Role of microRNA and Wnt signaling pathway in osteogenic differentiation of bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2013.10.029

Abstract

Abstract:

BACKGROUND: Differentially expressed microRNA plays an important role in proliferation and differentiation of osteoblasts. However, the underlying mechanism and possible targets are poorly understood.
OBJECTIVE: To review the recent research progress of the role of microRNA in osteogenic differentiation of bone marrow mesenchymal stem cells by Wnt signaling pathway.
METHODS: A computer-based online retrieval of CNKI and Pubmed databases was performed to search papers describing the role of microRNA in osteogenesis published during January 2000 to January 2012 using key words “microRNA, Wnt signaling pathway, osteogenesis”. In the same research field, papers published recently or in high impact factor journals were selected. A total of 205 papers were initially retrieved, and 33 were suitable for final analysis.
RESULTS AND CONCLUSION: MicroRNA is increasingly searched in the field of life science. Accumulative evidence exists that microRNA plays an important role in osteogenic differentiation via Wnt signaling pathway. A better understanding of the mechanism by which RNA regulates osteogenic differentiation is the basis for culture of tissue-engineered bone and shows great application prospect. At present, the research of microRNA is still in its infancy.

Key words: stem cells, stem cell review, bone marrow mesenchymal stem cells, Wnt signaling pathway, MicroRNA, osteogenesis, National Natural Science Foundation of China

CLC Number:

R318

Zou Han-lin, Guo Yong-fei, Liu Yan. Role of microRNA and Wnt signaling pathway in osteogenic differentiation of bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(10): 1896-1900.

