Role and mechanism of focal adhesion kinase in inducing osteogenic differentiation of mouse embryonic fibroblasts cells

doi:10.3969/j.issn.2095-4344.2969

Abstract

Abstract: BACKGROUND: Focal adhesion kinase (FAK) is regarded as a bridge molecule of “biomaterial/scaffold,” “seed cell,” and “growth factor” in bone tissue engineering. Exploration on the role and mechanism of focal adhesion kinase in inducing osteogenic differentiation of related seed cells is particularly important for the development and application of bone tissue engineering.
OBJECTIVE: To determine the role and mechanism of FAK in inducing osteogenic differentiation of immortalized mouse embryonic fibroblasts (iMEF).
METHODS: Under the same induction conditions, the iMEF cells with (iMEFFAK+/+ cells) or without FAK knockout (iMEFFAK-/- cells), treated with or without PI3K/AKT phosphorylation inhibitor LY294002 or ERK1/2 phosphorylation inhibitor U0126, were induced to differentiate into osteoblasts. The morphological changes of iMEFs (iMEFFAK+/+ and iMEFFAK-/-) at different induction periods were observed under a microscope. Runx2 protein levels and corresponding p-ERK1/2 and p-AKT levels were detected by western blot. RT-PCR technology was used to detect the transcription level of Runx2 gene. Finally, the induced iMEFs (iMEFFAK+/+ and iMEFFAK-/-) were stained with alizarin red staining for calcium nodules 3 weeks after osteogenesis induction.
RESULTS AND CONCLUSION: The osteogenic effect of iMEFFAK-/- cells was lower than that of iMEFFAK+/+ cells under the same induction conditions. Both the expression levels of Runx2 and the osteogenic effect of iMEFFAK+/+ cells and iMEFFAK-/- cells treated with LY294002 decreased significantly compared with the control group. Both the expression levels of Runx2 and the osteogenic effect of iMEFFAK+/+ cells and iMEFFAK-/- cells treated with U0126 decreased significantly compared with the control group. To conclude, silencing FAK expression can inhibit osteogenic differentiation of mouse embryonic fibroblasts by reducing the levels of PI3K/AKT, serine/threonine protein kinase, and ERK1/2 phosphorylation levels

Key words: focal adhesion kinase, immortalization, mouse, embryo, fibroblast, ERK1/2 , osteogenic differentiation

CLC Number:

Li Zhen, Huang Yonghui, Sun Jifu, Sun Haitao. Role and mechanism of focal adhesion kinase in inducing osteogenic differentiation of mouse embryonic fibroblasts cells[J]. Chinese Journal of Tissue Engineering Research, 2021, 25(2): 165-171.

