Transient gamma-secretase inhibition accelerates and enhances fracture repair likely via Notch signaling modulation

Approximately 10% of skeletal fractures result in healing complications and non-union, while most fractures repair with appropriate stabilization and without pharmacologic intervention. It is the latter injuries that cannot be underestimated as the expenses associated with their treatment and subsequent lost productivity are predicted to increase to over $74 billion by 2015. During fracture repair, local mesenchymal stem/progenitor cells (MSCs) differentiate to form new cartilage and bone, reminiscent of events during skeletal development. We previously demonstrated that permanent loss of gamma-secretase activity and Notch signaling accelerates bone and cartilage formation from MSC progenitors during skeletal development, leading to pathologic acquisition of bone and depletion of bone marrow derived MSCs. Here, we investigated whether transient and systemic gamma-secretase and Notch inhibition is capable of accelerating and enhancing fracture repair by promoting controlled MSC differentiation near the fracture site. Our radiographic, microCT, histological, cell and molecular analyses reveal that single and intermittent gamma-secretase inhibitor (GSI) treatments significantly enhance cartilage and bone callus formation via the promotion of MSC differentiation, resulting in only a moderate reduction of local MSCs. Biomechanical testing further demonstrates that GSI treated fractures exhibit superior strength earlier in the healing process, with single dose GSI treated fractures exhibiting bone strength approaching that of un-fractured tibiae. These data further establish that transient inhibition of gamma-secretase activity and Notch signaling temporarily increases osteoclastogenesis and accelerates bone remodeling, which coupled with the effects on MSCs likely explains the accelerated and enhanced fracture repair. Therefore, we propose that the Notch pathway serves as an important therapeutic target during skeletal fracture repair.     

Modulation of Notch1 signaling regulates bone fracture healing

Fracture healing involves interactions of different cell types, driven by various growth factors, and signaling cascades. Periosteal mesenchymal progenitor cells give rise to the majority of osteoblasts and chondrocytes in a fracture callus. Notch signaling has emerged as an important regulator of skeletal cell proliferation and differentiation. We investigated the effects of Notch signaling during the fracture healing process. Increased Notch signaling in osteochondroprogenitor cells driven by overexpression of Notch1 intracellular domain (NICD1) (αSMACreERT2 mice crossed with Rosa-NICD1) during fracture resulted in less cartilage, more mineralized callus tissue, and stronger and stiffer bones after 3 weeks. Periosteal cells overexpressing NICD1 showed increased proliferation and migration in vitro. In vivo data confirmed that increased Notch1 signaling caused expansion of alpha-smooth muscle actin (αSMA)-positive cells and their progeny including αSMA-derived osteoblasts in the callus without affecting osteoclast numbers. In contrast, anti-NRR1 antibody treatment to inhibit Notch1 signaling resulted in increased callus cartilage area, reduced callus bone mass, and reduced biomechanical strength. Our study shows a positive effect of induced Notch1 signaling on the fracture healing process, suggesting that stimulating the Notch pathway could be beneficial for fracture repair.     

Autocrine and Paracrine Actions of IGF-I Signaling in Skeletal Development

Insulin-like growth factor-I (IGF-I) regulates cell growth, survival, and differentiation by acting on the IGF-I receptor, (IGF-IR)-a tyrosine kinase receptor, which elicits diverse intracellular signaling responses. All skeletal cells express IGF-I and IGF-IR. Recent studies using tissue/cell-specific gene knockout mouse models and cell culture techniques have clearly demonstrated that locally produced IGF-I is more critical than the systemic IGF-I in supporting embryonic and postnatal skeletal development and bone remodeling. Local IGF-I/IGF-IR signaling promotes the growth, survival and differentiation of chondrocytes and osteoblasts, directly and indirectly, by altering other autocrine/paracrine signaling pathways in cartilage and bone, and by enhancing interactions among these skeletal cells through hormonal and physical means. Moreover, local IGF-I/IGF-IR signaling is critical for the anabolic bone actions of growth hormone and parathyroid hormone. Herein, we review evidence supporting the actions of local IGF-I/IGF-IR in the above aspects of skeletal development and remodeling.     