Figures/Tables 1

2.1 Wnt通路组成及信号传导机制 Wnt基因是鼠类乳腺癌病毒诱导的小鼠乳腺癌中克隆出的一种原癌基因，于1982年由Nusse等报道并称int-1基因，因其与果蝇的无翅基因Wingless(wg)为同源基因，故被合称为Wnt-1[6]。目前，在哺乳动物中已发现19种Wnt 基因，分别命名为Wntl、Wnt2、Wnt3、Wnt3a等[7]。该基因编码的Wnt蛋白由350-400个氨基酸残基组成，是一类富含L-半胱氨酸的分泌性糖蛋白[8]。根据Wnt和受体结合后激活信号转导途径的差异，将Wnt信号转导途径分为经典Wnt信号通路、Wnt/PCP通路(planner cell polarity pathway)和Wnt/Ca 通路。目前已证实骨形成和重建主要与经典的Wnt信号通路有关，是近年来对成骨分化影响的研究热点。经典Wnt信号通路又称β-Catenin依赖性通路，由Wnt糖蛋白(主要是Wnt1、Wnt3a、Wnt8a、Wnt10b等)、β-Catenin、卷轴受体(frizzled receptor)，以及低密度脂蛋白受体相关蛋白LRP5/6、胞内信号调节因子、糖原合成激酶、淋巴增强因子、T细胞因子等组成[9]。Wnt蛋白为Wnt/β-catenin经典通路的起始因子。在没有胞外Wnt信号时，靶细胞细胞质内的β-catenin和大肠腺瘤息肉蛋白、核心蛋白Axin、酪氨酸激酶和糖原合成激酶形成一个多蛋白的β-Catenin降解复合物，其中的酪氨酸激酶和糖原合成激酶可将β-Catenin氨基端Ser/Thr磷酸化，最终被泛素/蛋白激酶途径降解[10]，使得胞质内的β-Catenin维持在较低的浓度，使其无法激活下游靶基因的转录过程。Frizzled为细胞膜表面的一类具有七次跨膜结构的受体蛋白，其含有Wnt蛋白结合所必需的半胱氨酸富集结构域。LRP-5/6是Wnt的共受体，均为低密度脂蛋白受体超家族成员的单跨膜蛋白，分别含有1 615和 1 613个氨基酸残基[11-12]。当外界环境改变，诱导Wnt大量生成时，Wnt蛋白与Frz和LRP5/6复合物的特异结合能激活含PDZ结构域胞内信号调节因子，胞内信号调节因子释放信号抑制糖原合成激酶对β-Catenin的磷酸化-降解。最终导致β-Catenin在胞浆中稳定积累并进入细胞核内进而激活淋巴增强因子/T细胞因子，启动Wnt下游靶基因转录[13]。 2.2 经典Wnt通路对骨髓间充质干细胞向成骨细胞分化的影响经典Wnt通路的活动在前成骨细胞，骨衬细胞和骨细胞中均存在。近年来研究显示，增加Wnt信号通路的激活因子或删除分泌Wnt信号通路的抑制因子导致的经典Wnt通路的激活能够明显促进骨髓间充质干细胞向成骨细胞分化。Hill等[14]利用β-Catenin基因敲除的动物实验研究显示：β-Catenin水平的下调使骨髓间充质干细胞向成骨细胞的分化活性明显减弱，并影响骨骼的矿化过程。Gaur等[15]利用frizzled相关蛋白(SFRP1)基因敲除的小鼠的研究发现T细胞因子1上存在Runx2的激活位点，提示β-Catenin可能通过影响TCF-Runx2发挥成骨作用。Qiu等[16]通过对LRP5基因突变的研究发现，一方面，LRP5能够通过经典的Wnt通路促进人骨髓间充质干细胞向成骨细胞分化，另一方面可以抑制其向脂肪细胞分化。Diarra等[17]在小鼠类风湿性关节炎的逆向骨破坏模型中，通过口服Dkk1(Wnt信号通路的拮抗物)的特异性抗体，增加了成骨细胞的数量，骨形成的比率和骨保护素表达增强且减少破骨细胞的数量。以往认为糖皮质激素可以抑制骨的形成，近年来的研究表明糖皮质激素可以增强frizzled相关蛋白(SFRP1)的表达而抑制Wnt通路，进而抑制骨髓间充质干细胞向成骨细胞的分化[18]。Wang等[19]则发现Dkk1基因敲除的小鼠可以减弱糖皮质激素的上述作用，提示糖皮质激素也可能通过增强Dkk1抑制Wnt通路，从而抑制骨髓间充质干细胞向成骨细胞分化。然而也有报道指出，外源性Wnt1可以下调多种成骨标志物如碱性磷酸酶、骨涎蛋白、骨钙素等的表达[20]。Boer等[21]也通过对经慢病毒转染Wnt3a的研究发现，Wnt3a可以促进骨髓间充质干细胞的增殖，却强烈抑制骨髓间充质干细胞向成骨细胞的分化和多种成骨标志物的表达。那么是否经典Wnt通路在骨髓间充质干细胞向成骨细胞的分化仅仅发挥阳性作用，这一题尚未完全明了。Liu等[22]在其研究中发现低浓度的Wnt3a可以促进成骨细胞分化，而高浓度的Wnt3a则部分抑制成骨分化，这些研究表明Wnt对于骨髓间充质干细胞向成骨细胞的分化可能与细胞所处的微环境中Wnt蛋白的含量有关。 