Notch 信号通路：结构、疾病和治疗

The receptors and ligands of NOTCH signaling
NOTCH信号的受体和配体

D. melanogaster has only one NOTCH receptor⁹. C. elegans has two redundant NOTCH receptors, LIN-12 and GLP-1⁴. Mammals have four NOTCH paralogs, NOTCH1, NOTCH2, NOTCH3, and NOTCH4²¹, showing both redundant and unique functions. In humans, NOTCH1, NOTCH2, NOTCH3, and NOTCH4 are located on chromosomes 9, 1, 19, and 6, respectively. After transcription and translation, NOTCH precursors are generated in the endoplasmic reticulum (ER) and then translocated into the Golgi apparatus. In the ER, the NOTCH precursors are initially glycosylated at the EGF-like repeat domain. Glycosylations include O-fucosylation, O-glucosylation, and O-GlcNAcylation, which are catalyzed by the enzymes POFUT1, POGLUT1, and EOGT1, respectively⁴³. Subsequently, in the Golgi apparatus, O-fucose is extended by the Fringe family of GlcNAc transferases, while O-glucose is extended by the xylosyltransferases GXYLT1/2 and XXYLT1^44–46. The glycosylation of NOTCH is vital to its stability and function. Alteration of core glycosylation enzymes severely inhibits the activity of NOTCH signaling^47–51, making these enzymes vital for further research.
D.黑腹果蝇只有一个NOTCH受体⁹。C.线虫具有两个冗余的NOTCH受体，LIN-12和GLP-¹⁴。哺乳动物有四个NOTCH旁系同源物，NOTCH 1，NOTCH 2，NOTCH 3和NOTCH 4²¹，显示出冗余和独特的功能。在人类中，NOTCH 1、NOTCH 2、NOTCH 3和NOTCH 4分别位于染色体9、1、19和6上。在转录和翻译后，NOTCH前体在内质网（ER）中产生，然后易位到高尔基体中。在ER中，NOTCH前体最初在EGF样重复结构域处糖基化。糖基化包括O-岩藻糖基化、O-葡糖基化和O-GlcNAc酰化，其分别由酶POFUT 1、POGLUT 1和EOGT 1催化⁴³。 随后，在高尔基体中，0-岩藻糖通过GlcNAc转移酶的Fringe家族延伸，而0-葡萄糖通过木糖基转移酶GXYLT 1/2和XXYLT 1^{44 - 46延伸}。NOTCH的糖基化对它的稳定性和功能至关重要。核心糖基化酶的改变严重抑制NOTCH信号传导的活性^{47 - 51}，使得这些酶对于进一步研究至关重要。

The glycosylated NOTCH precursors undergo S1 cleavage in the Golgi apparatus before being transported to the cell membrane. The cleavage always occurs at a conserved site (heterodimerization domain) and is catalyzed by a furin-like protease, cutting NOTCH into a heterodimer (mature form). Here, we take mouse NOTCH1 as an example to illustrate the structure of mature NOTCH on the cell membrane.
糖基化的NOTCH前体在被转运至细胞膜之前在高尔基体中经历S1裂解。切割总是发生在保守位点（异源二聚化结构域），并由弗林蛋白酶样蛋白酶催化，将NOTCH切割成异源二聚体（成熟形式）。在此，我们以小鼠NOTCH1为例来说明细胞膜上成熟NOTCH的结构。

The extracellular domain (N-terminal) contains 36 EGF-like repeats and a negative regulatory region (NRR)⁴³. The 11th and 12th EGF-like repeats usually interact with ligands⁴³, although a new study found that many more motifs of the extracellular domain are involved in ligand binding⁵². The NRR domain is composed of three cysteine-rich Lin12-NOTCH repeats (LNRs) and a heterodimerization region critical for S2 cleavage. Located after the membrane-spanning region, the intracellular RBPJ association module (RAM) domain is responsible for interacting with transcription factors in the nucleus, and seven ankyrin repeat (ANK) domains are observed in the RAM domain. Nuclear localization sequences are located on both sides of the ANK domains. At the end of the intracellular domain (C-terminus), there are conserved proline/glutamic acid/serine/threonine-rich motifs (PEST domains) that contain degradation signals and are thus critical for the stability of the NOTCH intracellular domain (NICD). Mammalian NOTCH2-4 have similar structures to NOTCH1, diverging mainly in the number of EGF-like repeats, the glycosylation level of the EGF-like repeats, and the length of the PEST domains. The level of NOTCH receptors on the cell membrane is controlled by constitutive endocytosis, which is promoted by ubiquitin ligases. An appreciable amount of NOTCH receptors are ubiquitinated and degraded in the proteosome, while the rest are expressed on the cell membrane to transmit signals.
细胞外结构域（N-末端）含有36个EGF样重复和一个负调控区（NRR）⁴³。第11和第12个EGF样重复序列通常与配体相互作用⁴³，尽管一项新的研究发现细胞外结构域的更多基序参与配体结合⁵²。NRR结构域由三个富含半胱氨酸的Lin 12-NOTCH重复序列（LNR）和一个对S2裂解至关重要的异源二聚化区组成。位于跨膜区之后的细胞内RBPJ结合模块（RAM）结构域负责与核内转录因子相互作用，在RAM结构域中观察到7个锚蛋白重复（ANK）结构域。核定位序列位于ANK结构域的两侧。 在胞内结构域的末端（C-末端），存在保守的富含脯氨酸/谷氨酸/丝氨酸/苏氨酸的基序（PEST结构域），其包含降解信号，因此对于NOTCH胞内结构域（NICD）的稳定性是关键的。哺乳动物的NOTCH 2 -4与NOTCH 1具有相似的结构，主要在EGF样重复序列的数量、EGF样重复序列的糖基化水平和PEST结构域的长度上不同。细胞膜上NOTCH受体的水平由组成性内吞作用控制，而组成性内吞作用由泛素连接酶促进。相当数量的NOTCH受体在蛋白体中被泛素化和降解，而其余的在细胞膜上表达以传递信号。

