TGF-β signaling in health, disease and therapeutics
健康、疾病和治疗中的 TGF-β 信号转导

The studies on TGF-β started as early as the 1980s and have developed rapidly ever since. Although TGF-β was first found to be secreted by transformed cells,¹ it is widely produced by non-neoplastic tissues such as salivary glands, muscles, kidneys, liver, heart, brain, and embryos as well.^2,3,4 In fact, platelets have been identified as one of the most abundant sources of TGF-β among all normal tissues.⁵ The ubiquitous expression of TGF-β in health strongly indicates its critical and multiple roles in physiological conditions.
TGF-β的研究早在20世纪80年代就开始了，此后发展迅速。尽管 TGF-β 最初被发现是由转化细胞分泌的¹ ，但它也广泛由非肿瘤组织产生，如唾液腺、肌肉、肾脏、肝脏、心脏、大脑和胚胎。 ^{2 , 3 , 4}事实上，血小板已被确定为所有正常组织中最丰富的 TGF-β 来源之一。 ⁵ TGF-β 在健康中的普遍表达强烈表明其在生理条件下的关键和多重作用。

Accumulating evidence has suggested that TGF-β functions diversely among different cell types in a context-dependent manner. Generally, cell survival, metabolism, growth, proliferation, differentiation, adhesion, migration, and death are all under the regulation of TGF-β. Proper TGF-β signaling is critical to the normal functioning and homeostasis of healthy bodies while aberrant TGF-β signaling can lead to diseases of various categories. For this reason, numerous targeted therapies that can remedy dysregulated TGF-β activity have been developed with some demonstrating encouraging safety and efficacy in clinical trials.
越来越多的证据表明，TGF-β 在不同细胞类型中以环境依赖性方式发挥不同的功能。一般而言，细胞的存活、代谢、生长、增殖、分化、粘附、迁移和死亡均受TGF-β的调控。正确的 TGF-β 信号传导对于健康身体的正常功能和体内平衡至关重要，而异常的 TGF-β 信号传导则可能导致各种类型的疾病。出于这个原因，已经开发了许多可以纠正 TGF-β 活性失调的靶向疗法，其中一些疗法在临床试验中显示出令人鼓舞的安全性和有效性。

In this review, we focus on the mechanism, physiology, pathology, as well as therapeutics of TGF-β signaling, aiming to provide historical, current, and future perspectives on relevant topics.
在这篇综述中，我们重点关注 TGF-β 信号传导的机制、生理学、病理学以及治疗学，旨在提供相关主题的历史、当前和未来观点。

TGF-β was first reported in 1978 when De Larco and Todaro discovered the ‘sarcoma growth factors’ which were produced by transformed murine fibroblasts and were able to transform normal fibroblasts to anchorage-independent growth.¹ In 1981, Roberts et al. successfully isolated and purified TGF-β from non-neoplastic murine tissues,³ while at about the same time, Moses et al. independently accomplished the purification and characterization of the cytokine as well.⁶ Both groups also noticed that this relatively acid- and heat-stable polypeptide required disulfide bonds for activity and was sensitive to disulfide-reducing agent dithiothreitol. In 1983, studies by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels indicated that the 25,000-dalton TGF-β molecule in humans was actually composed of two 12,500-dalton subunits cross-linked by disulfide bonds.^7,8 Two years later, the amino-acid sequence of human TGF-β1, the first known TGF-β isoform, was revealed by Derynck et al. through direct protein sequencing and complementary deoxyribonucleic acid (DNA) cloning.² The sequencing established that the 112-amino-acid-long TGF-β1 monomer is initially synthesized as the C-terminal segment of a 390-amino-acid-long precursor polypeptide.² By the time of 1988, researchers had realized that TGF-β generally remained non-covalently associated with the N-terminal segment of its precursor when it was secreted.^9,10 TGF-β cannot bind to its receptors with its receptor-binding site being masked in this inactive form, however, certain treatments such as acidification could convert latent TGF-β complex into active TGF-β ligand.¹¹ In addition, the other two TGF-β isoforms in mammals, TGF-β2 and TGF-β3, were respectively identified in 1987¹² and 1988.^13,14 Although the three TGF-β isoforms are encoded by three different genes, their mature ligands show strong conservation of amino acid sequences.
TGF-β 于 1978 年首次报道，当时 De Larco 和 Todaro 发现了由转化的小鼠成纤维细胞产生的“肉瘤生长因子”，并且能够将正常成纤维细胞转化为不依赖贴壁的生长。 ¹ 1981 年，罗伯茨等人。成功地从非肿瘤性鼠组织中分离和纯化了 TGF-β， ³大约在同一时间，Moses 等人。并独立完成了细胞因子的纯化和表征。 ⁶两个小组还注意到，这种对酸和热相对稳定的多肽需要二硫键才能发挥活性，并且对二硫键还原剂二硫苏糖醇敏感。 1983年，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳研究表明，人体中25,000道尔顿的TGF-β分子实际上是由两个通过二硫键交联的12,500道尔顿的亚基组成。 ^{7 , 8}两年后，Derynck 等人揭示了人类 TGF-β1（第一个已知的 TGF-β 同工型）的氨基酸序列。通过直接蛋白质测序和互补脱氧核糖核酸（DNA）克隆。 ²测序确定，112 个氨基酸长的 TGF-β1 单体最初是作为 390 个氨基酸长的前体多肽的 C 端片段合成的。 ²到 1988 年，研究人员意识到 TGF-β 在分泌时通常与其前体的 N 末端片段保持非共价结合。^{9 , 10} TGF-β 不能与其受体结合，其受体结合位点以这种非活性形式被掩盖，但是，某些治疗（例如酸化）可以将潜在的 TGF-β 复合物转化为活性 TGF-β 配体。 ¹¹此外，哺乳动物中的另外两种 TGF-β 同工型 TGF-β2 和 TGF-β3 分别于 1987 年¹²和 1988 年被鉴定。 ^{13 , 14}尽管这三种 TGF-β 同工型由三个不同的基因编码，但它们的成熟配体显示出氨基酸序列的强烈保守性。

