Beta-catenin signaling plays a disparate role in different phases of fracture repair: implications for therapy to improve bone healing.
Beta-catenin signaling plays a disparate role in different phases of fracture repair: implications for therapy to improve bone healing.
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DOI：
10.1371/journal.pmed.0040249
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发表时间：
2007-07-31
期刊：
影响因子：
15.8
通讯作者：
Alman BA
中科院分区：
文献类型：
--
作者：
Chen Y;Whetstone HC;Lin AC;Nadesan P;Wei Q;Poon R;Alman BA
延迟骨折愈合会导致严重的残疾，通常需要额外的手术治疗。改善骨折修复的药物治疗将大大改善患者的预后。调节骨愈合的信号通路开始被解开，它们为药物治疗提供了线索。β-catenin信号通路激活T细胞因子（TCF）依赖性转录，已成为胚胎骨骼形成的关键调节因子，积极调节成骨细胞。然而，其在骨修复中的作用尚不清楚。本研究的目的是探讨β-catenin信号在骨修复中的作用。Western blot分析显示，β-catenin在骨愈合过程中显著上调。在表达TCF报告基因的转基因小鼠模型中，通过β-Gal活性测定观察胫骨骨折愈合过程中的激活，我们发现β-catenin介导的TCF依赖性转录在骨折修复过程中在骨和软骨形成过程中都被激活。通过逆转录- pcr，我们观察到在骨折修复过程中有几种WNT配体表达。DKK1 （WNT/β-catenin通路拮抗剂）抑制β-catenin信号传导和愈合过程，提示WNT配体调节β-catenin。由表达Cre重组酶的腺病毒诱导的有条件表达无β-catenin等位基因或稳定β-catenin等位基因的小鼠的愈合明显受到抑制。在表达成骨细胞特异性β-catenin零等位基因的小鼠中，骨折修复也受到抑制。与之形成鲜明对比的是，在表达一种激活形式的β-catenin的小鼠中，骨愈合显著增强，这种β-catenin的表达仅限于成骨细胞。用锂活化的β-连环蛋白治疗小鼠骨折愈合，但只有在骨折后开始治疗才能促进愈合。这些结果表明，β-catenin在骨折修复的不同阶段具有不同的功能。在早期阶段，β-连环蛋白的精确调控是多能间充质细胞分化成成骨细胞或软骨细胞所必需的。一旦这些未分化的细胞变成成骨细胞谱系，β-连环蛋白正向调节成骨细胞。这与之前报道的发育过程中β-连环蛋白的功能不同。通过锂离子处理激活β-catenin有可能促进骨折愈合，但只有在修复后期使用时，在间充质细胞已经成为成骨细胞谱系之后。在一项小鼠研究中，Benjamin Alman和他的同事发现β-catenin在骨折修复的不同阶段起着不同的作用；此外，锂活化β-catenin可促进骨折修复后期的愈合。大多数人一生中至少骨折过一根骨头。如果用石膏石膏或金属板和钉固定受损的骨头，大多数骨折会自然而迅速地愈合。骨损伤后不久，多能间充质细胞聚集在损伤部位。在这里，它们繁殖并分化成成骨细胞（形成骨骼的细胞）和软骨细胞（形成软骨的细胞，覆盖关节的致密结缔组织）。成骨细胞和软骨细胞通过制造新骨来修复骨折，这一过程被称为骨化。骨愈合包括两种类型的骨化。在膜内骨化过程中，间充质细胞和成骨祖细胞直接成骨，在骨折处形成坚硬的“骨痂”。在软骨内成骨过程中，间充质细胞分化为软骨细胞，在骨折部位形成软骨，成骨细胞转化为骨。最后，由两种类型的骨化形成的骨骼被重塑，使其与受损骨骼的原始形状和强度非常相似。不幸的是，骨折并不总是能有效愈合。如果愈合延迟，可能需要额外的手术来修复断裂。但是手术是有风险的，所以以药物为基础的促进骨修复的方法是非常有用的。为了开发这种治疗方法，研究人员需要了解在正常愈合过程中是什么控制了成骨细胞和软骨细胞的分化和活性。在这项研究中，研究人员研究了β-catenin信号通路在骨修复中的作用。这条通路调节胚胎发育过程中的骨形成，这一过程与骨愈合非常相似。β-连环蛋白通常在细胞内迅速降解。然而，如果一个被称为WNT家族的特定蛋白质家族的成员与细胞表面的WNT受体结合，β-连环蛋白就会进入细胞核，在那里它与一种叫做T细胞因子（TCF）的蛋白质相互作用。这种相互作用激活了许多基因的转录（将DNA复制成信使RNA，用于制造蛋白质），并改变了细胞的行为。研究人员首先测量了小鼠和人类骨骼中的β-连环蛋白水平。在这两个物种中，正在修复的骨头中产生的β-连环蛋白比完整的骨头多得多。然后，他们研究了TCF报告小鼠——TCF控制标记基因表达的动物。他们报道，β-连环蛋白介导的tcf依赖性转录在这些小鼠骨折后的骨和软骨形成过程中都被激活。接下来，研究人员制造了一种小鼠，可以诱导其在骨折的所有细胞中表达一种无活性形式的β-catenin或一种稳定（永久活性）形式的β-catenin。非活性β-catenin的表达减缓了愈合速度，但出乎意料的是，稳定β-catenin的表达也减缓了愈合速度。失活β-catenin的成骨细胞特异性表达也会延迟骨愈合，而稳定β-catenin的成骨细胞特异性表达则会促进这一过程。最后，用锂（可以防止β-连环蛋白的降解）治疗野生型小鼠，如果在骨折后给药，可以促进骨愈合，但如果在骨折前给药，则会干扰骨愈合。这些发现表明，β-catenin信号（研究人员表明，它主要由WNT信号激活）在骨修复的不同阶段具有不同的作用。在这个过程的早期，它控制着由多能间充质细胞形成的成骨细胞和软骨细胞的比例。因此，过多或过少的β-连环蛋白都会在这个阶段干扰骨愈合。随后，β-catenin促进成骨细胞的分化并增强其成骨的能力，因此在这一阶段，β-catenin过少会阻碍愈合，而β-catenin水平的增加会促进愈合。在测试影响β-连环蛋白信号的药物对人类骨愈合的影响之前，这些发现需要在人体中得到证实。然而，研究人员的最后发现是，如果在适当的时间给予锂，可以促进骨愈合，这尤其令人鼓舞；锂被广泛用于治疗一种形式的抑郁症，因此很容易在临床试验中进行测试。请通过本摘要的在线版本http://dx.doi.org/10.1371/journal.pmed.0040249访问这些网站。MedlinePlus百科全书包含关于骨折和骨折修复的页面（英语和西班牙语）维基百科有关于骨折和骨愈合的页面(注意：维基百科是一个免费的在线百科全书，任何人都可以编辑；英属哥伦比亚省高等教育、培训和技术部提供了有关膜内和软骨内成骨的动画。加拿大美国骨科医师学会有一个关于骨折的信息丰富的讨论。多伦多儿童医院（本研究的作者隶属于该医院）有一个名为SickKids的网站，其中有一个关于儿童生理学的页面，包括骨骼发育的图表
Delayed fracture healing causes substantial disability and usually requires additional surgical treatments. Pharmacologic management to improve fracture repair would substantially improve patient outcome. The signaling pathways regulating bone healing are beginning to be unraveled, and they provide clues into pharmacologic management. The β-catenin signaling pathway, which activates T cell factor (TCF)-dependent transcription, has emerged as a key regulator in embryonic skeletogenesis, positively regulating osteoblasts. However, its role in bone repair is unknown. The goal of this study was to explore the role of β-catenin signaling in bone repair. Western blot analysis showed significant up-regulation of β-catenin during the bone