Humans and mice have five acknowledged NOTCH ligands^21,53,54: delta-like ligand 1 (DLL1), delta-like ligand 3 (DLL3), delta-like ligand 4 (DLL4), Jagged-1 (JAG1), and Jagged-2 (JAG2), all of which present redundant and unique functions. For instance, DLL1 governs cell differentiation and cell-to-cell communication⁵⁴, DLL3 suppresses cell growth by inducing apoptosis⁵⁵, DLL4 activates NF-κΒ signaling to enhance vascular endothelial factor (VEGF) secretion and tumor metastasis⁵⁶, JAG1 enhances angiogenesis⁵⁴, and JAG2 promotes cell survival and proliferation⁵⁴.
人类和小鼠具有五种公认的NOTCH配体^21、53、54：δ样配体1（DLL 1）、δ样配体3（DLL 3）、δ样配体4（DLL 4）、Jagged-1（JAG 1）和Jagged-2（JAG 2），所有这些都呈现冗余和独特的功能。例如，DLL 1支配细胞分化和细胞间通讯⁵⁴，DLL 3通过诱导细胞凋亡抑制细胞生长⁵⁵，DLL 4激活NF-κ B信号传导以增强血管内皮因子（VEGF）分泌和肿瘤转移⁵⁶，JAG 1增强血管生成⁵⁴，并且JAG 2促进细胞存活和增殖⁵⁴。

The structures of the NOTCH ligands are partially similar to those of the receptors. The ligands are also transmembrane proteins, and the extracellular domains contain multiple EGF-like repeats, which determine the crosstalk with corresponding receptors. The levels and functions of the ligands are also controlled by ubiquitylation and endocytosis (discussed in the section “Ligand ubiquitylation”).
NOTCH配体的结构与受体的结构部分相似。配体也是跨膜蛋白，并且细胞外结构域包含多个EGF样重复，其决定与相应受体的串扰。配体的水平和功能也受泛素化和内吞作用控制（在“配体泛素化”部分讨论）。

The canonical NOTCH signaling pathway
经典的NOTCH信号通路

The mature NOTCH receptors on the cell membrane are heterodimers, with the heterodimerization domain being cleaved in the Golgi apparatus (S1 cleavage). Generally, binding to extracellular domains of NOTCH receptors allows ligands to initiate endocytosis. Such endocytosis induces receptors to change their conformation, exposing the enzymatic site for S2 cleavage⁵⁷. Receptors then experience S3 cleavage, changing into the effector form: NOTCH intracellular domain (NICD). NICD is degraded in the cytoplasm or transported into the nucleus to regulate the transcription of target genes (Fig. ​(Fig.22).
细胞膜上的成熟NOTCH受体是异源二聚体，其中异源二聚化结构域在高尔基体中被切割（S1切割）。通常，与NOTCH受体的细胞外结构域的结合允许配体引发内吞作用。这种内吞作用诱导受体改变其构象，暴露酶促位点用于S2裂解⁵⁷。然后受体经历S3裂解，转变为效应物形式：NOTCH胞内结构域（NICD）。NICD在细胞质中降解或转运到细胞核中，调节靶基因的转录（图2 -2）。

S2 cleavage is the only ligand-binding step and is thus vital for signal initiation. The structural basis of S2 cleavage is illustrated in Fig. ​Fig.2.2. The S2 site (metalloprotease site) is hidden by the LNR domain in the silent phase, referred to as the “autoinhibited conformation”⁵⁸. Once bound with ligands, the receptor extends the LNR domain and exposes the S2 site for cleavage^59–61. The core enzymes for S2 cleavage include a disintegrin and metalloprotease 10 (ADAM 10) and its isoforms ADAM 17 and ADAMTS1^62–64, which are popular targets for drug discovery. The product of S2 cleavage (larger part) is composed of the transmembrane domain and the intracellular domain, which is also called NOTCH extracellular truncation (NEXT)⁶⁵.
S2裂解是唯一的配体结合步骤，因此对于信号起始至关重要。S2裂解的结构基础如图2所示。2.第一章S2位点（金属蛋白酶位点）在沉默期被LNR结构域隐藏，称为“自抑制构象”⁵⁸。一旦与配体结合，受体延伸LNR结构域并暴露S2位点用于切割^{59 - 61}。S2裂解的核心酶包括解整合素和金属蛋白酶10（ADAM 10）及其同种型ADAM 17和ADAMTS^{162 - 64}，它们是药物发现的流行靶标。 S2裂解的产物（大部分）由跨膜结构域和胞内结构域组成，也称为NOTCH胞外截短（NEXT）⁶⁵。