The effects of TGF-β signaling in cell proliferation,^15,16 cell differentiation,^17,18 embryonic development,¹⁹ wound healing,²⁰ immune regulation,^21,22 tissue fibrosis,^23,24 and tumor development^25,26 have been studied shortly after the discovery of the cytokine. Meanwhile, the receptors in TGF-β signaling known as TGF-β receptor I (TβRI) and TβRII were also identified and characterized in the 1980s.^27,28,29 But it was not until the discovery of signaling mediators small (Sma) in Caenorhabditis elegans and mothers against decapentaplegic (Mad) in Drosophila melanogaster that the homologous small mothers against decapentaplegic (SMAD) proteins were identified as the canonical signal transducers of TGF-β signaling in humans in 1996.^30,31,32 Since then, the development of TGF-β research has been largely accelerated. In recent times, as studies on TGF-β signaling in both health and disease going deeper and further, a lot of TGF-β-targeting therapies have been developed and assessed for the treatment of various diseases,^{33,34,35,36,37,38,39} revealing a promising future for the studies in this area (Fig. 1).
TGF-β信号传导对细胞增殖、 ^{15、16细胞}分化^{、 17、18}胚胎发育、 ¹⁹伤口愈合、 ²⁰免疫调节^{、 21、22}组织纤维化^{、 23、24}和肿瘤发展^25、26的影响已被短期研究细胞因子被发现后。与此同时，TGF-β 信号传导中的受体，即 TGF-β 受体 I (TβRI) 和 TβRII 也在 20 世纪 80 年代被鉴定和表征。 ^{27 , 28 , 29}但直到发现了秀丽隐杆线虫中的小信号介导物（Sma）和果蝇中的抗十五麻痹母蛋白（Mad），同源的小母抗十五麻痹蛋白（SMAD）才被确定为典型的信号转导子。 1996 年人类中 TGF-β 信号转导的研究进展。 ^{30 , 31 , 32}从那时起，TGF-β 研究的发展大大加速。近年来，随着对健康和疾病中 TGF-β 信号传导的研究越来越深入，许多 TGF-β 靶向疗法已被开发和评估用于治疗各种疾病， ^{33 , 34 , 35 , 36 , 37、38、39揭示了}该领域研究的广阔前景（图1 ） 。

History of research on TGF-β signaling
TGF-β信号研究的历史

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During the biosynthesis of TGF-β, the precursor undergoes post-translational processing to become a latent complex which is the secretory form of TGF-β. The latent TGF-β complex still requires further activation to eventually become a mature cytokine before it can trigger signal transduction in cells (Fig. 2).
在 TGF-β 的生物合成过程中，前体经过翻译后加工成为潜在复合物，即 TGF-β 的分泌形式。潜在的TGF-β复合物仍需要进一步激活才能最终成为成熟的细胞因子，然后才能触发细胞内的信号转导（图2 ）。

Biosynthesis and activation of TGF-β. Each TGF-β monomer is initially synthesized as a precursor polypeptide. In the endoplasmic reticulum, TGF-β precursors lose their signal peptides and dimerize through disulfide bonds. The dimers then transit into the Golgi where they are cleaved by protease furin into mature cytokine segments and latency-associated peptides (LAPs) to form small latent complexes (SLCs). The secreted SLCs can further link to latent TGF-β-binding proteins (LTBPs) which target them into the extracellular matrix (ECM) for storage, or they can link to glycoprotein-A repetitions predominant protein (GARP) or leucine-rich repeat-containing protein 33 (LRRC33) which tethers them to the cell surface. Numerous factors such as acids, bases, reactive oxygen species (ROS), thrombospondin-1 (TSP-1), certain proteases, and integrins can release the mature cytokines from the latent complexes and thus are known as TGF-β activators
TGF-β 的生物合成和激活。每个 TGF-β 单体最初都是作为前体多肽合成的。在内质网中，TGF-β前体失去信号肽并通过二硫键形成二聚体。然后二聚体进入高尔基体，在那里它们被弗林蛋白酶裂解成成熟的细胞因子片段和潜伏相关肽（LAP），从而形成小的潜伏复合物（SLC）。分泌的 SLC 可以进一步连接到潜在的 TGF-β 结合蛋白 (LTBP)，将其靶向细胞外基质 (ECM) 进行储存，或者它们可以连接到糖蛋白 A 重复优势蛋白 (GARP) 或富含亮氨酸的重复蛋白含有将它们束缚在细胞表面的蛋白质 33 (LRRC33)。许多因子，如酸、碱、活性氧 (ROS)、血小板反应蛋白-1 (TSP-1)、某些蛋白酶和整合素，可以从潜在复合物中释放成熟的细胞因子，因此被称为 TGF-β 激活剂

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TGF-β biosynthesis and latency
TGF-β生物合成和潜伏期

Each TGF-β monomer is initially synthesized as a precursor polypeptide composed of a mature cytokine as its C-terminal segment, a signal peptide at the N-terminus, and a latency-associated peptide (LAP) in between.² The signal peptide leads the precursor into the endoplasmic reticulum lumen and promptly gets removed. The remainder of the precursor then dimerizes through three disulfide bonds and transits into the Golgi where it gets cleaved between the mature cytokine and LAP by protease furin.⁴⁰ However, the cytokine segment is still unable to bind its receptors after the cleavage, for it remains associated with LAP in a non-covalent way that masks its receptor-binding site and forms a small latent complex (SLC).⁴¹ In most cases, LAP is linked to latent TGF-β-binding protein (LTBP) through a disulfide bond, making the SLC into a large latent complex (LLC) when secreted.⁴² LTBP can further bind to fibrillin to target the LLC into the extracellular matrix (ECM) for storage.⁴³ Alternatively, LAP can also form disulfide linkage with leucine-rich repeat-containing protein 32 (LRRC32) or LRRC33 to tether SLC to the cell surface. Unlike LTBP which is widely expressed by many cell types, LRRC32, also known as glycoprotein-A repetitions predominant protein (GARP), is specifically detected in regulatory T cells (Tregs), platelets, and endothelium,⁴⁴ whereas high expression of LRRC33 is found in macrophages, dendritic cells (DCs), and B cells.⁴⁵
每个 TGF-β 单体最初合成为前体多肽，由成熟细胞因子作为其 C 端片段、N 端信号肽和其间的潜伏相关肽 (LAP) 组成。 ²信号肽引导前体进入内质网腔并迅速被去除。然后前体的其余部分通过三个二硫键二聚化并转移到高尔基体中，在那里它被蛋白酶弗林蛋白酶在成熟细胞因子和 LAP 之间裂解。 ⁴⁰然而，细胞因子片段在裂解后仍然无法结合其受体，因为它仍然以非共价方式与 LAP 结合，从而掩盖其受体结合位点并形成小的潜在复合物 (SLC)。 ⁴¹在大多数情况下，LAP 通过二硫键与潜在的 TGF-β 结合蛋白 (LTBP) 连接，使 SLC 在分泌时形成一个大的潜在复合物 (LLC)。 ⁴² LTBP 可以进一步与原纤维蛋白结合，将 LLC 靶向细胞外基质 (ECM) 进行储存。 ⁴³另外，LAP 还可以与富含亮氨酸重复序列的蛋白 32 (LRRC32) 或 LRRC33 形成二硫键，将 SLC 束缚在细胞表面。与在多种细胞类型中广泛表达的 LTBP 不同，LRRC32（也称为糖蛋白 A 重复优势蛋白 (GARP)）在调节性 T 细胞 (Treg)、血小板和内皮细胞中特异性检测到， ⁴⁴而 LRRC33 则高表达存在于巨噬细胞、树突状细胞 (DC) 和 B 细胞中。 ⁴⁵