Wnt and hedgehog signaling pathways in bone development

Cell-cell signaling is a major strategy that vertebrate embryos employ to coordinately control cell proliferation, differentiation, and survival in many developmental processes. Similar cell signaling pathways also control adult tissue regeneration and repair. We demonstrated in the developing skeletal system that the Wnt/beta-catenin signaling controls the differentiation of progenitor cells into either osteoblasts or chondrocytes. Genetic ablation of beta-catenin in the developing mouse embryo resulted in ectopic formation of chondrocytes at the expense of osteoblast differentiation during both intramembranous and endochondral ossification. Conversely, ectopic upregulation of the canonical Wnt signaling led to suppression of chondrocyte formation and enhanced ossification. As other signaling pathways also play critical roles in controlling skeletal development, to gain a full picture of the molecular regulatory network of skeletal development, we investigated how the Wnt/beta-catenin signaling is integrated with Indian hedgehog (Ihh) signaling in controlling various aspects of skeletal development. We found that Wnt signaling acts downstream of Ihh signaling and is required in osteoblasts after Osterix expression to promote osteoblast maturation during endochondral bone formation. Since similar controlling mechanisms of osteoblast proliferation and differentiation may be employed by adult mesenchymal progenitor cells during fracture repair, these studies suggest that, to enhance fracture repair or bone formation, Ihh signaling needs to be enhanced at early stages, whereas Wnt signaling should be upregulated slightly later in differentiated osteoblasts.     

MAPKs通路在MSCs的增殖及向成骨细胞分化过程中的作用

间充质干细胞（MSCs）是一种多潜能成体干细胞,在体外诱导剂的作用下能向成骨细胞分化。在MSCs向成骨细胞分化过程中,受到MAPKs、BMPs、Notch和Wnt等多种信号通路的调控。其中MAPKs信号通路研究比较深入,近年来研究表明在MAPKs信号通路的五种途径中,ERKs、p38MAPK和JNKs途径参与了成骨细胞增殖和分化的信号转导。现对MAPKs通路与其参与的MSCs增殖和成骨分化过程简要综述。     

Notch信号通路在骨髓间充质干细胞及骨代谢疾病中的功能研究进展

骨髓间充质干细胞是存在于骨髓基质中的一种多能干细胞,具有多向分化的潜能,其天然再生能力对骨骼的生长代谢和骨转换起着至关重要的作用。Notch信号通路是一条在进化中高度保守的信号转导通路,与骨髓间充质干细胞的增殖、分化与凋亡密切相关,影响人体骨骼发育,也是多种骨代谢疾病的重要调节通路。以往对Notch信号通路的研究主要集中在神经干细胞,对骨髓间充质干细胞的研究较少。本文通过查阅文献,阐述不同的影响因素介导Notch信号通路对骨髓间充质干细胞分化的调节,并总结了Notch信号通路在骨代谢疾病如Alagille综合征、Adams Oliver综合征、脊椎肋骨发育不良、HajduCheney综合征、骨折愈合中的研究近况。     

Stimulation of osteoblastic cell differentiation by Notch.

Notch is a transmembrane protein that plays a critical role in the determination of cellular differentiation pathways. Although its importance in the development of mesenchymal tissues has been suggested, its role in skeletal tissues has not been well investigated. Northern blot experiments showed the expression of Notch1 in MC3T3-E1 osteoblastic cells at early differentiation stages. When a Notch1 cytoplasmic domain (Notch-IC [NIC]) delivered by an adenovirus vector was expressed in osteoblastic MC3T3-E1 cells, a significant increase in calcified nodule formation was observed in long-term cultures. Activation of endogenous Notch in MC3T3-E1 by coculturing them with Delta-like-1 (Dll1)-expressing myeloma cells also resulted in a stimulation of calcified nodule formation. Not only affecting nodule formation, Notch activation also had effects on osteoblastic differentiation of multipotent mesenchymal cells. Osteoblastic differentiation of C3H10T1/2 cells induced by bone morphogenetic protein 2 (BMP-2) was significantly stimulated, whereas adipogenic differentiation was suppressed strongly, resulting in a dominant differentiation of osteoblastic cells. NIC expression in primary human bone marrow mesenchymal stem cells (hMSCs) also induced both spontaneous and stimulated osteoblastic cell differentiation. These observations suggest that osteoblastic cell differentiation is regulated positively by Notch and that Notch could be a unique and interesting target molecule for the treatment of osteoporosis.     

