Establishment of mesenchymal stem cell lines derived from the bone marrow of green fluorescent protein‑transgenic mice exhibiting a diversity in intracellular transforming growth factor‑β and bone morphogenetic protein signaling

Sawada,Shunsuke; Chosa,Naoyuki; Takizawa,Naoki; Yokota,Jun; Igarashi,Yasuyuki; Tomoda,Koichi; Kondo,Hisatomo; Yaegashi,Takashi; Ishisaki,Akira

doi:10.3892/mmr.2016.4794

Establishment of mesenchymal stem cell lines derived from the bone marrow of green fluorescent protein‑transgenic mice exhibiting a diversity in intracellular transforming growth factor‑β and bone morphogenetic protein signaling

Authors:
- Shunsuke Sawada
- Naoyuki Chosa
- Naoki Takizawa
- Jun Yokota
- Yasuyuki Igarashi
- Koichi Tomoda
- Hisatomo Kondo
- Takashi Yaegashi
- Akira Ishisaki
View Affiliations

Affiliations: Division of Cellular Biosignal Sciences, Department of Biochemistry, Iwate Medical University, Yahaba, Iwate 028‑3694, Japan, Department of Otolaryngology, Dentistry and Oral Surgery, Kansai Medical University, Hirakata, Osaka 573‑1010, Japan, Department of Prosthodontics and Oral Implantology, Iwate Medical University School of Dentistry, Morioka, Iwate 020‑8505, Japan, Division of Periodontology, Department of Conservative Dentistry, Iwate Medical University School of Dentistry, Morioka, Iwate 020‑8505, Japan
Published online on: January 18, 2016 https://doi.org/10.3892/mmr.2016.4794
Pages: 2023-2031
Copyright: © Sawada et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Cytokines and their intercellular signals regulate the multipotency of mesenchymal stem cells (MSCs). The present study established the MSC lines SG‑2, ‑3, and ‑5 from the bone marrow of green fluorescent protein (GFP)‑transgenic mice. These cell lines clearly expressed mouse MSC markers Sca‑1 and CD44, and SG‑2 and ‑5 cells retained the potential for osteogenic and adipogenic differentiation in the absence of members of the transforming growth factor (TGF)‑β superfamily. By contrast, SG‑3 cells only retained adipogenic differentiation potential. Analysis of cytokine and cytokine receptor expression in these SG cell lines showed that bone morphogenetic protein (BMP) receptor 1B was most highly expressed in the SG‑3 cells, which underwent osteogenesis in response to BMP, while TGF‑β receptor II was most highly expressed in SG‑3 and ‑5 cells. However, it was unexpectedly noted that phosphorylation of Smad 2, a major transcription factor, was induced by TGF‑β1 in SG‑2 cells but not in SG‑3 or ‑5 cells. Furthermore, TGF‑β1 clearly induced the expression of Smad‑interacting transcription factor CCAAT/enhancer binding protein‑β in SG‑2 but not in SG‑3 or ‑5 cells. These results demonstrated the establishment of TGF‑β‑responsive SG‑2 MSCs, BMP‑responsive SG‑3 MSCs and TGF‑β/BMP‑unresponsive SG‑5 MSCs, each of which was able to be traced by GFP fluorescence after transplantation into in vivo experimental models. In conclusion, the present study suggested that these cell lines may be used to explore how the TGF‑β superfamily affects the proliferation and differentiation status of MSCs in vivo.

