Cytotoxic T lymphocyte-associated antigen 4-Ig (CTLA4-Ig)-modified bone marrow-derived mesenchymal stem cells (MSCs-CTLA4) have excellent osteogenic function in xenografts, but their mechanism of action remains to be elucidated. As bidirectional signaling between erythropoietin-producing hepatocyte receptors B4 (EphB4) and ephrinB2 is vital for bone remodeling, this study aimed to fully characterize the role of MSCs-CTLA4 in promoting bone regeneration in xenotransplantation through EphB4/ephrinB2 and their cross talk with the Wnt/beta-catenin pathway.
Methods:
MSCs-CTLA4 were investigated for their osteogenic capacity through xenotransplantation in vivo. MSCs-CTLA4 were treated with ephrinB2-FC or FC under conditions of osteogenic induction and cultured with or without immune activation conditions established by phytohemagglutinin and peripheral blood mononuclear cells in vitro. Osteogenesis markers and the Wnt pathway-related molecules such as EphB4, runt-related transcription factor 2 (Runx2), collagen 1 (COL1), osteocalcin (OCN), alkaline phosphatase (ALP), calcium nodus, β-catenin, phospho-glycogen synthase kinase 3-beta (p-GSK-3β)-Ser9, and glycogen synthase kinase 3-beta (GSK-3β) were detected.
Results:
MSCs-CTLA4-based xenografts show better osteogenic capacity compared with MSC-based xenografts. EphB4 expression was reduced in MSCs compared with MSCs-CTLA4 under immune activation conditions. In ephrinB2-FC-treated cells, levels of osteogenesis markers were increased compared with FC-treated cells. The activity of GSK-3 was inhibited and the expression of β-catenin in MSCs was increased by ephrinB2-FC treatment.
Conclusions:
CTLA4 modification maintains EphB4 expression in MSCs under immune activation conditions, and EphB4 cross talk with the Wnt pathway promotes osteogenic differentiation of MSCs-CTLA4.
Get full access to this article
View all access options for this article.
References
1.
YamadaY., NakamuraS., ItoK., UmemuraE., HaraK., NagasakaT., AbeA., BabaS., FuruichiY., IzumiY., KleinO.D., and WakabayashiT.Injectable bone tissue engineering using expanded mesenchymal stem cells. Stem Cells, 31, 572, 2013.
2.
Ben-AriA., RivkinR., FrishmanM., GabermanE., LevdanskyL., and GorodetskyR.Isolation and implantation of bone marrow-derived mesenchymal stem cells with fibrin micro beads to repair a critical-size bone defect in mice. Tissue Eng Part A, 15, 2537, 2009.
3.
da Silva MeirellesL., ChagastellesP.C., and NardiN.B.Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci, 119, 2204, 2006.
4.
ZomorodianE., and EslaminejadM.B.Mesenchymal stem cells as a potent cell source for bone regeneration. Stem Cells Int, 2012, 980353, 2012.
5.
AnkrumJ.A., OngJ.F., and KarpJ.M.Mesenchymal stem cells: immune evasive, not immune privileged. Nat Biotechnol, 32, 252, 2014.
6.
TianH.T., ZhangB., TianQ., LiuY., YangS.H., and ShaoZ.W.Construction of self-assembled cartilage tissue from bone marrow mesenchymal stem cells induced by hypoxia combined with GDF-5. J Huazhong Univ Sci Technol Med Sci, 33, 700, 2013.
7.
YoshikawaT., NakajimaH., UemuraT., KasaiT., EnomotoY., TamuraT., NonomuraA., and TakakuraY.In vitro bone formation induced by immunosuppressive agent tacrolimus hydrate (FK506). Tissue Eng, 11, 609, 2005.
8.
PonceletA.J., VercruysseJ., SaliezA., and GianelloP.Although pig allogeneic mesenchymal stem cells are not immunogenic in vitro, intracardiac injection elicits an immune response in vivo. Transplantation, 83, 783, 2007.
