Aberrant Cell Signaling and Related Diseases Jimin Shao (邵吉民) E-mail: shaojimin@zju.edu.cn

Overview of cell signaling and signal transduction pathways Disorders of signal transduction and mechanisms of diseases

Overview of cell signaling and signal transduction pathways (1) Direct Intercellular Communication (Gap Junction Intercellular Communication) (2) Signaling by plasma-membrane bound molecules (3) Receptor-Mediated Intercellular communication (Cell-surface / Nuclear receptors ) Gap Junction

(4) Communication by Extracellular Vesicles(EVs) Tkach, M. and C. Thery (2016). "Communication by Extracellular Vesicles: Where We Are and Where We Need to Go." Cell 164(6): 1226-1232. Figure 1. EV-Mediated Effects Promoting Tumor Growth, Invasiveness, and Metastasis 在多细胞生物体内，远处的细胞可以通过传递单个分子或细胞外囊泡（EVs; 也叫作 exosomes和microvesicles等）交换信息。细胞可以通过细胞外囊泡（EVs）的分泌与邻近细胞或远处的细胞进行沟通。EVs具有脂质双分子层结构，包含跨膜蛋白、胞质蛋白和RNA等成分。根据它们的亚细胞起源不同可将EVs分成不同的类型（Colombo et al., 2014）。一部分EVs是通过从细胞质膜出芽释放的。这些EVs直径大约为100-1000 nm，在文献中通常被称作microvesicles, ectosomes或microparticles。其他类型的小泡外泌体是由多泡体（MVBs）内部产生，与质膜融合分泌而形成的。外泌体的直径一般小于150 nm，富含胞内体的成分。所有EVs都具有表面分子，使他们能够有针对性地到达受体细胞。一旦附着至靶细胞，EVs可以诱导经由受体配体相互作用的信号，或者可以通过内吞作用和/或吞噬作用与靶细胞膜内化或融合将其内容物递送到其细胞质中，由此改变受体细胞的生理状态。

2. Receptor-Mediated Signal Transduction Systems Cell Surface Receptors: - Ion Channel Linked Receptors - G-protein Coupled Receptors (GPCR) - Enzyme Linked Receptors Receptor tyrosine kinases Tyrosine-kinase-associated receptors Receptor serine/threonine kinases Receptor guanylyl cyclases etc. - Others Nuclear Receptors: - Steroid Receptors - Retinoid Receptors - Orphan Receptors

3. General process for transmembrane signal transduction Synthesis and secretion of signaling molecules (Ligands) Receptor binding and transmemberane signal transduction Intracellular signaling pathway Regulation of cellular metabolism, function, gene expression, etc. Down-regulation or termination of cellular responses As a general signal transduction process involves the following major approaches: Any of these steps has problem, the abnormality will occur. 7

4. Cell Signaling Functions Specific physiological functions for different cell types Responses to stress Metabolism Cell fate: Cell cycle, growth, differentiation, death. Cell mobility Interaction with microenvironment 5. Important signaling pathways and networks

Jason I. Herschkowitz and Xiaoyong Fu Jason I. Herschkowitz and Xiaoyong Fu. MicroRNAs Add an Additional Layer to the Complexity of Cell Signaling. Sci. Signal.4 (184), jc5. [DOI: 10.1126/scisignal.2002182]

Disorders of signal transduction systems and Mechanisms of diseases Causes: Gene mutation-- Function loss or gain Change of quantity or activity of signal proteins Epigenetic changes Autoimmune diseases Secondary changes

Pathogenesis: Down-regulation / interruption of signaling Signal Insufficiency (ligands) Receptors down-regulation / desensitization: decreased quantity, binding affinity, inhibitory Ab, cofactor disorders, function loss, etc. Defects in post-receptor pathways: adaptors, Signal transducers, Effectors (Enzymes, Transcription factors, etc) Up-regulation / over-activation of signaling Signal Excess Receptor up-regulation, hypersensitivity, stimulatory Ab, etc Signal transducers, TFs: over-expression, persistent activation Others

1. Aberrant Signal （1）信息分子过量：在脑缺血，癫痫及神经退行性疾病等多种病理过程中，可见细胞外液中谷氨酸/门冬氨酸含量显著增高，其结果是NMDA受体过度激活，胞外Ca2+内流使胞内Ca2+浓度持续增高，使信号转导途径中的多种酶持续激活而导致神经细胞兴奋性毒性 （2）信息分子不足：胰岛素作为一种重要的信息分子，在维持正常血糖水平中起重要作用，当胰岛素生成减少、机体产生抗胰岛素抗体或在应激状态下产生各种胰岛素拮抗因子时，均可导致胰岛素的相对或绝对不足，最终引起高血糖