View Figures

View References

1	Prockop DJ: Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 276:71–74. 1997. View Article : Google Scholar : PubMed/NCBI
2	Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S and Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science. 284:143–147. 1999. View Article : Google Scholar : PubMed/NCBI
3	Docheva D, Popov C, Mutschler W and Schieker M: Human mesenchymal stem cells in contact with their environment: Surface characteristics and the integrin system. J Cell Mol Med. 11:21–38. 2007. View Article : Google Scholar : PubMed/NCBI
4	Baksh D, Song L and Tuan RS: Adult mesenchymal stem cells: Characterization, differentiation and application in cell and gene therapy. J Cell Mol Med. 8:301–316. 2004. View Article : Google Scholar : PubMed/NCBI
5	Kassem M, Abdallah BM and Saeed H: Osteoblastic cells: Differentiation and transdifferentiation. Arch Biochem Biophys. 473:183–187. 2008. View Article : Google Scholar : PubMed/NCBI
6	Jones E and Yang X: Mesenchymal stem cells and bone regeneration: Current status. Injury. 42:562–568. 2011. View Article : Google Scholar : PubMed/NCBI
7	Proff P and Römer P: The molecular mechanism behind bone remodelling: A review. Clin Oral Investig. 13:355–362. 2009. View Article : Google Scholar : PubMed/NCBI
8	Lazar-Karsten P, Dorn I, Meyer G, Lindner U, Driller B and Schlenke P: The influence of extracellular matrix proteins and mesenchymal stem cells on erythropoietic cell maturation. Vox Sang. 101:65–76. 2011. View Article : Google Scholar
9	Yew TL, Chang MC, Hsu YT, Weng WH, Tsai CC, Chiu FY, Hung SC and He FY: Efficient expansion of mesenchymal stem cells from mouse bone marrow under hypoxic conditions. J Tissue Eng Regen Med. 7:984–993. 2013. View Article : Google Scholar
10	Weiss A and Attisano L: The TGF beta superfamily signaling pathway. Wiley Interdiscip Rev Dev Biol. 2:47–63. 2013. View Article : Google Scholar : PubMed/NCBI
11	Miyazawa K, Shinozaki M, Hara T, Furuya T and Miyazono K: Two major Smad pathways in TGF-beta superfamily signalling. Genes Cells. 7:1191–1204. 2002. View Article : Google Scholar : PubMed/NCBI
12	Heldin CH, Landström M and Moustakas A: Mechanism of TGF-beta signaling to growth arrest, apoptosis and epithelial-mesenchymal transition. Curr Opin Cell Biol. 21:166–176. 2009. View Article : Google Scholar : PubMed/NCBI
13	Meulmeester E and Ten Dijke P: The dynamic roles of TGF-β in cancer. J Pathol. 223:205–218. 2011. View Article : Google Scholar
14	Imamura T, Oshima Y and Hikita A: Regulation of TGF-β family signaling by ubiquitination and de ubiquitination. J Biochem. 154:481–489. 2013. View Article : Google Scholar : PubMed/NCBI
15	Herhaus L and Sapkota GP: The emerging roles of deubiquitylating enzymes (DUBs) in the TGFβ and BMP pathways. Cell Signal. 26:2186–2192. 2014. View Article : Google Scholar : PubMed/NCBI
16	Lee SY, Lee JH, Kim JY, Bae YC, Suh KT and Jung JS: BMP2 increases adipogenic differentiation in the presence of dexamethasone, which is inhibited by the treatment of TNF-α in human adipose tissue-derived stromal cells. Cell Physiol Biochem. 34:1339–1350. 2014. View Article : Google Scholar
17	Pountos I, Georgouli T, Henshaw K, Bird H, Jones E and Giannoudis PV: The effect of bone morphogenetic protein-2, bone morphogenetic protein-7, parathyroid hormone and platelet-derived growth factor on the proliferation and osteogenic differentiation of mesenchymal stem cells derived from osteoporotic bone. J Orthop Trauma. 24:552–556. 2010. View Article : Google Scholar : PubMed/NCBI