Signaling Pathways Affecting Skeletal Health

Skeletal health is dependent on the balance between bone resorption and formation during bone remodeling. Multiple signaling pathways play essential roles in the maintenance of skeletal integrity by positively or negatively regulating bone cells. During the last years, significant advances have been made in our understanding of the essential signaling pathways that regulate bone cell commitment, differentiation and survival. New signaling anabolic pathways triggered by parathyroid hormone, local growth factors, Wnt signaling, and calcium sensing receptor have been identified. Novel signals induced by interactions between bone cells-matrix (integrins), osteoblasts/osteocytes (cadherins, connexins), and osteoblasts/osteoclast (ephrins, Wnt-RhoA, semaphorins) have been discovered. Recent studies revealed the key pathways (MAPK, PI3K/Akt) that critically control bone cells and skeletal mass. This review summarizes the most recent knowledge on the major signaling pathways that control bone cells, and their potential impact on the development of therapeutic strategies to improve human bone health.     

Notch Regulation of Bone Development and Remodeling and Related Skeletal Disorders

Notch signaling mediates cell-to-cell interactions that are critical for embryonic development and tissue renewal. In the canonical signaling pathway, the Notch receptor is cleaved following ligand binding, resulting in the release and nuclear translocation of the Notch intracellular domain (NICD). NICD induces gene expression by forming a ternary complex with the DNA binding protein CBF1/Rbp-Jk, Suppressor of Hairless, Lag1, and Mastermind-Like (Maml). Hairy Enhancer of Split (Hes) and Hes related with YRPW motif (Hey) are classic Notch targets. Notch canonical signaling plays a central role in skeletal development and bone remodeling by suppressing the differentiation of skeletal cells. The skeletal phenotype of mice misexpressing Hes1 phenocopies partially the effects of Notch misexpression, suggesting that Hey proteins mediate most of the skeletal effects of Notch. Dysregulation of Notch signaling is associated with diseases affecting human skeletal development, such as Alagille syndrome, brachydactyly and spondylocostal dysostosis. Somatic mutations in Notch receptors and ligands are found in tumors of the skeletal system. Overexpression of NOTCH1 is associated with osteosarcoma, and overexpression of NOTCH3 or JAGGED1 in breast cancer cells favors the formation of osteolytic bone metastasis. Activating mutations in NOTCH2 cause Hajdu-Cheney syndrome, which is characterized by skeletal defects and fractures, and JAG1 polymorphisms, are associated with variations in bone mineral density. In conclusion, Notch is a regulator of skeletal development and bone remodeling, and abnormal Notch signaling is associated with developmental and postnatal skeletal disorders.     

MT1‐MMP and Type II Collagen Specify Skeletal Stem Cells and Their Bone and Cartilage Progeny

