Osteogenic differentiation is a complex and still poorly understood biological process regulated by intrinsic cellular signals and extrinsic microenvironmental cues. Following appropriate stimuli, mesenchymal stem cells (MSCs) differentiate into osteoblasts through a tightly regulated multistep process driven by several transcription factors and characterized by the expression of a number of bone-specific proteins. In this study, we describe a novel transcription factor that we named osteoblast inducer (ObI)-1, involved in MSC differentiation toward the osteogenic lineage. ObI-1 encodes for a nuclear protein subjected to proteasomal degradation and expressed during osteoblast differentiation both in a murine multipotent mesenchymal cell line (W20-17) and in primary murine MSCs. RNA interference-mediated knockdown of ObI-1 expression significantly impairs osteoblast differentiation and matrix mineralization with reduced expression of the osteogenic markers, Runt-related transcription factor 2 (Runx2) and osteopontin. Conversely, ObI-1 overexpression enhances osteogenic differentiation and bone-specific markers expression. ObI-1 stimulates bone morphogenetic protein (BMP)-4 expression and the consequent activation of the Smad pathway; treatment with a BMP receptor type I antagonist completely abolishes ObI-1-mediated stimulation of osteogenic differentiation. Collectively, our findings suggest that ObI-1 modulates osteogenic differentiation, at least in part, through the BMP signaling pathway, increasing Runx2 activation and leading to osteoblast commitment and maturation.
Get full access to this article
View all access options for this article.
References
1.
KomoriT. (2006). Regulation of osteoblast differentiation by transcription factors. J Cell Biochem, 99:1233–1239.
2.
KarsentyG, KronenbergHM and SettembreC. Genetic control of bone formation. Annu Rev Cell Dev Biol, 25:629–648.
3.
UristMR. (1965). Bone: formation by autoinduction. Science, 150:893–899.
4.
WangRN, GreenJ, WangZ, DengY, QiaoM, PeabodyM, ZhangQ, YeJ, YanZ, et al. (2014). Bone morphogenetic protein (BMP) signaling in development and human diseases. Genes Dis, 1:87–105.
5.
ChenG, DengC and LiYP. (2012). TGF-β and BMP signaling in osteoblast differentiation and bone formation. Int J Biol Sci, 8:272–288.
6.
SuzawaM, TakeuchiY, FukumotoS, KatoS, UenoN, MiyazonoK, MatsumotoT and FujitaT. (1999). Extracellular matrix-associated bone morphogenetic proteins are essential for differentiation of murine osteoblastic cells in vitro. Endocrinology, 140:2125–2133.
7.
XiaoG, GopalakrishnanR, JiangD, ReithE, BensonMD and FranceschiRT. (2002). Bone morphogenetic proteins, extracellular matrix, and mitogen-activated protein kinase signaling pathways are required for osteoblast-specific gene expression and differentiation in MC3T3-E1 cells. J Bone Miner Res, 17:101–110.
8.
AlmalkiSG and AgrawalDK. (2016). Key transcription factors in the differentiation of mesenchymal stem cells. Differentiation, 92:41–51.
9.
DucyP, ZhangR, GeoffroyV, RidallAL and KarsentyG. (1997). Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell, 89:747–754.
10.
KomoriT, YagiH, NomuraS, YamaguchiA, SasakiK, DeguchiK, ShimizuY, BronsonRT, GaoYH, et al. (1997). Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell, 89:755–764.
11.
ItoY, BaeSC and ChuangLS. (2015). The RUNX family: developmental regulators in cancer. Nat Rev Cancer, 15:81–95.
12.
OttoF, ThornellAP, CromptonT, DenzelA, GilmourKC, RosewellIR, StampGW, BeddingtonRS, MundlosS, et al. (1997). Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell, 89:765–771.
13.
LeeB, ThirunavukkarasuK, ZhouL, PastoreL, BaldiniA, HechtJ, GeoffroyV, DucyP and KarsentyG. (1997). Missense mutations abolishing DNA binding of the osteoblast-specific transcription factor OSF2/CBFA1 in cleidocranial dysplasia. Nat Genet, 16:307–310.
14.
DucyP, StarbuckM, PriemelM, ShenJ, PineroG, GeoffroyV, AmlingM and KarsentyG. (1999). A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development. Genes Dev, 13:1025–1036.
15.
GrafeI, AlexanderS, PetersonJR, SniderTN, LeviB, LeeB and MishinaY. (2017). TGF-β family signaling in mesenchymal differentiation. Cold Spring Harb Perspect Biol, 10:pii:a022202.
16.
LeeKS, HongSH and BaeSC. (2002). Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-beta and bone morphogenetic protein. Oncogene, 21:7156–7163.
17.
WuM, ChenG and LiYP. (2016). TGF-beta and BMP signaling in osteoblast, skeletal development, and bone formation, homeostasis and disease. Bone Res, 4:16009.
18.
FitzsimmonsREB, MazurekMS, SoosA and SimmonsCA. (2018). Mesenchymal stromal/stem cells in regenerative medicine and tissue engineering. Stem Cells Int, 2018:8031718.
19.