18	Yokota J, Chosa N, Sawada S, Okubo N, Takahashi N, Hasegawa T, Kondo H and Ishisaki A: PDGF-induced PI3K-mediated signal enhances TGF-β-induced osteogenic differentiation of human mesenchymal stem cells in the TGF-β-activated MEK-dependent manner. Int J Mol Med. 33:534–542. 2014.PubMed/NCBI
19	Ray P and Chapman SC: Cytoskeletal reorganaization drives mesenchymal condensation and regulates downstream molecular signaling. PLoS One. 10:e01347022015. View Article : Google Scholar
20	Sengle G, Carlberg V, Tufa SF, Charbonneau NL, Smaldone S, Carlson EJ, Ramirez F, Keene DR and Sakai LY: Abnormal activation of BMP signaling causes myopathy in Fbn2 null mice. PLoS Genet. 11:e10053402015. View Article : Google Scholar : PubMed/NCBI
21	Kramman R and Humphreys BD: Kidney pericytes: Roles in regeneration and fibrosis. Semin Nephrol. 34:374–383. 2014. View Article : Google Scholar
22	Touboul C, Vidal F, Pasquier J, Lis R and Rafii A: Role of mesenchymal cells in the natural history of ovarian cancer: A review. J Transl Med. 12:2712014. View Article : Google Scholar : PubMed/NCBI
23	Wise AF and Ricardo SD: Mesenchymal stem cells in kidney inflammation and repair. Nephrology (Carlton). 17:1–10. 2012. View Article : Google Scholar
24	Okabe M, Ikawa M, Kominami K, Nakanishi T and Nishimune Y: 'Green mice' as a source of ubiquitous green cells. FEBS Lett. 407:313–319. 1997. View Article : Google Scholar : PubMed/NCBI
25	Aomatsu E, Takahashi N, Sawada S, Okubo N, Hasegawa T, Taira M, Miura H, Ishisaki A and Chosa N: Novel SCRG1/BST1 axis regulates self-renewal, migration and osteogenic differentiation potential in mesenchymal stem cells. Sci Rep. 4:36522014. View Article : Google Scholar
26	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods. 25:402–408. 2001. View Article : Google Scholar
27	Gee CJ and Harris H: Tumorigenicity of cells transformed by Simian virus 40 and of hybrids between such cells and normal diploid cells. J Cell Sci. 36:223–240. 1979.PubMed/NCBI
28	Howell N: Suppression of transformation and tumorigenicity in interspecies hybrids of human SV40-transformed and mouse 3T3 cell lines. Cytogenet Cell Genet. 34:215–229. 1982. View Article : Google Scholar : PubMed/NCBI
29	Kahn P, Topp WC and Shin S: Tumorigenicity of SV40-transformed human and monkey cells in immunodeficient mice. Virology. 126:348–360. 1983. View Article : Google Scholar : PubMed/NCBI
30	Nitta M, Katabuchi H, Ohtake H, Tashiro H, Yamaizumi M and Okamura H: Characterization and tumorigenicity of human ovarian surface epithelial cells immortalized by SV40 large T antigen. Gynecol Oncol. 81:10–17. 2001. View Article : Google Scholar : PubMed/NCBI
31	Kang MK and Park NH: Extension of cell life span using exogenous telomerase. Methods Mol Biol. 371:151–165. 2007. View Article : Google Scholar : PubMed/NCBI
32	Lee MK, Hande MP and Sabapathy K: Ectopic mTERT expression in mouse embryonic stem cells does not affect differentiation but confers resistance to differentiation- and stress-induced p53-dependent apoptosis. J Cell Sci. 118:819–829. 2005. View Article : Google Scholar : PubMed/NCBI
33	Abraham S, Sweet T, Khalili K, Sawaya BE and Amini S: Evidence for activation of the TGF-beta1 promoter by C/EBP beta and its modulation by Smads. J Interferon Cytokine Res. 29:1–7. 2009. View Article : Google Scholar