healing process. Using a β-Gal activity assay to observe activation during healing of tibia fractures in a transgenic mouse model expressing a TCF reporter, we found that β-catenin-mediated, TCF-dependent transcription was activated in both bone and cartilage formation during fracture repair. Using reverse transcription-PCR, we observed that several WNT ligands were expressed during fracture repair. Treatment with DKK1 (an antagonist of WNT/β-catenin pathway) inhibited β-catenin signaling and the healing process, suggesting that WNT ligands regulate β-catenin. Healing was significantly repressed in mice conditionally expressing either null or stabilized β-catenin alleles induced by an adenovirus expressing Cre recombinase. Fracture repair was also inhibited in mice expressing osteoblast-specific β-catenin null alleles. In stark contrast, there was dramatically enhanced bone healing in mice expressing an activated form of β-catenin, whose expression was restricted to osteoblasts. Treating mice with lithium activated β-catenin in the healing fracture, but healing was enhanced only when treatment was started subsequent to the fracture. These results demonstrate that β-catenin functions differently at different stages of fracture repair. In early stages, precise regulation of β-catenin is required for pluripotent mesenchymal cells to differentiate to either osteoblasts or chondrocytes. Once these undifferentiated cells have become committed to the osteoblast lineage, β-catenin positively regulates osteoblasts. This is a different function for β-catenin than has previously been reported during development. Activation of β-catenin by lithium treatment has potential to improve fracture healing, but only when utilized in later phases of repair, after mesenchymal cells have become committed to the osteoblast lineage. In a study in mice Benjamin Alman and colleagues show that β-catenin functions differently in different stages of fracture repair; moreover, activation of β-catenin by lithium improves fracture healing when used in later phases of repair. Most people break at least one bone during their life. If the damaged bone is immobilized with a plaster cast or with metal plates and pins, most fractures heal naturally and quickly. Soon after a bone is damaged, cells called pluripotent mesenchymal cells collect at the injury site. Here, they multiply and change (differentiate) into osteoblasts (cells that make bone) and chondrocytes (cells that make cartilage, the dense connective tissue that covers joints). Osteoblasts and chondrocytes mend the fracture by making new bone, a process called ossification. Bone healing involves two types of ossification. In intramembranous ossification, mesenchymal cells and osteoblast progenitor cells make bone directly, forming a hard “callus” within the fracture. In endochondral ossification, mesenchymal cells differentiate into chondrocytes and make cartilage at the fracture site, which osteoblasts turn into bone. Finally, the bone made by both types of ossification is remodeled so that it closely resembles the damaged bone's original shape and strength. Unfortunately, fractures do not always heal efficiently. If healing is delayed, additional surgery may be needed to repair the break. But surgery can be risky, so drug-based ways of encouraging bone repair would be very useful. To develop such treatments, researchers need to understand what controls the differentiation and activity of osteoblasts and chondrocytes during normal healing. In this study, the researchers have investigated the role of the β-catenin signaling pathway in bone repair. This pathway regulates bone formation during embryonic development, a process that closely resembles bone healing. β-catenin is usually degraded