Skeletal formation is dependent on timely recruitment of skeletal stem cells and their ensuing synthesis and remodeling of the major fibrillar collagens, type I collagen and type II collagen, in bone and cartilage tissues during development and postnatal growth. Loss of the major collagenolytic activity associated with the membrane‐type 1 matrix metalloproteinase (MT1‐MMP) results in disrupted skeletal development and growth in both cartilage and bone, where MT1‐MMP is required for pericellular collagen dissolution. We show here that reconstitution of MT1‐MMP activity in the type II collagen‐expressing cells of the skeleton rescues not only diminished chondrocyte proliferation, but surprisingly, also results in amelioration of the severe skeletal dysplasia associated with MT1‐MMP deficiency through enhanced bone formation. Consistent with this increased bone formation, type II collagen was identified in bone cells and skeletal stem/progenitor cells of wildtype mice. Moreover, bone marrow stromal cells isolated from mice expressing MT1‐MMP under the control of the type II collagen promoter in an MT1‐MMP‐deficient background showed enhanced bone formation in vitro and in vivo compared with cells derived from nontransgenic MT1‐MMP‐deficient littermates. These observations show that type II collagen is not stringently confined to the chondrocyte but is expressed in skeletal stem/progenitor cells (able to regenerate bone, cartilage, myelosupportive stroma, marrow adipocytes) and in the chondrogenic and osteogenic lineage progeny where collagenolytic activity is a requisite for proper cell and tissue function.     

Skeletal (stromal) stem cells: An update on intracellular signaling pathways controlling osteoblast differentiation

Skeletal (marrow stromal) stem cells (BMSCs) are a group of multipotent cells that reside in the bone marrow stroma and can differentiate into osteoblasts, chondrocytes and adipocytes. Studying signaling pathways that regulate BMSC differentiation into osteoblastic cells is a strategy for identifying druggable targets for enhancing bone formation. This review will discuss the functions and the molecular mechanisms of action on osteoblast differentiation and bone formation; of a number of recently identified regulatory molecules: the non-canonical Notch signaling molecule Delta-like 1/preadipocyte factor 1 (Dlk1/Pref-1), the Wnt co-receptor Lrp5 and intracellular kinases. This article is part of a Special Issue entitled: Stem Cells and Bone.     

水蛭素通过上调VEGF/Notch1信号通路促进人骨髓间充质干细胞成骨分化

摘要：目的 观察水蛭素对人骨髓间充质干细胞（bone marrow mesenchymal stem cell,BMSC）成骨分化的影响。方法 BMSCs细胞分为正常培养的对照组、成骨诱导的诱导组以及加入不同浓度（1、10、20 ATU/mL）处理的水蛭素组。MTT检测细胞增值并筛选水蛭素最适作用浓度。流式细胞仪检测细胞凋亡。RT-PCR和Western blot分别检测成骨基因Runx2、Osterix、COL1A1的mRNA和蛋白表达。BCIP/NBT染色法检测细胞中的碱性磷酸酶水平。茜素红染色检测矿化结节。检测VEGF、Notch1、Jagged1和CBF1的mRNA和蛋白表达。结果 骨髓间充质干细胞经成骨诱导细胞增殖显著增加,中高浓度的水蛭素可以不同程度促进成骨诱导的BMSCs细胞增殖（P＜0.05）,并筛选出20 ATU/mL作为水蛭素的使用浓度。水蛭素抑制成骨诱导的BMSCs细胞凋亡,上调Runx2、Osterix、COL1A1的mRNA和蛋白表达,增加碱性磷酸酶水平,促进细胞中矿化结节的生成,并提升BMSCs细胞中VEGF、Notch1、Jagged1和CBF1的表达（P＜0.05）。结论 水蛭素可能通过上调VEGF/Notch1信号通路促进人骨髓间充质干细胞成骨分化。     

Notch信号通路在骨重建过程中的双向调节作用

Notch信号通路对骨形成及骨吸收的刺激和抑制作用都被广泛报道,其在成骨细胞、骨细胞、破骨细胞、骨髓间充质干细胞等的生成或分化中的作用出现了"矛盾"的结果,表现为对于骨形成-骨吸收偶联关系的双向调节作用,可见,Notch信号通路对骨重建过程的影响并非单一的促进或抑制作用,本文就Notch信号通路对成骨细胞、破骨细胞、骨髓间充质干细胞生成、分化及功能的双向调节作用做一综述,以期为相关骨代谢疾病的研究思路提供参考。     

