Research progress of Hedgehog signaling pathway regulating RANKL expression in osteoblasts

doi:10.3969/j.issn.2095-4344.2017.08.025

Abstract

Abstract:

BACKGROUND: Hedgehog signaling pathway is shown to contribute to the osteogenesis of bone marrow mesenchymal stem cells, and to play an important role in regulating the expression of RANKL in osteoblasts. Nowadays, the definite signal transduction pathway has been revealed gradually.
OBJECTIVE: To review the research progress of RANKL in osteoblasts that regulated by hedgehog signaling pathway.
METHODS: A computer-based online search in CNKI, Google Scholar and PubMed databases was performed with the key words of “Hedgehog, osteoblast, BMSCs, PTHrP, CREB, NFAT, RANKL” in English and Chinese, respectively. Literatures related to the expression of RANKL in osteoblasts that regulated by the Hedgehog signaling pathway were included initially and 37 eligible articles were extensively summarized for review.
RESULTS AND CONCLUSION: The Hedgehog signaling pathway plays an advanced role in the osteogenic differentiation of bone marrow mesenchymal stem cells, and also upregulates intracellular parathyroid hormone related protein, which activates its downstream signaling molecules cAMP response element binding protein and nuclear factor of activated T cells ulteriorly, to promote the expression of RANKL in osteoblasts and increase the differentiation and formation from osteoclast precursor cells to mature osteoclasts.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Osteoblasts, Mesenchymal Stem Cells, RANK Ligand, Tissue Engineering

CLC Number:

R318

Wu Xiu-tuan, Li Wen-liang, Xie Liu-rong, Liao Hong-bing. Research progress of Hedgehog signaling pathway regulating RANKL expression in osteoblasts[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(8): 1294-1300.