NEXT is further cleaved at the S3 site, releasing NICD, which can be translocated into the nucleus and function as a transcription factor. The enzyme responsible for S3 cleavage is γ-secretase, which contains the catalytic subunits presenilin1 or presenilin2 (PS1 or PS2)^66,67, APH-1, PEN-2, and nicastrin (NCT)⁶⁸. However, the classical substrates for γ-secretase contain NOTCH receptors and amyloid precursor protein (APP), the successive cleavage of which is related to Alzheimer’s disease^69–72. The structural basis for γ-secretase to recognize NOTCH or APP had remained unclear until recently, when Yigong Shi’s team elucidated the structural basis^31,32. In short, the transmembrane helix of NOTCH or APP closely interacts with the surrounding transmembrane helix of PS1 (the catalytic subunit of γ-secretase); thus, the hybrid β-sheet promotes substrate cleavages, although some differences exist between NOTCH and APP⁷³. Structural information would accelerate the discovery of substrate-specific inhibitors of NOTCH and APP. Additionally, S3 cleavage can occur both on the cell membrane and in the endosome after NEXT is endocytosed, termed the endocytosis-independent model and endocytic-activation model, respectively⁷⁴.
NEXT在S3位点进一步裂解，释放NICD，其可以易位到细胞核中并作为转录因子发挥作用。负责S3裂解的酶是γ-分泌酶，其含有催化亚基早老素1或早老素2（PS1或PS2）^66、67、APH-1、PEN-2和nicastrin（NCT）⁶⁸。然而，γ-分泌酶的经典底物含有NOTCH受体和淀粉样前体蛋白（APP），其连续裂解与阿尔茨海默病相关^{69 - 72}。γ-分泌酶识别NOTCH或APP的结构基础一直不清楚，直到最近，Yigong Shi的团队阐明了结构基础^31，32。 简而言之，NOTCH或APP的跨膜螺旋与周围PS1的跨膜螺旋（γ-分泌酶的催化亚基）密切相互作用;因此，尽管NOTCH和APP 73之间存在一些差异，但杂合β-折叠促进底物裂解。结构信息将加速NOTCH和APP的底物特异性抑制剂的发现。此外，在NEXT被内吞后，S3裂解可以发生在细胞膜上和内体中，分别称为内吞作用独立模型和内吞激活模型74。

After release from the cell membrane, NICD is translocated into the nucleus to regulate gene transcription, the mechanism of which may be related to the nuclear localization sequences of NICD and importins alpha 3, 4, and 7⁷⁵. However, the details of this translocation remain unclear. CBF-1/suppressor of hairless/Lag1 (CSL, also called recombination signal binding protein-J, RBPJ) is a ubiquitous transcription factor (TF) that recruits other co-TFs to regulate gene expression^76,77. The target genes of NOTCH signaling are largely determined by the Su (H) motif of CSL, which is responsible for DNA binding²¹. The canonical NOTCH target gene families are Hairy/Enhancer of Split (HES) and Hairy/Enhancer of Split related to YRPW motif (HEY)²¹.
从细胞膜释放后，NICD易位到细胞核中以调节基因转录，其机制可能与NICD和导入素α 3、4和7的核定位序列有关。然而，这种易位的细节仍然不清楚。CBF-1/无毛抑制因子/Lag 1（CSL，也称为重组信号结合蛋白-J，RBPJ）是一种普遍存在的转录因子（TF），其募集其他co-TF来调节基因表达^76，77。NOTCH信号传导的靶基因主要由CSL的Su（H）基序决定，其负责DNA结合²¹。典型的NOTCH靶基因家族是与YRPW基序（HEY）21相关的Hairy/裂解增强子（HES）和Hairy/裂解增强子。

In the traditional model of NICD regulating gene transcription^21,42,78,79, CSL recruits corepressor proteins and histone deacetylases (HDACs) to repress the transcription of target genes without NICD binding. NICD binding can change the conformation of the CSL-repressing complex, dissociating repressive proteins and recruiting activating partners to promote the transcription of target genes. The transcriptional coactivator Mastermind-like protein (MAML) is one of the core activating partners that can recognize the NICD/CSL interface, after which it recruits other activating partners. Drugs targeting MAML are under study.
在NICD调节基因转录的传统模型^{中21，42，78，79}，CSL招募辅阻遏蛋白和组蛋白脱乙酰酶（HDAC）来抑制靶基因的转录，而无需NICD结合。NICD结合可改变CSL-阻遏复合物的构象，解离阻遏蛋白并募集激活配偶体以促进靶基因的转录。转录辅激活因子类主蛋白（MAML）是识别NICD/CSL界面的核心激活配偶体之一，在识别NICD/CSL界面后，MAML会招募其他激活配偶体。针对MAML的药物正在研究中。

Recently, Kimble et al. used single-molecule fluorescence in situ hybridization to study the NOTCH transcriptional program in germline stem cells of C. elegans and found that NICD dictated the probability of transcriptional firing and thus the number of nascent transcripts⁸⁰. However, NICD did not orchestrate a synchronous transcriptional response in the nucleus, in contrast to that seen in the classical model. Gomez-Lamarca et al. found similar results in D. melanogaster⁸¹. NICD promoted the opening of chromatin and enhanced the recruitment of both the NICD-containing activating CSL complex and the NICD-free repressive CSL complex. Bray et al. proposed a new model to interpret their findings. In the NOTCH-off state, chromatin is compact, and only the NICD-free (repressing) CSL complex regulates transcription. In the NOTCH-on state, chromatin is loosened and bound to both NICD-containing (activating) and NICD-free (repressive) CSL complexes. Because the number of activating complexes is greater than that of repressive complexes after NICD enters the nucleus, NICD promotes the transcription of target genes. Bray et al. further reported that nucleosome turnover occurred frequently at NOTCH-responsive regions and depended on the Brahma SWI/SNF chromatin remodeling complex⁸². Consistently, Kimble et al. found that NOTCH signaling regulated the duration of the transcriptional burst but not the intensity of signaling or the time between bursts⁸³. Oncogenic NOTCH is also considered to enhance repositioning to promote the transcription of genes, such as MYC⁸⁴. In general, the new model from Bray et al. helps explain the flexibility of NOTCH signaling, although the details still require further elucidation.
最近，Kimble等利用单分子荧光原位杂交技术研究了C. elegans，并发现NICD决定了转录触发的概率，从而决定了新生转录本的数量⁸⁰。然而，NICD并没有在细胞核中协调同步转录反应，这与经典模型中所见相反。Gomez-Lamarca等在D. ⁸¹. history of life NICD促进了染色质的开放，并增强了含NICD的激活CSL复合物和无NICD的抑制CSL复合物的募集。Bray等人提出了一个新的模型来解释他们的发现。在NOTCH关闭状态下，染色质是紧凑的，只有无NICD（抑制）的CSL复合物调节转录。 在NOTCH-on状态下，染色质松动并结合到含NICD（激活）和无NICD（抑制）的CSL复合物上。由于NICD进入细胞核后，激活复合物的数量大于抑制复合物的数量，因此NICD促进了靶基因的转录。Bray等人进一步报道，核小体更新频繁发生在NOTCH反应区，并依赖于Brahma SWI/SNF染色质重塑复合物⁸²。同样，Kimble等人发现NOTCH信号调节转录爆发的持续时间，但不调节信号的强度或爆发之间的时间⁸³。致癌性NOTCH也被认为增强重新定位以促进基因（如MYC 84）的转录。一般来说，来自Bray等人的新模型。 有助于解释NOTCH信令的灵活性，尽管细节仍需要进一步阐明。