TGF-β activation TGF-β激活

The bioactivity of TGF-β is based on ligand-receptor interaction which requires the exposure of its receptor-binding site. Thus, the activation of TGF-β represents the release of mature cytokine from the latent complex. Numerous factors have been identified as TGF-β activators as introduced below. Notably, integrin-dependent activation is so far the best described and likely the most important mechanism, while TGF-β activation mediated by acids, bases, reactive oxygen species (ROS), thrombospondin-1 (TSP-1), proteases, and other TGF-β activators is collectively known as integrin-independent activation.
TGF-β的生物活性基于配体-受体相互作用，这需要暴露其受体结合位点。因此，TGF-β的激活代表成熟细胞因子从潜在复合物中的释放。如下所述，许多因子已被鉴定为 TGF-β 激活剂。值得注意的是，整合素依赖性激活是迄今为止描述最好的，也可能是最重要的机制，而 TGF-β 激活则由酸、碱、活性氧 (ROS)、血小板反应蛋白-1 (TSP-1)、蛋白酶和其他酶介导。 TGF-β激活剂统称为整合素非依赖性激活。

TGF-β activation by integrins
整合素激活 TGF-β

Integrins are heterodimeric transmembrane receptors each consisting of an α-subunit and a β-subunit. TGF-β activation by integrins requires the binding of the integrins to an RGD sequence in the LAP of TGF-β1 and TGF-β3. Therefore, latent TGF-β2 without the RGD motif is excluded from integrin-dependent activation.⁴⁶
整合素是异二聚体跨膜受体，每个受体由α亚基和β亚基组成。整联蛋白激活 TGF-β 需要整联蛋白与 TGF-β1 和 TGF-β3 的 LAP 中的 RGD 序列结合。因此，没有 RGD 基序的潜在 TGF-β2 被排除在整合素依赖性激活之外。 ⁴⁶

Among all integrins, αVβ6 and αVβ8 integrins are the best studied TGF-β activators. The expression of αVβ6 integrin is nearly restricted to epithelial cells and is upregulated in response to morphogenesis, wounding, inflammation, and tumorigenesis.⁴⁷ In contrast, αVβ8 integrin is widely expressed by epithelial cells,⁴⁸ fibroblasts,⁴⁹ macrophages,⁵⁰ DCs,⁵¹ Tregs,⁵² and different kinds of tumor cells.⁵³ The lack of αVβ6 and αVβ8 integrin activity reproduces the phenotypes of TGF-β1- and TGF-β3-null mice, indicating the central importance of integrin-dependent activation.^54,55
在所有整合素中，αVβ6 和 αVβ8 整合素是研究最多的 TGF-β 激活剂。 αVβ6 整合素的表达几乎仅限于上皮细胞，并且在形态发生、受伤、炎症和肿瘤发生时上调。 ⁴⁷相比之下，αVβ8 整合素广泛表达于上皮细胞、 ⁴⁸成纤维细胞、 ⁴⁹巨噬细胞、 ^{50 种}DC、 ^{51 种}Tregs、 ^{52 种}和不同种类的肿瘤细胞。 ⁵³缺乏 αVβ6 和 αVβ8 整合素活性会重现 TGF-β1 和 TGF-β3 缺失小鼠的表型，表明整合素依赖性激活的核心重要性。 ^{54 , 55}

Upon binding to the RGD motif in LAP, the mechanisms by which αVβ6 and αVβ8 integrins activate TGF-β are quite different. With latent TGF-β being tethered to ECM or cell membrane (through the binding of LAP to LTBP, GARP, or LRRC33 as mentioned before) and the cytoplasmic domain of integrin β6 subunit linking to the actin cytoskeleton, αVβ6 integrin can transmit contractile force which changes the conformation of LAP to release TGF-β ligand.^56,57 However, the cytoplasmic domain of integrin β8 subunit does not link to the actin cytoskeleton. One effective mechanism for αVβ8 integrin-mediated TGF-β activation requires the proteolytic activity of membrane type 1-matrix metalloproteinase (MT1-MMP, also known as MMP14).⁴⁸ Alternatively, membrane molecules such as GARP and LRRC33 which bind and present latent TGF-β on the surface of one cell can cooperate with the αVβ8 integrin expressed on a different cell to activate TGF-β in trans.^45,58,59 A recent study reveals that upon binding to αVβ8 integrin, the flexible membrane-presented latent complex can expose the active domain of the TGF-β ligand to its receptors for binding and signaling without the need to release diffusible cytokine.⁶⁰
与 LAP 中的 RGD 基序结合后，αVβ6 和 αVβ8 整合素激活 TGF-β 的机制截然不同。由于潜在的 TGF-β 被束缚在 ECM 或细胞膜上（通过前面提到的 LAP 与 LTBP、GARP 或 LRRC33 的结合）以及整合素 β6 亚基的胞质结构域与肌动蛋白细胞骨架相连，αVβ6 整合素可以传递收缩力，改变 LAP 的构象以释放 TGF-β 配体。 ^{56 , 57}然而，整合素 β8 亚基的胞质结构域并不与肌动蛋白细胞骨架相连。 αVβ8 整合素介导的 TGF-β 激活的一种有效机制需要 1 型膜基质金属蛋白酶（MT1-MMP，也称为 MMP14）的蛋白水解活性。 ⁴⁸另外，GARP 和 LRRC33 等膜分子在一个细胞表面结合并呈递潜在的 TGF-β，可以与不同细胞上表达的 αVβ8 整合素配合，反式激活 TGF-β。 ^{45 , 58 , 59}最近的一项研究表明，与 αVβ8 整合素结合后，柔性膜呈递的潜在复合物可以将 TGF-β 配体的活性结构域暴露于其受体，进行结合和信号传导，而无需释放可扩散的细胞因子。 ⁶⁰