Viral infections or other damages to pancreatic -cell (1) Aberrant Signal (Signal Insufficiency) Insulin receptor (IR): heterotetramer (2, 2) Insulin binding leads to change in conformation Activates IR -subunit PTK activity -subunit phosphorylates Tyr residues on cytoplasmic domains as well as downstream substrates (IRS) Activates downstream pathways; Biological effects Viral infections or other damages to pancreatic -cell insulin production hyperglycemia Diabetes (Type I) （2）信息分子过量：在脑缺血，癫痫及神经退行性疾病等多种病理过程中，可见细胞外液中谷氨酸/门冬氨酸含量显著增高，其结果是NMDA受体过度激活，胞外Ca2+内流使胞内Ca2+浓度持续增高，使信号转导途径中的多种酶持续激活而导致神经细胞兴奋性毒性 （1）信息分子不足：胰岛素作为一种重要的信息分子，在维持正常血糖水平中起重要作用，当胰岛素生成减少、机体产生抗胰岛素抗体或在应激状态下产生各种胰岛素拮抗因子时，均可导致胰岛素的相对或绝对不足，最终引起高血糖

(2) Aberrant Signal (Signal Excess) ischemia, epilepsy, neurodegenerative diseases extracellular glutamate/aspartic acid NMDAR activation (N-methyl-D-aspartate receptor: Ion Channel Linked Receptor) Ca2+ influx [Ca2+]i , activation of enzymes excitatory intoxication （1）信息分子过量：在脑缺血，癫痫及神经退行性疾病等多种病理过程中，可见细胞外液中谷氨酸/门冬氨酸含量显著增高，其结果是NMDA受体过度激活，胞外Ca2+内流使胞内Ca2+浓度持续增高，使信号转导途径中的多种酶持续激活而导致神经细胞兴奋性毒性

2. Aberrant Receptor in Cell Signaling

Disturbance of receptors can occur in: gene level (genetics or epigenetics); processes of protein synthesis, post-translational modification, conformation, oligomerization, translocation, endocytosis, degradation, etc. Receptor alterations in number, structure, function, and regulation can result in: down-regulation: decrease in number of receptors desensitization: decreased response to ligand stimulation up regulation: increase in number of receptors hypersensitivity: increased response to ligand stimulation, or self-activation without ligands Receptor diseases: receptor alterations --- changes of ligand-receptor signaling --- abnormal cellular effects --- diseases

（1）Receptor Gene Mutation Genetic insulin-resistant diabetes: IR gene mutations Disturbances in synthesis transfer to the membrane affinity to insulin PTK activity proteolysis Insulin + IR Activate RPTK IRS PI3K Ras/Raf/ MEK/ERK Glycogen Synthesis, Cell Transport proliferation & Utilization 胰岛素受体(insulin receptor, IR)为酪氨酸蛋白激酶型受体。胰岛素与IR结合导致受体的酪氨酸蛋白激酶（protein tyrosine kinase , PTK）激活，该酶通过胰岛素受体底物（IRS）激活PI-3K及Ras－Raf－MEK－ERK等多条信号转导通路：①促进葡萄糖转运蛋白4(GLUT4)转位到膜上，从而增加外周组织摄取葡萄糖的能力；②使无活性的糖原合酶转为激活的形式，增加糖原的合成；③使基因表达增强，蛋白质合成增加、促进细胞的增殖等。 遗传性的胰岛素抵抗性糖尿病包括Leprechaunism综合征、Rabson-Mendenhall 综合征和A型胰岛素抵抗症。患者一般有家族史，除有严重高血糖和高胰岛素血症外，多数患者还伴有黑色棘皮及多毛症，面容丑陋。迄今全世界已报道了该病患者约50多种胰岛素受体的基因突变，呈明显的异质性，以点突变为主，分布于受体的胞外区和PTK区。突变可导致受体合成障碍、受体往细胞膜运输受阻、受体与胰岛素亲和力下降、PTK活性降低及受体降解加快等，使得靶细胞对胰岛素反应丧失。 Type II Diabetes