The noncanonical NOTCH signaling pathway
非经典NOTCH信号通路

Pathways other than canonical signaling pathway are also able to initiate signaling, classified as noncanonical NOTCH signaling pathways. Although the mature NOTCH receptors on the cell membrane are capable of ligand binding, some are endocytosed for renewal. Endocytosed NOTCH receptors can return to the cell membrane, be degraded in lysosomes or activated in endosomes (ligand-independent activation)^74,85. Interestingly, endosome trafficking can also be regulated by NOTCH signaling⁸⁶. Endosomes have been proven to contain ADAM and γ-secretase⁸⁷. Ligand-independent activation of NOTCH signaling is vital to T cell development⁸⁸. One example of ligand-independent activation is T cell receptor (TCR)-mediated self-amplification⁸⁷. The activated TCR/CD3 complex can activate the signaling axis of LCK-ZAP70-PLCγ-PKC. PKC then activates ADAM and γ-secretase on the endosome to initiate S2 and S3 cleavage and thus NOTCH signaling. Activated NOTCH signaling can further upregulate immune-related genes to amplify the immune response.
除了规范信号传导途径之外的途径也能够启动信号传导，被分类为非规范NOTCH信号传导途径。尽管细胞膜上的成熟NOTCH受体能够与配体结合，但有些被内吞以进行更新。内吞的NOTCH受体可以返回到细胞膜，在溶酶体中降解或在内体中活化（配体非依赖性活化）^74，85。有趣的是，内体运输也可以通过NOTCH信号传导调节⁸⁶。已证实内体含有ADAM和γ-分泌酶⁸⁷。NOTCH信号传导的配体非依赖性激活对T细胞发育至关重要⁸⁸。配体非依赖性活化的一个实例是T细胞受体（TCR）介导的自身扩增⁸⁷。 活化的TCR/CD 3复合物可激活LCK-ZAP 70-PLCγ-PKC信号轴。然后PKC激活内体上的ADAM和γ-分泌酶以启动S2和S3切割，从而启动NOTCH信号传导。激活的NOTCH信号可以进一步上调免疫相关基因以放大免疫应答。

Independent of CSL, NICD can interact with the NF-κB, mTORC, PTEN, AKT, Wnt, Hippo, or TGF-β pathways at the cytoplasmic and/or nuclear level to regulate the transcription of target genes^34,89–96. The crosstalk between NICD and NF-κB affects the malignant properties of cervical cancer⁸⁹, colorectal cancer⁹⁷, breast cancer⁹⁸, and small-cell lung cancer cells⁹⁹. Targeting the NF-κB pathway could be an effective way to block noncanonical NOTCH signaling.
独立于CSL，NICD可以在细胞质和/或细胞核水平上与NF-κB、mTORC、PTEN、AKT、Wnt、Hippo或TGF-β途径相互作用，以调节靶基因的转录^{34，89 - 96}。NICD和NF-κB之间的串扰影响宫颈癌⁸⁹、结肠直肠癌⁹⁷、乳腺癌⁹⁸和小细胞肺癌细胞^{99的恶性性质}。靶向NF-κB通路可能是阻断非经典NOTCH信号传导的有效途径。

In addition to those mentioned above, there is a newly identified mechanism of noncanonical activation. In the classical model, S3 cleavage is necessary for NOTCH receptors to release NICD and thus regulate the transcription of target genes. However, membrane-tethered NOTCH may activate the PI3K-AKT pathway, promoting the transcription of interleukin-10 and interleukin-12¹⁰⁰. In blood flow-mediated NOTCH signaling, the transmembrane domain instead of NICD recruits other partners to promote the formation of an endothelial barrier³⁵. NOTCH3 itself can promote the apoptosis of tumor endothelial cells, independent of cleavage and transcription regulation¹⁰¹. The JAG1 intracellular domain can promote tumor growth and epithelial–mesenchymal transition (EMT) without binding to NOTCH receptors¹⁰². These noncanonical mechanisms provide this ancient signaling pathway with more unique functions while massively increasing its complexity.
除了上面提到的那些，还有一个新发现的非典型激活机制。在经典模型中，S3切割对于NOTCH受体释放NICD并因此调节靶基因的转录是必需的。然而，膜栓系的NOTCH可以激活PI 3 K-AKT通路，促进白细胞介素-10和白细胞介素-12 100的转录。在血流介导的NOTCH信号传导中，跨膜结构域而不是NICD募集其他配偶体以促进内皮屏障的形成³⁵。NOTCH 3本身可以促进肿瘤内皮细胞的凋亡，不依赖于切割和转录调节¹⁰¹。JAG 1胞内结构域可以促进肿瘤生长和上皮-间质转化（EMT），而不结合NOTCH受体¹⁰²。 这些非经典机制为这个古老的信号通路提供了更多独特的功能，同时大大增加了它的复杂性。

