吉林大学学报(医学版)  2019, Vol. 45 Issue (04): 971-975 DOI: 10.13481/j.1671-587x.20190441
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生长板软骨的胚基来源于中胚层的间充质细胞[1]，分为静止区(储备区)、增殖区和肥大区[2]，是骨纵向生长的主要分化区域。由经典形态发生素和其他系统及组织特异性因子引发的主要信号通路与生长板软骨的生长发育有密切关联。来自内外界的各种信号刺激能够通过复杂的信号级联反应汇聚于生长板软骨细胞的主要转录因子SRY-盒包含蛋白9(sex determining region Y-box 9, Sox9)上，从而保证软骨系统的正常生长发育[3]。目前已发现多种信号通路参与调控生长板软骨的生长和发育，如转化生长因子β(transforming growth factor-β, TGF-β)/骨形态发生蛋白(bone morphogenetic protein，BMP)信号通路[4]、WNT/β连环蛋白(β-catenin)信号通路[5]、成纤维细胞生长因子(fibroblast growth factor, FGF)信号通路[6]、刺猬因子(Hedgehog，HH)信号通路[7]和Notch信号通路[8]等。目前国内已有综述[9]报道了参与生长板软骨的系统调节因子和局部调节因子，但其对参与生长板软骨的信号通路的介绍还不够全面及完整，且近年来信号通路控制生长板软骨的研究又有新的进展。本文作者全面地介绍控制生长板软骨生长发育的信号通路，阐述异常信号通路导致的相应软骨异常，旨在为临床生长板软骨遗传性疾病的诊断和治疗提供理论依据。
1 TGF-β/ BMP信号通路TGF-β超家族包括TGF-β、BMP和生长分化因子(growth and differentiation factors, GDFs)、活化素和抑制素(activins/inhibins)以及其他亚类[10]。TGF-β受体有3种，分别为Ⅰ型、Ⅱ型和Ⅲ型。TGF-β超家族配体与跨膜丝氨酸/苏氨酸受体结合后，形成异聚配体-受体复合物。其中与配体结合的Ⅱ型受体磷酸化Ⅰ型受体，Ⅰ型受体募集并磷酸化调节性Smad转录因子(regulatory Smad, R-Smads), R-Smads与Smad4形成复合物后进入细胞核调节基因的表达。R-Smads分为2类，一类为由TGF-β激活的AR-Smads，包括Smad2和Smad3；另一类是由BMP等激活的BR-Smads，包括Smad1、Smad5和Smad9(Smad8)。抑制性Smads包括Smad6和Smad7，可与激活的Ⅰ型受体结合，抑制或调节TGF-β家族的信号转导[11]。Sox9属于Sox基因家族中的重要成员，是软骨生长和发育的主要调节转录因子[12]，BMP/TGF-β信号通路能通过调节Sox9的表达，从而调节软骨的发育[13]。
活化素受体样激酶2(activin receptor-like kinase 2, ALK2)可与BMP、TGF-β和活化素配体结合。小鼠软骨细胞中缺失ALK2会明显减弱BMP信号通路的转导，并造成颅面缺损、轴突骨缺损和胸椎后凸，但是生长板软骨无明显变化[14-15]。小鼠软骨细胞中活化素受体样激酶3(activin receptor-like kinase 3, ALK3)和活化素受体样激酶6(activin receptor-like kinase 6, ALK6)的同时缺失会导致软骨广泛性发育不良，无生长板形成[16]。软骨细胞过表达TGF-β Ⅱ型受体，骨骺生长板中促进软骨细胞肥大的基因表达明显增强，导致关节软骨的进行性丧失。小鼠软骨细胞中Smad3基因缺失，软骨细胞肥大过早，肥大区长度增加。小鼠软骨细胞特异性缺失Smad1/5/9，表现为严重的生长板软骨发育不良[17]。Smad1/5双突变小鼠的表型与ALK3/ALK6受体突变小鼠的表型非常相似，表现为致死性软骨发育不良，而大多数Smad9缺陷小鼠可有正常生长板和软骨形成[17]。特异性敲除小鼠软骨细胞中BMP2基因，其生长板软骨细胞的分化和成熟发生障碍[18]，而敲除BMP4基因仅引起轻度软骨表型异常[19]。因此，BMP2是软骨细胞的主要配体。Smad7基因缺失的小鼠体型矮小，生长板中血管长入迟缓并伴随肥大细胞层变薄[20]。Smad6基因缺失的小鼠呈现出类似Smad7基因缺失发育障碍的表型[21]。小鼠软骨细胞和肢芽间充质中TGF-β活化激酶1(TGF-β activated kinase 1, TAK1)特异性缺失导致严重的软骨发育不良、次级骨化中心形成受损、关节形成不全和BMP信号转导减弱[22]。
2 WNT/β-catenin信号通路WNT信号通路能够调节软骨发育和软骨细胞分化。多数WNT信号通过经典的β-连环蛋白(β-catenin)通路发挥作用。当WNT配体不存在时，β-catenin结合在由糖原合成酶激酶3(glycogen synthase kinase 3β, GSK3β)、axin和结肠腺瘤样息肉蛋白(adenomatosis polyposis coli, APC)组成的分解复合物中。GSK3β磷酸化β-catenin，从而触发其泛素化和降解。当WNT配体与受体结合后，GSK3β介导的β-catenin磷酸化过程被抑制。随后β-catenin转移至核内，参与调节下游基因的表达[23]。