34	Ramji DP and Foka P: CCAAT/enhancer-binding proteins: Structure, function and regulation. Biochem J. 365:561–575. 2002. View Article : Google Scholar : PubMed/NCBI
35	Zahnow CA: CCAAT/enhancer-binding protein beta: Its role in breast cancer and associations with receptor tyrosine kinases. Expert Rev Mol Med. 11:e122009. View Article : Google Scholar : PubMed/NCBI
36	Lekstrom-Himes J and Xanthopoulos KG: Biological role of the CCAAT/enhancer-binding protein family of transcription factors. J Biol Chem. 273:28545–28548. 1998. View Article : Google Scholar : PubMed/NCBI
37	Akira S, Isshiki H, Sugita T, Tanabe O, Kinoshita S, Nishio Y, Nakajima T, Hirano T and Kishimoto T: A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J. 9:1897–1906. 1990.PubMed/NCBI
38	Williams SC, Cantwell CA and Johnson PF: A family of C/EBP-related proteins capable of forming covalently linked leucine zipper dimers in vitro. Genes Dev. 5:1553–1567. 1991. View Article : Google Scholar : PubMed/NCBI
39	Katz S, Kowenz-Leutz E, Müller C, Meese K, Ness SA and Leutz A: The NF-M transcription factor is related to C/EBP beta and plays a role in signal transduction, differentiation and leukemogenesis of avian myelomonocytic cells. EMBO J. 12:1321–1332. 1993.PubMed/NCBI
40	Haas SC, Huber R, Gutsch R, Kandemir JD, Cappello C, Krauter J, Duyster J, Ganser A and Brand K: ITD- and FL-induced FLT3 signal transduction leads to increased C/EBP beta-LIP expression and LIP/LAP ratio by different signalling modules. Br J Haematol. 148:777–790. 2010. View Article : Google Scholar
41	Gutsch R, Kandemir JD, Pietsch D, Cappello C, Meyer J, Simanowski K, Huber R and Brand K: CCAAT/enhancer-binding protein beta inhibits proliferation in monocytic cells by affecting the retinoblastoma protein/E2F/cyclin E pathway but is not directly required for macrophage morphology. J Biol Chem. 286:22716–22729. 2011. View Article : Google Scholar : PubMed/NCBI
42	Nerlov C: The C/EBP family of transcription factors: A paradigm for interaction between gene expression and proliferation control. Trends Cell Biol. 17:318–324. 2007. View Article : Google Scholar : PubMed/NCBI
43	Tsukada J, Yoshida Y, Kominato Y and Auron PE: The CCAAT/enhancer (C/EBP) family of basic-leucine zipper (bZIP) transcription factors is a multifaceted highly-regulated system for gene regulation. Cytokine. 54:6–19. 2011. View Article : Google Scholar : PubMed/NCBI
44	Pham TH, Langmann S, Schwarzfischer L, El Chartouni C, Lichtinger M, Klug M, Krause SW and Rehli M: CCAAT enhancer-binding protein beta regulates constitutive gene expression during late stages of monocyte to macrophage differentiation. J Biol Chem. 282:21924–21933. 2007. View Article : Google Scholar : PubMed/NCBI
45	Liu S, Croniger C, Arizmendi C, Harada-Shiba M, Ren J, Poli V, Hanson RW and Friedman JE: Hypoglycemia and impaired hepatic glucose production in mice with a deletion of the C/EBP beta gene. J Clin Invest. 103:207–213. 1999. View Article : Google Scholar : PubMed/NCBI
46	Croniger CM, Millward C, Yang J, Kawai Y, Arinze IJ, Liu S, Harada-Shiba M, Chakravarty K, Friedman JE, Poli V and Hanson RW: Mice with a deletion in the gene for CCAAT/enhancer-binding protein beta have an attenuated response to cAMP and impaired carbohydrate metabolism. J Biol Chem. 276:629–638. 2001. View Article : Google Scholar
47	Poli V: The role of C/EBP isoforms in the control of inflammatory and native immunity functions. J Biol Chem. 273:29279–29282. 1998. View Article : Google Scholar : PubMed/NCBI
