Smads在骨形态发生蛋白诱导成骨中的作用

Smads蛋白是骨形态发生蛋白细胞内信号传导中起重要作用的转录因子,在骨形态发生蛋白诱导骨形成中发挥着关键作用.骨形态发生蛋白可以激活多种Smads蛋白并使其转移至细胞核,通过直接与特定DNA序列相结合、与核调节因子形成复合体、募集转录共活化或共抑制因子等3种方式调节目的基因的转录,最终诱导骨形成.该文就Smads蛋白的定义与分类、Smads信号转导通路及其在骨形态发生蛋白诱导成骨中的作用机制、BMP/Smads信号调节通路与其他通路之间的关系等相关研究作一综述.     

骨形态发生蛋白信号传导和功能调节的结构基础

骨形态发生蛋白(BMP)是已知最具特异性的骨形成诱导因子.BMP通过先后结合并活化细胞膜表面Ⅱ型受体(BMP-RⅡ)和Ⅰ型受体(BMP-RⅠ),进而激活Smad蛋白细胞内信号传导通路,最终诱导细胞核内的靶基因转录及蛋白表达这一经典信号传导途径来发挥其生物活性.BMP信号传导途径中某些关键步骤和功能调节机制仍不明确.某些BMP晶体结构的发现,使得对其信号传导过程的认识空前提高,尤其在信号传导复合物形成和细胞外功能调控等方面.该文就BMP配体与受体间作用、细胞外拮抗物和细胞膜水平调节三个方面的相关研究作一综述.     

调控骨形态发生蛋白诱导成骨的相关转录因子

骨形态发生蛋白（Bone morphogenetic protein,BMPs）是一种确切具有诱导成骨活性的生长因子,其主要生物学作用是诱导未分化的间充质细胞分化形成软骨和骨。迄今为止,至少发现了15种BMP,除BMP1外,其他BMPs均属于转化生长因子β（transforming growth factor-beta,TGF-β）超家族成员,它们可通过旁分泌和自分泌的形式诱导骨、软骨及与骨有关的结缔组织的形成,还能诱导骨源和非骨源性细胞的成骨分化。转录因子是一种核蛋白,是一类可以通过与下游靶基因DNA结合,从而调控下游基因转录的分子。随着对BMPs诱导成骨研究的深入,一些与BMPs相关的转录因子日益受到关注,对它们的研究也成为热点。根据目前的研究结果发现,调控BMPs诱导成骨的相关转录因子可分为两类,包括正调控因子（如Cbfαl、Osterix、Dlx5、Hoxc8和Msx2等）和负调控因子（如CIZ、AJ18、Tob和c-Ski等）,本综述对它们的研究进展分别作以介绍。     

软骨内成骨的调控

骨骼的纵向生长不仅需要激素的调节，同时也需要旁分泌的局部调节因子的调节，因而调节因子可分为系统性调节因子和局部调节因子。系统性调节因子包括生长激素、甲状腺素、雌激素、雄激素、维生素D3、视黄酸及皮质激素等。局部调节因子包括骨形态发生蛋白（BMPs）、成纤维细胞生长因子（FGFs）、Ihh／甲状旁腺素相关肽（PTHrP）及类视黄醇等，它们与核内转录因子相互作用，调节与控制软骨内成骨的进程。现对近年来软骨内成骨的主要调节因子研究综述如下。     

骨形态发生蛋白分子信号转导的研究进展

骨形态发生蛋白在骨、软骨、神经组织、心脏、肾和肺等不同组织发挥着多种生理作用，其信号转导主要通过丝氨酸／苏氨酸激酶受体和Smads蛋白来实现，同时受胞外拮抗剂、假受体和I—Smads蛋白等因素的调节．对骨形态发生蛋白信号转导机制的深入了解，为将来实现对骨形态发生蛋白生理作用的药物调控奠定了基础。     

