Notch signaling in differentiation and function of dendritic cells

Hematopoietic stem cells give rise to multiple lineages of cells. This process is governed by a tightly controlled signaling network regulated by cytokines and a direct cell-cell contact. Notch signaling represents one of the major pathways activated during direct interaction between hematopoietic progenitor cells and bone marrow stroma. A critical role of Notch signaling in differentiation of T- and B-lymphocytes has now been established. Until recently, the role of Notch signaling in the development of myeloid cells and particular dendritic cells remained unclear. In this review, we discuss recent exciting findings that shed light on the critical role of Notch in differentiation and the function of dendritic cells and its impact on immune responses.     

Wnt和Notch信号通路在肿瘤干细胞自我更新中的作用

肿瘤干细胞是一类能够导致肿瘤发生的具有自我更新能力的细胞，它与干细胞具有很多相似性，其中最重要的一点是自我更新能力。它们具有相似的自我更新调节通路，如：Wnt，Notch和Shh(Sonic hedgehog)。Wnt和Notch信号通路通过其受体和配体的相互作用在自我更新的增殖和分化中都起着重要的作用，两者均能促进干细胞增殖而抑制其分化，但各自侧重不同。此外，Wnt和Notch信号通路之间相互作用、协调共同完成干细胞的自我更新。对肿瘤干细胞的Wnt和Notch信号通路研究将为未来肿瘤的靶向治疗提供新的方向。     

成骨信号通路在非创伤性股骨头坏死中的作用

文题释义： 非创伤性股骨头坏死：是一种由多种非创伤因素引起的股骨头血液供应中断或受损,导致成骨细胞和骨细胞的凋亡,骨髓干细胞、骨细胞、脂肪细胞的增殖代谢紊乱,进而出现股骨头局部骨质变性、坏死,骨小梁断裂,最终导致股骨头塌陷和髋关节功能障碍的疾病,目前其发病机制仍不明确,是在国内外常见且尚未解决的难治性骨代谢性疾病之一。 Wnt/β-catenin信号通路：广泛存在于多细胞动物中,是一条在物种进化中高度保守的信号通路,因为有β-catenin参与信号转导故又被称为经典Wnt通路。其构成主要包括：细胞外因子、卷曲蛋白、胞质蛋白、低密度脂蛋白受体相关蛋白5/6、糖原合成酶激酶3β、T细胞因子/淋巴增强因子等。 背景：近年来非创伤股骨头坏死的发病率逐年增高,具有起病隐秘、病情发展迅速、致残率高的特点,给患者及其家庭乃至社会带来极大的负担。若能明确其发病机制,对非创伤股骨头坏死的早期有效治疗有重要意义。 目的：综述国内外相关文献,总结成骨信号通路在非创伤性股骨头坏死发病机制中的研究进展。 方法：以“非创伤性股骨头坏死,成骨,信号通路,发病机制”或“non-traumatic osteonecrosis of femoral head,Osteogenesis,Signaling pathways,pathogenesis,Wnt/β-catenin,PPARγ,TGF-β/Smad,PI3K/AKT,MAPK,Notch”等作为检索词,检索PubMed、Embase、Medline、中国知网、万方、维普等数据库中有关非创伤性股骨头坏死相关成骨信号通路机制及应用的研究文献。 结果与结论：近年来,成骨信号通路在非创伤性股骨头坏死中的作用日益受到重视,非创伤性股骨头坏死发生发展中骨髓间充质干细胞的异常分化亦成为研究的焦点。成骨信号通路转导异常造成的骨髓间充质干细胞异常分化、成骨分化抑制、骨质破坏增加、骨代谢平衡破坏,可能是导致非创伤性股骨头坏死的主要原因。将Wnt/β-catenin、PPARγ、TGF-β/Smad、PI3K/AKT、MAPK、Notch等成骨信号通路作为干预非创伤性股骨头坏死的治疗靶点或许是一种可行的办法,尽管目前已有大量体外及动物实验研究证实成骨信号通路可能具有调控骨髓间充质干细胞分化并逆转股骨头坏死的潜能,但其具体的作用机制尚未明确,且目前相关临床应用研究仍较少,故探索成骨信号通路的作用机制、加快临床的应用研究将是未来研究的方向。 ORCID: 0000-0001-7591-6337(许灿宏) 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     

调控骨髓间充质干细胞成骨分化的Wnt/β-catenin信号通路及相关因素

背景:骨髓间充质干细胞诱导成骨而抑制成脂分化是骨质疏松症防治的关键,也是骨组织修复工程种子细胞的来源,Wnt信号通路对骨形成起着重要作用.目的:综述Wnt/β-catenin信号通路调控骨髓间充质干细胞成骨分化的相关因素及分子机制.方法:应用计算机检索CNKI、PubMed及万方医学数据库建库至2020年2月发表的相关...     

