Human amniotic fluid mesenchymal stem cells (hAFMSCs) are promising for therapeutic applications in bone damage. Calcium sensing receptor (CaSR), a G protein-coupled receptor, plays a physiological role in the regulation of bone metabolism. Thus, the bone CaSR could be targeted by calcimimetic agonists, which may be potentially helpful in treating bone diseases. The aim of our study was to characterize CaSR expression in hAFMSCs and to assess the activity of calcimimetic R-568 during in vitro osteogenesis. Using western blotting, immunofluorescence, and flow cytometry, we consistently observed constitutive CaSR in osteo-differentiating hAFMSCs. Notably, both R-568 and calcium significantly enhanced hAFMSC osteogenic differentiation after exposure to osteogenic medium. To provide further evidence of the involvement of CaSR in osteogenesis, we correlated its expression with that of established osteogenic markers, that is, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteopontin (OPN), and novel, not yet completely defined regulators of osteogenesis. Among these are β-catenin and Slug, which are mediators of Wnt signaling, and nuclear factor of activated T cells c1 (NFATc1), which plays a critical role in calcium/calcineurin signaling. Taken together, our results demonstrate that CaSR is expressed in hAFMSCs, positively correlates with osteogenic markers, and is activated by R-568. Notably, downregulation of CaSR by RNA interference supports the conclusion that CaSR activation plays a central role in hAFMSC osteogenesis. Thus, this study provides significant information on the mechanisms of hAFMSC osteogenesis, which could provide additional molecular basis for the use of calcimimetics in bone regenerative medicine.
Get full access to this article
View all access options for this article.
References
1.
De CoppiP, BartschG, SiddiquiMM, XuT, SantosCC, PerinL, MostoslavskyG, SerreAC, SnyderEY, et al. (2007). Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol, 25:100–106.
2.
ShawSWS, DavidAL and De CoppiP. (2011). Clinical applications of prenatal and postnatal therapy using stem cells retrieved from amniotic fluid. Curr Opin Obstet Gynecol, 23:109–116.
3.
TrohatouO, AnagnouNF and RoubelakisMG. (2013). Human amniotic fluid stem cells as an attractive tool for clinical applications. Curr Stem Cell Res Ther, 8:125–132.
4.
HiguchiA, ShenPY, ZhaoJK, ChenCW, LingQD, ChenH, WangHC, BingJT and HsuST. (2011). Osteoblast differentiation of amniotic fluid-derived stem cells irradiated with visible light. Tissue Eng Part A, 17:2593–2602.
5.
AntonucciI, IezziI, MorizioE, MastrangeloF, PantaloneA, Mattioli-BelmonteM, GiganteA, SaliniV, CalabreseG, et al. (2009). Isolation of osteogenic progenitors from human amniotic fluid using a single step culture protocol. BMC Biotechnol, 9:9
6.
MaraldiT, RiccioM, RescaR, PisciottaA, La SalaGB, FerrariA, BruzzesiG, MottaA, MigliaresiC, MarzonaL and De PolA. (2011). Human amniotic fluid stem cells seeded in fibroin scaffold produce in vivo mineralized matrix. Tissue Eng Part A, 17:2833–2843.
7.
RodriguesMT, LeeB-K, LeeSJ, GomesME, ReisRL, AtalaA and YooJJ. (2012). The effect of differentiation stage of amniotic fluid stem cells on bone regeneration. Biomaterials, 33:6069–6078.
8.
PeisterA, DeutschER, KolambkarY, HutmacherDW and GuldbergRE. (2009). Amniotic fluid stem cells produce robust mineral deposits on biodegradable scaffolds. Tissue Eng Part A, 15:3129–3138.
9.
MariePJ. (2010). The calcium-sensing receptor in bone cells: a potential therapeutic target in osteoporosis. Bone, 46:571–576.
10.
DvorakMM, SiddiquaA, WardDT, CarterDH, DallasSL, NemethEF and RiccardiD. (2004). Physiological changes in extracellular calcium concentration directly control osteoblast function in the absence of calciotropic hormones. Proc Natl Acad Sci U S A, 101:5140–5145.
11.
QuinnSJ, YeCP, DiazR, KiforO, BaiM, VassilevP and BrownE. (1997). The Ca2+-sensing receptor: a target for polyamines. Am J Physiol, 273:C1315–C1323.
12.