Runx2基因参与骨代谢相关通路的研究进展

随着老龄化时代的到来,骨质疏松症成为人们研究的热点,而Runx2基因作为一种成骨分化特异性转录因子,参与多种信号通路调控成骨分化的过程。本文仅就Runx2基因参与转化生长因子-β(transforming growth factor-β,TGF-β)、骨形态发生蛋白(bone morphogenetic proteins,BMPs)通路、Notch信号通路及Wnt信号通路的研究进展进行综述。     

骨诱导活性研究进展

骨诱导理论是骨愈合机制中继自身成骨作用和骨传导理论后提出的又一学说。无论有无骨移植,骨诱导活性均在骨愈合机制中发挥重要作用。以往对骨诱导的理解只局限于骨形态发生蛋白及其异位诱导成骨现象。随着骨诱导理论的发展,现已发现其他相关骨生长因子在骨愈合机制中也具有诱导成骨作用。目前时这些骨生长因子的研究已进入体内如何与受体作用以表达细胞内外信号转导和它们之间如何相互作用而产生或引起成骨性细胞分化、增殖等诱导作用的分子学机制阶段,且已利用重组基因技术生产这些骨生长因子并在临床应用中取得初步成果。     

神经营养因子家族及其受体在重组人骨形态发生蛋白-2诱导成骨中的表达

[目的]通过观察重组人骨形态发生蛋白-2（rhBMP-2）诱导成骨过程中神经营养因子（NTFs）家族及受体的表达，探讨神经营养因子在BMP骨诱导中的作用。[方法]建立小鼠右侧股后部异位成骨模型，实验组植入rhBMP-2胶原复合物，对照组仅植入相同体积的胶原海绵。分别于术后7、14和21d取材，进行组织学、免疫组化及RT-PCR检测。[结果]组织学证实rhBMP-2胶原复合物具有良好的骨诱导能力，而对照组未见成骨。术后第7d，在大量软骨形成期NGF和TrkA阳性染色达到高峰，成纤维细胞、成软骨细胞、软骨细胞、肥大软骨细胞和成骨细胞中均有阳性表达；BDNF在软骨细胞及软骨基质中有阳性染色，其受体TrkB仅在成软骨细胞和软骨细胞中有表达；NT-3与NGF表达相似，其受体TrkC仅在成软骨细胞和软骨细胞中有阳性染色。术后第14d，NGF和TrkA阳性表达局限于部分成骨细胞、骨样细胞和成骨样细胞；NT-3在软骨细胞、成骨细胞中明显表达。术后21d，只有少量成骨细胞、成骨样细胞中有NGF和TrkA的表达。RT—PCR检测结果显示，NTFs mRNA于术后第7d表达最高，与免疫组化中蛋白质的表达结果相一致。[结论]NTFs及其受体在rhBMP-2诱导成骨过程明显表达，提示NTFs可能通过直接和间接的方式协同BMP的诱导成骨过程。     

成骨诱导的兔骨髓基质干细胞成骨活性的表达及维持

目的观察成骨诱导的兔骨髓基质干细胞(BMSCs)体内、外环境下成骨活性的表达及维持。方法观察BMSCs在体外成骨诱导培养条件下的成骨分化特性;构建兔BMSCs与活骨组织共培养模型模拟体内“成骨环境”,将成骨诱导的MSCs置于共培养及普通传代培养条件下进行传代培养,观察经成骨诱导的BMSCs在体外及模拟体内的培养条件下细胞的表型维持情况。结果药物成骨诱导培养的BMSCs,其ALP活性及骨钙素均显著高于普通培养组(P<0.05);经过诱导培养的BMSCs,其Ⅰ型胶原、骨钙素免疫组化阳性。RT-PCR法半定量测定Ⅰ型胶原mRNA,成骨诱导培养的Ⅰ型胶原mRNA表达量明显高于普通传代培养对照组。药物成骨诱导后的细胞在体外普通传代培养传5代后,细胞碱性磷酸酶(ALP)活性、骨钙素水平及Ⅰ型胶原表达稳定维持在较高水平,保持其成骨细胞的表型;在共培养条件下,ALP活性、骨钙素水平Ⅰ型胶原表达保持在高水平,且ALP活性、骨钙素水平在大部分时间点均高于普通传代培养。结论药物成骨诱导培养呈现促BMSCs向成骨方向转化的特点,能使ALP、骨钙素及Ⅰ型胶原表达短期内达到高水平;经成骨诱导的BMSCs在体外或模拟的体内传代培养条件下,均能维持成骨表型,保持成骨活力。     