The interaction of the Wnt and Notch pathways modulates natural killer versus T cell differentiation

The Wnt and Notch signaling pathways have been independently shown to play a critical role in regulating hematopoietic cell fate decisions. We previously reported that induction of Notch signaling in human CD34(+)CD38(-) cord blood cells by culture with the Notch ligand Delta 1 resulted in more cells with T or natural killer (NK) lymphoid precursor phenotype. Here, we show that addition of Wnt3a to Delta 1 further increased the percentage of CD34(-)CD7(+) and CD34(-)CD7(+)cyCD3(+) cells with increased expression of CD3 epsilon and preT alpha. In contrast, culture with Wnt3a alone did not increase generation of CD34(-)CD7(+) precursors or expression of CD3 epsilon or preT alpha gene. Furthermore, Wnt3a increased the amount of activated Notch1, suggesting that Wnt modulates Notch signaling by affecting Notch protein levels. In contrast, addition of a Wnt signaling inhibitor to Delta 1 increased the percentage of CD56(+) NK cells. Overall, these results demonstrate that regulation of Notch signaling by the Wnt pathway plays a critical role in differentiation of precursors along the early T or NK differentiation pathways. Disclosure of potential conflicts of interest is found at the end of this article.     

微重力对骨髓间充质干细胞成骨分化的影响

骨髓间充质干细胞（BMSCs）是一种多能成体于细胞,是组织工程重要的种子细胞来源之一.微重力对BMSCs成骨分化具有抑制作用,可使骨量减少和骨微结构改变,从而导致骨质疏松症.这一过程受到多条信号通路的调控,如MAPK信号通路、Notch信号通路和Wnt/β-catenin信号通路等,它们协同调节微重力下BMSCs向成骨细胞方向的分化.研究微重力对BMSCs成骨分化的影响,可以阐明骨质流失机理,为相关疾病的治疗提供新的靶点,促进我国太空宇航事业的发展.     

Modeling notch signaling in normal and neoplastic hematopoiesis: global gene expression profiling in response to activated notch expression

In normal hematopoiesis, proliferation is tightly linked to differentiation in ways that involve cell-cell interaction with stromal elements in the bone marrow stem cell niche. Numerous in vitro and in vivo studies strongly support a role for Notch signaling in the regulation of stem cell renewal and hematopoiesis. Not surprisingly, mutations in the Notch gene have been linked to a number of types of malignancies. To better define the function of Notch in both normal and neoplastic hematopoiesis, a tetracycline-inducible system regulating expression of a ligand-independent, constitutively active form of Notch1 was introduced into murine E14Tg2a embryonic stem cells. During coculture, OP9 stromal cells induce the embryonic stem cells to differentiate first to hemangioblasts and subsequently to hematopoietic stem cells. Our studies indicate that activation of Notch signaling in flk+ hemangioblasts dramatically reduces their survival and proliferative capacity and lowers the levels of hematopoietic stem cell markers CD34 and c-Kit and the myeloid marker CD11b. Global gene expression profiling of day 8 hematopoietic progenitors in the absence and presence of activated Notch yield candidate genes required for normal hematopoietic differentiation, as well as putative downstream targets of oncogenic forms of Notch including the noncanonical Wnts Wnt4 and 5A. Disclosure of potential conflicts of interest is found at the end of this article.     

Regulation of Myelopoiesis by CD137L Signaling

CD137 ligand (CD137L) has emerged as a powerful regulator of myelopoiesis that links emergency situations, such as infections, to the generation of additional myeloid cells, and to their activation and maturation. CD137L is expressed on the cell surface of hematopoietic stem and progenitor cells (HSPC) and antigen presenting cells (APC) as a transmembrane protein. The signaling of CD137L into HSPC induces their proliferation and differentiation to monocytes and macrophages, and in monocytes CD137L signaling induces differentiation to potent dendritic cells (DC). CD137L signaling is initiated by CD137 which is expressed by T cells, once they become activated. Some of these activated, CD137-expressing T cells migrate from the site of infection to the bone marrow where they interact with HSPC to induce myelopoiesis, or they induce monocyte to DC differentiation locally at the site of infection. Therapeutically, induction of CD137L signaling can be utilized to reinitiate myeloid differentiation in acute myeloid leukemia cells, and to generate potent DC for immunotherapy.     