TGF-β activation by acids and bases
酸和碱激活 TGF-β

It has long been noticed that acidification can unmask the activity of freshly secreted TGF-β.⁶¹ Sharply defined parameters for human TGF-β activation by acids and bases show that the transition from latency of all three isoforms occurred between pH 2.5 and 4, and between pH 10 and 12.⁶² Thus, extremely acidic environments such as the microenvironments in tumor tissues and the resorption lacunae of osteoclasts are possibly conducive to local TGF-β activation.^63,64 A study on lung fibrosis even suggests that physiologic concentrations of lactic acid are sufficient enough to activate TGF-β in a pH-dependent manner.⁶⁵
人们早就注意到酸化可以揭示新分泌的 TGF-β 的活性。 ⁶¹酸和碱对人 TGF-β 激活的明确定义的参数表明，所有三种亚型的潜伏期转变发生在 pH 2.5 至 4 之间以及 pH 10 至 12 之间。 ⁶²因此，极端酸性环境（例如肿瘤中的微环境）组织和破骨细胞的吸收腔隙可能有利于局部 TGF-β 的激活。 ^{63 , 64}一项关于肺纤维化的研究甚至表明，乳酸的生理浓度足以以 pH 依赖性方式激活 TGF-β。 ⁶⁵

TGF-β activation by ROS ROS 激活 TGF-β

TGF-β1 is the only isoform that can be directly activated by ROS, for a unique methionine residue at the amino acid position 253 of its LAP is required for oxidation-triggered conformational change.⁶⁶ However, ROS can induce other TGF-β activators such as TSP-1⁶⁷ and MMPs⁶⁸ to activate all three isoforms in an indirect manner. ROS-mediated TGF-β activation prevails in tissues exposed to asbestos,^69,70 ultraviolet,⁶⁸ and ionizing radiation.⁷¹ High glucose intake can also induce ROS production and consequentially increase TGF-β activation to play roles in the development of fibrotic diseases and inflammatory diseases.^72,73 Moreover, in T cells, ROS can be elevated during apoptosis or upon stimulation by T cell receptor (TCR) and cluster of differentiation 28 (CD28) to contribute to the immunosuppression mediated by activated TGF-β.^74,75
TGF-β1 是唯一可以被 ROS 直接激活的异构体，因为其 LAP 253 位氨基酸上的独特蛋氨酸残基是氧化触发的构象变化所必需的。 ⁶⁶然而，ROS 可以诱导其他 TGF-β 激活剂，例如 TSP-1 ⁶⁷和 MMP ⁶⁸以间接方式激活所有三种亚型。 ROS 介导的 TGF-β 激活在暴露于石棉、 ^{69 、 70}紫外线、 ⁶⁸和电离辐射的组织中普遍存在。 ⁷¹高葡萄糖摄入还可以诱导 ROS 产生，从而增加 TGF-β 激活，从而在纤维化疾病和炎症性疾病的发展中发挥作用。 ^{72 , 73}此外，在 T 细胞中，ROS 在细胞凋亡期间或在受到 T 细胞受体 (TCR) 和分化簇 28 (CD28) 刺激后会升高，从而有助于激活的 TGF-β 介导的免疫抑制。 ^{74 , 75}

TGF-β activation by TSP-1
TSP-1 激活 TGF-β

TSP-1 is a multi-functional ECM protein not only abundant in platelet α-granules but also secreted by fibroblasts, endothelial cells, macrophages, T cells, and many other cell types.⁷⁶ The KRFK sequence in TSP-1 can recognize the LSKL sequence in LAP to competitively disrupt its interaction with the receptor-binding site of the TGF-β ligand. Since the LSKL sequence in LAP is conserved among TGF-β isoforms, it is suggested that the direct binding of TSP-1 to latent complex is capable of activating all three TGF-β isoforms through this protease- and cell-independent mechanism.⁷⁷ Interestingly, TSP-1 can also bind to the mature TGF-β ligand to form a complex that retains the biological activity of the cytokine.⁷⁸ ROS,⁶⁷ glucose,⁷⁹ angiotensin II,⁸⁰ hypoxia,⁸¹ wounding,⁸² inflammation,⁸³ pathogens,^84,85,86 and many other factors can all induce TSP-1 to function as a TGF-β activator in wound healing,^67,82 cardiovascular diseases,^81,86 renal diseases,⁷⁹ fibrotic diseases,^87,88 inflammatory diseases,⁸³ infectious diseases,⁸⁹ and tumors.⁹⁰
TSP-1是一种多功能ECM蛋白，不仅在血小板α颗粒中丰富，而且由成纤维细胞、内皮细胞、巨噬细胞、T细胞和许多其他细胞类型分泌。 ⁷⁶ TSP-1 中的 KRFK 序列可以识别 LAP 中的 LSKL 序列，从而竞争性破坏其与 TGF-β 配体的受体结合位点的相互作用。由于 LAP 中的 LSKL 序列在 TGF-β 同工型中是保守的，因此表明 TSP-1 与潜在复合物的直接结合能够通过这种独立于蛋白酶和细胞的机制激活所有三种 TGF-β 同工型。 ⁷⁷有趣的是，TSP-1 还可以与成熟的 TGF-β 配体结合形成复合物，保留细胞因子的生物活性。 ⁷⁸ ROS、 ⁶⁷葡萄糖、 ⁷⁹血管紧张素 II、 ⁸⁰缺氧、 ⁸¹受伤、 ⁸²炎症、 ⁸³病原体^{、 84、85、86和}许多其他因素都可以诱导 TSP-1 在伤口愈合中充当 TGF-β 激活剂， ^{67 、 82}心血管疾病、 ^{81 、 86}肾脏疾病、 ⁷⁹纤维化疾病、 ^{87 、 88}炎症性疾病、 ⁸³传染病、 ⁸⁹和肿瘤。 ⁹⁰