EngelhardtJ.J., SullivanT.J., and AllisonJ.P.CTLA-4 overexpression inhibits T cell responses through a CD28-B7-dependent mechanism. J Immunol, 177, 1052, 2006.
11.
QureshiO.S., ZhengY., NakamuraK., AttridgeK., ManzottiC., SchmidtE.M., BakerJ., JefferyL.E., KaurS., BriggsZ., HouT.Z., FutterC.E., AndersonG., WalkerL.S.K., and SansomD.M.Trans-endocytosis of CD80 and CD86: a molecular basis for the cell-extrinsic function of CTLA-4. Science, 332, 600, 2011.
12.
DaiF., ShiD., HeW., WuJ., LuoG., YiS., XuJ., and ChenX.hCTLA4-gene modified human bone marrow-derived mesenchymal stem cells as allogeneic seed cells in bone tissue engineering. Tissue Eng, 12, 2583, 2006.
13.
ArthurA., ZannettinoA., PanagopoulosR., KoblarS.A., SimsN.A., StylianouC., MatsuoK., and GronthosS.EphB/ephrin-B interactions mediate human MSC attachment, migration and osteochondral differentiation. Bone, 48, 533, 2011.
14.
MartinT.J., AllanE.H., HoP.W.M., GooiJ.H., QuinnJ.M.W., GillespieM.T., KrasnoperovV., and SimsN.A.Communication between ephrinB2 and EphB4 within the osteoblast lineage. Adv Exp Med Biol, 658, 51, 2009.
15.
ZhaoC., IrieN., TakadaY., ShimodaK., MiyamotoT., NishiwakiT., SudaT., and MatsuoK.Bidirectional ephrinB2-EphB4 signaling controls bone homeostasis. Cell Metab, 4, 111, 2006.
16.
MatsuoK., and IrieN.Osteoclast–osteoblast communication. Arch Biochem Biophys, 473, 201, 2008.
17.
JaiswalN., HaynesworthS.E., CaplanA.I., and BruderS.P.Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem, 64, 295, 1997.
18.
PennisiA., LingW., LiX., KhanS., ShaughnessyJ.D., BarlogieB., and YaccobyS.The ephrinB2/EphB4 axis is dysregulated in osteoprogenitors from myeloma patients and its activation affects myeloma bone disease and tumor growth. Blood, 114, 1803, 2009.
19.
SponerP., KuceraT., Diaz-GarciaD., and FilipS.The role of mesenchymal stem cells in bone repair and regeneration. Eur J Orthop Surg Traumatol, 24, 257, 2014.
20.
HoffH., KolarP., AmbachA., RadbruchA., and Brunner-WeinzierlM.C.CTLA-4 (CD152) inhibits T cell function by activating the ubiquitin ligase Itch. Mol Immunol, 47, 1875, 2010.
21.
TakeharaM., MurakamiM., InobeM., TanakaK., ChikumaS., SaitoI., KanegaeY., YasunamiY., NakanoM., YamashitaK., TodoS., and UedeT.Long-term acceptance of allografts by in vivo gene transfer of regulatable adenovirus vector containing CTLA4IgG and loxP. Hum Gene Ther, 12, 415, 2001.
22.
MartinC., PlatM., TerquiM., CondeF., MelchiorB., CoulonF., Nerriere-DaguinV., NeveuI., NaveilhanP., SoulillouJ.P., VanhoveB., and BrachetP.Transgeneic pigs expressing CTLA4-Ig in neurons and xenotransplantation for brain repair. Xenotransplantation, 10, 492, 2003.
23.
BasheyA., MedinaB., CorringhamS., PasekM., CarrierE., VroomanL., StreicherH., LowyI., SolomonS.W., MorrisL.E., HollandK., MasonJ.R., SoifferR.J., and BallE.D.Clinical trial of therapeutic blockade of CTLA4 with ipilimumab in patients with relapse of malignancy following allogeneic hematopoietic transplantation. Blood, 110, 491a, 2007.
24.