骨髓间充质干细胞Notch1信号通路异常与骨髓瘤骨病

背景：多发性骨髓瘤骨病的发病机制目前尚未完全明确,骨髓间充质干细胞向成骨细胞分化障碍参与其中,而Notch1信号通路在间充质干细胞的增殖分化中起重要作用。 目的：探讨Notch1信号通路在多发性骨髓瘤骨病中的作用。 方法：分离培养多发性骨髓瘤患者和正常人骨髓间充质干细胞,Real-time PCR和Western blot检测成骨诱导分化前后Notch1和成骨基因Runx2的表达,以及Von Kossa染色鉴定钙质沉积程度。在多发性骨髓瘤患者间充质干细胞成骨诱导分化过程中,加入Notch1信号通路抑制剂DAPT和安慰剂,48 h后real-time PCR和western blot鉴定Notch1信号通路下游分子Hes1和成骨指标Runx2表达,2周后Von Kossa染色鉴定钙质沉积程度。 结果与结论：成骨诱导48 h后,间充质干细胞的Notch1表达减低,但是骨髓瘤患者间充质干细胞的降低幅度小于正常对照间充质干细胞;48 h后Runx2的表达在骨髓瘤患者间充质干细胞的表达明显弱于正常对照间充质干细胞;2周后,Von Kossa染色鉴定钙质沉积程度,骨髓瘤患者间充质干细胞明显弱于正常对照间充质干细胞;48 h后Hes1表达在DAPT组明显低于安慰剂组;而Runx2的表达在DAPT组明显高于安慰剂组。2周后 DAPT组钙质沉积明显强于安慰剂组。实验说明多发性骨髓瘤患者的间充质干细胞中,Notch1信号通路失活缺陷可能抑制其向成骨细胞分化。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

过表达Notch1胞内域对c-Kit+骨髓间充质干细胞分化的影响

BACKGROUND: Activation of Notch signaling plays a critical role in stem cell differentiation, and this effect seems to be cell-type dependent. Little is reported on the role of activation of Notch1 signaling in the differentiation of c-Kit+ bone marrow mesenchymal stem cells. OBJECTIVE: To analyze the influence of activation of Notch1 signaling on the differentiation of c-Kit+ bone marrow mesenchymal stem cells. METHODS: The Notch1 intracellular domain (N1-ICD) was obtained from the cDNA library by PCR and cloned into BamHI/AgeI digested adenoviral GV314 plasmid to construct N1-ICD overexpressing shuttle plasmid, and the positive clones were verified by sequencing. N1-ICD shuttle plasmid and helper plasmids pBHGloxΔE1,3 Cre were used to co-transfect HEK293T cells to obtain N1-ICD overexpressing adenoviral particles (N1-ICD-Ad). The c-Kit+ subpopulation were isolated from bone marrow mesenchymal stem cells of the Sprague-Dawley rat femur via magnetic activated cell sorting. After transfection of the c-Kit+ BMSCs with N1-ICD-Ad adenovirus, we assessed the activation of Notch1 signaling and differentiation in c-Kit+ bone marrow mesenchymal stem cells by quantitative RT-PCR and immunofluorescent staining. RESULTS AND CONCLUSION: N1-ICD coding sequence was successfully generated from the cDNA library, and then was cloned into the linearized adenoviral vectors GV314. The resistant clones were verified by sequencing. With the assistance of packaging plasmids, recombinant N1-ICD-Ad adenovirus plasmids were successful packaged in HEK293T cells, and its title was 2×1012 PFU/L. c-Kit+ bone marrow mesenchymal stem cells with the purity of 91.6% were successfully isolated from the bone marrow mesenchymal stem cells of the Sprague-Dawley rat femur. Compared with the blank and negative controls, N1-ICD-Ad infection in the c-Kit+ bone marrow mesenchymal stem cells led to substantial accumulation of N1-ICD in the cytoplasm and nuclei, significantly unregulated expressions of Hes1 (a downstream gene of Notch) and cardiomyocyte differentiation genes Nkx2.5 and cTnT, significantly increased the expression of von Willebrand factor, an endothelial cell differentiation gene, and mildly increased the expression of smooth muscle 22α, a smooth muscle cell differentiation gene. These experimental results indicate that the activation of Notch1 signaling contributes to multi-lineages differentiation of c-Kit+ bone marrow mesenchymal stem cells, and the construction of N1-ICD overexpressing adenoviral vector makes the foundation for further research on the role of Notch1 signaling in stem cell biology.      