β-catenin可通过调节Sox9的表达来维持软骨细胞的分化[24]。小鼠胚胎软骨细胞中β-catenin的失活，导致软骨增殖区Sox9表达增加，小鼠患有严重的侏儒症，并且小鼠软骨细胞增殖减少、软骨细胞肥大延迟和软骨内骨形成受损[25]。低密度脂蛋白受体相关蛋白5/6(LDL receptor related protein 5/6, LRP5/6)为WNT信号通路的辅助受体，LRP5/6基因敲除导致小鼠胚胎期股骨和胫骨生长板发育异常[26]。β-catenin相关蛋白1(β-catenin interacting protein 1, ICAT)能够抑制软骨细胞中的β-catenin，使胫骨生长板增殖区和肥大区减少[27]。
3 FGF信号通路FGF家族的配体可以结合4种不同的FGF受体(FGF receptor, FGFR)：FGFR1、FGFR2、FGFR3和FGFR4。配体与受体结合后，FGFR发生同源二聚化和转自磷酸化，使对接的衔接蛋白FRS2ɑ(FGFR substrate 2ɑ)磷酸化，从而促进其他衔接蛋白的结合以及不同信号级联反应的激活，包括丝裂原活化蛋白激酶(mitogen-activated protein kinases，MAPKs)、磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase, PI3K)、磷脂酶Cγ(PLCγ)和酪氨酸激酶-信号转导及转录激活因子(JAK-STAT)。FGF9和FGF18是软骨内骨形成的重要调节因子，能促进和维持印度刺猬因子(Indian Hedgehog, IHH)和转录受体相关转录因子2(Runt-related transcription factor-2, Runx2)的表达，参与生长板软骨的血管化。FGFR3信号通过活化STAT1、ERK1/2和p38 MAPK信号通路，调节生长板的形成和软骨内骨化。FGFR3还可以通过调节BMP、WNT、IHH和甲状旁腺激素相关蛋白(parathyroid hormone-related protein, PTHrP)等其他信号通路，间接调控生长板软骨细胞的生长和发育[28]。小鼠FGFR3的缺失导致软骨细胞增殖增加，肥大区增厚，而FGFR3过表达则表现为生长板非常短小，软骨发育不全，以致造成严重的侏儒症[29]。FGFR1和FGFR2在生长板形成的过程中发挥着重要作用。小鼠FGFR1和FGFR2基因缺失，可导致生长板的长度变短，增殖区软骨细胞数量减少[30]。FGF信号可以激活丝裂原活化细胞外调节激酶1(mitogen-activated extracellular regulated kinase 1, MEK1)，使MEK1磷酸化并激活细胞外调节蛋白激酶1/2(extracellular signal-regulated kinase, ERK1/2)。软骨细胞中MEK1的激活，可通过抑制肥大细胞的分化进而导致软骨发育不全。FGFR3信号可通过活化ERK1/2调节生长板的形成和软骨内骨化。软骨细胞特异性敲除ERK1/2基因会引起严重的软骨发育不全，而肥大软骨细胞中ERK1/2基因敲除则会引起肥大区增厚以及软骨细胞终末分化障碍[31]。MEK1和ERK1/2也可以被迅速加速性纤维肉瘤(rapidly accelerated fibrosarcoma, RAF)激酶激活。软骨细胞中特异性敲除a-RAF和b-RAF不会影响软骨内骨发育。然而，软骨细胞中c-RAF的特异性敲除会引起肥大区扩张，软骨细胞凋亡减少，生长板血管侵入受损，体内ERK1/2磷酸化水平降低[32]。
4 HH信号通路脊椎动物体内有3种HH配体：音猬因子(Sonic Hedgehog，SHH)、IHH和沙漠刺猬因子(Desert Hedgehog，DHH)。在HH配体不存在的情况下，G蛋白偶联受体Smoothened(Smo)会被跨膜蛋白受体Patched(Ptc)抑制。当HH配体与Ptc结合时，Smo的抑制被解除，Gli转录因子被激活[33]，从而调节下游基因的表达。SHH缺失的小鼠，在出生时死亡[34]。IHH由肥大软骨细胞产生，在软骨细胞分化中起关键作用。IHH基因缺失的小鼠在胚胎期显示出严重的骨骼生长迟缓，软骨细胞增殖降低，软骨细胞过早和异常肥大[35]。DHH在软骨和骨中不表达。
5 Notch信号通路Notch信号通路在软骨形成中起重要作用。Notch受体结合Delta或Jagged后，分裂形成NEXT(Notch extracellular truncation)片段，随后该片段被γ-secretase蛋白酶切割产生Notch细胞内区(Notch intracellular domain, NICD)。NICD转移到细胞核中，与转录因子重组结合蛋白J (recombinationbinding protein-J, RBPJ)和转录激活因子MAML1(mastermind-like-1)结合。NICD、RBPJ和MAML1三者形成的复合物促进HES和HEY家族的碱性螺旋-环-螺旋蛋白(basic helix-loop-helix, bHLH)转录因子的表达，从而抑制下游靶基因的表达[36]。
在Notch信号功能增强的小鼠模型中，Notch作用于Sox9和Runx2的上游，抑制软骨细胞的增殖[37]。NICD在软骨细胞中的特异性过表达会引起软骨细胞增殖降低以及软骨细胞肥大障碍，而这些细胞中Notch功能的丧失增加了肥大区的厚度, Notch通过抑制Sox9的表达来负性调节中轴骨中软骨细胞的分化[38]。研究[39]显示:Notch信号通路通过调节Sox9的表达以调节软骨细胞的肥大。骨软骨祖细胞中Notch受体的缺失促进了软骨细胞的分化[40]。以上研究表明:Notch信号通路主要通过调控细胞生长和发育的转录因子的表达，从而调控生长板软骨的发育。