TGF-β activation by proteases
蛋白酶激活 TGF-β

Many proteases have been proved capable of directly activating TGF-β in vitro. However, the function of an individual protease seems redundant in vivo, as deficiency of a single species generally leads to no significant signs of impaired TGF-β activation.⁹¹ Among these proteases, MMPs such as MMP-2, MMP-9, and MMP-13 are conducive to the TGF-β activation in wound healing,⁹² cardiovascular diseases,⁹³ renal diseases,⁹⁴ fibrotic diseases,⁹⁵ and tumors.⁹⁶ Interestingly, although the activation by MMPs works for all three TGF-β isoforms, latent TGF-β2 and TGF-β3 appear much more sensitive to MMP-9 treatment than latent TGF-β1.⁹⁶ Moreover, a serine protease known as plasmin plays an important role in the TGF-β activation mediated by macrophages^97,98 and endothelial cells.^99,100
许多蛋白酶已被证明能够在体外直接激活TGF-β。然而，单个蛋白酶的功能在体内似乎是多余的，因为单个物种的缺陷通常不会导致 TGF-β 激活受损的明显迹象。 ⁹¹在这些蛋白酶中，MMP-2、MMP-9 和 MMP-13 等 MMP 有利于伤口愈合、 ⁹²心血管疾病、 ⁹³肾脏疾病、 ⁹⁴纤维化疾病、 ⁹⁵和肿瘤中的 TGF-β 激活。 ⁹⁶有趣的是，尽管 MMP 的激活作用适用于所有三种 TGF-β 同工型，但潜在的 TGF-β2 和 TGF-β3 似乎对 MMP-9 治疗比潜在的 TGF-β1 更敏感。 ⁹⁶此外，一种称为纤溶酶的丝氨酸蛋白酶在巨噬^{细胞97、98}和内皮细胞介导的 TGF-β 激活中发挥着重要作用。 ^{99 , 100}

TGF-β signal is transmitted into the cells by TβRI (also known as activin receptor-like kinase 5, ALK5) and TβRII both of which are enzyme-linked receptors with dual specificity of serine/threonine kinase and tyrosine kinase. Studies have revealed that TGF-β1 and TGF-β3 bind TβRII prior to TβRI due to higher affinity, while TGF-β2 binds poorly to both receptors.^12,101,102 TβRIII, also known as β-glycan, lacks the motifs to directly mediate TGF-β signal transduction. However, TβRIII is able to bind TGF-β especially TGF-β2 with high affinity and thus acts as a co-receptor that presents the ligand to the receptors and further enhances their binding.^{101,103,104,105,106,107} The ligand-receptor interaction subsequently activates the intracellular signaling of TGF-β through a canonical pathway and several non-canonical pathways.
TGF-β信号通过TβRI（也称为激活素受体样激酶5，ALK5）和TβRII传递到细胞中，这两种受体都是具有丝氨酸/苏氨酸激酶和酪氨酸激酶双重特异性的酶联受体。研究表明，由于亲和力较高，TGF-β1 和 TGF-β3 在 TβRII 之前先与 TβRII 结合，而 TGF-β2 与这两种受体的结合都很差。 ^{12 , 101 , 102} TβRIII，也称为β-聚糖，缺乏直接介导TGF-β信号转导的基序。然而，TβRIII能够以高亲和力结合TGF-β尤其是TGF-β2，因此充当共受体，将配体呈递给受体并进一步增强它们的结合。 ^{101 , 103 , 104 , 105 , 106 , 107}配体-受体相互作用随后通过经典途径和几个非经典途径激活TGF-β的细胞内信号传导。

Canonical TGF-β signaling
典型的 TGF-β 信号传导

The canonical TGF-β signaling is mediated by transcription factors SMADs and thus is also known as the SMAD signaling. Notably, the canonical pathway is under the regulation of various factors that can control the intensity and manner of cellular responses at different levels (Fig. 3).
典型的 TGF-β 信号传导由转录因子 SMAD 介导，因此也称为 SMAD 信号传导。值得注意的是，经典途径受到多种因素的调节，这些因素可以在不同水平上控制细胞反应的强度和方式（图3 ）。

Canonical TGF-β signaling. TGF-β can initially bind to its co-receptor TGF-β receptor III (TβRIII) or directly bind to its receptor TβRII which subsequently recruits TβRI to form a TGF-β-TβRI-TβRII complex. TβRII then actives TβRI through phosphorylation, leading to its dissociation with signaling inhibitor FK506-binding protein 1A (FKBP12) and interaction with signaling effectors receptor-activated SMADs (R-SMADs). R-SMADs which are presented to TβRI by adaptor protein SMAD anchor for receptor activation (SARA) get activated through phosphorylation and undergo oligomerization with common-partner SMAD (co-SMAD). The SMAD oligomers then translocate into the nucleus where they function as transcription factors (TFs), mediating the transcriptional activation or repression of target genes by binding to specific DNA sequences known as SMAD-binding elements (SBEs) and generally in cooperation with other TFs as well as transcriptional cofactors. In this way, TGF-β signaling can activate the expression of inhibitory SMADs (I-SMADs) which in turn function to attenuate the transcriptional regulation mediated by TGF-β signaling through several mechanisms. Moreover, many protein kinases (PKs), protein phosphatases (PPs), and (E3) ubiquitin ligases can also modulate canonical TGF-β signaling through various post-translational modifications of SMADs. (TFBS, TF-binding site)
典型的 TGF-β 信号传导。 TGF-β 最初可以与其共受体 TGF-β 受体 III (TβRIII) 结合，或直接与其受体 TβRII 结合，后者随后招募 TβRI 形成 TGF-β-TβRI-TβRII 复合物。然后，TβRII 通过磷酸化激活 TβRI，导致其与信号传导抑制剂 FK506 结合蛋白 1A (FKBP12) 解离，并与信号传导效应器受体激活的 SMAD (R-SMAD) 相互作用。 R-SMAD 通过接头蛋白 SMAD 受体激活锚 (SARA) 呈递给 TβRI，通过磷酸化被激活，并与共同伙伴 SMAD (co-SMAD) 发生寡聚化。然后 SMAD 寡聚体易位到细胞核中，在细胞核中充当转录因子 (TF)，通过与称为 SMAD 结合元件 (SBE) 的特定 DNA 序列结合并通常与其他 TF 配合来介导靶基因的转录激活或抑制，如以及转录辅助因子。通过这种方式，TGF-β信号传导可以激活抑制性SMADs (I-SMADs)的表达，而抑制性SMADs进而通过多种机制减弱TGF-β信号传导介导的转录调节。此外，许多蛋白激酶 (PK)、蛋白磷酸酶 (PP) 和 (E3) 泛素连接酶也可以通过 SMAD 的各种翻译后修饰来调节经典 TGF-β 信号传导。 （TFBS，TF 结合位点）