How faithfully does intramembranous bone regeneration recapitulate embryonic skeletal development?

Postnatal intramembranous bone regeneration plays an important role during a wide variety of musculoskeletal regeneration processes such as fracture healing, joint replacement and dental implant surgery, distraction osteogenesis, stress fracture healing, and repair of skeletal defects caused by trauma or resection of tumors. The molecular basis of intramembranous bone regeneration has been interrogated using rodent models of most of these conditions. These studies reveal that signaling pathways such as Wnt, TGFβ/BMP, FGF, VEGF, and Notch are invoked, reminiscent of embryonic development of membranous bone. Discoveries of several skeletal stem cell/progenitor populations using mouse genetic models also reveal the potential sources of postnatal intramembranous bone regeneration. The purpose of this review is to compare the underlying molecular signals and progenitor cells that characterize embryonic development of membranous bone and postnatal intramembranous bone regeneration.     

Notch和Wnt信号通路对神经干细胞增殖分化的影响

Notch和Wnt信号通路是调节神经干细胞(neural stem cells,NSCs)增殖、分化的重要通路,Notch信号通路的靶基因Hes1、Hes5及HES相关蛋白等分化抑制信号,通过旁侧抑制机制阻止NSCs的分化,并促进其自我更新;通过NICD与CSL DNA结合蛋白的直接结合,形成GFAP的转录激活复合物,上调GFAP的表达,从而促进NSCs向星形胶质细胞的分化。Wnt信号通过Wnt/β-catenin信号通路对细胞周期素D1和D2的转录调节,调控NSCs细胞周期的进程,使其量增殖;然而,过表达的Wnt3a和Wnt7a蛋白能够抑制NSCs的增殖,促进NSCs向神经元方向分化。     

Analysis of the human fetal liver hematopoietic microenvironment

In the adult, hematopoietic stem cells (HSCs) are resident in the bone marrow (BM) compartment and are in direct association with the BM stromal microenvironment. However, human adult HSCs are largely quiescent and undergo limited self-renewal. This is in contrast to the higher frequency of cycling HSCs undergoing self-renewal during fetal development when hematopoiesis is transiently localized to the fetal liver (FL), suggesting that FL provides a more conducive microenvironment to support HSCs. Here, we provide phenotypic and molecular characterization of primary human FL stromal cells capable of supporting human repopulating progenitors. Qualitative and quantitative analysis revealed several properties unique to FL stromal cells compared to adult BM-derived stroma that included a greater than 10-fold enhanced proliferative capacity of FL stromal vs adult BM, and a 2-fold increase in the number of N-cadherin- and osteopontin-expressing cells. Supportive of extrinsic influences likely to modulate HSC expansion, global gene expression microarray analysis revealed that FL stroma has higher expression of regulators of the Wnt signaling pathway compared to adult BM stroma, which demonstrated an increased expression of the Notch signaling pathway. Our results suggest that human FL stromal cells provide a unique microenvironment to HSCs compared to adult BM stroma by controlling Wnt signaling of HSCs during human fetal hematopoietic development, while Notch signaling is tightly regulated by the HSC microenvironment in the adult. We propose that the human HSC niche is ontogenically controlled during human development to provide appropriate expansion of fetal HSCs and subsequent maintenance of adult HSCs.     

CTL epitope distribution patterns in the Gag and Nef proteins of HIV-1 from subtype A infected subjects in Kenya: Use of multiple peptide sets increases the detectable breadth of the CTL response

Background

The early B lymphopoiesis in mammals is regulated through close interactions with stromal cells and components of the intracellular matrix in the bone marrow (BM) microenvironment. Although B lymphopoiesis has been studied for decades, the factors that are implicated in this process, both autocrine and paracrine, are inadequately explored. Wnt signaling is known to be involved in embryonic development and growth regulation of tissues and cancer. Wnt molecules are produced in the BM, and we here ask whether canonical Wnt signaling has a role in regulating human BM B lymphopoiesis.