Figures/Tables 1

2.1 Hedgehog信号通路在哺乳动物中，Hedgehog基因编码3种同源形式蛋白，即音速刺猬蛋白(Sonic hedgehog, SHedgehog)、印度刺猬蛋白(Indian hedgehog, IHedgehog)、沙漠刺猬蛋白(Desert hedgehog, DHedgehog) [3]。SHedgehog在神经系统、皮肤、消化道中广泛表达[4]，IHedgehog主要在骨、软骨、消化道、胰腺中表达，DHedgehog主要在生殖器中表达，也在外周神经和胰腺中表达[5-6]。Hedgehog信号传递受靶细胞膜上2种受体Patched(Ptc)和Smoothened (Smo)的控制。Ptc由肿瘤抑制基因Patched编码，是12次跨膜蛋白，能与配体直接结合，对Hedgehog信号起负调控作用[7]；Smo由原癌基因Smoothened编码，是7次跨膜蛋白，与G蛋白偶联受体同源，是Hedgehog信号传递所必须的受体[8]。在缺乏Hedgehog蛋白的情况下，Smo的活性被Ptc所抑制，而Hedgehog蛋白存在时，Hedgehog与 Ptc结合，解除Ptc对Smo的抑制作用，使得Smo被蛋白激酶A、CKI、GSK3磷酸化，同时Smo分子的C-末端发生构象改变，由闭合的无活性的状态转变为开放的有活性的状态，进而激活下游通路[9-11]。磷酸化的Smo通过与Cos2之间的动态相互作用将Cos2/Fu复合物招募到质膜上，从而诱导Fu二聚化。二聚化的Fu通过自我磷酸化激活，进而磷酸化其底物Cos2与Sufu，将Hedgehog信号传递至下游。这一过程将促使全长的转录因子Gli进入细胞核内启动目的基因的表达。Gli蛋白是分子量较大的多功能转录因子，定位于细胞核和细胞浆，将信号传递至核内。该蛋白家族成员只有在维持全长时才具有转录激活子的功能，启动下游靶基因的转录；当其羧基端被蛋白酶体水解后，就形成了转录抑制子，抑制下游靶基因的转录。 2.2 Hedgehog信号通路参与骨髓间充质干细胞成骨向分化的调节既往研究中，Hedgehog信号通路在骨代谢中的作用最初主要表现为促进骨生成，对胚胎期骨骼发育至关重要，若胚胎发育阶段Hedgehog信号蛋白缺乏将导致次级骨化中心和生长板的缺失，从而导致各种骨骼相关性疾病的发生[12]。有研究发现，SHedgehog可通过上调核心结合因子2(Runt-related transcription factor 2，Runx2)、特异性转录因子表达而促进骨髓间充质干细胞的成骨细胞向分化，增加成骨细胞的形成，并阻断骨髓间充质干细胞的成脂向分化，其中，特异性转录因子是作为Runx2的下游靶物介导成骨细胞分化，而Tian等[13-14]研究发现，成骨细胞样细胞经SHedgehog处理后，特异性转录因子与Runx2基因表达均有提高，但特异性转录因子基因表达却早于Runx2，这表明特异性转录因子可能并不是单独由Runx2调控，或有可能作为SHedgehog促进成骨细胞分化的直接作用靶点。Shimoyama等[15]研究发现，IHedgehog可以通过激活下游转录因子Gli2而刺激骨髓间充质干细胞碱性磷酸酶与骨钙素表达以及钙化作用。Gli2缺失可以明显抑制IHedgehog对骨髓间充质干细胞成骨向分化的诱导作用，而且IHedgehog或Gli2过表达均可上调参与成骨分化的重要转录因子Runx2的表达，并激活Runx2的成骨功能。通过免疫共沉淀分析显示，Gli2和Runx2之间存在物理交互，骨髓间充质干细胞中Runx2缺乏时，即使IHedgehog或Gli2过表达也不能增强骨髓间充质干细胞中碱性磷酸酶活性。由此可知，Gli2在IHedgehog通过调节Runx2诱导骨髓间充质干细胞成骨向分化过程中具有重要作用，Runx2缺乏将抑制IHedgehog或Gli2过表达对骨髓间充质干细胞成骨向分化的促进作用。在Tu等[16]的实验中，Runx2过表达可以促使Runx2基因缺失胚胎发育过程中的正常骨形成，却不能促使IHedgehog基因缺失胚胎发育过程中的正常骨形成，说明IHedgehog诱导骨髓间充质干细胞成骨向分化除了Runx2外，很有可能还需要其他因子的参与。Hedgehog家族成员可在多种组织和细胞中上调骨形态发生蛋白的表达，也有可能是IHedgehog通过上调骨形态发生蛋白的表达而促进骨髓间充质干细胞的成骨向分化。总之，Hedgehog调节骨髓间充质干细胞成骨向分化的分子机制较复杂且尚未完全清楚，仍有待充分阐明。 2.3 Hedgehog信号通路参与成骨细胞RANKL表达的调节而近几年的研究却发现，在已分化成熟的成骨细胞中Hedgehog信号活性增强却能间接导致破骨细胞增殖分化的活动明显增强。美国Ohba等[17]研究人员发现，小鼠出生后随着成骨细胞的不断分化成熟，Hedgehog信号通路的活性也随之逐渐下降。选择性上调Hedgehog信号通路的活性时，会引起骨形成的增加，但形成的骨质非常脆弱、疏松，同时也出现明显的骨吸收，最终导致突变老鼠产生严重的骨质缺乏。这说明，增强成骨细胞Hedgehog信号通路的活性，成骨改建活动与骨吸收活动均有所增强，但是骨吸收的速度要强于骨形成的速度。其中，骨形成可能是由于Hedgehog信号通路活性增强后，促进成骨细胞的增殖分化和减缓成骨细胞凋亡；而骨吸收则是成熟的成骨细胞中Hedgehog信号通路活性增强，进而促进与破骨细胞增殖分化密切相关的RANKL的表达而加快骨的吸收。 2.3.1 Hedgehog信号通路通过上调甲状旁腺激素相关蛋白调节RANKL的表达有研究表明，Hedgehog信号通路对成骨细胞RANKL基因表达的调控，离不开甲状旁腺相关蛋白的介导作用[18]。