KanayaK., TsuchidaY., InobeM., MurakamiM., HiroseT., KonS., KawaguchiS., WadaT., YamashitaT., IshiiS., and UedeT.Combined gene therapy with adenovirus vectors containing CTLA4Ig and CD40Ig prolongs survival of composite tissue allografts in rat model. Transplantation, 75, 275, 2003.
25.
LenschowD.J., ZengY., ThistlethwaiteJ.R., MontagA., BradyW., GibsonM.G., LinsleyP.S., and BluestoneJ.A.Long-term survival of xenogeneic pancreatic islet grafts induced by CTLA4lg. Science, 257, 789, 1992.
26.
PasqualeE.B.EPH receptor signalling casts a wide net on cell behaviour. Nat Rev Mol Cell Biol, 6, 462, 2005.
27.
ZhangM., YanY., LimY.B., TangD.Z., XieR., ChenA., TaiP., HarrisS.E., XingL.P., QinY.X., and ChenD.BMP-2 modulates beta-catenin signaling through stimulation of Lrp5 expression and inhibition of beta-TrCP expression in osteoblasts. J Cell Biochem, 108, 896, 2009.
28.
ZhangJ.H., ChenY.Q., and TangT.T.Hyaluronic acid mixed with BMP-2 gene transfected bone marrow derived mesenchymal stem cells for repair of diaphyseal defects in rabbits. Tissue Eng, 12, 1080, 2006.
29.
SumanasingheR.D., BernackiS.H., and LoboaE.G.Osteogenic differentiation of human mesenchymal stem cells in collagen matrices: effect of uniaxial cyclic tensile strain on bone morphogenetic protein (BMP-2) mRNA expression. Tissue Eng, 12, 3459, 2006.
30.
HuJ.J., LiuY.W., HeM.Y., JinD., ZhaoH., and YuB.Proteomic analysis on effectors involved in BMP-2-induced osteogenic differentiation of beagle bone marrow mesenchymal stem cells. Proteome Sci, 12, 13, 2014.
31.
KatoM., PatelM.S., LevasseurR., LobovI., ChangB.H., GlassD.A.2nd, HartmannC., LiL., HwangT.H., BraytonC.F., LangR.A., KarsentyG., and ChanL.Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor. J Cell Biol, 157, 303, 2002.
32.
BennettC.N., OuyangH., MaY.L., ZengQ., GerinI., SousaK.M., LaneT.F., KrishnanV., HankensonK.D., and MacDougaldO.A.Wnt10b increases postnatal bone formation by enhancing osteoblast differentiation. J Bone Miner Res, 22, 1924, 2007.
33.
MicleaR.L., KarperienM., BoschC.A., van der HorstG., van der ValkM.A., KobayashiT., KronenbergH.M., RawadiG., AkcakayaP., LowikC.W., FoddeR., WitJ.M., and Robanus-MaandagE.C.Adenomatous polyposis coli-mediated control of beta-catenin is essential for both chondrogenic and osteogenic differentiation of skeletal precursors. BMC Dev Biol, 9, 26, 2009.
34.
DayT.F., GuoX., Garrett-BealL., and YangY.Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev Cell, 8, 739, 2005.
35.
HillT.P., SpaterD., TaketoM.M., BirchmeierW., and HartmannC.Canonical Wnt/beta-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell, 8, 727, 2005.
36.
KennellJ.A., and MacDougaldO.A.Wnt signaling inhibits adipogenesis through beta-catenin-dependent and -independent mechanisms. J Biol Chem, 280, 24004, 2005.
37.
HarwoodA.J.Regulation of GSK-3: a cellular multiprocessor. Cell, 105, 821, 2001.
38.
BaronR., and KneisselM.WNT signaling in bone homeostasis and disease: from human mutations to treatments. Nat Med, 19, 179, 2013.
39.
BaronR., and RawadiG.Targeting the Wnt/beta-catenin pathway to regulate bone formation in the adult skeleton. Endocrinology, 148, 2635, 2007.