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TGF-β-activated SMAD signaling
TGF-β 激活 SMAD 信号传导

TGF-β ligand initially binds to TβRII monomer to promote its homodimerization or directly binds to pre-existing TβRII homodimer to recruit TβRI for assembly.^{108,109,110,111} This forms a heteromeric TGF-β-TβRI-TβRII complex in which low-affinity TβRI requires high-affinity TβRII to bind TGF-β ligand and constitutively active TβRII requires phosphorylating TβRI to transduce intracellular signal.¹¹² The phosphorylation of TβRI occurs in its juxtamembrane GS domain at several serine and threonine residues, triggering conformational changes that transform the GS domain from a site that binds the signaling inhibitor known as immunophilin FK506-binding protein 1A (FKBP12) into a binding site for the signaling effectors known as receptor-activated SMADs (R-SMADs).¹¹³
TGF-β配体最初与TβRII单体结合以促进其同二聚化，或直接与预先存在的TβRII同二聚体结合以募集TβRI进行组装。 ^{108 , 109 , 110 , 111}这形成异聚 TGF-β-TβRI-TβRII 复合物，其中低亲和力 TβRI 需要高亲和力 TβRII 才能结合 TGF-β 配体，而组成型活性 TβRII 需要磷酸化 TβRI 来转导细胞内信号。 ¹¹² TβRI 的磷酸化发生在其近膜 GS 结构域的几个丝氨酸和苏氨酸残基处，引发构象变化，将 GS 结构域从结合信号抑制剂（称为亲免素 FK506 结合蛋白 1A (FKBP12)）的位点转变为亲免素 FK506 结合蛋白 1A (FKBP12) 的结合位点。信号传导效应器称为受体激活 SMAD (R-SMAD)。 ¹¹³

R-SMADs, including SMAD2 and SMAD3, consist of a globular Mad homology 1 (MH1) domain at the N-terminus, a globular MH2 domain at the C-terminus, and a highly flexible long linker region in between. R-SMADs are retained in cytoplasm and presented to TβRI by the adaptor protein known as SMAD anchor for receptor activation (SARA).¹¹⁴ The R-SMAD MH2 domain then gets phosphorylated at two serine residues in the extreme C-terminal SXS motif by the TβRI kinase domain which is located immediately downstream of the TβRI GS domain.¹¹³ Activated R-SMADs undergo homo-oligomerization or hetero-oligomerization through their MH2 domains upon phosphorylation, and they can also oligomerize with SMAD4, the common-partner SMAD (co-SMAD) which lacks the SXS motif for phosphorylation by TβRI kinase. Notably, studies have suggested that SMAD heterotrimers containing two R-SMADs and one SMAD4 are likely more common and stable than other SMAD oligomers.^{115,116,117,118,119} Although different SMAD oligomers can vary in function, they all act to regulate the transcription of target genes by binding to DNA after translocating into the nucleus. The MH1 domains of SMAD4, SMAD3, and a specific SMAD2 splicing variant recognize the nucleic acid sequence GTCT or its reverse complement AGAC in double-stranded DNA which are known as the canonical SMAD-binding elements (SBEs).¹²⁰ Other SBEs such as the 5GC SBEs including GGCGC and GGCCG have also been discovered, indicating a relatively loose DNA-binding specificity of the SMAD oligomers.¹²¹ However, the binding to a single SBE is so weak that SMAD oligomers generally require interacting with replications of SBE copies as well as other DNA-binding sequence-specific transcription factors to function.^119,120,122 In fact, many SBE repeats are enriched at the binding sites for SMAD-interacting transcription factors, exactly increasing the binding accessibility, specificity, and affinity of SMAD oligomers associated with specific transcription factors.^123,124,125 Despite a large number of SMAD-interacting transcription factors indicating a huge amount of potential gene targets for canonical TGF-β signaling, the dominant effects are generally determined by the master transcription factors in specific cell types and contexts which contribute to the complexity and variability of cellular responses to TGF-β.¹²⁵ 重试    错误原因

Regulation of SMAD signaling by inhibitory SMADs (I-SMADs) 重试    错误原因

TGF-β and many other factors can induce the expression of SMAD6 and SMAD7 which function to inhibit TGF-β signaling and thus are known as I-SMADs.^126,127 Unlike R-SMADs, I-SMADs lack the N-terminal MH1 domain and the C-terminal SXS motif, however, they retain the C-terminal MH2 domain which can competitively bind to activated receptor TβRI to inhibit the phosphorylation of R-SMADs.^128,129 Through some extra mechanisms, SMAD7 confers greater abilities in suppressing TGF-β signaling than SMAD6 does.¹³⁰ For example, SMAD7 recruits E3 ubiquitin ligases such as SMAD ubiquitination regulatory factors (SMURFs) and neural precursor cell expressed, developmentally downregulated 4-like (NEDD4L) to TβRI, R-SMADs, and co-SMAD to mediate the proteasomal and lysosomal degradation of these TGF-β signaling components.^{131,132,133,134,135} SMAD7 can also trigger the dephosphorylation of TβRI by recruiting protein phosphatase 1 (PP1) to the receptor.¹³⁶ Moreover, with its MH2 domain, SMAD7 can oligomerize with R-SMADs to compete with co-SMAD¹³³ and can bind to specific DNA sequences to disrupt the formation of the transcriptional SMAD-DNA complex.¹³⁷ Taken together, TGF-β signaling induces I-SMADs to form a negative feedback loop of itself.
TGF-β和许多其他因子可以诱导SMAD6和SMAD7的表达，其功能是抑制TGF-β信号传导，因此被称为I-SMAD。 ^{126 , 127}与 R-SMAD 不同，I-SMAD 缺乏 N 端 MH1 结构域和 C 端 SXS 基序，但保留了 C 端 MH2 结构域，可以竞争性地与激活的受体 TβRI 结合，抑制 R 的磷酸化-SMAD。 ^{128 , 129}通过一些额外的机制，SMAD7 比 SMAD6 具有更强的抑制 TGF-β 信号传导的能力。 ¹³⁰例如，SMAD7 招募 E3 泛素连接酶，例如 SMAD 泛素化调节因子 (SMURF) 和神经前体细胞表达的、发育下调的 4-like (NEDD4L) 至 TβRI、R-SMAD 和 co-SMAD，以介导蛋白酶体和溶酶体降解这些 TGF-β 信号传导成分。 ^{131 , 132 , 133 , 134 , 135} SMAD7 还可以通过将蛋白磷酸酶1 (PP1)募集至受体来触发TβRI的去磷酸化。 ¹³⁶此外，凭借其 MH2 结构域，SMAD7 可以与 R-SMAD 寡聚，与 co-SMAD 竞争¹³³ ，并且可以结合特定的 DNA 序列，破坏转录 SMAD-DNA 复合物的形成。 ¹³⁷总之，TGF-β 信号传导诱导 I-SMAD 形成自身的负反馈循环。