精原干细胞增殖和分化阶段小鼠睾丸基因的差异表达

目的 分析精原干细胞增殖和分化阶段小鼠睾丸组织基因表达谱的变化,初步探讨精原干细胞增殖和分化的调控机制. 方法 48只雄性昆明白小鼠采用间隔24 d二次腹腔注射白消安(10 mg/kg)制备小鼠精子再生模型,8只作为对照组.根据精子再生过程生精上皮的组织形态学变化以及精原细胞增殖情况选取精原干细胞处于增殖和分化阶段的睾丸组织,运用基因表达谱芯片检测2个阶段的睾丸组织基因表达差异,对差异表达基因进行生物信息学分析. 结果 检测到睾丸组织差异表达基因911个.上调608个(增殖期/分化期)、下调303个.差异表达基因分别涉及生物学过程、分子功能和分子组成.84个信号通路功能改变差异有统计学意义(P<0.05),包括Notch和wnt信号通路.与干细胞相关的差异基因有56个,上调40个、下调16个.部分干细胞的阳性标记物(如CA9、Stra8、hgb1、Oct4和Thy1)和部分生长因子(如Fgf2、Csf1和Pdgfa)上调. 结论 小鼠精原干细胞增殖和分化过程的调控涉及许多基因(分属不同信号通路)的差异表达,这些基因和通路对精原干细胞增殖的作用还有待进一步研究.     

Notch signaling inhibition induces G0/G1 arrest in murine Leydig cells

As a highly evolutionarily conserved signaling pathway, Notch widely participates in cell‐fate decisions and the development of various tissues and organs. In male reproduction, research on the Notch signaling pathway has mainly concentrated on germ cells and Sertoli cells. Leydig cells are the primary producers of testosterone and play important roles in spermatogenesis and maintaining secondary sexual characteristics. In this study, we used TM3 cells, a murine adult Leydig cell line, to investigate the expression profiles of Notch receptors and ligands and observe the effect of Notch signaling on the proliferation of TM3 cells. We found that Notch 1–3 and the ligands Dll‐1 and Dll‐4 were expressed in TM3 cells, Notch 1–3 and the ligand Dll‐1 were expressed in testis interstitial Leydig cells, and Notch signaling inhibition suppressed the proliferation of TM3 cells and induced G0/G1 arrest. Inhibition of Notch signaling increased the expression of p21^Waf1/Cip1 and p27. Overall, our results suggest that Notch inhibition suppresses the proliferation of TM3 cells and P21^Waf1/Cip1, and p27 may contribute to this process.     

中药调控Notch信号通路治疗骨质疏松症的研究

骨质疏松症（osteoporosis,OP）作为临床骨科方面常见的骨代谢性疾病，其发病原因主要是由于成骨细胞与破骨细胞共同介导的骨形成与骨吸收失去动态平衡关系所致，造成机体单位体积内的骨量减少、骨密度下降以及骨微结构改变，从而导致OP的发生和发展。机体内的骨代谢过程受多条信号传导通路的调控，神经源性位点缺口同源蛋白（Notch）信号通路作为重要的骨重建通路在调控与维持骨代谢稳定方面发挥着至关重要的作用，通过影响其通路蛋白的表达量，可以直接或间接地调控相关骨细胞的增殖、分化和凋亡，从而维持“骨平衡”的动态关系。近年来，运用中医药手段防治OP在临床上取得了良好的疗效，海内外学者对中药调控Notch信号通路防治OP的作用机制进行了多层次、多维度的探究，发现中药、Notch信号通路和OP三者之间存在明显的相关性。基于此，本文通过对中药单体及复方调控Notch信号通路防治OP的相关研究文献进行收集，整理、分析并总结目前的研究现状，以期为中药防治OP提供参考，为新药的研发和中医药特色诊疗方法的应用提供新的思路。     