QuerquesF, CantilenaB, CozzolinoC, EspositoMT, PassaroF, ParisiS, LombardoB, RussoT and PastoreL. (2015). Angiotensin receptor I stimulates osteoprogenitor proliferation through TGFβ-mediated signaling. J Cell Physiol, 230:1466–1474.
20.
JinS, ChoiH, KwonJT, KimJ, JeongJ, KimJ, HamS, ChoBN, YooYJ and ChoC. (2015). Identification and characterization of reproductive KRAB-ZF genes in mice. Gene, 565:45–55.
21.
EspositoMT, Di NotoR, MirabelliP, GorreseM, ParisiS, MontanaroD, Del VecchioL and PastoreL. (2009). Culture conditions allow selection of different mesenchymal progenitors from adult mouse bone marrow. Tissue Eng Part A, 15:2525–2536.
22.
MariottiE, MirabelliP, AbateG, SchiattarellaM, MartinelliP, FortunatoG, Di NotoR and Del VecchioL. (2008). Comparative characteristics of mesenchymal stem cells from human bone marrow and placenta: CD10, CD49d, and CD56 make a difference. Stem Cells Dev, 17:1039–1041.
23.
BoergermannJH, KopfJ, YuPB and KnausP. (2010). Dorsomorphin and LDN-193189 inhibit BMP-mediated Smad, p38 and Akt signalling in C2C12 cells. Int J Biochem Cell Biol, 42:1802–1807.
24.
LeeYC, ChengCJ, BilenMA, LuJF, SatcherRL, Yu-LeeLY, GallickGE, MaitySN and LinSH. (2011). BMP4 promotes prostate tumor growth in bone through osteogenesis. Cancer Res, 71:5194–5203.
25.
KamitaniT, KitoK, NguyenHP and YehET. (1997). Characterization of NEDD8, a developmentally down-regulated ubiquitin-like protein. J Biol Chem, 272:28557–28562.
26.
ParisiS, PassaroF, AloiaL, ManabeI, NagaiR, PastoreL and RussoT. (2008). Klf5 is involved in self-renewal of mouse embryonic stem cells. J Cell Sci, 121:2629–2634.
27.
AloiaL, ParisiS, FuscoL, PastoreL and RussoT. (2010). Differentiation of embryonic stem cell 1 (Dies1) is a component of bone morphogenetic protein 4 (BMP4) signaling pathway required for proper differentiation of mouse embryonic stem cells. J Biol Chem, 285:7776–7783.
AmablePR, TeixeiraMV, CariasRB, GranjeiroJM and BorojevicR. (2013). Identification of appropriate reference genes for human mesenchymal cells during expansion and differentiation. PLoS One, 8:e73792.
30.
QuirozFG, PosadaOM, Gallego-PerezD, Higuita-CastroN, SarassaC, HansfordDJ, Agudelo-FlorezP and LópezLE. (2010). Housekeeping gene stability influences the quantification of osteogenic markers during stem cell differentiation to the osteogenic lineage. Cytotechnology, 62:109–120.
31.
VandesompeleJ, De PreterK, PattynF, PoppeB, Van RoyN, De PaepeA and SpelemanF. (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol, 3:RESEARCH0034.
32.
PryszczLP, Huerta-CepasJ and GabaldónT. (2011). MetaPhOrs: orthology and paralogy predictions from multiple phylogenetic evidence using a consistency-based confidence score. Nucleic Acids Res, 39:e32.
33.
FiltzTM, VogelWK and LeidM. (2014). Regulation of transcription factor activity by interconnected post-translational modifications. Trends Pharmacol Sci, 35:76–85.
34.
Hammond-MartelI, YuH and Affar elB. (2012). Roles of ubiquitin signaling in transcription regulation. Cell Signal, 24:410–421.
35.
BellowsCG, AubinJE and HeerscheJNM. (1991). Initiation and progression of mineralization of bone nodules formed in vitro: the role of alkaline phosphatase and organic phosphate. Bone, 14:27–40.
36.
ChenJ, SinghK, MukherjeeBB and SodekJ. (1993). Developmental expression of osteopontin (OPN) mRNA in rat tissues: evidence for a role for OPN in bone formation and resorption. Matrix, 13:113–123.
37.
CanalisE, EconomidesAN and GazzerroE. (2003). Bone morphogenetic proteins, their antagonists, and the skeleton. Endocr Rev, 24:218–235.
38.
LeeK-S, KimH-J, LiQ-L, ChiX-Z, UetaC, KomoriT, WozneyJM, KimE-G, ChoiJ-Y, RyooH-M and BaeS-C. (2000). Runx2 is a common target of transforming growth factor beta1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12. Mol Cell Biol, 20:8783–8792.
39.
CauleyJA. (2013). Public health impact of osteoporosis. J Gerontol A Biol Sci Med Sci, 68:1243–1251.
40.
BalestrieriML, LuSJ, De NigrisF, GiovaneA, Williams-IgnarroS, D'ArmientoFP, FengQ, FioritoC, TestaG, et al. (2010). Therapeutic angiogenesis in diabetic apolipoprotein E-deficient mice using bone marrow cells, functional hemangioblasts and metabolic intervention. Atherosclerosis, 209:403–414.
