Rap 1b as a Mitogenic Signalin Thyroid

Rap 1b 作为促有丝分裂信号蛋白甲状腺

• 批准号: 6895421
• 负责人: DANIEL L ALTSCHULER
• 金额: $ 29.37万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6895421
• 关键词: biological signal transduction; carcinogenesis; cell proliferation; cholera toxin; comparative genomic hybridization; cyclic AMP; genetic recombination; genetically modified animals; guanine nucleotide binding protein; hyperplasia; karyotype; laboratory mouse; mitogens; neoplasm /cancer pharmacology; neoplastic process; protein structure function; protooncogene; tamoxifen; thyroid gland; thyroid inhibitor; thyroid neoplasm; transfection

DESCRIPTION (provided by applicant): cAMP stimulates proliferation of numerous cell types, particularly of differentiated endocrine origin, by acting synergistically with other growth factors. Thus, downstream targets of cAMP represent attractive candidates for new oncogenes. We have implicated the G-protein Rap1b as a critical target of cAMP mitogenic signaling by showing that Rap1b-expressing cells display enhanced DNA synthesis and are endowed with tumorigenic properties. Additionally, in thyroid follicular cells, which depends on increases in cellular cAMP by thyrotropin (TSH) for mitogenesis, DNA synthesis stimulated by TSH or cAMP requires Rap1b-GTP binding (activation) and its phosphorylation by the cAMP-dependent protein kinase PKA. Thus, we have proposed that Rap1b can be viewed as conditional oncogene--its role in cell proliferation is linked to the mitogenic signaling program elicited by cAMP. We now present new results showing that a GTPase-deficient (G12V) and phosphomimetic (S179D) Rap1b mutant, Rap1b-G12V-S179D, promotes mitogenesis independently of cAMP. Moreover, a dominant negative version of the Rap1b activator Epac, N-Epac, inhibits the mitogenic effect of cAMP but it has no effect on the mitogenic action of Rap1b-G12V-S179D. These results prove conditionality of the cAMP mitogenic signal on cAMP/Epac activation and cAMP/PKA phosphorylation of Rap1b, which is bypassed by Rap1b- G12V-S179D; they implicate activation and phosphorylation of Rab1b as the main biochemical events in the transduction of the cAMP mitogenic signal. However, it is necessary to verify whether Rap1b behaves as a conditional oncogene in vivo, linked to pathophysiological conditions with increased TSH levels. We have generated mice with thyroid-specific expression of an active Raplb mutant, "floxed" by LoxP sites, which, upon stimulation of CRE activity, will cease to express the active Rap1b mutant and produce a dominant negative Rap1b. This is the first example of a mouse model containing an "in vivo/in lession" built-in control system permitting the switch of expression of a stimulatory to an inhibitory G protein, encoded by a single gene. Mice expressing an active Rap1b mutant show no discernable alteration of thyroid morphology and function under physiological conditions, but, under pathologically sustained cAMP signaling, their thyroids are enlarged, display enhanced DNA synthesis, and present multiple adenomas, and, even carcinomas. These results suggest that Rap1b also behaves as a conditional oncogene in vivo. We propose to exploit these mice to understand the multi-step tumorigenic process in thyroid. The specific aims of this proposal are: 1: To establish whether thyroid-specific expression of Rap1b relays a cAMP-dependent mitogenic response. 2: To determine the effect of Rap1b in the establishment and/or progression of thyroid tumorigenesis. 3: To determine whether Rap1b action is required for cAMP-mediated hyperplasia. The long-term goal of this proposal is the identification of mitogenic signals relayed by Rap1b involved in cAMP-dependent cell proliferation in endocrine systems.

描述（由申请人提供）：cAMP通过与其他生长因子协同作用，刺激多种细胞类型的增殖，特别是分化内分泌来源的细胞。因此，cAMP的下游靶点代表了新的癌基因的有吸引力的候选者。我们发现g蛋白Rap1b是cAMP有丝分裂信号传导的关键靶点，表达Rap1b的细胞显示出增强的DNA合成并具有致瘤特性。此外，在甲状腺滤泡细胞中，有丝分裂发生依赖于促甲状腺素（TSH）增加细胞cAMP， TSH或cAMP刺激的DNA合成需要Rap1b-GTP结合（激活）并通过cAMP依赖性蛋白激酶PKA使其磷酸化。因此，我们提出Rap1b可以被视为条件癌基因——它在细胞增殖中的作用与cAMP引发的有丝分裂信号程序有关。我们现在提出的新结果表明，gtpase缺陷（G12V）和同源磷酸化（S179D） Rap1b突变体Rap1b-G12V-S179D独立于cAMP促进有丝分裂发生。此外，Rap1b激活子Epac的显性阴性版本N-Epac抑制cAMP的有丝分裂作用，但对Rap1b- g12v - s179d的有丝分裂作用没有影响。这些结果证明了cAMP有丝分裂信号对cAMP/Epac激活和cAMP/PKA磷酸化的条件性，Rap1b- G12V-S179D绕过了cAMP/Epac激活和cAMP/PKA磷酸化；它们暗示Rab1b的激活和磷酸化是cAMP有丝分裂信号转导的主要生化事件。然而，有必要验证Rap1b是否在体内作为一种条件癌基因，与TSH水平升高的病理生理状况有关。我们已经培养了具有甲状腺特异性表达活性Rap1b突变体的小鼠，该突变体被LoxP位点“封闭”，在CRE活性刺激下，将停止表达活性Rap1b突变体，并产生显性阴性Rap1b。这是第一个包含“体内/病变”内建控制系统的小鼠模型，该控制系统允许由单个基因编码的刺激G蛋白表达转换为抑制G蛋白表达。表达活性Rap1b突变体的小鼠在生理条件下甲状腺形态和功能没有明显的改变，但在病理持续的cAMP信号传导下，它们的甲状腺增大，DNA合成增强，并出现多发性腺瘤，甚至癌。这些结果表明Rap1b在体内也表现为条件癌基因。我们打算利用这些小鼠来了解甲状腺的多步骤致瘤过程。这项建议的具体目标是：