Rap 1b as a Mitogenic Signalin Thyroid

说明(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，特别是分化的内分泌来源的细胞。因此，cAMP下游靶点代表了新癌基因的有吸引力的候选基因。我们已经暗示G蛋白Rap1b是cAMP有丝分裂信号的关键靶点，因为我们证明了Rap1b表达的细胞显示出增强的DNA合成，并被赋予了致瘤特性。此外，在依赖促甲状腺激素(TSH)增加细胞内cAMP进行有丝分裂的甲状腺滤泡细胞中，TSH或cAMP刺激的DNA合成需要Rap1b-GTP结合(激活)和cAMP依赖的蛋白激酶PKA的磷酸化。因此，我们认为Rap1b可以被视为条件癌基因--它在细胞增殖中的作用与cAMP诱导的有丝分裂信号程序有关。我们现在公布的新结果表明，GTPase缺陷(G12V)和模拟磷酸(S179D)的Rap1b突变体Rap1b-G12V-S179D促进有丝分裂，而不依赖cAMP。此外，Rap1b激活剂EPAC的显性阴性版本N-EPAC抑制cAMP的促有丝分裂作用，但对Rap1b-G12V-S179D的促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Rap1b的制约作用，而Rap1b-G12V-S179D绕过了这一过程，提示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在体内是否表现为条件癌基因，与TSH水平升高的病理生理条件有关。我们已经产生了一种甲状腺特异表达活性Raplb突变体的小鼠，该突变体被loxP位点“漂浮”，一旦Cre活性被刺激，它将停止表达活性的Rap1b突变体，并产生显性的阴性Rap1b。这是第一个含有“体内/体内”控制系统的小鼠模型，该系统允许由单个基因编码的刺激性G蛋白的表达切换到抑制性G蛋白的表达。表达活性Rap1b突变体的小鼠在生理条件下没有明显的甲状腺形态和功能变化，但在病理上持续的cAMP信号下，它们的甲状腺增大，DNA合成增强，并出现多发性腺瘤，甚至癌症。这些结果表明，Rap1b在体内也是一种条件癌基因。我们建议利用这些小鼠来了解甲状腺中多步骤的肿瘤形成过程。这项建议的具体目标是： 1：确定甲状腺特异表达的Rap1b是否传递cAMP依赖的有丝分裂反应。 2：探讨Rap1b在甲状腺肿瘤形成和/或发展中的作用。 3：确定cAMP介导的增殖是否需要Rap1b作用。 这项建议的长期目标是鉴定由Rap1b传递的有丝分裂信号，该信号参与内分泌系统中cAMP依赖的细胞增殖。

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.