Growth differentiation factor‑5 induces tenomodulin expression via phosphorylation of p38 and promotes viability of murine mesenchymal stem cells from compact bone

Qu,Yanlong; Zhou,Li; Lv,Bing; Wang,Chunlei; Li,Pengwei

doi:10.3892/mmr.2017.8325

Growth differentiation factor‑5 induces tenomodulin expression via phosphorylation of p38 and promotes viability of murine mesenchymal stem cells from compact bone

Authors:
- Yanlong Qu
- Li Zhou
- Bing Lv
- Chunlei Wang
- Pengwei Li
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Affiliations: Department of Orthopedics, The Third Ward, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, P.R. China
Published online on: December 20, 2017 https://doi.org/10.3892/mmr.2017.8325
Pages: 3640-3646
Copyright: © Qu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Growth differentiation factor (GDF)‑5 serves a role in tissue development and tenomodulin serves an important role in the development of tendons. The effects of GDF‑5 on mesenchymal stem cells (MSCs), particularly with regards to tendon bioengineering, are poorly understood. The present study aimed to investigate the effects of GDF‑5 on cell viability and tenomodulin expression in MSCs from murine compact bone. MSCs were isolated from murine compact bones and confirmed by flow cytometric analysis. In addition, the adipogenic, osteoblastic and chondrocyte differentiation capabilities of the MSCs were determined. MSCs were treated with GDF‑5 and the effects of GDF‑5 on MSC viability were determined. The mRNA and protein expression levels of tenomodulin were detected by reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. MSCs from murine compact bone were successfully isolated. GDF‑5 had optimal effects on cell viability at 100 ng/ml (+36.9% of control group without GDF‑5 treatment, P<0.01) and its effects peaked after 6 days of treatment (+56.6% of control group, P<0.001). Compared with the control group, treatment with 100 ng/ml GDF‑5 for 4 days enhanced the mRNA expression levels of tenomodulin (3.56±0.94 vs. 1.02±0.25; P<0.05). In addition, p38 was activated by GDF‑5, as determined by enhanced expression levels of phosphorylated p38 (p‑p38). The GDF‑5‑induced protein expression levels of p‑p38 and tenomodulin were markedly inhibited following treatment with SB203580, an inhibitor of p38 mitogen‑activated protein kinase. These results suggested that GDF‑5 treatment may increase tenomodulin protein expression via phosphorylation of p38 in MSCs from murine compact bone. These findings may aid the future development of tendon bioengineering.