Figures/Tables 11

References

[1] 马远征,王以朋,刘强,等.中国老年骨质疏松症诊疗指南(2018)[J]. 中国实用内科杂志,2019,39(1):38-61.
[2] 鲁先闯, 张志峰,黄健. 骨髓间充质干细胞修复软骨缺损的应用前景[J]. 中国组织工程研究, 2015,19(19):3093-3088.
[3] CAPLAN AI. Mesenchymal Stem Cells: Time to Change the Name! Stem Cells Transl Med. 2017;6(6):1445-1451.
[4] ZAJDEL A, KALUCKA M, KOKOSZKA-MIKOLAJ E, et al. Osteogenic differentiation of human mesenchymal stem cells from adipose tissue and Wharton’s jelly of the umbilical cord. Acta Biochim Pol. 2017; 64(2):365-369.
[5] ARTHUR A, ZANNETTINO A, GRONTHOS S. The therapeutic applications of multipotential mesenchymal/stromal stem cells in skeletal tissue repair. J Cell Physiol. 2009;218(2):237-245.
[6] HUANG E, BI Y, JIANG W, et al. Conditionally immortalized mouse embryonic fibroblasts retain proliferative activity without compromising multipotent differentiation potential. PLoS One. 2012; 7(2):e32428.
[7] Nelson FR, Brighton CT, Ryaby J, et al. Use of physical forces in bone healing. J Am Acad Orthop Surg. 2003; 11(5):344-354.
[8] 廉静. Notch信号通路在BMP2诱导iMEF细胞成骨分化中的作用及机制研究[D]. 重庆:重庆医科大学,2016.
[9] KOMORI T. Regulation of proliferation, differentiation and functions of osteoblasts by Runx2. Int J Mol Sci. 2019;20(7):1694.
[10] KOMORI T. Molecular mechanism of runx2-dependent bone development. Mol Cells. 2020;43(2):168-175.
[11] 陈力莹,范锟铻,王金水,等. Wnt-1基因对人表皮干细胞的分化与增殖的影响研究[J]. 中华损伤与修复杂志,2012,7(1):29-33.
[12] 施彦,舒斌,杨荣华,等. Wnt和Notch信号通路在大鼠创面愈合模型中的表达作用[J].中华损伤与修复杂志,2014,9(2):31-34.
[13] GU H, HUANG Z, YIN X, et al. Role of c-Jun N-terminal kinase in the osteogenic and adipogenic differentiation of human adipose-derived mesenchymal stem cells. Exp Cell Res. 2015; 339(1):112-121.
[14] KAMAMOTO M, MACHIDA J, MIYACHI H, et al. A novel mutation in the C-terminal region of RUNX2/CBFA1 distal to the DNA-binding runt domain in a Japanese patient with cleidocranial dysplasia. Int J Oral Maxillofac Surg. 2011; 40(4):434-437.
[15] FRANCESCHI RT, XIAO G. Regulation of the osteoblast-specific transcription factor, Runx2: responsiveness to multiple signal transduction pathways. J Cell Biochem. 2003; 88(3):446-454.
[16] SCHLAEPFER DD, HAUCK CR, SIEG DJ. Signaling through focal adhesion kinase. Prog Biophys Mol Biol. 1999;71(3-4):435-478.
[17] ZHAO X, GUAN JL. Focal adhesion kinase and its signaling pathways in cell migration and angiogenesis. Adv Drug Deliv Rev. 2011;63(8): 610-615.
[18] MITRA SK, SCHLAEPFER DD. Integrin-regulated FAK-Src signaling in normal and cancer cells. Curr Opin Cell Biol. 2006; 18(5):516-523.
[19] ZACHARY I, ROZENGURT E. Focal adhesion kinase (p125FAK): a point of convergence in the action of neuropeptides, integrins, and oncogenes. Cell. 1992;71(6):891-894.
[20] HAKUNO D, TAKAHASHI T, LAMMERDING J, et al. Focal adhesion kinase signaling regulates cardiogenesis of embryonic stem cells. J Biol Chem. 2005;280(47):39534-39544.
[21] SHEN TL, PARK AY, ALCARAZ A, et al. Conditional knockout of focal adhesion kinase in endothelial cells reveals its role in angiogenesis and vascular development in late embryogenesis. J Cell Biol. 2005; 169(6):941-952.
[22] SUN X, ZHENG W, QIAN C, et al. Focal adhesion kinase promotes BMP2-induced osteogenic differentiation of human urinary stem cells via AMPK and Wnt signaling pathways. J Cell Physiol. 2020;235(5): 4954-4964.
[23] FURUTA Y, ILIC D, KANAZAWA S, et al. Mesodermal defect in late phase of gastrulation by a targeted mutation of focal adhesion kinase, FAK. Oncogene. 1995;11(10):1989-1995.
[24] ILIC D, FURUTA Y, KANAZAWA S, et al. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature.1995; 377(6549):539-544.