Thyroid proliferation & secretion of thyroxine  (2) Autoimmune diseases-thyropathy Gs AC cAMP Thyroid proliferation & secretion of thyroxine  Gq PLC IP3 DAG Ca2+ PKC TSH-R（GPCR) 30~35 residues Stimulatory Ab hyperthyroidism Blocking Ab TSH-R 295~302 385~395 residues Binding of TSH to R↓ Stimulatory Ab mimic the effect of TSH and produces increased thyroxine; More than 10% of elderly people suffer from autoimmune diseases, in which their immune system attacks their own cells. One explanation for this class of disorders is based upon aging of suppressor T cells. As this cell population ages, their inhibition of the activation of other T and B cells by self-antigens declines. The activated immune cells participate in an attack on other tissues, producing the symptoms of the autoimmune diseases. Some diseases that affect young individuals also have strong autoimmune components. In addition to type 1 diabetes, autoimmuthyroidism is another example of autoimmune disorder. In normal condition, binding of thyroid stimulating factor (TSH) to receptor will initiate AC (through Gs) or/and PLC (through Gq) signaling, and thus regulates the growth of thyroid gland and secretion of thyroxine. Production of various thyroid autoantibodies will lead to deregulation. There are two types of antibody to thyroid receptor: stimulatory antibodies mimic the function of TSH and cause the hyperthyroidism. Inhibitory antibodies will antagonize the TSH and lead to hypothyroidism, such as chronic lymphocytic thyroiditis hypothyroidism

桥本病 (Hashimoto’s thyroditis,慢性淋巴细胞性甲状腺炎) Graves病（弥漫性毒性甲状腺肿） 刺激性抗体模拟TSH 的作用 促进甲状腺素分泌和甲状腺腺体生长 女性>男性 甲亢、甲状腺弥漫性肿大、突眼 桥本病 (Hashimoto’s thyroditis,慢性淋巴细胞性甲状腺炎) 阻断性抗体与TSH受体结合 减弱或消除了TSH的作用 抑制甲状腺素分泌 甲状腺功能减退、黏液性水肿

(3)Secondary Abnormality in Receptors Heart failure, Myocardial hypertrophy -adrenergic receptors (GPCR) down regulated or desensitized Reaction to catecholamines Myocardial contraction Alleviate Accelerate myocardial lesion heart failure The pathological processes for these types of disorder are caused secondarily by changes of environments where the receptors are exposed to. For example, 1, 2 and 1adrenergic receptors mediate cell signaling involved both in the normal regulation of cardiac function and in pathological process of heart failure. Among these receptors, 1 is the most prominent one for normal regulation of cardiac function, accounts for 70% to 80% in healthy individuals. It is down-regulated both in terms of number (50% of the normal control) and of sensitivity (desensitized) to the stimulation of catecholamine in the patients or animal models with heart failure. This is actually a compensate reaction to the stimulation of overproduced catecholamine. It can alleviate the cardiac lesion, and at the same time, aggravates the process of heart failure by depressing the contractility. （一）原发性受体信号转导异常与疾病 （二）继发性受体信号转导异常与疾病 心力衰竭患者及动物的心脏对异丙肾上腺素引起的正性肌力反应明显减弱，即β受体对儿茶酚胺刺激发生了减敏(desensitization)反应。心力衰竭时，β受体下调，特别是β1受体数量减少，可降至50%以下；β2受体数量变化不明显，但对配体的敏感性亦有降低。β受体减敏是对过量儿茶酚胺刺激的代偿反应，可抑制心肌收缩力，减轻心肌的损伤，但也是促进心力衰竭发展的原因之一

Receptor diseases: 分类 累及的受体 主要临床特征 遗传性受体病 膜受体异常 家族性高胆固醇血症 LDL受体     分类 累及的受体 主要临床特征 遗传性受体病　 　　膜受体异常　　家族性高胆固醇血症   LDL受体 血浆LDL升高，脂质代谢紊乱，动脉粥样硬化 　　　　　　　　　家族性肾性尿崩症 ADH V2型受体(GPCR) 男性发病，多尿、口渴和多饮 　　　　　　　　 c视网膜色素变性　　 视紫质 进行性视力减退 Cccccccccccccccc 遗传性色盲 视锥细胞视蛋白 色觉异常 11111111111111 严重联合免疫缺陷症 IL-2受体γ链 T细胞减少或缺失，反复感染 Cccccccccccccccc II型糖尿病 胰岛素受体(RTK) 高血糖,血浆胰岛素正常或升高 ccc核受体异常 ccc雄激素抵抗综合征 雄激素受体 不育症，睾丸女性化 cccccccccccccccc维生素D抵抗性佝偻病 维生素D受体 佝偻病骨损害，秃发，继发性甲状旁腺素增高 Cccccccccccccccc甲状腺素抵抗综合征 β甲状腺素受体 甲状腺功能减退，生长迟缓 cccccccccccccccc雌激素抵抗综合征 雌激素受体 骨质疏松，不孕症 Ccccccccccccccc 糖皮质激素抵抗综合征 糖皮质激素受体 多毛症,性早熟,低肾素性高血压

