts include a signal sequence for secretion, several highly charged amino acid residues, and
6|Pagemany glycosylation sites. Wnt proteins also display a characteristic distribution of 22 cysteineresidues. overexpression in tissue culture cells, several different N-linked glycosylatedintermediate Wnt protein products are observed in cell lysates [30-32], suggesting that Wntprotein 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 andextracellular matrix (ECM), the reason being that Wnt proteins are lipid modified by theattachment of a palmitate on the first conserved cysteine residue within the protein family and ona 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 Wntgene products (proteins with the same function in different species) are often very highlyconserved.  Based on their ability to induce secondary body axis in Xenopus embryos, theWnt 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 secondarybody axis .
7|Page2.2 Wnt Secretion and Extracellular TransportTwo recent genetic screens have identiﬁed the multipass transmembrane protein Wntless(Wls)/Evenness interrupted (Evi)/Mom-3 as acting in the secretory pathway to promote therelease of Wnts from producing cells . A model of Wnt secretion and realease is shown infigure 1.Exogenously derived lipoproteins termed ―argosomes‖ are implicated in moving Wntsand other lipid-modified proteins such as Hedgehogs . A model is proposed whereinpalmitoylated proteins associate with lipoprotein particles on the extracellular face of cells.Traffic of Wnt proteins from one cell to the next requires this association . In addition,transcytosis may regulate Wnt movement. It has been proposed that the retromer complexpromotes the association of secreted Wnts with other proteins required for ligand transport, suchas lipoprotein particles. Figure 1 : 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).
8|PageThe hydrophobic Wnt protein either remains associated with the plasma membrane or binds directly to lipoproteinparticles or HSPGs to facilitate spreading and gradient formation (2). After the release of Wnt, Wls is internalizedthrough AP-2/clathrin-mediated endocytosis (3) and is transported back to the TGN through a retromer-dependenttrafficking step (4). An alternative possibility is that Wnt is not released at the plasma membrane, but isreinternalized together with Wls (5). Dissociation of Wnt from Wls may take place in endosomes, after which Wntis released (possibly in association with lipoprotein particles) through a recycling endosomal pathway (6). Also inthis scenario, Wls is recycled back to the TGN through a retromer-dependent transport step (7). Wnt may also bereinternalized through a Reggie-1/Flotillin-2 dependent pathway (8), which may lead to the release of a moremobile, micelle-like form of Wnt or to the association of Wnt with lipoprotein particles (6).2.3 Wnt ReceptionThe seven-pass transmembrane proteinFrizzled (Fz) protein was ﬁrst receptor foundto transduce a Wnt signal . . Fz proteinscontain a large extracellular domaincontaining a conserved motif comprised of10 cysteine residues called the cysteine-richdomain (CRD) that has been shown to bind Figure 2 : Structure of Frizzleds and FRPs. Frizzledto 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 acytoplasmic 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 withwith 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-passtransmembrane proteins of the low-density lipoprotein (LDL) family called Lrp5 and -6 totransduce the canonical Wnt signal. Lrp5 and -6 proteins have a relatively small intracellulardomain and a large extracellular domain containing several potential protein interactiondomains .
9|PageThere are other proteins with known Wnt-bindingdomains that can serve as receptors for Wnt ligands.The single-pass tyrosine kinase Ror2, althoughstructurally distinct from Fz receptors, is involvedin other forms of Wnt signaling. Another well-characterized Wnt-binding domain is the Wntinhibitory factor (WIF) module, which is also foundin the cell surface atypical receptor tyrosine kinase Figure 3: Different receptors that Wnt proteins can bind to.Ryk . http://www.stanford.edu/group/nusselab/cgi- bin/wnt/receptors2.4 Wnt Signal Transduction PathwaysWnt proteins signal through the canonical and the non-canonical pathways which are composedof three independent signal transduction pathways (the Wnt/β-catenin pathway, theWnt/Ca+2 pathways, or the Wnt/planar polarity pathway) that are used to regulate the expressionof different genes. The Wnt/β-catenin pathway is commonly referred to as the canonicalpathway. It promotes cell fate determination, proliferation, and survival by increasing β-catenin levels and altering gene expression through Lef/Tcf transcription factors . The non-canonical Wnt/Ca+2 pathway stimulates heterotrimeric G proteins, increases intracellularcalcium levels, decreases cyclic GMP levels, and activates protein kinase C to induce NF-AT and other transcription factors . The non-canonical Wnt/planar polarity pathway activatesRho/Rac GTPases and Jun N-terminal kinase to modulate cytoskeletal organization and geneexpression. Distinct Wnt ligands probably act through specific Frizzled (Fzd) receptors to initiateeach .