2.3 微小RNA经Wnt信号通路在成骨分化中的调控作用微小RNA(miR)是一类在生物进化过程中高度保守的由18-22个核苷酸组成的单链非编码RNA，其首先由RNA聚合酶Ⅱ转录成初级miR，再由核糖核苷酸酶Ⅲ剪切，最终由Dicer酶完成miR前体到成熟miR的转化[23]。miR结合于mRNA 3’端的非编码区(3’UTR)，通过抑制靶mRNA的翻译或降解靶mRNA起作用，从而调控靶标基因的表达[24]。有差异性表达的微小RNA在成骨细胞的增殖和分化中发挥广泛而又重要的作用，但其作用机制、作用靶点还不甚清楚，是近年来的研究热点。 Wienholds等[25]在斑马鱼的胚胎组织中发现组织特异性表达微小RNA共有120个，其中微小RNA140仅表达于软骨源性组织；进一步的研究发现微小RNA140也特异性的表达于小鼠胚胎软骨组织。Lewis和Chen等[26-27]通过靶基因突变技术和双荧光素酶报告系统进行检测和分析，发现微小RNA140可能的作用靶点为促使软骨细胞肥大的调节因子——组蛋白脱乙酰基酶4。组蛋白脱乙酰基酶4和Runx2关系密切，它们是在软骨细胞肥大和骨细胞分化中起关键作用的转录因子。Colno[28]则也发现组蛋白脱乙酰基酶4是近来被确定为在骨骼肌肉发育中发挥重要作用的微小RNA-1的靶点，微小RNA-1通过转录后调节作用抑制组蛋白脱乙酰基酶4提高骨骼肌肉的分化。微小RNA140可能也起到相类似的作用。 Huang等[29]发现在骨髓间充质干细胞向脂肪细胞分化的过程中，miR204及其同系物miR211的表达上调，而影响成骨分化的重要转录因子Runx2的表达受抑制。通过双荧光素酶报告基因系统显示miR204能够靶向作用于Runx2的3’UTR区，过表达miR204能够降低Runx2的转录水平，从而阻遏骨髓间充质干细胞向成骨细胞分化，并能促进骨髓间充质干细胞向脂肪细胞分化。Kapinas等[30]在研究hFOB1.19细胞向成骨细胞分化的过程中发现，新鲜分化成骨细胞的胞外基质中miR-29a的含量较高。靶标分析验证miR-29a的激活位点位于淋巴增强因子/T细胞因子。miR-29a的转录可能与经典Wnt信号通路的激活形成一个正反馈环路。miR-29a的作用靶点为Dkk1等Wnt通路拮抗剂，miR-29a的过表达能够使包括Dkk1、Kremen2、sFRP2在内的多种Wnt通路拮抗物的表达显著下调，从而激活Wnt通路；而Wnt通路的激活又能级联放大激活淋巴增强因子/T细胞因子，而进一步促进miR-29a的表达。糖原合成激酶、核心蛋白Axin等都是经典Wnt通路的重要的节点因子。Liu等[31]为验证微小RNA与糖原合成激酶、Axin等可能存在的靶向抑制作用。利用软件初步筛选出可能存在位点匹配的微小RNA18种。再将18种微小RNA通过病毒载体和糖原合成激酶、Axin3’端的非编码区共同转染至293T细胞。通过qPCR和Western blot的检测验证微小RNA的生物学功能。发现miR-15b/16、miR-107对Axin有明显的靶向作用，而糖原合成激酶的作用靶点则与miR-29b，miR-129，miR-124，miR-101等微小RNA相关度较高。说明微小RNA与经典Wnt信号通路存在广泛的交叉作用关系。 Zhang等[32]在促进鼠类MC3T3成骨细胞系和MOL-A5成骨前体细胞系向成骨细胞分化的过程中，通过高通量基因芯片技术筛选其微小RNA表达谱。结果发现在2种细胞系中共同显著上调或下调的微小RNA有38种，通过靶点预测分析其中有13种与DKK-1-mRNA的3’UTR区相关，尤以miR-335-5p上调最为显著。miR-335-5p在调节成骨分化的过程中发挥重要作用。miR-335-5p能够特异性结合于Dkk1-mRNA的3’UTR区，通过转录后调节作用阻遏Dkk1的表达。Dkk1是最为人熟知的骨形成调节因子之一，Dkk1能够钝化Wnt信号通路中重要的LRP5/6受体，从而抑制经典Wnt信号通路的激活，并且近年的研究证明其与地塞米松引起的骨形成抑制相关。miR-335-5p下调Dkk1水平使糖原合成激酶磷酰化，从而提高β-catenin的促转录作用，通过增强经典Wnt信号通路促进成骨分化。Zhou等[33]报道miR-371-373是人胚胎干细胞特异性微小RNA，miR-371-373可以有效的激活经典Wnt信号通路，其作用靶点为T细胞因子/淋巴增强因子1。Zhou等还发现，miR372-373可以影响包括Dkk1在内的4种经典Wnt信号通路相关蛋白基因的表达，提示miR372-373可能通过形成TCF/LEF1- miR372-373- Dkk1的反馈环路发挥对干细胞分化的调节作用。"