41.
BruderSP, JaiswalN and HaynesworthSE. (1997). Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem, 64:278–294.
42.
NautaAJ and FibbeWE. (2007). Immunomodulatory properties of mesenchymal stromal cells. Blood, 110:3499–3506.
43.
JheonAH, GanssB, CheifetzS and SodekJ. (2001). Characterization of a novel KRAB/C2H2 zinc finger transcription factor involved in bone development. J Biol Chem, 276:18282–18289.
44.
KirkinV and DikicI. (2007). Role of ubiquitin- and Ubl-binding proteins in cell signaling. Curr Opin Cell Biol, 19:199–205.
45.
MurataniM and TanseyWP. (2003). How the ubiquitin-proteasome system controls transcription. Nat Rev Mol Cell Biol, 4:192–201.
46.
ConawayRC, BrowerCS and ConawayJW. (2002). Emerging roles of ubiquitin in transcription regulation. Science, 17:1254–1258.
47.
JonasonJH, XiaoG, ZhangM, XingL and ChenD. (2009). Post-translational regulation of Runx2 in bone and cartilage. J Dent Res, 88:693–703.
48.
ZaidiSK, SullivanAJ, van WijnenAJ, SteinJL, SteinGS and LianJB. (2002). Integration of Runx and Smad regulatory signals at transcriptionally active subnuclear sites. Proc Natl Acad Sci U S A, 99:8048–8053.
49.
BrazilDP, ChurchRH, SuraeS, GodsonC and MartinF. (2015). BMP signalling: agony and antagony in the family. Trends Cell Biol, 25:249–264.
50.
WagnerDO, SieberC, BhushanR, BorgermannJH, GrafD and KnausP. (2010). BMPs: from bone to body morphogenetic proteins. Sci Signal, 3:mr1.
51.
TsujiK, BandyopadhyayA, HarfeBD, CoxK, KakarS, GerstenfeldL, EinhornT, TabinCJ and RosenV. (2006). BMP2 activity, although dispensable for bone formation, is required for the initiation of fracture healing. Nat Genet, 38:1424–1429.
52.
HuangZ, WangD, Ihida-StansburyK, JonesPL and MartinJF. (2009). Defective pulmonary vascular remodeling in Smad8 mutant mice. Hum Mol Genet, 18:2791–2801.
53.
PuglisiR, MontanariM, ChiarellaP, StefaniniM and BoitaniC. (2004). Regulatory role of BMP2 and BMP7 in spermatogonia and Sertoli cell proliferation in the immature mouse. Eur J Endocrinol, 151:511–520.
54.
BabittJL, HuangFW, WrightingDM, XiaY, SidisY, SamadTA, CampagnaJA, ChungRT, SchneyerAL, et al. (2006). Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression. Nat Genet, 38:531–539.
55.
WeaverM, YinglingJM, DunnNR, BellusciS and HoganBL. (1999). Bmp signaling regulates proximal-distal differentiation of endoderm in mouse lung development. Development, 126:4005–4015.
56.
LeeJH, BhangDH, BeedeA, HuangTL, StrippBR, BlochKD, WagersAJ, TsengYH, RyeomS, KimCF, et al. (2014). Lung stem cell differentiation in mice directed by endothelial cells via a BMP4-NFATc1-thrombospondin-1 axis. Cell, 156:440–455.
57.
RosendahlA, PardaliE, SpeletasM, Ten DijkeP, HeldinCH and SiderasP. (2002). Activation of bone morphogenetic protein/Smad signaling in bronchial epithelial cells during airway inflammation. Am J Respir Cell Mol Biol, 27:160–169.
58.
SongY, ColemanL, ShiJ, BeppuH, SatoK, WalshK, LoscalzoJ and ZhangYY. (2008). Inflammation, endothelial injury, and persistent pulmonary hypertension in heterozygous BMPR2-mutant mice. Am J Physiol Heart Circ Physiol, 295:H677–H690.
59.
HåkelienAM, BryneJC, HarstadKG, LorenzS, PaulsenJ, SunJ, MikkelsenTS, MyklebostO and Meza-ZepedaLA. (2014). The regulatory landscape of osteogenic differentiation. Stem Cells, 32:2780–2793.
60.
CuiJ, YinY, MaQ, WangG, OlmanV, ZhangY, ChouWC, HongCS, ZhangC, et al. (2015). Comprehensive characterization of the genomic alterations in human gastric cancer. Int J Cancer, 137:86–95.
61.
DuanM, HaoJ, CuiS, WorthleyDL, ZhangS, WangZ, ShiJ, LiuL, WangX, et al. (2018). Diverse modes of clonal evolution in HBV-related hepatocellular carcinoma revealed by single-cell genome sequencing. Cell Res, 28:359–373.
62.
LandoM, FjeldboCS, WiltingSM, SnoekBC, AarnesEK, ForsbergMF, KristensenGB, SteenbergenRD and LyngH. (2015). Interplay between promoter methylation and chromosomal loss in gene silencing at 3p11-p14 in cervical cancer. Epigenetics, 10:970–980.
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.