10 | P a g e Figure 4: Wnt Signaling Pathways and their ImplicationsThe canonical Wnt signaling pathway (see Figure 5) is activated when Wnts interact withLrp/Fzd receptor complexes as shown in the middle portion of figure 4. Receptor engagementactivates an unknown kinase(s) (K) that phosphorylates the cytoplasmic tail of Lrp5/6. Thesephosphorylated residues (P) serve as docking sites for Axin and the APC, Dsh, β-catenincomplex. A GSK3β binding protein (GBP) is also mobilized after receptor ligation and excludesGSK3β from the proximal receptor complex. β-Catenin thereby escapes phosphorylation eventsthat 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
11 | P a g eactin cytoskeleton (lower left of this figure), some β-catenin molecules travel to the nucleuswhere they interact with Lef1/Tcf transcription factors to either increase or decrease theexpression of specific target genes. β-Catenin displaces nuclear co-repressors (CoR) fromLef1/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 withother transcription factors (TF). Soluble antagonists block the canonical Wnt signaling pathwayand promote β-catenin degradation via two mechanisms. Figure 5 : The Canonical Wnt Signaling PathwaySoluble frizzled related proteins (Sfrp) bind free Wnt molecules and compete with surfacereceptors (top right of figure 5). In contrast, Dkks interact with extracellular domains in Lrp5/6
12 | P a g eand recruit them to complexes containing Krm (top left of figure 5). This trimolecular complex isinternalized to lysosomes where Lrp5/6 are either degraded or recycled to the surface. Dkktherefore decreases Lrp5/6 cell surface expression to regulate Wnt signaling . Figure 6 : Nuclear activity of ß-cateninAccumulated ß-catenin then translocates to the nucleus, replaces Groucho from TCF, andactivates target genes. ß-catenin forms a complex with TCF and the transcription factors Brg1and CBP. Lgs and Pygo also bind to -catenin, possibly driving its nuclear localization in additionto playing a direct role in transcriptional activation. Negative regulation of signaling is providedby NLK (Nemo-like kinase) which phosphorylates TCF, and ICAT (inhibitor of catenin) andChibby, which are antagonists of ß-catenin. In addition to TCF, two other DNA-binding proteinshave been shown to associate with ß-catenin: Pitx2 and Prop1. In the case of Prop1, ß-catenincan act as a transcriptional activator or repressor of specific genes, depending on the co-factors
13 | P a g epresent. The participation of any particular ß-catenin complex in transcriptional regulation ishighly cell type-dependent.The non-canonical pathway can be broadly classified into 2 branches based upon phenotypicresponse; the Planar Cell Polarity (PCP) pathway and the Wnt/ Ca2+ pathway . Howeversome authors have classified the pathways as Wnt/calcium signaling, Wnt/PCP signaling,Wnt/JNK signaling and Wnt/Rho signaling . The PCP pathway is involved in cellularasymmetry, and it is this cellular asymmetry that controls the rigid architectural orientation ofepithelial tissues and sensory organs (e.g. inner ear cochlea), as well as the morphology and themigratory processes of mesodermal cells undergoing gastrulation. Activation of PCP signalingoccurs 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 theactivation of the GTPases Rho and Rac. Activated Rac subsequently stimulates JNK activation.The second branch of non-canonical signaling – the Wnt/Ca2+ pathway – is characterized byWnt-Fzd-induced PLC (phospholipase C) activation and the resultant increase of cytoplasmicCa2+ levels. These Ca2+ fluxes activate several Ca2+-responsive proteins, such as PKC (proteinkinase C) and CaMKII(calcium/calmodulin-dependent kinase II). CaMKII has been shown toactivate the transcription factor NFAT, TAK1 (TGF-beta activated kinase), and NLK (Nemo-likekinase, 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 isinvolved in regulating cellular adhesion, cytoskeletal rearrangements, and other developmentalprocesses, such as dorsoventral patterning and tissue separation in embryos .