[25] WANG J, XIANG B, DENG J, et al. Inhibition of viability, proliferation, cytokines secretion, surface antigen expression, and adipogenic and osteogenic differentiation of adipose-derived stem cells by seven-day exposure to 0.5 t static magnetic fields. Stem Cells Int. 2016; 2016:7168175.
[26] 胡新永,吕原,杨华清,等.糖皮质激素对膝关节周围成骨细胞一氧化氮合酶和骨重建的影响[J].中华损伤与修复杂志,2008, 3(4):439-444.
[27] HSU LW, GOTO S, NAKANO T, et al. The effect of exogenous histone H1 on rat adipose-derived stem cell proliferation, migration, and osteogenic differentiation in vitro. J Cell Physiol. 2012;227(10): 3417-3425.
[28] HU P, ZHU X, ZHAO C, et al. Fak silencing impairs osteogenic differentiation of bone mesenchymal stem cells induced by uniaxial mechanical stretch. J Dent Sci.2019;14(3):225-233.
[29] HU J, LIAO H, MA Z, et al. Focal adhesion kinase signaling mediated the enhancement of osteogenesis of human mesenchymal stem cells induced by extracorporeal shockwave. Sci Rep.2016; 6:20875.
[30] 扎拉嘎胡,徐忠伟,兰晓霞,等. 层粘连蛋白促脐带间充质干细胞向成骨细胞分化与黏着斑激酶表达[J]. 中国组织工程研究,2011, 15(6):29-33.
[31] XIE H, CAO T, FRANCO-OBREGON A, et al. Graphene-Induced Osteogenic Differentiation Is Mediated by the Integrin/FAK Axis. Int J Mol Sci. 2019;20(3):574.
[32] YUAN C, GOU X, DENG J, et al. FAK and BMP-9 synergistically trigger osteogenic differentiation and bone formation of adipose derived stem cells through enhancing Wnt-beta-catenin signaling. Biomed Pharmacother. 2018; 105:753-757.
[33] WU J, ZHAO J, SUN L, et al. Long non-coding RNA H19 mediates mechanical tension-induced osteogenesis of bone marrow mesenchymal stem cells via FAK by sponging miR-138. Bone. 2018;108: 62-70.
[34] LIU Y, MA Y, ZHANG J, et al. MBG-Modified beta-TCP Scaffold Promotes Mesenchymal Stem Cells Adhesion and Osteogenic Differentiation via a FAK/MAPK Signaling Pathway. ACS Appl Mater Interfaces. 2017; 9(36):30283-30296.
[35] THORPE LM, YUZUGULLU H, ZHAO JJ. PI3K in cancer: divergent roles of isoforms, modes of activation and therapeutic targeting. Nat Rev Cancer. 2015; 15(1):7-24.
[36] YU JS, CUI W. Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination. Development. 2016;143(17):3050-3060.
[37] GU YX, DU J, SI MS, et al. The roles of PI3K/Akt signaling pathway in regulating MC3T3-E1 preosteoblast proliferation and differentiation on SLA and SLActive titanium surfaces. J Biomed Mater Res A. 2013; 101(3):748-754.
[38] CHEN LL, HUANG M, TAN JY, et al. PI3K/AKT pathway involvement in the osteogenic effects of osteoclast culture supernatants on preosteoblast cells. Tissue Eng Part A. 2013;19(19-20):2226-2232.
[39] ZHANG Y, YANG JH. Activation of the PI3K/Akt pathway by oxidative stress mediates high glucose-induced increase of adipogenic differentiation in primary rat osteoblasts. J Cell Biochem. 2013;114(11): 2595-2602.
[40] GARTY J, KAUPPI M, KAUPPI A. Differential responses of certain lichen species to sulfur-containing solutions under acidic conditions as expressed by the production of stress-ethylene. Environ Res. 1995; 69(2):132-143.
[41] TWIGG SR, VORGIA E, MCGOWAN SJ, et al. Reduced dosage of ERF causes complex craniosynostosis in humans and mice and links ERK1/2 signaling to regulation of osteogenesis. Nat Genet. 2013; 45(3): 308-313.
[42] BAI B, HE J, LI YS, et al. Activation of the ERK1/2 signaling pathway during the osteogenic differentiation of mesenchymal stem cells cultured on substrates modified with various chemical groups. Biomed Res Int. 2013;2013:361906.
[43] XU L, MENG F, NI M, et al. N-cadherin regulates osteogenesis and migration of bone marrow-derived mesenchymal stem cells. Mol Biol Rep. 2013;40(3):2533-2539.
[44] MIRAOUI H, OUDINA K, PETITE H, et al. Fibroblast growth factor receptor 2 promotes osteogenic differentiation in mesenchymal cells via ERK1/2 and protein kinase C signaling. J Biol Chem. 2009; 284(8):4897-4904.