NOTCH and somitogenesis NOTCH与体节发生

The somitogenesis of vertebrates occurs in a strict order and is regulated by the segmentation clock. It is closely related to the expression of oscillating genes regulated by NOTCH, Wnt and FGF signaling^159–162. NOTCH signaling triggers an excitatory signal, causing presomitic mesoderm (PSM) to transition into a self-sustaining cyclic oscillation state^163,164. The gene oscillation period is consistent with the half-life of HES7¹⁶⁵ and induces lunatic fringe (Lfng) transcription. LFNG, as a glycosyl transferase that can modify the extracellular domain of NOTCH after translation and periodically blocks the cleavage of NOTCH receptors, causes the formation of cyclic NICD^166–168. PSM is a group of self-sustaining oscillating cells, but the synchronous oscillation between depends on the transmission of NOTCH signaling^169–171. LFNG inhibits the activation of NOTCH signaling in neighboring cells by regulating the function of DLL1^164,172,173. In Lfng-knockout mice, PMS oscillation fails to synchronize, but PMS oscillation amplitude and period remain unaffected¹⁷⁰. This finding further demonstrates that LFNG is a key coupling factor for synchronous oscillations between cells.
脊椎动物的体节发生有严格的顺序，并受到体节时钟的调控。它与由NOTCH、Wnt和FGF信号传导调节的振荡基因的表达密切相关^{159 - 162}。NOTCH信号传导触发兴奋性信号，导致分裂前中胚层（PSM）转变成自持循环振荡状态^163、164。基因振荡周期与HES 7 165的半衰期一致，并诱导疯狂边缘（Lfng）转录。LFNG作为糖基转移酶，可以在翻译后修饰NOTCH的细胞外结构域并周期性地阻断NOTCH受体的切割，导致环状NICD^{166 - 168的形成}。 PSM是一组自维持振荡小区，但是它们之间的同步振荡取决于NOTCH信令^{169 - 171的传输}。LFNG通过调节DLL 1的功能来抑制相邻细胞中NOTCH信号传导的激活^{164、172、173}。在Lfng敲除小鼠中，PMS振荡未能同步，但PMS振荡幅度和周期保持不受影响¹⁷⁰。这一发现进一步证明LFNG是细胞间同步振荡的关键耦合因子。

NOTCH and skeleton 缺口和骨架

In the growth and development of MPC, NOTCH signaling regulates and inhibits the production of osteoblasts¹⁵¹, chondrocytes^174–178, and osteoclasts^179,180 through different ligands and receptors (NOTCH1, NOTCH2, JAG1, DLL1) as well as the downstream target gene (SRY-related high-mobility group box 9, SOX9). In addition, the latest research shows that inhibiting glucose metabolism can guide NOTCH to regulate MPC¹⁵⁰, proving the complex role of NOTCH signaling in the skeletal microenvironment. In the mouse model, the absence of NOTCH signaling leads to depletion of MPC and nonunion of fractures¹⁸¹, consistent with the finding that activated JAG1-NOTCH signaling reduces MPC senescence and cell cycle arrest. Interestingly, using γ-secretase inhibitors intermittently and temporarily for fractures significantly promotes cartilage and bone callus formation, as well as superior strength¹⁸². This indicates that NOTCH signaling exerts its function in a temporally and spatially dependent manner.
在MPC的生长和发育中，NOTCH信号通过不同的配体和受体（NOTCH 1、NOTCH 2、JAG 1、DLL 1）以及下游靶基因（SRY相关高迁移率族蛋白9，SOX 9）调节和抑制成骨细胞151、软骨细胞174 - 178和破骨细胞179、180的产生。此外，最新研究表明，抑制葡萄糖代谢可以引导NOTCH调节MPC¹⁵⁰，证明了NOTCH信号在骨骼微环境中的复杂作用。在小鼠模型中，NOTCH信号传导的缺乏导致MPC的消耗和骨折不愈合¹⁸¹，这与活化的JAG 1-NOTCH信号传导减少MPC衰老和细胞周期停滞的发现一致。 有趣的是，间歇性和暂时性使用γ-分泌酶抑制剂治疗骨折可显著促进软骨和骨痂形成，以及上级强度¹⁸²。这表明NOTCH信号以时间和空间依赖的方式发挥其功能。

NOTCH and cardiomyogenesis
NOTCH与心肌发生

During heart wall formation, NOTCH signaling regulates the ratio of cardiomyocytes to noncardiomyocytes by inhibiting myogenesis, further promoting atrioventricular canal remodeling and maturation, EMT development and heart valve formation^183–185. In the endocardium layer, the DLL4-NOTCH1-mediated Hey1/2-Bmp2-Tbx2 signaling axis is a complex negative feedback regulation loop, where overexpressed Tbx2 can in turn inhibit upstream Hey expression^186–189. In embryos lacking key NOTCH signaling molecules such as Notch1, Rbpj, Hey1/Heyl, or Hey2, EMT development is hindered, and endocardial cells are activated but fail to scatter and invade heart glia¹⁹⁰. NOTCH signaling affects the expression of the cadherin 5¹⁹⁰ and the TGF-β family member bone morphogenetic protein 2 (BMP2)^186,189. In addition, by downregulating VEGFR2, a key negative regulator of EMT within atrioventricular canals (AVCs), NOTCH signaling further induces EMT. Studies have found that active NOTCH1 is most highly expressed in endocardial cells at the base of the trabecular membrane. Bone morphogenetic protein 10 (BMP10)¹⁹¹ and Neuregulin 1 (NRG1)¹⁹² are key molecules of NOTCH signaling that regulate the proliferation, differentiation, and correct folding of cardiomyocytes during trabecular development.
在心脏壁形成期间，NOTCH信号传导通过抑制肌生成来调节心肌细胞与非心肌细胞的比率，进一步促进房室管重塑和成熟、EMT发育和心脏瓣膜形成^{183 - 185}。在内分泌层中，DLL 4-NOTCH 1介导的Hey 1/2-Bmp 2-Tbx 2信号传导轴是一个复杂的负反馈调节环，其中过表达的Tbx 2可以反过来抑制上游Hey表达^{186 - 189}。在缺乏关键NOTCH信号分子如Notch 1、Rbpj、Hey 1/Hey 1或Hey 2的胚胎中，EMT发育受阻，内皮细胞被激活，但不能分散和侵入心脏胶质细胞。 NOTCH信号传导影响钙粘蛋白5¹⁹⁰和TGF-β家族成员骨形态发生蛋白2（BMP 2）^{186、189的表达}。此外，通过下调VEGFR 2（房室管（AVC）内EMT的关键负调节因子），NOTCH信号传导进一步诱导EMT。研究发现，活性NOTCH 1在小梁膜基底的内皮细胞中表达最高。骨形态发生蛋白10（BMP 10）¹⁹¹和神经调节蛋白1（NRG 1）¹⁹²是NOTCH信号传导的关键分子，其在小梁发育期间调节心肌细胞的增殖、分化和正确折叠。