References

[1] Pham L, Beyer K, Jensen ED,et al. Bone morphogenetic protein 2 signaling in osteoclasts is negatively regulated by the BMP antagonist, twisted gastrulation.J Cell Biochem. 2011;112(3):793-803.

[2] Park SJ, Jung SH, Jogeswar G,et al. The transcription factor snail regulates osteogenic differentiation by repressing Runx2 expression. Bone. 2010;46(6):1498-1507.

[3] Phinney DG, Prockop DJ. Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair--current views.Stem Cells. 2007; 25(11):2896-2902.

[4] Mason JJ, Williams BO. SOST and DKK: Antagonists of LRP Family Signaling as Targets for Treating Bone Disease. J Osteoporos. 2010;2010. pii: 460120.

[5] Mizuno Y, Yagi K, Tokuzawa Y,et al. miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation. Biochem Biophys Res Commun. 2008;368(2): 267-272.

[6] Nusse R, Theunissen H, Wagenaar E,et al. The Wnt-1 (int-1) oncogene promoter and its mechanism of activation by insertion of proviral DNA of the mouse mammary tumor virus. Mol Cell Biol. 1990;10(8):4170-4179.

[7] Wodarz A, Nusse R. Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol. 1998;14:59-88.

[8] Rao TP, Kühl M. An updated overview on Wnt signaling pathways: a prelude for more. Circ Res. 2010;106(12): 1798-1806.

[9] Cadigan KM, Liu YI. Wnt signaling: complexity at the surface. J Cell Sci. 2006;119(Pt 3):395-402.

[10] Liu C, Li Y, Semenov M,et al. Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell. 2002;108(6):837-847.

[11] He X, Semenov M, Tamai K,et al. LDL receptor-related proteins 5 and 6 in Wnt/beta-catenin signaling: arrows point the way. Development. 2004;131(8):1663-1677.

[12] Zeng X, Huang H, Tamai K,et al. Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions. Development. 2008;135(2):367-375.

[13] Mulholland DJ, Dedhar S, Coetzee GA,et al. Interaction of nuclear receptors with the Wnt/beta-catenin/Tcf signaling axis: Wnt you like to know. Endocr Rev. 2005;26(7):898-915.

[14] Hill TP, Später D, Taketo MM,et al. Canonical Wnt/beta- catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell. 2005;8(5):727-738.

[15] Gaur T, Lengner CJ, Hovhannisyan H,et al. Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression. J Biol Chem. 2005;280(39):33132- 331340.

[16] Qiu W, Andersen TE, Bollerslev J,et al. Patients with high bone mass phenotype exhibit enhanced osteoblast differentiation and inhibition of adipogenesis of human mesenchymal stem cells. J Bone Miner Res. 2007;22(11): 1720-1731.

[17] Diarra D, Stolina M, Polzer K,et al. Dickkopf-1 is a master regulator of joint remodeling.Nat Med. 2007;13(2):156-163.

[18] Wang FS, Lin CL, Chen YJ,et al. Secreted frizzled-related protein 1 modulates glucocorticoid attenuation of osteogenic activities and bone mass. Endocrinology. 2005;146(5): 2415-2423.

[19] Wang FS, Ko JY, Yeh DW,et al. Modulation of Dickkopf-1 attenuates glucocorticoid induction of osteoblast apoptosis, adipocytic differentiation, and bone mass loss. Endocrinology. 2008;149(4):1793-1801.

[20] Harada S, Rodan GA. Control of osteoblast function and regulation of bone mass. Nature. 2003;423(6937):349-355.

[21] de Boer J, Siddappa R, Gaspar C,et al. Wnt signaling inhibits osteogenic differentiation of human mesenchymal stem cells. Bone. 2004;34(5):818-826.

[22] Liu G, Vijayakumar S, Grumolato L,et al. Canonical Wnts function as potent regulators of osteogenesis by human mesenchymal stem cells. J Cell Biol. 2009;185(1):67-75.

[23] Hutvágner G, McLachlan J, Pasquinelli AE,et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA.Science. 2001; 293(5531):834-838.

[24] Nakajima N, Takahashi T, Kitamura R,et al. MicroRNA-1 facilitates skeletal myogenic differentiation without affecting osteoblastic and adipogenic differentiation. Biochem Biophys Res Commun. 2006;350(4):1006-1012.

[25] Wienholds E, Kloosterman WP, Miska E,et al. MicroRNA expression in zebrafish embryonic development. Science. 2005;309(5732):310-311.

[26] Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell. 2005;120(1):15-20.

[27] Chen JF, Mandel EM, Thomson JM,et al. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet. 2006;38(2): 228-233.

[28] Colnot C.Cellular and molecular interactions regulating skeletogenesis. J Cell Biochem. 2005;95(4):688-697.

[29] Huang J, Zhao L, Xing L,et al. MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation. Stem Cells. 2010;28(2):357-364.

[30] Kapinas K, Kessler C, Ricks T,et al. miR-29 modulates Wnt signaling in human osteoblasts through a positive feedback loop.J Biol Chem. 2010;285(33):25221-25231.

[31] Liu Y, Huang T, Zhao X,et al. MicroRNAs modulate the Wnt signaling pathway through targeting its inhibitors. Biochem Biophys Res Commun. 2011;408(2):259-264.

[32] Zhang J, Tu Q, Bonewald LF,et al. Effects of miR-335-5p in modulating osteogenic differentiation by specifically downregulating Wnt antagonist DKK1.J Bone Miner Res. 2011;26(8):1953-1963.

[33] Zhou AD, Diao LT, Xu H,et al. β-Catenin/LEF1 transactivates the microRNA-371-373 cluster that modulates the Wnt/β-catenin-signaling pathway. Oncogene. 2012;31(24): 2968-2978.