6 影响生长板软骨发育的其他信号通路和因素除了上述信号通路外，PTHrP信号通路、视黄酸(retinoic acid, RA)信号通路、雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)信号通路和胰岛素样生长因子(insulin-like growth factor, IGF)信号通路对生长板软骨的生长和发育也有着至关重要的作用。PTHrP在静止区软骨细胞中表达，能维持软骨细胞的增殖，而生长板中距离静止区更远的软骨细胞不接受PTHrP信号。有研究[41]表明：小鼠中PTHrP基因的缺失加速了软骨细胞的肥大和矿化。RA信号通路能够促进生长板软骨的生长以及软骨细胞的分化[42]。mTOR信号是软骨细胞增殖和肥大的重要协调因子，mTOR参与形成2种不同的复合物：mTOR complex 1(mTORC1)和mTOR complex 1(mTORC2)。最近的一项关于mTOR信号通路研究[43]表明：mTORC1促进早期软骨细胞的增殖并抑制其晚期的终末分化。IGF信号通路在软骨内骨化中具有非常重要的作用[44]。除了信号通路外，研究[45]表明生物钟也能调节生长板软骨的稳态。
7 展望生长板软骨的形成是一个动态的、协调一致的过程。各个信号通路相互作用才能保证生长板软骨的正常形成和发育。目前针对各种生长板软骨的疾病，例如生长板软骨发育不良、软骨细胞肥大障碍、初级和次级骨化中心形成受损等，治疗方案及功效远远不能满足人们的预期，仍需进一步研究。今后不仅要研究调控生长板软骨生长发育的信号通路，更需要将现有的知识运用到临床当中，解决临床中存在的各种生长板软骨发育异常以及由其异常而导致的骨骼疾病。
| [1] | DE LUCA F. Regulatory role for growth hormone in statural growth:IGF-dependent and IGF-independent effects on growth plate chondrogenesis and longitudinal bone growth[J]. Pediatr Endocrinol Rev, 2018, 16(Suppl 1): 33–38. |
| [2] | 李松. 下颌髁状突软骨与生长板软骨生长发育的比较研究[J]. 昆明医科大学学报, 2014, 35(2): 1–4. |
| [3] | BONAFE L, CORMIER-DAIRE V, HALL C, et al. Nosology and classification of genetic skeletal disorders:2015 revision[J]. Am J Med Genet A, 2015, 167A(12): 2869–2892. |
| [4] | GARRISON P, YUE SN, HANSON J, et al. Spatial regulation of bone morphogenetic proteins (BMPs) in postnatal articular and growth plate cartilage[J]. PLoS One, 2017, 12(5): e0176752. DOI:10.1371/journal.pone.0176752 |
| [5] | AICHER S, KAKKANAS A, COHEN L, et al. Differential regulation of the Wnt/β-catenin pathway by hepatitis C virus recombinants expressing core from various genotypes[J]. Sci Rep, 2018, 8(1): 11185. DOI:10.1038/s41598-018-29078-2 |
| [6] | KIMURA T, OZAKI T, FUJITA K, et al. Proposal of patient-specific growth plate cartilage xenograft model for FGFR3 chondrodysplasia[J]. Osteoarthr Cartilage, 2018, 26(11): 1551–1561. DOI:10.1016/j.joca.2018.07.015 |
| [7] | HARAGUCHI R, KITAZAWA R, IMAI Y, et al. Growth plate-derived hedgehog-signal-responsive cells provide skeletal tissue components in growing bone[J]. Histochem Cell Biol, 2018, 149(4): 365–373. DOI:10.1007/s00418-018-1641-5 |
| [8] | ZHENG YX, LIU CC, NI L, et al. Cell type-specific effects of Notch signaling activation on intervertebral discs:Implications for intervertebral disc degeneration[J]. J Cell Physiol, 2018, 233(7): 5431–5440. DOI:10.1002/jcp.26385 |
| [9] | 徐真然, 罗飞宏. 生长板的局部调控新进展[J]. 医学综述, 2018, 24(19): 3772–3776. DOI:10.3969/j.issn.1006-2084.2018.19.006 |