Regulation of SMAD signaling by transcriptional cofactors
转录辅助因子对 SMAD 信号传导的调节

Transcriptional cofactors are actively recruited to the transcriptional SMAD complex to regulate its activity. Notably, many of these transcriptional cofactors have histone modification activity and thus enable TGF-β signaling to trigger epigenetic changes. Histone acetyltransferases (HATs) such as p300, cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB)-binding protein (CBP), p300/CBP-associated factor (PCAF), and general control non‐repressed protein 5 (GCN5) act as the transcriptional coactivators of SMADs by increasing the accessibility to DNA.^{138,139,140,141} The interaction between p300/CBP and doubly phosphorylated R-SMADs requires SMAD4 for stabilization and is critical for SMAD-mediated transcriptional activation. Other SMAD coactivators include melanocyte-specific gene 1 (MSG1),¹⁴² zinc finger E-box-binding homeobox 1 (ZEB1),^143,144 and the histone methyltransferase (HMT) known as SET domain-containing protein 7 (SETD7).¹⁴⁵ Contrary to HATs, histone deacetylases (HDACs) generally act as the transcriptional corepressors of SMADs by decreasing the accessibility to DNA. SMAD3 can directly recruit HDAC4 and HDAC5 to gene promoters to inhibit the function of transcription factors via histone deacetylation.¹⁴⁶ SMADs can also associate with HDACs through interaction with other corepressors such as TGF-β-induced factor (TGIF),¹⁴⁷ ecotropic viral integration site 1 (EVI1),^148,149 Sloan-Kettering Institute proto-oncogene (SKI),^150,151,152 as well as SKI-related novel gene N (SNO).¹⁵³ Other transcriptional corepressors of SMADs include cellular-myelocytomatosis viral oncogene (MYC),¹⁵⁴ SMAD nuclear-interacting protein 1 (SNIP1),¹⁵⁵ ZEB2,^143,156 and HMTs such as suppressor of variegation 3-9 homolog 1 (SUV39H1) and SET domain bifurcated 1 (SETDB1) which can both trigger the methylation of histone 3 lysine 9 (H3K9) at gene promoters.^157,158
转录辅助因子被积极招募到转录 SMAD 复合物中以调节其活性。值得注意的是，许多转录辅助因子具有组蛋白修饰活性，从而使 TGF-β 信号传导能够触发表观遗传变化。组蛋白乙酰转移酶 (HAT)，例如 p300、环磷酸腺苷 (cAMP) 反应元件结合蛋白 (CREB) 结合蛋白 (CBP)、p300/CBP 相关因子 (PCAF) 和一般控制非抑制蛋白 5 (GCN5) ）通过增加 DNA 的可及性，充当 SMAD 的转录共激活剂。 ^{138 , 139 , 140 , 141} p300/CBP 和双磷酸化 R-SMAD 之间的相互作用需要 SMAD4 来稳定，并且对于 SMAD 介导的转录激活至关重要。其他 SMAD 共激活剂包括黑素细胞特异性基因 1 (MSG1)、 ¹⁴²锌指 E 盒结合同源框 1 (ZEB1) ^{、 143、144}和被称为含 SET 结构域的蛋白 7 (SETD7) 的组蛋白甲基转移酶 (HMT)。 ¹⁴⁵与 HAT 相反，组蛋白脱乙酰酶 (HDAC) 通常通过降低 DNA 的可及性来充当 SMAD 的转录辅阻遏物。 SMAD3可以直接将HDAC4和HDAC5招募到基因启动子处，通过组蛋白脱乙酰化来抑制转录因子的功能。^{第146章​​151、152以及}SKI相关新基因N(SNO)。^第153章SMAD 的其他转录辅阻遏物包括细胞骨髓细胞瘤病毒癌基因 (MYC)、^{第 154 章}SMAD 核相互作用蛋白 1 (SNIP1)、^{第 155 章}ZEB2、^{第 143 章、第 156 章和}HMT，例如杂色抑制因子 3-9 同源物 1 (SUV39H1) 和 SET结构域分叉 1 (SETDB1) 均可触发基因启动子处组蛋白 3 赖氨酸 9 (H3K9) 的甲基化。^{157 , 158}