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1	Sharma P and Maffulli N: Biology of tendon injury: Healing, modeling, and remodeling. J Musculoskelet Neuronal Interact. 6:181–190. 2006.PubMed/NCBI
2	DeFranco MJ, Derwin K and Iannotti JP: New therapies in tendon reconstruction. J Am Acad Orthop Surg. 12:298–304. 2004. View Article : Google Scholar : PubMed/NCBI
3	Park A, Hogan MV, Kesturu GS, James R, Balian G and Chhabra AB: Adipose-derived mesenchymal stem cells treated with growth differentiation factor-5 express tendon-specific markers. Tissue Eng Part A. 16:2941–2951. 2010. View Article : Google Scholar : PubMed/NCBI
4	Zhou S, Yates KE, Eid K and Glowacki J: Demineralized bone promotes chondrocyte or osteoblast differentiation of human marrow stromal cells cultured in collagen sponges. Cell Tissue Bank. 6:33–44. 2005. View Article : Google Scholar : PubMed/NCBI
5	Chamberlain G, Fox J, Ashton B and Middleton J: Concise review: Mesenchymal stem cells: Their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells. 25:2739–2749. 2007. View Article : Google Scholar : PubMed/NCBI
6	Howard D, Buttery LD, Shakesheff KM and Roberts SJ: Tissue engineering: Strategies, stem cells and scaffolds. J Anat. 213:66–72. 2008. View Article : Google Scholar : PubMed/NCBI
7	da Silva Meirelles L, Chagastelles PC and Nardi NB: Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci. 119:2204–2213. 2006. View Article : Google Scholar : PubMed/NCBI
8	Zhu H, Guo ZK, Jiang XX, Li H, Wang XY, Yao HY, Zhang Y and Mao N: A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone. Nat Protoc. 5:550–560. 2010. View Article : Google Scholar : PubMed/NCBI
9	Oshin AO, Caporali E, Byron CR, Stewart AA and Stewart MC: Phenotypic maintenance of articular chondrocytes in vitro requires BMP activity. Vet Comp Orthop Traumatol. 20:185–191. 2007.PubMed/NCBI
10	Aspenberg P: Stimulation of tendon repair: Mechanical loading, GDFs and platelets. A mini-review. Int Orthop. 31:783–789. 2007. View Article : Google Scholar : PubMed/NCBI
11	Buxton P, Edwards C, Archer CW and Francis-West P: Growth/differentiation factor-5 (GDF-5) and skeletal development. J Bone Joint Surg Am. 83-A Suppl 1:S23–S30. 2001.
12	Daans M, Luyten FP and Lories RJ: GDF5 deficiency in mice is associated with instability-driven joint damage, gait and subchondral bone changes. Ann Rheum Dis. 70:208–213. 2011. View Article : Google Scholar : PubMed/NCBI
13	Bolt P, Clerk AN, Luu HH, Kang Q, Kummer JL, Deng ZL, Olson K, Primus F, Montag AG, He TC, et al: BMP-14 gene therapy increases tendon tensile strength in a rat model of Achilles tendon injury. J Bone Joint Surg Am. 89:1315–1320. 2007. View Article : Google Scholar : PubMed/NCBI
14	Docheva D, Hunziker EB, Fässler R and Brandau O: Tenomodulin is necessary for tenocyte proliferation and tendon maturation. Mol Cell Biol. 25:699–705. 2005. View Article : Google Scholar : PubMed/NCBI
15	Aslan H, Kimelman-Bleich N, Pelled G and Gazit D: Molecular targets for tendon neoformation. J Clin Invest. 118:439–444. 2008. View Article : Google Scholar : PubMed/NCBI
16	Shukunami C, Takimoto A, Miura S, Nishizaki Y and Hiraki Y: Chondromodulin-I and tenomodulin are differentially expressed in the avascular mesenchyme during mouse and chick development. Cell Tissue Res. 332:111–122. 2008. View Article : Google Scholar : PubMed/NCBI
17	Shukunami C, Takimoto A, Oro M and Hiraki Y: Scleraxis positively regulates the expression of tenomodulin, a differentiation marker of tenocytes. Dev Biol. 298:234–247. 2006. View Article : Google Scholar : PubMed/NCBI
18	Pisani DF, Pierson PM, Massoudi A, Leclerc L, Chopard A, Marini JF and Dechesne CA: Myodulin is a novel potential angiogenic factor in skeletal muscle. Exp Cell Res. 292:40–50. 2004. View Article : Google Scholar : PubMed/NCBI
19	Shukunami C and Hiraki Y: Chondromodulin-I and tenomodulin: The negative control of angiogenesis in connective tissue. Curr Pharm Des. 13:2101–2112. 2007. View Article : Google Scholar : PubMed/NCBI
20	Mendias CL, Bakhurin KI and Faulkner JA: Tendons of myostatin-deficient mice are small, brittle, and hypocellular. Proc Natl Acad Sci USA. 105:pp. 388–393. 2008; View Article : Google Scholar : PubMed/NCBI
21	Popov C, Burggraf M, Kreja L, Ignatius A, Schieker M and Docheva D: Mechanical stimulation of human tendon stem/progenitor cells results in upregulation of matrix proteins, integrins and MMPs and activation of p38 and ERK1/2 kinases. BMC Mol Biol. 16:62015. View Article : Google Scholar : PubMed/NCBI
22	Schwartz AJ, Sarver DC, Sugg KB, Dzierzawski JT, Gumucio JP and Mendias CL: p38 MAPK signaling in postnatal tendon growth and remodeling. PLoS One. 10:e01200442015. View Article : Google Scholar : PubMed/NCBI
23	Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop Dj and Horwitz E: Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 8:315–317. 2006. View Article : Google Scholar : PubMed/NCBI
24	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 : PubMed/NCBI
25	Pereira RF, Halford KW, O' Hara MD, Leeper DB, Sokolov BP, Pollard MD, Bagasra O and Prockop DJ: Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice. Proc Natl Acad Sci USA. 92:pp. 4857–4861. 1995; View Article : Google Scholar : PubMed/NCBI
26	Prockop DJ: Marrow stromal cells as stem cells for non hematopoietic tissues. Science. 276:71–74. 1997. View Article : Google Scholar : PubMed/NCBI
27	Varma MJ, Breuls RG, Schouten TE, Jurgens WJ, Bontkes HJ, Schuurhuis GJ, van Ham SM and van Milligen FJ: Phenotypical and functional characterization of freshly isolated adipose tissue-derived stem cells. Stem Cells Dev. 16:91–104. 2007. View Article : Google Scholar : PubMed/NCBI
28	Phinney DG, Kopen G, Isaacson RL and Prockop DJ: Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: Variations in yield, growth, and differentiation. J Cell Biochem. 72:570–585. 1999. View Article : Google Scholar : PubMed/NCBI
29	Yin Z, Chen X, Chen JL and Ouyang HW: Stem cells for tendon tissue engineering and regeneration. Expert Opin Biol Ther. 10:689–700. 2010. View Article : Google Scholar : PubMed/NCBI
30	Farng E, Urdaneta AR, Barba D, Esmende S and McAllister DR: The effects of GDF-5 and uniaxial strain on mesenchymal stem cells in 3-D culture. Clin Orthop Relat Res. 466:1930–1937. 2008. View Article : Google Scholar : PubMed/NCBI
31	Nakamura K, Shirai T, Morishita S, Uchida S, Saeki-Miura K and Makishima F: p38 mitogen-activated protein kinase functionally contributes to chondrogenesis induced by growth/differentiation factor-5 in ATDC5 cells. Exp Cell Res. 250:351–363. 1999. View Article : Google Scholar : PubMed/NCBI
32	Coleman CM and Tuan RS: Functional role of growth/differentiation factor 5 in chondrogenesis of limb mesenchymal cells. Mech Dev. 120:823–836. 2003. View Article : Google Scholar : PubMed/NCBI
33	Watanabe H, de Caestecker MP and Yamada Y: Transcriptional cross-talk between Smad, ERK1/2 and p38 mitogen-activated protein kinase pathways regulates transforming growth factor-beta-induced aggrecan gene expression in chondrogenic ATDC5 cells. J Biol Chem. 276:14466–14473. 2001. View Article : Google Scholar : PubMed/NCBI
34	Zaidi SH, Huang Q, Momen A, Riazi A and Husain M: Growth differentiation factor 5 regulates cardiac repair after myocardial infarction. J Am Coll Cardiol. 55:135–143. 2010. View Article : Google Scholar : PubMed/NCBI