48	Zwergal A, Quirling M, Saugel B, Huth KC, Sydlik C, Poli V, Neumeier D, Ziegler-Heitbrock HW and Brand K: C/EBP beta blocks p65 phosphorylation and thereby NF-kappa B-mediated transcription in TNF-tolerant cells. J Immunol. 177:665–672. 2006. View Article : Google Scholar : PubMed/NCBI
49	Cappello C, Zwergal A, Kanclerski S, Haas SC, Kandemir JD, Huber R, Page S and Brand K: C/EBP beta enhances NF-kappaB-associated signalling by reducing the level of IkappaB-alpha. Cell Signal. 21:1918–1924. 2009. View Article : Google Scholar : PubMed/NCBI
50	Liu Y, Nonne macher MR and Wigdahl B: CCAAT/enhancer-binding proteins and the pathogenesis of retrovirus infection. Future Microbiol. 4:299–321. 2009. View Article : Google Scholar : PubMed/NCBI
51	Spooner CJ, Guo X, Johnson PF and Schwartz RC: Differential roles of C/EBP beta regulatory domains in specifying MCP-1 and IL-6 transcription. Mol Immunol. 44:1384–1392. 2007. View Article : Google Scholar
52	Abraham M, Shapiro S, Karni A, Weiner HL and Miller A: Gelatinases (MMP-2 and MMP-9) are preferentially expressed by Th1 vs. Th2 cells. J Neuroimmunol. 163:157–164. 2005. View Article : Google Scholar : PubMed/NCBI
53	Guo J, Zhang H, Xiao J, Wu J, Ye Y, Li Z, Zou Y and Li X: Monocyte chemotactic protein-1 promotes the myocardial homing of mesenchymal stem cells in dilated cardiomyopathy. Int J Mol Sci. 14:8164–8178. 2013. View Article : Google Scholar : PubMed/NCBI
54	Belema-Bedada F, Uchida S, Martire A, Kostin S and Braun T: Efficient homing of multipotent adult mesenchymal stem cells depends on FROUNT-mediated clustering of CCR2. Cell Stem Cell. 2:566–575. 2008. View Article : Google Scholar : PubMed/NCBI
55	Gerard C and Rollins BJ: Chemokines and disease. Nat Immunol. 2:108–115. 2001. View Article : Google Scholar : PubMed/NCBI
56	Rot A and von Andrian UH: Chemokines in innate and adaptive host defense: Basic chemokinese grammar for immune cells. Annu Rev Immunol. 22:891–928. 2004. View Article : Google Scholar : PubMed/NCBI
57	Dwyer RM, Potter-Beirne SM, Harrington KA, Lowery AJ, Hennessy E, Murphy JM, Barry FP, O'Brien T and Kerin MJ: Monocyte chemotactic protein-1 secreted by primary breast tumors stimulates migration of mesenchymal stem cells. Clin Cancer Res. 13:5020–5027. 2007. View Article : Google Scholar : PubMed/NCBI
58	Schenk S, Mal N, Finan A, Zhang M, Kiedrowski M, Popovic Z, McCarthy PM and Penn MS: Monocyte chemotactic protein-3 is a myocardial mesenchymal stem cell homing factor. Stem Cells. 25:245–251. 2007. View Article : Google Scholar
59	Zhou YL, Zhang HF, Li XL, Di RM, Yao WM, Li DF, Feng JL, Huang J, Cao KJ and Fu M: Increased stromal-cell-derived factor 1 enhances the homing of bone marrow derived mesenchymal stem cells in dilated cardiomyopathy in rats. Chin Med J (Engl). 123:3282–3287. 2010.
60	Zhuang Y, Chen X, Xu M, Zhang LY and Xiang F: Chemokine stromal cell-derived factor 1/CXCL12 increases homing of mesenchymal stem cells to injured myocardium and neovascularization following myocardial infarction. Chin Med J (Engl). 122:183–187. 2009.
61	Abbott JD, Huang Y, Liu D, Hickey R, Krause DS and Giordano FJ: Stromal cell-derived factor-1 alpha plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury. Circulation. 110:3300–3305. 2004. View Article : Google Scholar : PubMed/NCBI
62	Mozid AM, Arnous S, Sammut EC and Mathur A: Stem cell therapy for heart diseases. Br Med Bull. 98:143–159. 2011. View Article : Google Scholar : PubMed/NCBI