骨代谢信号通路的研究进展

骨代谢包括骨形成及骨吸收两方面,受到多种因素的调控,其分子机制涉及遗传基因、信号通路、激素及旁分泌因子等多方面作用,而信号通路在其中起到了重要的调控作用。在目前的骨代谢信号通路研究中,BMP/Smads、Wnt/β-catenin及OPG/RANKL/RANK等3条通路已经得到较为深入的研究,并且公认为在骨代谢中起到了关键的信号调节作用,其中BMP/Smads及、Wnt/β-catenin通路主要影响骨形成,而OPG/RANKL/RANK则主要影响骨吸收。最新的研究表明,低氧/低氧诱导因子-la通路、PDGF、TGF-beta和FGF通路、AKt2选择通路、G蛋白信号通路、硫酸已酰肝素和硫酸软骨素通路、黏着斑激酶及胞外信号调节激酶通路等同样对成骨细胞及破骨细胞的分化增殖起到调节作用,并且发现BMP/Smads通路和Wnt/β-catenin通路之间存在着相互调节作用。对骨代谢信号通路的深入研究,可为骨代谢疾病尤其是骨质疏松症提供潜在的以细胞为基础的治疗新途径。     

BMP signaling components are expressed in human fracture callus

Of the various growth factors involved in the healing response after a fracture, bone morphogenetic proteins (BMPs) are emerging as key modulators. BMPs exert their effects by binding to a complex of type I and type II receptors leading to the phosphorylation of specific downstream effector proteins called Smads. The current study examined the presence of BMP signaling components in human callus obtained from five nascent malunions undergoing fracture fixation. These callus samples represented various stages of bone healing and a mixture of endochondral and intramembraneous bone healing. We performed immunohistochemistry on the callus, using antibodies for BMP (BMP-2,-3,-4,-7), their receptors (BMPR-IA, -IB, -II), and phosphorylated BMP receptor-regulated Smads (pBMP-R-Smads). Active osteoblasts showed fairly consistent positive staining for all BMPs that were examined, with the immunoreactivity most intense for BMP-7 and BMP-3. Immunostaining for BMPs in osteoblasts appeared to colocalize with the expression of BMPR-IA, -IB, and -II. Positive immunostaining for pBMP-R-Smads suggests that the BMP receptors expressed in these cells are activated. Staining for BMPs in cartilage cells was variable. The immunostaining appeared stronger in more mature cells, whereas staining for BMP receptors in cartilage cells was less ubiquitous. However, the expression of pBMP-R-Smads in cartilage cells suggests active signal transduction. Fibroblast-like cells also had a variable staining pattern. Overall, our findings indicate the presence of BMPs, their various receptors, and activated forms of receptor-regulated Smads in human fracture callus. To the best of our knowledge, this is the first study that documents the expression of these proteins in human fracture tissue. Complete elucidation of the roles of BMP in bone formation will hopefully lead to improved fracture healing care.     