rapidly in cells. However, if a member of a particular family of proteins known as the WNT family binds to a WNT receptor on the surface of a cell, β-catenin moves into the cell's nucleus where it interacts with a protein called T cell factor (TCF). This interaction activates the transcription (the copying of DNA into messenger RNA, which is used to make proteins) of numerous genes and alters the behavior of the cell. The researchers first measured β-catenin levels in mouse and human bones. In both species, much more β-catenin was made in bones undergoing repair than in intact bones. Then they studied TCF reporter mice—animals in which TCF controls the expression of a marker gene. β-catenin-mediated TCF-dependent transcription, they report, was activated during both bone and cartilage formation after a fracture in these mice. Next, the researchers made mice that could be induced to express an inactive form of β-catenin or a stabilized (permanently active) form of β-catenin in all the cells in a bone fracture. Expression of inactive β-catenin slowed the rate of healing but, unexpectedly, so did expression of stabilized β-catenin. Osteoblast-specific expression of inactive β-catenin also delayed bone healing, whereas osteoblast-specific expression of stabilized β-catenin enhanced the process. Finally, treatment of wild-type mice with lithium (which prevents the degradation of β-catenin) enhanced bone healing if given after a fracture, but interfered with it if given before. These findings indicate that β-catenin signaling (which, the researchers show, is mainly activated by WNT signaling) has different effects at different stages of bone repair. Early in the process, it controls the ratio of osteoblasts and chondrocytes made from the pluripotent mesenchymal cells. Consequently, too much or too little β-catenin interferes with bone healing at this stage. Later on, β-catenin promotes the differentiation of osteoblasts and enhances their ability to make bone, and so too little β-catenin at this stage prevents healing, whereas increased β-catenin levels stimulate healing. These findings need to be confirmed in people before testing agents that affect β-catenin signaling for their effects on human bone healing. Nevertheless, the researchers' final discovery that lithium improves bone healing if given at the right time is particularly encouraging; lithium is widely used to treat one form of depression so could be readily tested in clinical trials. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040249. MedlinePlus encyclopedia contains pages on broken bones and on bone fracture repair (in English and Spanish) Wikipedia has pages on bone fracture and on bone healing (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages) The UK National Health Service Direct encyclopedia provides pages on broken bones Animations of intramembranous and endochondral ossification are available from the Ministry of Advanced Education, Training and Technology, Province of British Columbia, Canada The American Academy of Orthopedic Surgeons has an informative discussion of fractures The Hospital for Sick Children in Toronto (where the authors of this study are affiliated) has a Web site called SickKids, which contains a page on child physiology, including diagrams of bone development
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影响因子：
64.8
作者：
Glinka, A;Wu, W;Niehrs, C
通讯作者：
Niehrs, C
DOI：
10.1073/pnas.102657399
发表时间：
2002-05-14
影响因子：
11.1
作者：
Cheon, SS;Cheah, AYL;Alman, BA
通讯作者：
Alman, BA
DOI：
10.1073/pnas.0505259102
发表时间：
2005-11-29
影响因子：
11.1
作者：
Clément-Lacroix, P;Ai, MR;Rawadi, G
通讯作者：
Rawadi, G
影响因子：
64.5
作者：
Gong, YQ;Slee, RB;Warman, ML
通讯作者：
Warman, ML
影响因子：
4.8
作者：
Gregory, CA;Perry, AS;Prockop, DJ
通讯作者：
Prockop, DJ