| [10] | SHI C, IURA A, TERAJIMA M, et al. Deletion of BMP receptor type IB decreased bone mass in association with compromised osteoblastic differentiation of bone marrow mesenchymal progenitors[J]. Sci Rep, 2016, 6: 24256. DOI:10.1038/srep24256 |
| [11] | SAMSA WE, ZHOU X, ZHOU G. Signaling pathways regulating cartilage growth plate formation and activity[J]. Semin Cell Dev Biol, 2017, 62: 3–15. DOI:10.1016/j.semcdb.2016.07.008 |
| [12] | LEFEBVRE V, DVIR-GINZBERG M. SOX9 and the many facets of its regulation in the chondrocyte lineage[J]. Connect Tissue Res, 2017, 58(1): 2–14. DOI:10.1080/03008207.2016.1183667 |
| [13] | HISCOCK T W, TSCHOPP P, TABIN C J. On the formation of digits and joints during limb development[J]. Dev Cell, 2017, 41(5): 459–465. DOI:10.1016/j.devcel.2017.04.021 |
| [14] | VAN DINTHER M, VISSER N, DE GORTER DJ, et al. ALK2 R206H mutation linked to fibrodysplasia ossificans progressiva confers constitutive activity to the BMP type Ⅰ receptor and sensitizes mesenchymal cells to BMP-induced osteoblast differentiation and bone formation[J]. J Bone Miner Res, 2010, 25(6): 1208–1215. |
| [15] | RIGUEUR D, BRUGGER S, ANBARCHIAN T, et al. The type Ⅰ BMP receptor ACVR1/ALK2 is required for chondrogenesis during development[J]. J Bone Miner Res, 2015, 30(4): 733–741. DOI:10.1002/jbmr.2385 |
| [16] | YOON BS, OVCHINNIKOV DA, YOSHⅡ I, et al. Bmpr1a and Bmpr1b have overlapping functions and are essential for chondrogenesis in vivo[J]. Proc Natl Acad Sci U S A, 2005, 102(14): 5062–5067. DOI:10.1073/pnas.0500031102 |
| [17] | RETTING K N, SONG B, YOON B S, et al. BMP canonical Smad signaling through Smad1 and Smad5 is required for endochondral bone formation[J]. Development, 2009, 136(7): 1093–1104. DOI:10.1242/dev.029926 |
| [18] | ZHANG H, BRADLEY A. Mice deficient for BMP2 are nonviable and have defects in amnion/chorion and cardiac development[J]. Development, 1996, 122(10): 2977–2986. |
| [19] | SHU B, ZHANG M, XIE R, et al. BMP2, but not BMP4, is crucial for chondrocyte proliferation and maturation during endochondral bone development[J]. J Cell Sci, 2011, 124(Pt 20): 3428–3440. |
| [20] | ESTRADA K D, WANG W G, RETTING K N, et al. Smad7 regulates terminal maturation of chondrocytes in the growth plate[J]. Dev Biol, 2013, 382(2): 375–384. DOI:10.1016/j.ydbio.2013.08.021 |
| [21] | ESTRADA K D, RETTING K N, CHIN A M, et al. Smad6 is essential to limit BMP signaling during cartilage development[J]. J Bone Miner Res, 2011, 26(10): 2498–2510. DOI:10.1002/jbmr.v26.10 |