GarrettJE, CapuanoIV, HammerlandLG, HungBCP, BrownEM, SC HebertEN and FullerF. (1995). Molecular cloning and functional expression of human parathyroid calcium receptor cDNAs. J Biol Chem, 270:12919–12925.
13.
RiccardiD, ParkJ, LeeWS, GambaG, BrownEM and HebertSC. (1995). Cloning and functional expression of a rat kidney extracellular calcium/polyvalent cation-sensing receptor. Proc Natl Acad Sci U S A, 92:131–135.
14.
HebertSC, ChengS and GeibelJ. (2004). Functions and roles of the extracellular Ca2+-sensing receptor in the gastrointestinal tract. Cell Calcium, 35:239–247.
15.
BrownEM. (2007). The calcium-sensing receptor: physiology, pathophysiology and CaR-based therapeutics. Subcell Biochem, 45:139–167.
16.
BonominiM, GiardinelliA, MorabitoC, Di SilvestreS, Di CesareM, Di PietroN, SirolliV, FormosoG, AmorosoL, MariggiòMA and PandolfiA. (2012). Calcimimetic R-568 and its enantiomer S-568 increase nitric oxide release in human endothelial cells. PLoS One, 7:e30682
17.
HouseMG, KohlmeierL, ChattopadhyayN, KiforO, YamaguchiT, LeboffMS, GlowackiJ and BrownEM. (1997). Expression of an extracellular calcium-sensing receptor in human and mouse bone marrow cells. J Bone Miner Res, 12:1959–1970.
18.
KamedaT, ManoH, YamadaY, TakaiH, AmizukaN, KoboriM, IzumiN, KawashimaH, OzawaH, et al. (1998). Calcium-sensing receptor in mature osteoclasts, which are bone resorbing cells. Biochem Biophys Res Commun, 422:419–422.
19.
YamaguchiT, ChattopadhyayN, KiforO, YeC, PeterM, SandersJL, BrownEM, VassilevPM and JenniferL. (2001). Expression of extracellular calcium-sensing receptor in human osteoblastic MG-63 cell line. Am J Physiol Cell Physiol, 280:C382–C393.
20.
Di TomoP, PipinoC, LanutiP, MorabitoC, PierdomenicoL, SirolliV, BonominiM, MisciaS, MariggiòMA, et al. (2013). Calcium sensing receptor expression in ovine amniotic fluid mesenchymal stem cells and the potential role of R-568 during osteogenic differentiation. PLoS One, 8:e73816
21.
YamaguchiT. (2008). The calcium-sensing receptor in bone. J Bone Miner Metab, 26:301–311.
22.
BrownEM, ChattopadhyayN and YanoS. (2004). Calcium-sensing receptors in bone cells. J Musculoskelet Neuronal Interact, 4:412–413.
23.
GabusiE, ManferdiniC, GrassiF, PiacentiniA, CattiniL, FilardoG, LambertiniE, PivaR, ZiniN, FacchiniA and LisignoliG. (2011). Extracellular calcium chronically induced human osteoblasts effects: specific modulation of osteocalcin and collagen type XV. J Cell Physiol, 227:3151–3161.
24.
AhlstromM, PekkinenM, RiehleU and Lamberg-AllardtC. (2008). Extracellular calcium regulates parathyroid hormone-related peptide expression in osteoblasts and osteoblast progenitor cells. Bone, 42:483–490.
BarradasAMC, FernandesHAM, GroenN, ChaiYC, SchrootenJ, van de PeppelJ, van LeeuwenJPTM, van BlitterswijkCA and de BoerJ. (2012). A calcium-induced signaling cascade leading to osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells. Biomaterials, 33:3205–3215.
27.
RobertsSJ, GerisL, KerckhofsG, DesmetE, SchrootenJ and LuytenFP. (2011). The combined bone forming capacity of human periosteal derived cells and calcium phosphates. Biomaterials, 32:4393–4405.
28.
McCullenSD, ZhanJ, OnoratoML, BernackiSH and LoboaEG. (2010). Effect of varied ionic calcium on human adipose-derived stem cell mineralization. Tissue Eng Part A, 16:1971–1981.
GregoryCA, GunnWG, PeisterA and ProckopDJ. (2004). An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal Biochem, 329:77–84.
33.
AparicioS, DotySB, CamachoNP, PaschalisEP, SpevakL, MendelsohnR and BoskeyAL. (2002). Optimal methods for processing mineralized tissues for Fourier transform infrared microspectroscopy. Calcif Tissue Int, 70:422–429.
34.