自身免疫性受体病 cccccccccccccccc重症肌无力   nAch受体 活动后肌无力 cccccccccccccccc自身免疫性甲状腺病 刺激性TSH受体(GPCR) 抑制性TSH受体 甲亢和甲状腺肿大 甲状腺功能减退 ccccccccccccccccII型糖尿病 胰岛素受体 高血糖，血浆胰岛素正常或升高 cccccccccccccccc艾迪生病 ACTH受体 色素沉着，乏力，血压低 继发性受体异常 ccccccccccccccc心力衰竭 肾上腺素能受体 心肌收缩力降低 ccccccccccccccc帕金森病 多巴胺受体 肌张力增高或强直僵硬 ccccccccccccccc肥胖 血糖升高 ccccccccccccccc肿瘤 生长因子受体 细胞过度增殖

3. Aberrant G-protein in Cell Signaling

(1) G-protein gene mutation Pituitary tumor: Gs gene mutation At Arg201 or Gln227 GTPase activity Persistent activation of Gs Persistent activation of AC cAMP Pituitary proliferation and secretion Acromegaly in adults Gigantism in children Hypothalamus GHRH Pituitary gland GHRH-R Gs Ac cAMP GH 生长激素(GH)是腺垂体分泌的多肽激素，其功能是促进机体生长。GH的分泌受丘脑下部GH释放激素（GHRH）和生长抑素的调节，GHRH与垂体细胞上的受体结合后经激活Gs，导致AC活性升高和cAMP积聚，促进腺垂体分泌GH。在分泌GH过多的垂体腺瘤中，有30％～40％是由于编码Gsα的基因点突变，其特征是Gsα的精氨酸201为半胱氨酸或组氨酸所取代，或谷氨酰胺227为精氨酸或亮氨酸所取代，这些突变抑制了GTP酶活性，使Gsα处于持续激活状态，AC活性升高，cAMP含量增加，垂体细胞生长和分泌功能活跃。故在这些垂体腺瘤中，信号转导障碍的关键环节是Gsα过度激活导致的GHRH和生长抑素对GH分泌的调节失衡。GH分泌增多可刺激骨骼过度生长，在成人引起肢端肥大症，在儿童引起巨人症。 Adenohypophysis 由于某些信号转导蛋白的过度表达，或基因突变使某一信号蛋白成为异常的不受控制的激活状态，或者某种抗受体抗体能够持续性刺激受体，都能使细胞内特定信号转导通路过度激活，导致细胞增殖、分化、凋亡或功能代谢的异常。

(2) G-protein modification Cholera toxin （霍乱毒素） intestinal epithelia Gs ribosylation at Arg201 Inactivation of GTPase Persistent activation of Gs and Ac, cAMP Secretion of chloride into the lumen, Inhibition of sodium uptake from the lumen, Large volumes of fluid into the lumen of the gut Diarrhea and dehydration Circulation failure (1) Cholera Ribosylation of Gs at arginine reduces the activity of GTPase, which results in prolonged activation of Gs. Prolonged Gs activity will lead to sequentially stimulation of adenylate cylase activity, elevation of cAMP, secretion of chloride into the gut, inhibition of sodium uptake from the lumen of the gut. The high ionic strength in gut (hypertonic) will cause water flows from body into lumen of gut and lead to diarrhea, dehydration and even failure of circulation. (2) Pertussis Pertussis toxin ribosylates Gi on a cysteine and thus inhibits the release and activation of Gi subunit. The major function of Gi is to turn off adenylate cyclase and maintain a balanced regulation down stream. Therefore, blockage of Gi activation will lead to overreaction to catecholamines, such as hypersensitivity to histamine, vasodilation and hypotension, insulin oversecretion and hypoglycemia, and so on.

