Regulation of Mouse Somatostatin Receptor Subtype 5 Gene

R 03研究计划的目的是扩大我们对小鼠生长抑素受体亚型5基因（sst 5）表达调控的基本分子机制的理解。目的是鉴定和克隆与sst 5启动子相互作用的细胞因子，并表征其在垂体细胞中的功能重要性。特别强调的是，将被放置在TtT 97促甲状腺细胞的sst 5启动子活性的重要细胞因子。这些实验将是先前由K 08指导临床科学家资助的工作的直接延伸 发展奖。 这项工作的总体目标是更好地了解下丘脑-垂体-甲状腺轴和生长抑素能系统之间的相互作用及其对垂体促甲状腺激素调节的联合作用。使用小鼠TtT-97促甲状腺肿瘤模型，我们以前已经证明，甲状腺激素治疗减少肿瘤大小与增强表达的sst 5基因。作为K 08奖的一部分进行的研究最初通过调查sst 5基因的基础启动子活性及其受甲状腺激素的调节来表征这些体内观察结果。小鼠sst 5基因已被克隆，其转录起始位点被定位，其结构被鉴定为具有3个外显子和2个内含子。在瞬时转染实验中，在各种垂体细胞系和原代TtT 97 thryotrope文化的功能启动子活性进行了评估。重要序列 已经使用缺失和突变策略鉴定了基础启动子活性。-67和-47之间的区域似乎包含一个对垂体细胞中sst 5基因的基础转录调节至关重要的核苷酸序列。该区域的功能重要性已经通过DNA酶I保护分析来鉴定DNA蛋白质相互作用而得到证实。对这些关键的sst 5启动子区域的序列分析揭示了核苷酸同源性， 已知转录因子的潜在结合位点，包括Ptx 1和Egr-1。这些因子和其他因子在调节小鼠sst 5启动子中的作用将是R 03提案的主题。这些数据不仅将增强我们对生长抑素能系统和垂体调节的基本分子机制的理解，而且对神经内分泌肿瘤的发病机制和治疗具有长期的临床应用。生长抑素类似物奥曲肽已被用于治疗各种神经内分泌肿瘤，生长抑素能系统的分子机制的描述将在这些疾病的临床应用。对生长抑素受体功能和调节的进一步了解将有助于开发更有效的生长抑素类似物用于神经内分泌肿瘤的诊断和治疗。此外，了解这些受体转录调控的分子机制， 有助于我们操纵特定肿瘤中生长抑素受体状态的能力，并增强对生长抑素类似物治疗的临床反应。

The goal of this R03 research proposal is to expand our understanding of the basic molecular mechanism(s) regulating expression of the mouse somatostatin receptor subtype 5 gene (sst5). The aim is to identify and clone cellular factors that interact with the sst5 promoter and characterize their functional importance in pituitary cells. Specific emphasis will be placed on the cellular factors important for sst5 promoter activity in TtT97 thyrotropes. These experiments will be a direct extension of the work previously funded by a K08 Mentored Clinical Scientist Development Award. The overall goal of this work is to gain a better understanding of the interaction between the hypothalamicpituitary-thyroid axis and the somatostatinergic system and their combined effect on pituitary thyrotrope regulation. Using the murine TtT-97 thyrotropic tumor model, we have previously demonstrated that treatment with thyroid hormone reduces tumor size in association with enhanced expression of the sst5 gene. Studies performed as part of the K08 Award have initially characterized these in vivo observations by investigating basal promoter activity of the sst5 gene and its regulation by thyroid hormone. The mouse sst5 gene has been cloned, its transcription start site localized and its structure identified as having 3 exons and 2 introns. Functional promoter activity has been assessed in transient transfection experiments in a variety of pituitary cell lines and primary TtT97 thryotrope cultures. Sequences important for basal promoter activity have been identified using a deletion and mutational strategy. The region between -67 and -47 appear to contain a nucleotide sequence that is critical for basal trancriptional regulation of the sst5 gene in pituitary cells. The functional importance of this region has been confirmed by DNase I protection analysis to identify DNA protein interactions. Sequence analysis of these critical sst5 promoter regions revealed nucleotide homology to potential binding sites for known transcription factors, including Ptx 1 and Egr-1. The role of these factors and others in regulation of the mouse sst5 promoter will be the subject of this R03 proposal. Not only will these data enhance our understanding of the basic molecular mechanisms of the somatostatinergic system and pituitary regulation, but have long range clinical applications regarding the pathogenesis and treatment of neuroendocrine tumors. The somatostatin analog, octreotide, has been used to treat a variety of neuroendocrine tumors and description of the molecular mechanisms of the somatostatinergic system will be of clinical application to these diseases. Further knowledge of somatostatin receptor function and regulation will potentially contribute to the development of more efficacious somatostatin analogs for the diagnosis and treatment of neuroendocrine tumors. Additionally, understanding the molecular mechanism(s) underlying transcriptional regulation of these receptors may contribute to our ability to manipulate somatostatin receptor status in specific tumors and enhance clinical response to somatostatin analog therapy.