| [22] | GAO L, SHEU T J, DONG YF, et al. TAK1 regulates SOX9 expression in chondrocytes and is essential for postnatal development of the growth plate and articular cartilages[J]. J Cell Sci, 2013, 126(Pt 24): 5704–5713. |
| [23] | KAWANO Y, KYPTA R. Secreted antagonists of the Wnt signalling pathway[J]. J Cell Sci, 2003, 116(Pt 13): 2627–2634. |
| [24] | BHATTARAM P, PENZO-MÉNDEZ A, KATO K, et al. SOXC proteins amplify canonical WNT signaling to secure nonchondrocytic fates in skeletogenesis[J]. J Cell Biol, 2014, 207(5): 657–671. DOI:10.1083/jcb.201405098 |
| [25] | AKIYAMA H, LYONS J P, MORI-AKIYAMA Y, et al. Interactions between Sox9 and beta-catenin control chondrocyte differentiation[J]. Genes Dev, 2004, 18(9): 1072–1087. DOI:10.1101/gad.1171104 |
| [26] | CHAO B N, BALDWIN W H, HEALEY J F, et al. Characterization of a genetically engineered mouse model of hemophilia A with complete deletion of the F8 gene[J]. J Thromb Haemost, 2016, 14(2): 346–355. DOI:10.1111/jth.13202 |
| [27] | ZHU M, CHEN M, ZUSCIK M, et al. Inhibition of beta-catenin signaling in articular chondrocytes results in articular cartilage destruction[J]. Arthritis Rheum, 2008, 58(7): 2053–2064. DOI:10.1002/art.v58:7 |
| [28] | HUNG I H, SCHOENWOLF G C, LEWANDOSKI M, et al. A combined series of Fgf9 and Fgf18 mutant alleles identifies unique and redundant roles in skeletal development[J]. Dev Biol, 2016, 411(1): 72–84. DOI:10.1016/j.ydbio.2016.01.008 |
| [29] | ORNITZ D M, MARIE P J. Fibroblast growth factor signaling in skeletal development and disease[J]. Genes Dev, 2015, 29(14): 1463–1486. DOI:10.1101/gad.266551.115 |
| [30] | KARUPPAIAH K, YU K, LIM J, et al. FGF signaling in the osteoprogenitor lineage non-autonomously regulates postnatal chondrocyte proliferation and skeletal growth[J]. Development, 2016, 143(10): 1811–1822. DOI:10.1242/dev.131722 |
| [31] | MATSUSHITA T, CHAN Y Y, KAWANAMI A, et al. Extracellular signal-regulated kinase 1(ERK1) and ERK2 play essential roles in osteoblast differentiation and in supporting osteoclastogenesis[J]. Mol Cell Biol, 2009, 29(21): 5843–5857. DOI:10.1128/MCB.01549-08 |
| [32] | LIU ES, RAIMANN A, CHAE BT, et al. c-Raf promotes angiogenesis during normal growth plate maturation[J]. Development, 2016, 143(2): 348–355. DOI:10.1242/dev.127142 |
| [33] | LEE R T, ZHAO Z, INGHAM P W. Hedgehog signalling[J]. Development, 2016, 143(3): 367–372. DOI:10.1242/dev.120154 |