14 | P a g e Figure 7 : Non- canonical pathways2.5 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).
15 | P a g e 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 . 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 . 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 . 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 .2.6 Wnt Signaling in Bone formation:The Wnt signal transduction pathway has been implicated in bone formation: patients sufferingfrom osteoporosis–pseudoglioma syndrome have an inactivating mutation in the Wnt co-receptor LRP5 , whereas an activating LRP5 mutation is associated with high bone masssyndrome  . Analysis of LRP5-deficient mice revealed a decreased numberof osteoblasts suggesting that Wnt signaling stimulates bone formation at the level ofosteoprogenitor proliferation .
16 | P a g eFigure 8 : Role of canonical Wnt signaling in skeletal development. Mesenchymal stem cells have the abilityto differentiate into chondrocytes or osteoblasts, depending on the environmental cues. Canonical Wnt signalingis regulated to control the lineage progression between chondrocyte and osteoblast. If there is inadequatecanonical Wnt signaling, differentiation towards chondrocyte lineage is encouraged. However, the maturation ofchondrocytes requires the presence of canonical Wnt signaling. Canonical Wnt signaling is also required for theprogression of osteoblast progenitor cells toward osterix positive osteoblasts and then osteocalcin-positiveosteoblasts. 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 minussigns indicate inhibitory effects of physiological canonical Wnt signalingThe effects of Wnt signaling on chondrogenesis are complex and have been implicated in theregulation of chondrogenic differentiation and hypertrophy . Chondrocyte-specificinactivationof β-catenin using Col2a1-Cre transgene leads to decreased chondrocyte proliferationand delayed hypertrophic chondrocyte differentiation . In another loss-of-functionmodel, Dermo 1-Cre transgene was used to delete the β-catenin gene in the mesenchymalprecursors of both chondrocytes and osteoblasts. It was shown that there is a significant delayin chondrocyte maturation in conditional knockout embryos . At the same time Activationof β-cateninin limb and head mesenchyme repressed the expression of Sox9, a factor essentialfor chondrogenesis, thereby preventing mesenchymal cells from differentiating into skeletalprecursors . In the absence of β-catenin, the expression of early osteoblast markers, suchas collagen I, osterix, and osteocalcin was greatly diminished .
17 | P a g e Figure 9 : 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 toabnormal development of the kidney, pituitary gland, female reproductive system, and mammarygland. Wnt4 is conserved throughout vertebrates and was originally studied in non-mammalianvertebrates such as zebrafish, chicken and xenopus . Several studies have tried to gauge thevaried functions of this protein, giving the impression that this is indeed a highly conservedprotein essential for proper organ formation and development. Some of the findings are asfollows: 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
18 | P a g e role of Wnt-4 signaling in controlling mesenchyme to epithelium transformation and kidney tubule induction is well established . 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 . 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 . 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 . 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 . 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
19 | P a g e also in malignant tumor tissue, implies that changes in WNT4 signaling are associated with abnormal cell proliferation in human breast cells . 7. Screening of the multiple adult human tissues Figure 10 . 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 . 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 . In mouse spleen, Wnt1and Wnt-4 signaling regulates differentiation of the thymocytes, the number of which is decreased in compound mutants .
20 | P a g e Figure 11 . 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 ofcalcium homeostasis in vertebrates. However, although continuous infusion of PTH inducesbone loss, intermittent administration of PTH results in bone formation . Bindingof PTH and PTHrP to PTH1R activates two signaling pathways in osteoblasts: the PKA pathwaywhich is responsible for the majority of the calciotropic and skeletal actions of PTH, the PKCpathway leading to accumulation of 1, 4, 5-inositol triphosphate and increased intracellularcalcium. This pathway has been found to regulate IGF-binding protein-5 .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 wasmeasured in whole cell lysates by Western blot was seen .