D'AlimonteI, NargiE, LannuttiA, MarchisioM, PierdomenicoL, CostanzoG, Di IorioP, BalleriniP, GiulianiP, CaciagliF and CiccarelliR. (2013). Adenosine A1 receptor stimulation enhances osteogenic differentiation of human dental pulp-derived mesenchymal stem cells via WNT signaling. Stem Cell Res, 11:611–624.
35.
D'AlimonteI, LannuttiA, PipinoC, Di TomoP, PierdomenicoL, CianciE, AntonucciI, MarchisioM, RomanoM, et al. (2013). Wnt signaling behaves as a “Master Regulator”. in the osteogenic and adipogenic commitment of human amniotic fluid mesenchymal stem cells. Stem Cell Rev, 9:642–654.
36.
LanutiP, FuhrmannS, LachmannR, MarchisioM, MisciaS and KernF. (2009). Simultaneous characterization of phospho-proteins and cell cycle in activated T cell subsets. Int J Immunopathol Pharmacol, 22:689–698.
37.
LanutiP, SantilliF, MarchisioM, PierdomenicoL, VitacolonnaE, SantavenereE, IaconeA, DavìG, RomanoM and MisciaS. (2012). A novel flow cytometric approach to distinguish circulating endothelial cells from endothelial microparticles: relevance for the evaluation of endothelial dysfunction. J Immunol Methods, 380:16–22.
38.
HénautL, BoudotC, MassyZA, Lopez-FernandezI, DupontS, MaryA, DrüekeTB, KamelS, BrazierM and MentaverriR. (2014). Calcimimetics increase CaSR expression and reduce mineralization in vascular smooth muscle cells: mechanisms of action. Cardiovasc Res, 101:256–265.
39.
CutronaMB, BeznoussenkoGV, FusellaA, MartellaO, MoralP and MironovAA. (2013). Silencing of mammalian Sar1 isoforms reveals COPII-independent protein sorting and transport. Traffic, 14:691–708.
40.
BaiM, TrivediS and BrownEM. (1998). Dimerization of the extracellular calcium-sensing receptor (CaR) on the cell surface of CaR-transfected HEK293 cells. J Biol Chem, 273:23605–23610.
41.
AntonucciI, PantaloneA, TeteS, SaliniV, BorlonganCV, HessD and StuppiaL. (2012). Amniotic fluid stem cells: a promising therapeutic resource for cell-based regenerative therapy. Curr Pharm Des, 18:1846–1863.
42.
ChangW, TuC, BajraR, KomuvesL, MillerS, StrewlerG and ShobackD. (1999). Calcium sensing in cultured chondrogenic RCJ3.1C5.18 cells. Endocrinology, 140:1911–1919.
43.
ChattopadhyayN, YanoS, Tfelt-HansenJ, RooneyP, KanuparthiD, BandyopadhyayS, RenX, TerwilligerE and BrownEM. (2004). Mitogenic action of calcium-sensing receptor on rat calvarial osteoblasts. Endocrinology, 145:3451–3462.
44.
YamaguchiT, OlozakI, ChattopadhyayN, ButtersRR, KiforO, ScaddenDT and BrownEM. (1998). Expression of extracellular calcium (Ca2+o)-sensing receptor in human peripheral blood monocytes. Biochem Biophys Res Commun, 246:501–506.
45.
MartinoNA, Lange-ConsiglioA, CremonesiF, ValentiniL, CairaM, GuaricciAC, AmbruosiB, SciorsciRL, LacalandraGM, ReshkinSJ and Dell'AquilaME. (2011). Functional expression of the extracellular calcium sensing receptor (CaSR) in equine umbilical cord matrix size-sieved stem cells. PLoS One, 6:e17714
46.
LamBS, CunninghamC and AdamsGB. (2011). Pharmacologic modulation of the calcium-sensing receptor enhances hematopoietic stem cell lodgment in the adult bone marrow. Blood, 117:1167–1175.
47.
PenolazziL, LisignoliG, LambertiniE, TorreggianiE, ManferdiniC, LolliA, VecchiatiniR, CiardoF, GabusiE, et al. (2011). Transcription factor decoy against NFATc1 in human primary osteoblasts. Int J Mol Med, 28:199–206.
48.
FromiguéO, HaÿE, BarbaraA and MariePJ. (2010). Essential role of nuclear factor of activated T cells (NFAT)-mediated Wnt signaling in osteoblast differentiation induced by strontium ranelate. J Biol Chem, 285:25251–25258.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