NOTCH and the vasculature
Notch和脉管系统

NOTCH4 and DLL4 are specifically expressed on vascular endothelial cells (ECs)^184,193. Deficiencies in NOTCH signaling result in serious defects in the vasculature of the embryo and yolk sac during embryonic development¹⁹⁴ as well as abnormal development of multiple organs, such as the retinal vasculature^195,196 and uterine blood vessels¹⁹⁷ in rats. At the cellular level, the vascular system mainly includes ECs, pericytes and vascular smooth muscle cells (VSMCs). Under stressors such as hypoxia, resting ECs quickly transform into a state of active growth and high plasticity and then dynamically transform between tip cells (TCs) and stalk cells (SCs) through lateral inhibition rather than direct lineage changes^154,155. This cascade reaction between DLL4-mediated NOTCH signaling and VEGFA-VEGFR2 signaling induces ECs near dominant TCs to maintain a high level of NOTCH signaling, inhibiting their differentiation into TCs^198,199. NOTCH signaling activates the Wnt pathway through feedback regulation to maintain the connection between ECs, promoting vascular stability²⁰⁰. In addition, DLL4-NOTCH can maintain arterial blood–retinal barrier homeostasis by inhibiting transcytosis²⁰¹. NOTCH signaling is also important for the development of VSMCs^202,203. Blocking Notch signaling in neural crest cells, especially NOTCH2 and NOTCH3, results in vascular dysplasia, aortic defects, and even bleeding^{202,204–206}. The regulation of the downstream transcription factors PAX1, SCX, and SOX9 by NOTCH signaling is vital for regulating the differentiation of progenitor cells in the sclera toward VSMCs²⁰⁷.

NOTCH signaling acts decisively in the arteriovenous differentiation of endothelial cells^208,209. NOTCH signaling induces the expression of the arterial marker ephrin B2 and inhibits that of the venous marker EphB4, thereby regulating the number and diameter of arteriovenous vessels^210,211. In mice with dysfunctional mutations of NOTCH signaling molecules such as Notch1, Dll4, Hey1, or Hey2, the arterial subregion is defective, while venous differentiation is hyperactive, leading to unexpected bleeding^210,212,213. Before blood perfusion, active NOTCH signaling on the arterial side can be detected. High levels of VEGF, ERK/MAP kinase and Wnt pathway components increase DLL4 expression^214,215, and the transcription factors Fox1C and Fox2C promote DLL4 activation²¹⁶. Interestingly, ECs can sense and respond to laminar flow through NOTCH1, similar to the shear stress response, transforming the hemodynamic mechanical force into an intracellular signal, which is necessary for vascular balance^217,218.
NOTCH信号传导在内皮细胞的动静脉分化中起决定性作用^208、209。NOTCH信号传导诱导动脉标志物肝配蛋白B2的表达并抑制静脉标志物EphB4的表达，从而调节动静脉血管210、211的数量和直径。在具有NOTCH信号分子如Notch 1、Dll 4、Hey 1或Hey 2的功能失调突变的小鼠中，动脉亚区是有缺陷的，而静脉分化是过度活跃的，导致意外出血^{210、212、213}。在血液灌注之前，可以检测到动脉侧的活性NOTCH信号传导。 高水平的VEGF、ERK/MAP激酶和Wnt途径组分增加DLL 4表达^214、215，并且转录因子Fox 1C和Fox 2C促进DLL 4活化²¹⁶。有趣的是，EC可以感测并响应通过NOTCH 1的层流，类似于剪切应力响应，将血液动力学机械力转化为血管平衡所必需的细胞内信号^217，218。