| [34] | CHIANG C, LITINGTUNG Y, LEE E, et al. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function[J]. Nature, 1996, 383(6599): 407–413. DOI:10.1038/383407a0 |
| [35] | AMANO K, DENSMORE M J, LANSKE B. Conditional deletion of Indian Hedgehog in limb mesenchyme results in complete loss of growth plate formation but allows mature osteoblast differentiation[J]. J Bone Miner Res, 2015, 30(12): 2262–2272. DOI:10.1002/jbmr.2582 |
| [36] | ENGIN F, LEE B. NOTCHing the bone:insights into multi-functionality[J]. Bone, 2010, 46(2): 274–280. DOI:10.1016/j.bone.2009.05.027 |
| [37] | WANG C C, INZANA J A, MIRANDO A J, et al. NOTCH signaling in skeletal progenitors is critical for fracture repair[J]. J Clin Invest, 2016, 126(4): 1471–1481. DOI:10.1172/JCI80672 |
| [38] | CHEN S, TAO J N, BAE Y J, et al. Notch gain of function inhibits chondrocyte differentiation via Rbpj-dependent suppression of Sox9[J]. J Bone Miner Res, 2013, 28(3): 649–659. DOI:10.1002/jbmr.1770 |
| [39] | KOHN A, RUTKOWSKI T P, LIU Z Y, et al. Notch signaling controls chondrocyte hypertrophy via indirect regulation of Sox9[J]. Bone Res, 2015, 3: 15021. DOI:10.1038/boneres.2015.21 |
| [40] | RUTKOWSKI T P, KOHN A, SHARMA D, et al. HES factors regulate specific aspects of chondrogenesis and chondrocyte hypertrophy during cartilage development[J]. J Cell Sci, 2016, 129(11): 2145–2155. DOI:10.1242/jcs.181271 |
| [41] | VORTKAMP A, LEE K, LANSKE B, et al. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein[J]. Science, 1996, 273(5275): 613–622. DOI:10.1126/science.273.5275.613 |
| [42] | CUNNINGHAM T J, DUESTER G. Mechanisms of retinoic acid signalling and its roles in organ and limb development[J]. Nat Rev Mol Cell Biol, 2015, 16(2): 110–123. DOI:10.1038/nrm3932 |
| [43] | YAN B, ZHANG Z M, JIN D D, et al. mTORC1 regulates PTHrP to coordinate chondrocyte growth, proliferation and differentiation[J]. Nat Commun, 2016, 7: 11151. DOI:10.1038/ncomms11151 |
| [44] | MURRAY P G, HANSON D, COULSON T, et al. 3-M syndrome:a growth disorder associated with IGF2 silencing[J]. Endocr Connect, 2013, 2(4): 225–235. DOI:10.1530/EC-13-0065 |
| [45] | REALE M E, WEBB I C, WANG X, et al. The transcription factor Runx2 is under circadian control in the suprachiasmatic nucleus and functions in the control of rhythmic behavior[J]. PLoS One, 2013, 8(1): e54317. DOI:10.1371/journal.pone.0054317 |