4. Aberrant intracellular Signaling The intracellular signaling involves: various messengers, transducers, transcription factors, and etc. Disorders can occur in any of these settings: -- Calcium overload is a general pathological process in various diseases; -- The level of NO is positively correlated with ischemic injury; -- Stimulation of NF-B is seen in various inflammatory responses; --Aberrant intracellular Signaling in carcinogenesis, and etc.

5. Multiple Abnormalities in Signaling Pathways

Multifactor Aberrancies in Cancer ---Enhancement of proliferating signals Ligands (GFs): e.g. EGF Receptors (overexpression, activation of TPK): e.g. EGFR Intracellular signal transducers： Ras gene mutation Ras-GTPase Ras activation Raf MEK ERK (MAPK) Proliferation TUMOR Cancer is characterized by successive changes in cell behavior caused by accumulated genetic alterations. In development of cancer, cell division cycle becomes deregulated, causing over-proliferation and tumor growth. Seven types of proteins that control cell growth and differentiation are growth factors, receptors, intracellular transducers, transcription factors, anti-apoptosis proteins, cell cycle control proteins and DNA repair proteins. Excess or deficiency in these proteins can lead to uncontrolled growth and malignant transformation of various cancers. Ras is a central participant in signaling pathways controlling cell division and cell growth. Ras mediates its effects in part by initiating a cascade of protein kinases that ultimately activates specific transcription factors to turn on transcription of target genes. Ras is active when bound to GTP, but inactive when bound to GDP. The oncogenic form of Ras always has GTP bound, and is therefore always active. Ras pathway is controlled by GAP (turn off Ras) and SOS. GAP binds only the GTP-bound form of Ras. Oncogenic Ras binds GAP poorly. The C terminal of SOS is inhibitory; if it is removed, SOS stimulates Ras all the time. Mutated Ras is estimated to cause 20-30% of human cancers including half of all colon cancers and one quarter of lung cancers. Many breast cancers involve mutated Ras. So do almost all pancreatic cancers. Additionally, mutated proteins involved above and below the level of Ras in these pathways can lead to chronic up-regulation of the pathways even in the absence of mutations in Ras itself.

---Deficits in proliferation-inhibiting signals TGF- + TGF-R PSTK activation Smad-phosphorylation P21/P27/P15 expression Cdk4 inhibition Cell cycle arrests Inhibits cell proliferation (pro-apoptosis) Cancers Gene mutation Negative regulation

Major signaling pathways relevant to cancer (Hanahan D, Weinberg RA. Cell 2011, 144:646) (Weinberg RA. the biology of cancer. 2013)

Stimulants and Pathological Effects 6. Relationship between Stimulants and Pathological Effects --Same Stimulant Induces Different Responses (the same stimuli can act on different receptors) --Different Signals Induces the Same Pathologic Response (different receptors use the same pathway or by cross-talk)

GPCR, RTK, Cytokine Receptors --Different receptors use same pathways GPCR, RTK, Cytokine Receptors PLC Ras PI-3K PKC Raf PKB MEK ERK

--Cross talk: hypertension leads to myocardial hypertrophy

7. Principles for Treatment of Aberrant Signaling-related Diseases Stratagy: Regulate the level of extracellular molecules Regulate the structure and the function of receptors Regulate the level and modifications of modification enzymes, messengers, signal transducers, transcription factors, effectors, etc Target therapy: Breast cancer: EGFR overexpression –Herceptin (mAB) Chronic myeloid leukemia (CML): Bcr-Abl (abnormal tyrosine kinase) — Gleevec (small compound inhibitor)

教学目标: 思考题： 1.掌握细胞信号转导异常的基本原因和类型； 2.掌握与细胞信号转导障碍相关的疾病的发生发展机制； 3.了解细胞信号转导在疾病临床诊疗中的应用。 思考题： 试述主要的细胞信号通路及其调节机制。 简述生长因子导致细胞增殖的信号转导过程。 受体异常有哪几种类型，举例相关疾病予以说明？ II型糖尿病中胰岛素受体有何异常？ 霍乱发生的分子机制。 举例说明肿瘤与信号转导异常的关系。 举例说明心血管疾病中的信号转导异常。 试述Graves病和桥本病发病机制的异同。 试述垂体腺瘤引起肢端肥大症机制。 举例说明G蛋白介导的细胞信号转导障碍性疾病的机制。