NOTCH and the hemopoietic system
NOTCH与造血系统

NOTCH signaling is important in the differentiation, development, and function of hematopoietic system cells, both lymphocytes and myeloid cells. In early embryonic development, the hematopoietic endothelium forms hematopoietic stem cells through NOTCH-dependent endothelial-to-hematopoietic transition²¹⁹. NOTCH signaling is fundamental in maintaining the number and stemness of hematopoietic stem cells²²⁰. In lymphocyte development, the absence of NOTCH1 or CSL in early hematopoietic progenitor cells (HPCs) leads to thymic T cell development retardation and B cell accumulation, with HES1 being the key mediator²²¹. Naïve thymocytes highly express NOTCH and immediately downregulate NOTCH1 expression once they successfully pass β-selection. Some scholars propose that NOTCH-mediated T cell development is initiated in the prethymic niche^222,223. For example, bone mesenchymal cells outside the thymus can cross-link with HPCs through NOTCH ligands on the surface to promote the generation of T cell lineages^224,225. Shreya S et al. induced the production of HSPC-derived CD7+ progenitor T cells with DLL4 and VCAM-1 in vitro engineering, and these cells further differentiated into mature T cells after thymus transplantation²²⁶. Regarding B cells, the development of splenic marginal zone B (MZB) cells depends on DLL1-NOTCH2 signaling^227,228. In addition, it was found that active NOTCH2 signaling can mediate the lineage conversion of follicular B cells into MZB cells so that mature B cell subpopulations can quickly and dynamically transform based on the needs of the immune system^229,230. The development of innate lymphoid cells (ILCs) was recently found to be NOTCH-dependent^231–233, and the response of different subtypes of ILCs to NOTCH signaling is heterogeneous^234,235. It is interesting that ILCs can activate MZB cells through DLL1 to enhance antibody production²³⁶. Regarding myeloid cells, NOTCH signaling is significant in the development of macrophages^237,238, dendritic cells^239,240, granulocytes²⁴¹, etc.
NOTCH信号传导在造血系统细胞（淋巴细胞和骨髓细胞）的分化、发育和功能中是重要的。在早期胚胎发育中，造血内皮通过NOTCH依赖性内皮向造血转化形成造血干细胞²¹⁹。NOTCH信号传导在维持造血干细胞220的数量和干细胞性中是基本^的。在淋巴细胞发育中，早期造血祖细胞（HPC）中缺乏NOTCH 1或CSL导致胸腺T细胞发育迟缓和B细胞积累，其中HES 1是关键介体²²¹。幼稚胸腺细胞高度表达NOTCH，一旦它们成功通过β-选择，立即下调NOTCH 1表达。 一些学者提出NOTCH介导的T细胞发育起始于胸腺前龛222，223。例如，胸腺外的骨髓间充质细胞可以通过表面上的NOTCH配体与HPC交联以促进T细胞谱系的产生224、225。Shreya S等人用DLL4和VCAM-1体外工程诱导HSPC衍生的CD7+祖T细胞的产生，并且这些细胞在胸腺移植后进一步分化为成熟T细胞226。关于B细胞，脾边缘区B（MZ B）细胞的发育取决于DLL1-NOTCH 2信号传导227，228。 此外，发现主动NOTCH 2信号传导可以介导滤泡B细胞向MZ B细胞的谱系转化，使得成熟的B细胞亚群可以根据免疫系统的需要快速且动态地转化^229、230。最近发现先天性淋巴样细胞（ILC）的发育是NOTCH依赖性的^{231 - 233}，并且不同亚型的ILC对NOTCH信号传导的应答是异质的^234，235。有趣的是，ILC可以通过DLL 1激活MZB细胞以增强抗体产生²³⁶。 关于骨髓细胞，NOTCH信号传导在巨噬细胞^237、238、树突细胞^239、240、粒细胞²⁴¹等的发育中是重要的。

NOTCH and the liver

NOTCH signaling plays a key role in determining the fate of biliary tract cells and directing the correct morphogenesis of the biliary tree. Active NOTCH signaling, especially mediated by NOTCH2 and JAG1, promotes the expression of transcription factors enriched in bile duct cells, induces the differentiation of hepatocytes toward bile duct cells, and promotes the formation of the bile duct plates^152,153. The expression of SOX9, a downstream molecule of NOTCH signaling, is synchronized with the asymmetric development of the bile duct^152,242, with a mouse model of liver-specific deletion of Sox9 echoing this finding. Interestingly, delayed biliary tract development caused by liver-specific deletion of Sox9 eventually resolves in a spontaneous manner, proving that SOX9 plays a major role in timing regulation through the development of the biliary tract²⁴³.

The liver has a strong compensatory regeneration ability, where NOTCH signaling responds quickly with significant upregulation, and the transformation of hepatocytes into bile duct-like cells can be observed (Fig. ​(Fig.3c).3c). Similarly, high levels of dual-phenotype hepatocytes can also be observed in liver slices of patients with early liver diseases. Additionally, in a mouse orthotopic liver transplantation model, a high level of NOTCH1 (NICD and HES1) signaling was found to have a protective effect on hepatocytes during ischemia–reperfusion injury, regulating macrophage immunity²⁴⁴. In incomplete liver injury, NOTCH signaling mediates the proliferation and differentiation of facultative progenitor cells, thereby promoting biliary tract repair. Such damage repair can be induced mainly by NOTCH2^245,246, consistent with the discovery of the role of NOTCH2 signaling in the differentiation and selection of liver progenitor cells during liver development.

NOTCH and the gastrointestinal tract

Studies have shown that NOTCH signaling prevents embryonic epithelial cells from differentiating into secretory lineages²⁴⁷, with Hes1 being the main negative regulator²⁴⁸. Highly activated NOTCH signaling promotes the differentiation of intestinal stem cells toward intestinal epithelial cells²⁴⁹. Inhibiting NOTCH signaling increases the differentiation of secretory goblet cells²⁵⁰. Additionally, the lateral inhibition of NOTCH/DLL1 and the synergy of the Wnt signaling pathway²⁵⁰ drive Paneth cell differentiation and subsequent crypt formation²⁵¹. NOTCH signaling is also essential in the lineage selection of gastric stem cells²⁵² and necessary to maintain the homeostasis of gastric antral stem cells²⁵³. Activated NOTCH signaling in differentiated mature gastric epithelial cells induces their dedifferentiation²⁵⁴. NOTCH signaling is also vital to the proliferation of pancreatic progenitor cells and their correct differentiation into mature pancreatic cells^255,256. DLL1 and DLL4 are specifically expressed in β cells, while JAG1 is expressed in α cells²⁵⁷. The DLL1-NOTCH-HES1 signaling axis promotes the growth and fate selection of multipotent pancreatic progenitor cells, while JAG1 competes with DLL1 to induce opposite effects²⁵⁸.