转化生长因子-β超家族对新骨生成的调节作用

目的　对近年来国内外有关转化生长因子 - β(TGF- β)超家族对新骨形成作用的研究进行综述。方法　广泛查阅相关文献 ,对 TGF- β、骨形成蛋白 (BMPs)及激活素 (ACT)在新骨形成 ,尤其是牵张性新骨形成(DO)作用进行分析综合。结果　 TGF- β、BMPs及 ACT对新骨形成均有促进作用 ,其作用机制各有特点。BMPs启动间充质细胞向骨细胞系分化 ,TGF- β刺激骨形成前体细胞生长分化 ,ACT增强 BMPs成骨 ,本身也促进成骨细胞增殖和胶原合成。结论　 TGF- β超家族对新骨形成的调控 ,有助于缩短 DO的周期     

Direct stimulation of osteoclastic bone resorption by bone morphogenetic protein (BMP)-2 and expression of BMP receptors in mature osteoclasts

Bone morphogenetic proteins (BMPs) play an important role in various kinds of pattern formation and organogenesis during vertebrate development. In the skeleton, BMPs induce the differentiation of cells of chondrocytic and osteoblastic cell lineage and enhance their function. However, the action of BMPs on osteoclastic bone resorption, a process essential for pathophysiological bone development and regeneration, is still controversial. In this study, we examine the direct effect of BMPs on osteoclastic bone-resorbing activity in a culture of highly purified rabbit mature osteoclasts. BMP-2 caused a dose- and time-dependent increase in bone resorption pits excavated by the isolated osteoclasts. BMP-4 also stimulated osteoclastic bone resorption. The increase in osteoclastic bone resorption induced by BMP-2 was abolished by the simultaneous addition of follistatin, a BMP/activin binding protein that negates their biological activity. Just as it increased bone resorption, BMP-2 also elevated the messenger RNA expressions of cathepsin K and carbonic anhydrase II, which are key enzymes for the degradation of organic and inorganic bone matrices, respectively. Type IA and II BMP receptors (BMPRs), and their downstream signal transduction molecules, Smad1 and Smad5, were expressed in isolated osteoclasts as well as in osteoblastic cells, whereas type IB BMPR was undetectable. BMPs directly stimulate mature osteoclast function probably mediated by BMPR-IA and BMPR-II and their downstream molecules expressed in osteoclasts. The results presented here expand our understanding of the multifunctional roles of BMPs in bone development.     

Activation of the TGF-beta/Smad signaling pathway in focal segmental glomerulosclerosis

BACKGROUND: Although the pathogenetic relevance of transforming growth factor-beta (TGF-beta) to glomerulosclerosis is well established, it is not known whether a signal transduction cascade of TGF-beta is involved in the development of focal segmental glomerulosclerosis (FSGS), nor is it clear how TGF-beta 1 is activated during the course of FSGS formation. METHODS: We examined the expression patterns of TGF-beta 1, thrombospondin-1 (TSP-1), TGF-beta type II receptor (TGF-beta IIR), phosphorylated Smad2/Smad3, and podocyte-specific epitopes [Wilms' tumor protein-1 (WT-1) and glomerular epithelial protein-1 (GLEPP-1)] in 15 renal biopsy specimens with idiopathic FSGS and six renal biopsies with no detectable abnormalities by means of immunohistochemistry. The mRNA expression patterns of TGF-beta 1, TGF-beta IIR, and TSP-1 were further evaluated by in situ hybridization in seven biopsies. RESULTS: In the controls, immunostaining for TGF-beta 1, TSP-1, TGF-beta IIR, and phosphorylated Smad2/Smad3 was almost negligible, but an apparent signal for TGF-beta 1, TSP-1, and TGF-beta IIR mRNAs was observed in the visceral glomerular epithelial cells (GEC). In the cases of FSGS, the expression levels of TGF-beta 1, TSP-1, and TGF-betaIIR proteins and mRNAs and phosphorylated Smad2/Smad3 were significantly increased, particularly in the GEC of the sclerotic segments, wherein WT-1 and GLEPP-1 were not detected. CONCLUSION: These results suggest that damage to podocyes may stimulate TGF-beta 1, TSP-1, and TGF-beta IIR expression in GEC, thereby activating the Smad signaling pathway and, in so doing, leading to overproduction of the extracellular matrix (ECM). Thus, a signal transduction cascade of the TGF-beta/Smad signaling pathway, which is activated in the GEC, appears to be involved in the development of FSGS.     