甲状旁腺激素相关蛋白是一种旁分泌因子，由关节周围的骨细胞和软骨细胞分泌，而其受体主要由成骨细胞、前肥大软骨细胞、早期肥大软骨细胞以及一些骨髓细胞分泌。成骨细胞经过甲状旁腺激素相关蛋白处理培养后表现为RANKL的表达量增加，而骨保护素的表达量下降；然而用Hedgehog信号通路抑制剂环杷明处理后，其骨保护素基因表达未见明显改变，反而甲状旁腺激素相关蛋白和RANKL的表达量却明显减少[19]，这说明Hedgehog信号通路在调节已分化成熟的成骨细胞RANKL表达时，需要有甲状旁腺激素相关蛋白的介导作用，通过激活其下游一连串的信号分子而促进RANKL的表达。增强SHedgehog信号活性能增加正常小鼠颅骨成骨细胞RANKL和甲状旁腺激素相关蛋白的表达，而甲状旁腺激素相关蛋白基因敲除小鼠成骨细胞的SHedgehog信号活性增强却不能增加RANKL的表达[19]，也证实了甲状旁腺激素相关蛋白在Hedgehog信号通路调节成骨细胞RANKL表达中的关键作用。 RANKL和骨保护素主要由成骨细胞表达，是调节破骨细胞增殖分化的主要调节因子，两者的动态平衡调控着破骨细胞的增殖与分化，而且两者均受Hedgehog信号通路调节。在选择性上调小鼠成骨细胞Hedgehog信号通路活性的实验中，成骨细胞中RANKL和骨保护素的表达均有增加[20]，但RANKL的表达更为明显，而且甲状旁腺激素相关蛋白的表达量也有所增加[9]，这些相关因子的增加也早于成骨细胞标记物的出现，表明RANKL的分泌表达以及Hedgehog信号通路对甲状旁腺激素相关蛋白表达的调节作用并不依赖于成骨细胞分化的程度。总之，增强Hedgehog信号通路活性而导致的骨吸收主要是通过甲状旁腺激素相关蛋白提高了RANKL与骨保护素的比例[21]，从而促进破骨细胞的增殖分化。 2.3.2 甲状旁腺激素相关蛋白调节RANKL表达的作用机制 Mak等[19]通过基因修饰敲除小鼠Hedgehog信号的膜结合性功能抑制性分子Ptch，建立成骨细胞Hedgehog信号增强的小鼠模型，结果发现，小鼠破骨细胞形成增多，骨量减少，出现骨质疏松症，而且该症状的出现与蛋白激酶A、环磷酸腺苷应答元件结合蛋白被激活密切相关。同时，Kim等[22-24]的研究也表明，甲状旁腺激素相关蛋白诱导成骨细胞RANKL表达过程中，cAMP/PKA/CREB信号调控轴是重要的轴路之一，其中环磷酸腺苷应答元件结合蛋白是该下游调节轴中的枢纽，而且蛋白激酶A被激活后也同时抑制了骨保护素的表达。环磷酸腺苷应答元件结合蛋白作为一种转录因子，经小鼠ORMDL3基因进一步研究，环磷酸腺苷应答元件结合蛋白信号分子的作用机制得以揭示，即环磷酸腺苷应答元件结合蛋白激活后将与目标基因启动子区域上的CRE位点或CRE半位点结合，促进基因的转录翻译[25]。蛋白激酶A在骨形成中扮演着重要角色，其位于细胞质基质中，是一种惰性酶，由2个调节亚基和2个酶催化亚基组成。甲状旁腺激素相关蛋白与成骨细胞膜上的受体结合后，通过激活细胞膜上的腺苷酸环化酶，使其分解ATP产生环磷酸腺苷，环磷酸腺苷水平提高后与蛋白激酶A上的调节亚基结合，将蛋白激酶A激活，紧接着蛋白激酶A上的酶催化亚基被释放，然后进入到细胞核，促使环磷酸腺苷应答元件结合蛋白磷酸化并与RANKL基因的远端增强子结合而发挥作用[26-27]。激活cAMP/PKA/CREB信号调控轴需要甲状旁腺激素相关蛋白作为引物启动，而甲状旁腺激素相关蛋白本身又受到Hedgehog信号的调控，因此，上述信号分子构成了一条串联信号轴，调控成骨细胞RANKL的表达。近几年的实验研究中发现，在受甲状旁腺激素相关蛋白激活作用的成骨细胞中，环磷酸腺苷应答元件结合蛋白结合到RANKL基因增强子上的CRE结合位点，并激活其活性，该过程需要有活化T细胞核因子的协同参与，当活化T细胞核因子缺乏时，甲状旁腺激素相关蛋白对RANKL基因增强子活性的增强作用将受到抑制，而甲状旁腺激素相关蛋白诱导的环磷酸腺苷应答元件结合蛋白磷酸化水平却没有受到明显影响[28-29]，说明活化T细胞核因子在甲状旁腺激素相关蛋白激活RANKL基因活化表达的作用中是不可或缺的。活化T细胞核因子是一种转录因子，其家族成员包括：活化T细胞核因子1(NFATc2)、活化T细胞核因子2(NFATc1)、活化T细胞核因子3(NFATc4)、活化T细胞核因子4(NFATc3)和活化T细胞核因子5。活化T细胞核因子发挥作用的过程可分为2个步骤: ①活化T细胞核因子的激活与核质穿梭；②DNA结合和反式激活。活化T细胞核因子的激活与核质穿梭主要是通过钙调磷酸酶与组成型激酶的动力学相互作用来调控。活化T细胞核因子在激活靶基因时常常与其他转录因子发生协同作用，在静息状态下，活化T细胞核因子处于一个高度磷酸化的状态位于胞浆内，当磷酸化的环磷酸腺苷应答元件结合蛋白与细胞核内的RANKL基因增强子结合时，活化T细胞核因子经过钙调磷酸酶去磷酸化后，由胞浆被转运至细胞核，与环磷酸腺苷应答元件结合蛋白共同调节RANKL基因的表达。 Takami等[30]经实验发现，激活成骨细胞膜上的Ca2+通道载体可同时增加RANKL和骨保护素的表达，但RANKL的表达水平是骨保护素的2倍，表明增加成骨细胞内Ca2+浓度可提高RANKL/OPG的比例，从而促进破骨细胞的增殖分化，其作用机制与促进活化T细胞核因子过表达密切相关。甲状旁腺激素相关蛋白是参与形成高钙血症的一个重要因子，引起血中Ca2+浓度提高[31-33]，而成骨细胞膜上存在Ca2+感受器，当细胞外Ca2+浓度提高时，可引起Ca2+向胞内转运，导致细胞内Ca2+水平升高[34]。成骨细胞中的Ca2+浓度升高达到阈值时，可激活CN/NFAT信号调控轴，增加活化T细胞核因子的表达水平和转录活性，促进活化T细胞核因子转运并聚集于细胞核内，"

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