Results

Examination of the mRNA expression pattern of Wnt ligands, Fzd receptors and Wnt antagonists revealed that BM B progenitor cells and stromal cells express a set of ligands and receptors available for induction of Wnt signaling as well as antagonists for fine tuning of this signaling. Furthermore, different B progenitor maturation stages showed differential expression of Wnt receptors and co-receptors, β-catenin, plakoglobin, LEF-1 and TCF-4 mRNAs, suggesting canonical Wnt signaling as a regulator of early B lymphopoiesis. Exogenous Wnt3A induced stabilization and nuclear accumulation of β-catenin in primary lineage restricted B progenitor cells. Also, Wnt3A inhibited B lymphopoiesis of CD133⁺CD10^- hematopoietic progenitor cells and CD10⁺ B progenitor cells in coculture assays using a supportive layer of stromal cells. This effect was blocked by the Wnt antagonists sFRP1 or Dkk1. Examination of early events in the coculture showed that Wnt3A inhibits cell division of B progenitor cells.

Conclusion

These results indicate that canonical Wnt signaling is involved in human BM B lymphopoiesis where it acts as a negative regulator of cell proliferation in a direct or stroma dependent manner.     

Notch1 expression in early lymphopoiesis influences B versus T lineage determination.

Notch receptors regulate fate decisions in many cells. One outcome of Notch signaling is differentiation of bipotential precursors into one cell type versus another. To investigate consequences of Notch1 expression in hematolymphoid progenitors, mice were reconstituted with bone marrow (BM) transduced with retroviruses encoding a constitutively active form of Notch1. Although neither granulocyte or monocyte differentiation were appreciably affected, lymphopoiesis was dramatically altered. As early as 3 weeks following transplantation, mice receiving activated Notch1-transduced BM contained immature CD4+ CD8+ T cells in the BM and exhibited a simultaneous block in early B cell lymphopoiesis. These results suggest that Notch1 provides a key regulatory signal in determining T lymphoid versus B lymphoid lineage decisions, possibly by influencing lineage commitment from a common lymphoid progenitor cell.     

BMP-9 induced osteogenic differentiation of mesenchymal stem cells: molecular mechanism and therapeutic potential

Promoting osteogenic differentiation and efficacious bone regeneration have the potential to revolutionize the treatment of orthopaedic and musculoskeletal disorders. Mesenchymal Stem Cells (MSCs) are bone marrow progenitor cells that have the capacity to differentiate along osteogenic, chondrogenic, myogenic, and adipogenic lineages. Differentiation along these lineages is a tightly controlled process that is in part regulated by the Bone Morphogenetic Proteins (BMPs). BMPs 2 and 7 have been approved for clinical use because their osteoinductive properties act as an adjunctive treatment to surgeries where bone healing is compromised. BMP-9 is one of the least studied BMPs, and recent in vitro and in vivo studies have identified BMP-9 as a potent inducer of osteogenic differentiation in MSCs. BMP-9 exhibits significant molecular cross-talk with the Wnt/ β-catenin and other signaling pathways, and adenoviral expression of BMP-9 in MSCs increases the expression of osteogenic markers and induces trabecular bone and osteiod matrix formation. Furthermore, BMP-9 has been shown to act synergistically in bone formation with other signaling pathways, including Wnt/ β-catenin, IGF, and retinoid signaling pathways. These results suggest that BMP-9 should be explored as an effective bone regeneration agent, especially in combination with adjuvant therapies, for clinical applications such as large segmental bony defects, non-union fractures, and/or spinal fusions.     

Non-canonical Wnt signaling pathways in hematopoiesis

Hematopoietic stem cells (HSCs) are a rare population of cells that are responsible for life-long generation of blood cells of all lineages. In order to maintain their numbers, HSCs must establish a balance between the opposing cell fates of self-renewal and initiation of hematopoietic differentiation. Multiple signaling pathways have been implicated in the regulation of HSC cell fate. One such set of pathways are those activated by the Wnt family of ligands. The function of the canonical Wnt signaling pathway, which utilizes β-catenin to regulate gene expression, has been extensively studied in hematopoiesis. However, there is a growing body of evidence that the other Wnt signaling pathways, termed non-canonical, also play an important role. In this review, we will discuss the regulation of hematopoiesis by the Wnt signaling pathways, focusing on the potential functions of non-canonical Wnt signaling pathways.