Regulation of SMAD signaling by SMAD modifications
通过 SMAD 修饰调节 SMAD 信号传导

Post-translational modifications can also regulate the functions of SMADs. Apart from TβRI kinase which phosphorylates R-SMADs in their C-terminal SXS motif to mediate their activation, many other protein kinases such as mitogen-activated protein kinase kinase kinase 1 (MAPKKK1),¹⁵⁹ p38 MAPK,¹⁶⁰ c-Jun N-terminal kinase (JNK),¹⁶¹ extracellular signal-regulated kinase (ERK),^162,163,164 rat sarcoma (RAS) homolog (Rho)-associated coiled-coil-containing protein kinase (ROCK),¹⁶⁰ glycogen synthase kinase (GSK)-3β,^165,166,167 calcium/calmodulin-dependent protein kinase II (CAMK2),¹⁶⁸ protein kinase C (PKC),¹⁶⁹ PKG,¹⁷⁰ and several cyclin-dependent kinases (CDKs)^167,171,172 can phosphorylate R-SMADs as well as co-SMAD at many different sites to enhance or attenuate SMAD activity. Meanwhile, the various phosphorylation of SMADs can be reversed by phosphatases. Several nuclear phosphatases known as the small C-terminal domain phosphatases (SCPs) can specifically dephosphorylate the linker region and MH1 domain of R-SMADs,^173,174 whereas protein phosphatase, magnesium/manganese-dependent 1A (PPM1A),¹⁷⁵ myotubularin-related protein 4 (MTMR4),¹⁷⁶ and protein phosphatase 2A (PP2A)¹⁷⁷ catalyze the dephosphorylation of the C-terminal SXS motif to terminate the signaling and promote the dissociation and cytoplasmic localization of SMADs.
翻译后修饰也可以调节 SMAD 的功能。除了磷酸化 R-SMAD C 端 SXS 基序以介导其激活的 TβRI 激酶外，还有许多其他蛋白激酶，如丝裂原激活蛋白激酶激酶 1 (MAPKKK1)、 ¹⁵⁹ p38 MAPK、 ¹⁶⁰ c-Jun N 端^第161章细胞外信号调节激酶（ERK），^{第162章，第163章，第164章}大鼠肉瘤（RAS）同源物（Rho）相关卷曲螺旋蛋白激酶（ROCK），^第160章糖原合酶激酶（GSK）- 3β ^{、 165、166、167钙}/钙调蛋白依赖性蛋白激酶 II (CAMK2)、 ¹⁶⁸蛋白激酶 C (PKC)、 ¹⁶⁹ PKG、 ¹⁷⁰和几种细胞周期蛋白依赖性激酶 ( ^{CDK ) 167、171、172}可以磷酸化 R-SMAD以及在许多不同位点的 co-SMAD 以增强或减弱 SMAD 活性。同时，SMADs的各种磷酸化可以被磷酸酶逆转。 几种被称为小 C 端结构域磷酸酶 (SCP) 的核磷酸酶可以特异性地使 R-SMAD 的接头区域和 MH1 结构域去磷酸化， ^{173 , 174}而蛋白磷酸酶，镁/锰依赖性 1A (PPM1A)， ¹⁷⁵肌管蛋白相关蛋白 4 (MTMR4)、 ¹⁷⁶和蛋白磷酸酶 2A (PP2A) ¹⁷⁷催化 C 端 SXS 基序的去磷酸化，以终止信号传导并促进 SMAD 的解离和细胞质定位。

Furthermore, SMADs can be ubiquitinated and deubiquitinated respectively by E3 ubiquitin ligases and deubiquitylating enzymes (DUBs). The E3 ubiquitin ligases that can mediate SMAD ubiquitination include SMURFs,^{135,178,179,180} NEDD4L,^134,181 WW domain-containing proteins (WWPs),^182,183,184 really interesting new gene (RING) finger protein 111 (RNF111),¹⁸⁵ C-terminus of heat shock protein (HSP) 70-interacting protein (CHIP),¹⁸⁶ itchy (ITCH) E3 ubiquitin ligase,¹⁸⁷ and S-phase kinase-associated protein (SKP)-cullin-F-box (SCF) E3 ubiquitin ligase complex.^188,189 The ubiquitination generally leads to the proteasomal degradation of SMADs, but in some cases, it also exerts non-degradative effects on SMAD activity.¹⁹⁰ Notably, the degradative ubiquitination of R-SMADs by NEDD4L requires the phosphorylation of the R-SMAD linker by CDK8/9 and GSK-3 in sequence to create binding sites for the E3 ubiquitin ligase.^171,181,191
此外，SMAD 可以分别被 E3 泛素连接酶和去泛素化酶 (DUB) 泛素化和去泛素化。能够介导 SMAD 泛素化的 E3 泛素连接酶包括 SMURF ^{、 135、178、179、180 NEDD4L 、 134、181含}WW 结构域的蛋白（WWP） ^{、 182、183、184真正}有趣的新基因（RING）指蛋白 111（RNF111） )、 ¹⁸⁵热休克蛋白 (HSP) C 末端 70 相互作用蛋白 (CHIP)、 ¹⁸⁶痒 (ITCH) E3 泛素连接酶、 ¹⁸⁷和 S 期激酶相关蛋白 (SKP)-cullin-F-box (SCF) ) E3 泛素连接酶复合物。 ^{188 , 189}泛素化通常会导致 SMAD 的蛋白酶体降解，但在某些情况下，它也会对 SMAD 活性产生非降解作用。 ¹⁹⁰值得注意的是，NEDD4L 对 R-SMAD 的降解性泛素化需要 CDK8/9 和 GSK-3 按顺序磷酸化 R-SMAD 接头，以创建 E3 泛素连接酶的结合位点。 ^{171 , 181 , 191}

Non-canonical TGF-β signaling
非典型 TGF-β 信号传导

Apart from the SMAD-dependent pathway, TGF-β can also signal through SMAD-independent pathways to activate ERK signaling, Rho guanosine triphosphatase (GTPase) signaling, p38 MAPK signaling, JNK signaling, nuclear factor-κB (NF-κB) signaling, phosphatidylinositol 3-kinase (PI3K)/AKR mouse thymoma proto-oncogene (AKT) signaling, as well as Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. These non-canonical TGF-β signaling pathways are involved in an extensive range of cellular events, greatly expanding the participation of TGF-β signaling in health and disease (Fig. 4).
除了 SMAD 依赖性途径外，TGF-β 还可以通过 SMAD 独立途径发出信号，激活 ERK 信号、Rho 鸟苷三磷酸酶 (GTPase) 信号、p38 MAPK 信号、JNK 信号、核因子-κB (NF-κB) 信号、磷脂酰肌醇 3-激酶 (PI3K)/AKR 小鼠胸腺瘤原癌基因 (AKT) 信号传导，以及 Janus 激酶 (JAK)/信号转导器和转录激活子 (STAT) 信号传导。这些非常规的TGF-β信号通路参与了广泛的细胞事件，极大地扩展了TGF-β信号传导在健康和疾病中的参与（图4 ）。