21 | P a g e Figure 12: PTH regulation of Wnt-4Furthermore, PTH increased the level of β-catenin expression in mouse osteoblastic cells(MC3T3-E1) via both PKA and PKC signaling pathways . More recently, PTH was shown toactivate β-catenin signaling in osteoblasts in vitro and in vivo by direct recruitment of LRP6 toPTH/PTH1R complex. In vivo studies confirmed that intermittent PTH treatment led to anincrease in amount of β catenin in osteoblasts (immunohistochemical analysis with antibodyto β-catenin) with a concurrent increase in bone formation in rat .Partridge et al. also demonstrated a link between PTH and Wnt4 expression in bone . Invivo microarray analysis of intermittent and continuous PTH 1–34 showed
22 | P a g ethat PTH regulated Wnt4 in bone. PTH was shown to stimulate Wnt4 primarily throughthe PKA pathway and Wnt4 treatment in osteoblasts induced early expression of bone markergenes and stimulated key canonical Wnt pathway genes. This finding suggested cross-talkbetween the Wnt signaling cascades .Previous studies at Dr. Partridge’s lab at the Department of Basic Science & CraniofacialBiology at NYU-CD has showed that Wnt-4 is significantly regulated by PTH in vivo afteranabolic and catabolic protocols in bone lining osteoblasts. As pointed out earlier Partridge etal. demonstrated that PTH stimulated Wnt-4 expression in cultured osteoblastic cells and thatthis stimulation is PKA dependent and is a primary response to PTH. It has also been shownthat exogenous Wnt-4 enhances bone marker gene expression during osteoblastdifferentiation by activating the non-canonical Wnt/Ca2+ and Wnt/PCP signaling pathwaysbut 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 tissuethrough a novel p38 non-canonical Wnt signaling pathway that is a known pathwayassociated with osteogenic differentiation . Therefore it can be hypothesized that non-canonical Wnt signaling plays a significant role in bone and that Wnt-4 may be an importantmolecule in PTH’s anabolic effect.Real time TR-PCR results have shown that PTH stimulates Wnt-4 mRNA expression in allphases of osteoblastic differentiation but is greatest in the mineralization phase and maximalstimulation 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 bonemarker genes at proliferation day 7, but enhances bone marker gene expression (runx2,osterix, osteocalcin, alkaline phosphatase, MMP-13) significantly in the absence of
23 | P a g edifferentiation-promoting factors such as ascorbate. Continuous treatment of rat primaryosteobalsts with Wnt-4 in an osteogenic environment showed that there was increasedosteocalcin mRNA and alkaline phosphatase expression in late stage differentiation withincreased mineralized nodules. In addition there was increased relative expression of runx2and osterix mRNA during osteoblast differentiation. It was also found that Wnt-4 acutetreatment in the early stages of primary cell differentiation was much more effective atstimulating the relative gene expression of runx2, osterix, osteocalcin, alkaline phosphatase,and collagen-1a mRNA expression. Together these suggest that Wnt-4 may promote thedifferentiation of osteoblasts as well as uncommitted cells in the bone environment as part ofPTH’s anabolic effect. Figure 13: Effect of WNT-4 on osteoblast proliferation & differentiation
24 | P a g e 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) .Figure 14.  Cell-intrinsic differences among stem cells Figure 15.  The effect of canonical Wnt on a particularinfluence the biological function of Wnts. (a) Different stem cell type of stem cell is context-dependent. (a) In atypes differentially respond to canonical Wnt signaling and microenvironment X, Wnt activity is modulated by theundergo 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 indistinct cell-intrinsic determinants (indicated in the figure by microenvironment Y, in which Wnts are modulated by thedifferently colored cells). (b) A stem cell type of a given cell factor(s) Y, responds to Wnt signaling by adopting a specificlineage (indicated in various grades of green) can integrate cell fate rather than by self-renewing. Thus, the biologicalcanonical Wnt signaling in different ways, depending on its cell- activity of Wnt in a particular microenvironment is influencedintrinsic properties which change over time. At different stages of by the convergence of Wnt signaling with other signaldevelopment, Wnt promotes stem cell self-renewal or lineage transduction pathways.commitment, or the cell loses its ability to respond to Wnt.
25 | P a g eSome 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 . 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 . 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   . 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  . 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 .