NOTCH and the nervous system

NOTCH signaling negatively regulates neurogenic phenotypes^259–262. Its absence induces differentiation of neural stem cells toward neurons at the cost of glial cell production, in both D. melanogaster and vertebrates^263–266. There are two mainstream models: the classic lateral inhibition model that is similar to vascular development²⁶⁷ and the model involving oscillatory expression of HES1, NEUROG2 and DLL1²⁶⁸. In addition, NOTCH signaling promotes the differentiation of most glial cell subtypes, except for oligodendrocytes. In the peripheral nervous system, the interaction between NOTCH signaling and Hairy2 is vital for the development of neural crest cells, although the specific regulatory mechanism remains unclear²⁶⁹. Active NOTCH signaling blocks the occurrence and stratification of the trigeminal nerve, leading to disorders of brain development. Furthermore, NOTCH signaling drives intestinal neural crest cells to develop into precocious glial cells in Hirschsprung disease^270,271. These results indicate that NOTCH signaling participates in neural crest differentiation, but further exploration is required²⁷².

Diseases associated with abnormal expression of NOTCH signaling related to mutations

NOTCH as a tumor suppressor in cancers
NOTCH作为癌症的肿瘤抑制因子

NOTCH may be involved in many cancers as a protumor effector, but it can also act as a tumor suppressor in others, such as squamous cell carcinoma (SCC) and neuroendocrine tumors⁴²³（图 .​(Fig.4,4, Table ​Table2).2). Antitumor mechanisms include regulating transcription factors with malignant effects, activating downstream suppressive genes, inhibiting the cell cycle, etc. In light of studies regarding its antitumor effects, the traditional opinion of NOTCH as an oncogene has been challenged⁴¹⁴.

Cleavage inhibitors 切割抑制剂

Antibody-drug conjugates

Given the severe adverse events of inhibiting the overall NOTCH pathway, antibodies targeting different receptors and ligands have been explored to achieve precise targeting of NOTCH signaling^578,579. There are five ligands and four receptors in the NOTCH signaling pathway²¹. Although the roles of each component are not completely clear, functions related to specific diseases have been confirmed, making them potential targets⁴¹.

Transcription blockers 转录阻断剂

Activating the transcription of target genes is the last step of NOTCH signaling. Therapies targeting downstream mediators of NOTCH signaling remain unexplored. NOTCH transcription depends on the NOTCH ternary complex (NTC), which contains the DNA-binding protein CSL (also called CBF-1/RBPJ, Su (H), or Lag-1), NICD and MAML1^620,621. RIN1, a small molecule inhibitor of RBPJ, causes proliferation of hematologic cancer cell lines in vitro⁶²². IMR-1, a small molecule inhibitor of MAML1, inhibits the growth of NOTCH-dependent cell lines in vitro⁶²³. CB-103, an orally active small molecule altering NTC function, produces loss-of-function NOTCH phenotypes and inhibits the growth of human breast cancer and leukemia xenografts, notably without causing the dose-limiting intestinal toxicity of other NOTCH inhibitors⁶²⁴. Such novel drugs may represent new agents for NOTCH-based diseases.
激活靶基因的转录是NOTCH信号传导的最后一步。靶向NOTCH信号传导的下游介质的疗法仍然未被探索。NOTCH转录依赖于NOTCH三元复合物（NTC），其含有DNA结合蛋白CSL（也称为CBF-1/RBPJ、Su（H）或Lag-1）、NICD和MAML 1^620、621。RIN 1，RBPJ的小分子抑制剂，在体外引起血液癌细胞系的增殖⁶²²。IMR-I，MAMLl的小分子抑制剂，在体外抑制NOTCH依赖性细胞系的生长⁶²³。 CB-103是一种改变NTC功能的口服活性小分子，产生功能丧失的NOTCH表型并抑制人乳腺癌和白血病异种移植物的生长，特别是不会引起其他NOTCH抑制剂的剂量限制性肠毒性⁶²⁴。这些新药物可能代表基于NOTCH的疾病的新药剂。

NOTCH signaling agonists NOTCH信号激动剂

NOTCH signaling can both accelerate and suppress the development of diseases, which unsurprisingly applies in cancers^625,626. That is, enhancing NOTCH signaling can be a targeted therapy strategy. Some chrysin and hesperetin compounds have been used to activate NOTCH signaling in anaplastic thyroid cancer with NOTCH1 deficiency^627,628. Inhibitory effects on established tumor cell lines were found, although the underlying mechanism remains unclear. The negative regulatory region (NRR) can autoinhibit the metalloprotease cleavage of NOTCH to enhance its signaling. Some activating antibodies of NOTCH receptors induce conformational changes in the NRR, making it accessible to ADAM metalloproteinases, thus facilitating activation of NOTCH signaling⁶²⁹.
NOTCH信号传导可以加速和抑制疾病的发展，这毫不奇怪地适用于癌症^625，626。也就是说，增强NOTCH信号传导可以是靶向治疗策略。一些白杨素和橙皮素化合物已被用于激活NOTCH 1缺乏的未分化甲状腺癌中的NOTCH信号^627，628。发现对已建立的肿瘤细胞系有抑制作用，但其潜在机制仍不清楚。负调控区（NRR）可以自身抑制金属蛋白酶切割NOTCH以增强其信号传导。NOTCH受体的一些活化抗体诱导NRR的构象变化，使其可接近ADAM金属蛋白酶，从而促进NOTCH信号转导的活化⁶²⁹。

This work was supported by the National Natural Science Foundation of China (No. 62131009, 82072597, 81874120, and 82073370).
这项工作得到了国家自然科学基金（编号：62131009、82072597、81874120、82073370）的资助。