Notch信号转导通路对软骨发育分化的影响

Notch信号转导通路广泛存在于多种动物细胞中,对调节细胞的增殖、存活、分化和凋亡等过程有重要意义,其受体和配体都是跨膜蛋白,因此是介导细胞间通讯的一种重要方式。软骨的分化发育起始自间充质细胞的凝集,Notch家族在软骨形成过程中的表达呈多样性,并对软骨的发育分化呈现时间和空间的调控。在此就Notch信号转导通路的组成、分布、转导机制及其对软骨分化发育的影响进行综述。     

Sprouty2 regulates endochondral bone formation by modulation of RTK and BMP signaling

Skeletal development is regulated by the coordinated activity of signaling molecules that are both produced locally by cartilage and bone cells and also circulate systemically. During embryonic development and postnatal bone remodeling, receptor tyrosine kinase (RTK) superfamily members play critical roles in the proliferation, survival, and differentiation of chondrocytes, osteoblasts, osteoclasts, and other bone cells. Recently, several molecules that regulate RTK signaling have been identified, including the four members of the Sprouty (Spry) family (Spry1–4). We report that Spry2 plays an important role in regulation of endochondral bone formation. Mice in which the Spry2 gene has been deleted have defective chondrogenesis and endochondral bone formation, with a postnatal decrease in skeletal size and trabecular bone mass. In these constitutive Spry2 mutants, both chondrocytes and osteoblasts undergo increased cell proliferation and impaired terminal differentiation. Tissue-specific Spry2 deletion by either osteoblast- (Col1-Cre) or chondrocyte- (Col2-Cre) specific drivers led to decreased relative bone mass, demonstrating the critical role of Spry2 in both cell types. Molecular analyses of signaling pathways in Spry2^−/− mice revealed an unexpected upregulation of BMP signaling and decrease in RTK signaling. These results identify Spry2 as a critical regulator of endochondral bone formation that modulates signaling in both osteoblast and chondrocyte lineages.     

Downregulation of Notch Modulators,Tetraspanin 5 and 10, Inhibits Osteoclastogenesis in Vitro

Genetic studies in human and mice have pinpointed an essential role of Notch signaling in osteoblast and osteoclast differentiation during skeletal development and bone remodeling. However, the factors and pathways regulating Notch activation in bone cells remain largely unknown. In this in vitro study, we have provided evidence that two of the TspanC8 subfamily members of tetraspanins, Tspan-5 and Tspan-10, are up-regulated during osteoclast differentiation and knockdown of their expression by shRNAs dramatically inhibits osteoclastogenesis. Loss of Tspan-5 and Tspan-10 in osteoclast lineage cells results in attenuation of ADAM10 maturation and Notch activation. Therefore, these two tetraspanins play a critical role in osteoclast formation, at least in part, by modulating Notch signaling pathway.     

Osteogenic and chondrogenic potential of biomembrane cells from the PMMA‐segmental defect rat model

A layer of cells (the “biomembrane”) has been identified in large segmental defects between bone and surgically placed methacrylate spacers or antibiotic‐impregnated cement beads. We hypothesize that this contains a pluripotent stem cell population with potential valuable applications in orthopedic tissue engineering. Objectives using biomembranes harvested from rat segmental defects were to: (1) Culture biomembrane cells in specialized media to direct progenitor cells along bone or cartilage cell differentiation lineages; (2) evaluate harvested biomembranes for mesenchymal stem cell markers, and (3) define relevant gene expression patterns in harvested biomembranes using microarray analysis. Culture in osteogenic media produced mineralized nodules; culture in chondrogenic media produced masses containing chondroitin sulfate/sulfated proteoglycans. Molecular analysis of biomembrane cells versus control periosteum showed significant upregulation of key genes functioning in mesenchymal stem cell differentiation, development, maintenance, and proliferation. Results identified significant upregulation of WNT receptor signaling pathway genes and significant upregulation of BMP signaling pathway genes. Findings confirm that the biomembrane has a pluripotent stem cell population. The ability to heal large bone defects is clinically challenging, and novel tissue engineering uses of the biomembrane hold great promise in treating non‐unions, open fractures with large bone loss and/or infections, and defects associated with tumor resection. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1198–1212, 2012