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 . 7. In a recent study from Nusses 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) .2.9 Role of Wnt Signaling in Mesenchymal Stem Cells:Mesenchymal stem cells (MSCs), otherwise termed as mesenchymal progenitor cells or marrowstromal cells are adherent, fibroblast-like population with the potential for extensive self-renewaland multilineage differentiation. Under appropriate culture conditions, MSCs are capable ofgiving rise to osteoblasts, adipocytes, chondrocytes and myoblasts. Their multipotency, ease ofisolation and ready availability make MSCs particularly suited for tissue engineering and genetherapy applications .MSCs express a number of Wnt ligands such as Wnt-2, Wnt-4, Wnt-5a, Wnt-11 and several Wntreceptors as well as various co-receptors and Wnt inhibitors . Exogenous application of Wnt-3a to cell cultures expands the multi-potential population of MSCs and this proliferative ispresumably achieved by the up-regulation of cyclin D1 and c-myc both of which drive cell cycleprogression to promote growth  .
27 | P a g e Figure 16: Wnt-4 signaling in the bone as part of PTH’s anabolic effect
28 | P a g eIn previous studies done in Dr. Partridge’s lab by Bergenstock et al., it was found that Wnt-4increased cell proliferation and CFU-F numbers by inhibiting mouse BMSSC apoptosis andstimulating their rate of growth. mBMSSCs treated with Wnt-4 in osteogenic and non-osteogenic cultures showed a significant increase in osteocalcin bone marker gene expressionand a significant reduction of adipocyte marker genes (ap2, pparϒ and C/EBPα). It was foundthat Wnt-4 stimulated the Wnt/ß-catenin pathway in proliferating mBMSSCs after Wnt-4treatment. Wnt-4 did not alter ß-catenin expression but stimulated the phosphorylation ofCamKII (non-canonical Wnt/Ca2+) in differentiating mBMSSCs while inhibiting JNKphophorylation (non-canonical Wnt/PCP) in proliferating mBMSSCs. This suggests thedivergent effect of Wnt-4 depending on the developmental stage of the cell. Since Wnt-4 isregulated by PTH in bone, this suggests two functions for Wnt-4 in the stem cell environmentas part of PTH’s anabolic effects. The first being to act on stem cells and still uncommittedosteoprogenitor cells to expand cell numbers which would eventually be released intoosteogenic differentiation.The mechanism of signaling switch, that determines whether canonical or non-canonical Wntsincrease stem cell proliferation or stimulate differentiation in certain tissues is not wellunderstood. Now the question arises how Wnt-4 stimulates the canonical and non-canonicalpathways. One paper suggests that the balance and coordination betweennuclear/transcriptionally active beta-catenin and cytoplasmic/cytoskeletal beta-catenin couplescanonical and non-canonical Wnt signaling. Another suggestion is that the coactivators CBP andp300, part of the Wnt signaling network of proteins, plays the integrator role .
29 | P a g eCREB binding protein (CBP) and p300 arekey regulators of RNA polymeraseII-mediatedtranscription that encode highly related proteinaccetlytransferases that bind a variety oftranscriptional regulators and other proteins.As pointed out earlier, research done in thislab has pointed out that continuous treatmentof mBMSSCs with Wnt-4 activates Wnt/beta-catenin pathway in the proliferation stage in asignificant manner and then the Wnt/Ca2+pathway is activated in differentiation, but Figure 17 : A. model of coactivator usage. Antagonizing the CBP/beta-catenin interaction leads to the downregulation of genesbeta-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 tonot 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 aproliferation 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 theCBP 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 theproliferation to differentiation. p300/beta-catenin arm.A recent paper has described that that CBP/beta-catenin mediated transcription is critical forstem 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 decreasein cellular potency. A subset of the gene expression cassette (e.g. Oct4, surviving, etc.) is criticalfor the maintenance of potency and proliferation, other genes such as hNkd and axin2 that areregulated in this manner are negative regulators that stop proliferation, exit cell cycle and initiate
30 | P a g ethe process of differentiation . The shift from the canonical to the non-canonical pathwayrelies on the activation of the PKC pathway. PKC phosphorylation of Ser89 of p300 increasesthe affinity of p300 for beta-catenin both in vivo an in vitro and thus the switch occurs and thedifferentiation pathway is initiated (shown in Figure 17) . If this is true in the case of Wnt-4is 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 PKAdependent manner and it is a primary response to PTH. It has been found to promote osteoblastdifferentiation by enhancing bone marker gene expression by activating the non-canonicalpathways.In mBMSSCs, Wnt-4 has been found to stimulate proliferation by activating the canonicalpathway 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 theosteoblast lineage in human BMSSCs and human dental pulp stem cells (hDPSCs).
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