骨形成蛋白2基因修饰的老年大鼠骨髓间充质干细胞成骨分化能力的研究

目的观察年龄因素对骨髓间充质干细胞(marrow mesenchymal stem cells, MSCs)成骨分化能力的影响;了解基因治疗对老年大鼠MSCs成骨分化能力的影响. 方法 1月龄(幼年组)、9月龄(成年组)及24月龄(老年组)雄性Wistar大鼠各6只,取MSCs经体外分离、培养及携带骨形成蛋白2(bone morphogenetic protein 2,BMP-2)基因的腺病毒载体(Ad-BMP-2)转染后,定量检测BMP-2、碱性磷酸酶(alkaline phosphate,ALP)表达,以及成骨细胞标志性蛋白:Ⅰ型胶原、骨涎蛋白(bone sialoprotein,BSP)和骨桥素(osteopontin, OPN)的表达.将转染的各组MSCs分别与磷酸三钙(tricalcium phosphate, TCP)复合后植入裸鼠体内,3周后取材,比较各组诱导异位成骨能力. 结果 ELISA检测表明BMP-2基因修饰的MSCs可以有效表达BMP-2,且表达量在各年龄组间差异无统计学意义(P>0.05);各组ALP于诱导后第9天达高峰,但组间差异均无统计学意义(P＞0.05);诱导后第7天,RT-PCR半定量检测示各组均有成骨细胞特征性蛋白,即:Ⅰ型胶原、OPN及BSP的明显表达,表达量在各组间差异无统计学意义(P>0.05);BMP-2基因转染的MSCs与TCP复合后可诱导裸鼠体内异位成骨,各组成骨量差异无统计学意义(P>0.05). 结论 BMP-2基因修饰的老年大鼠MSCs可以恢复成骨分化能力,基因治疗可能为老年性骨骼疾病提供一种新的治疗途径.     

Localization of Smads, the TGF-beta family intracellular signaling components during endochondral ossification.

Members of the transforming growth factor-beta (TGF-beta) family transduce signals from the cell membrane to the nucleus via specific type I and type II receptors and Smad proteins. Smad1 and Smad5 mediate intracellular signaling of bone morphogenetic protein (BMP), whereas Smad2 and Smad3 transduce TGF-beta signaling. Smad4 is a common mediator required for both pathways. Smad6 and Smad7 inhibit signaling by members of the TGF-beta superfamily. Here, we examined the expression of Smad1 to Smad7 proteins during endochondral ossification of epiphyseal plate of growing rats using immunohistochemical techniques. The expression of Smad proteins was correlated with the expression of TGF-beta1 and its receptors, and BMP-2/4 and BMP receptors. The results show that TGF-beta1 and BMP-2/4 were actively expressed in chondrocytes that are undergoing proliferation and maturation, which overlaps with expression of their corresponding type I and type II receptors. The Smads, however, exhibited a distinct expression pattern, respectively. For example, Smad1 and Smad5 were highly expressed in proliferating chondrocytes and in those chondrocytes that are undergoing maturation. The TGF-beta/activin-restricted Smads were also expressed in a nearly complementary fashion; Smad2 was strongly expressed in proliferating chondrocytes, whereas Smad3 was strongly observed in maturing chondrocytes. Smad4 was broadly expressed in all zones of epiphyseal plate. Inhibitory Smads, Smad6 and Smad7, were strongly expressed in the zone of cartilage that contained mature chondrocytes. Our findings show a colocalization of the pathway-restricted and inhibitory Smads with activating ligands or ligands whose action they antagonize and their receptors in various zones of epiphyseal growth plate, suggesting that TGF-beta superfamily Smad signaling pathways plays a morphogenic role during endochondral bone formation.