The Role of GCM2 in Parathyroid Gland Homeostasis

描述（由申请人提供）：这份重新提交的资助提案是 ARRA R01 资助 2 年的结果，该资助产生了新的数据，证明甲状旁腺细胞特异性转录因子 Gcm2（神经胶质细胞缺失 2）在甲状旁腺早期胚胎学发育后的关键作用，以及该蛋白质如何在成熟甲状旁腺中发挥作用，以控制甲状旁腺细胞的功能和存活。长期目标是利用 Gcm2 作用的知识来开发治疗甲状旁腺功能亢进症患者的新策略。本应用的目的是确定消耗的程度 Gcm2 会导致基因工程小鼠模型中甲状旁腺细胞的存活率降低。核心假设是，甲状旁腺中 Gcm2 的表达对于维持甲状旁腺细胞质量是终生所必需的，而 Gcm2 的缺乏将导致甲状旁腺细胞死亡。我们建议使用我们开发的小鼠模型，以时间控制的方式有条件地基因删除 Gcm2 基因，以鉴定调节 Gcm2 表达的基因。 Gcm2 以及受 Gcm2 作用控制的细胞，并揭示 Gcm2 缺失对成熟甲状旁腺细胞的影响。具有条件性 Gcm2 等位基因的小鼠还将使我们能够确定晚年 Gcm2 消融可以在多大程度上“拯救”患有甲状旁腺疾病（类似于人类甲状旁腺功能亢进症）的小鼠。在强有力的初步数据的指导下，该假设将通过追求两个具体目标来进行检验：1）确定Sonic Hedgehog信号传导功能和其他转录因子途径对GCM2基因转录的作用。根据我们的初步数据，我们预计 Sonic Hedgehog 在抑制非甲状旁腺细胞中 Gcm2 的表达中发挥着重要作用； 2)确定Gcm2的条件性缺失对正常甲状旁腺转录体以及对功能亢进的小鼠甲状旁腺的功能和大小的影响。我们预计，正常甲状旁腺中 Gcm2 的缺失将揭示受 Gcm2 调节的基因，并导致甲状旁腺萎缩。此外，使用完善的原发性甲状旁腺功能亢进症小鼠模型，我们预计在增生性或腺瘤性甲状旁腺中定时删除Gcm2将导致甲状旁腺细胞存活率降低和甲状旁腺功能亢进症消退。试剂和小鼠模型已在手，并且技术已在申请人的实验室中建立为可行的。该方法具有创新性，因为它利用了新颖的小鼠模型，应用了增强型酵母1杂交筛选等新技术来发现调节Gcm2表达的基因，并使用RNA-seq开发了Gcm2依赖性转录体的综合目录。这项拟议的研究意义重大，因为它有望增进我们对甲状旁腺细胞生物学的理解，并最终确定分子靶标，从而开发出针对人类原发性和三发性甲状旁腺功能亢进症的新疗法。

DESCRIPTION (provided by applicant): This resubmitted proposal for funding is the result of 2 years of ARRA R01 funding that generated novel data demonstrating a critical role for the parathyroid cell-specific transcription factor Gcm2 (glial cell missing 2) after early embryologica development of the parathyroid glands as well how this protein functions in mature parathyroid glands to control function and survival of parathyroid cells. The long-term goal is use knowledge of the role of Gcm2 to develop novel strategies for medical treatment of patients with hyperparathyroidism. The objective in this application is to identify the extent to which depletion of Gcm2 leads to reduced survival of parathyroid cells in genetically engineered mouse models. The central hypothesis is that expression of Gcm2 in the parathyroid is necessary throughout life to maintain parathyroid cell mass, and that lack of Gcm2 will induce parathyroid cell death. We propose to use mouse models that we have developed to genetically delete the Gcm2 gene conditionally, in a temporally controlled manner, to identify the genes that regulate expression of Gcm2 as well as those that are controlled by Gcm2 action, and to uncover the effect of loss of Gcm2 on mature parathyroid cells. Mice with conditional Gcm2 alleles will also allow us to determine the extent to which ablation of Gcm2 late in life can "rescue" mice that have parathyroid disorders that replicate human hyperparathyroidism. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) determine the role of Sonic hedgehog signaling function and other transcription factor pathway on transcription of the GCM2 gene. Based on our preliminary data, we expect Sonic hedgehog plays a major role in repressing expression of Gcm2 in non-parathyroid cells; and 2) determine the effect of conditional deletion of Gcm2 on the transcriptosome of normal parathyroid glands and on function and size of hyperfunctioning mouse parathyroid glands. We expect that deletion of Gcm2 in normal parathyroid glands will reveal genes that are regulated by Gcm2 and will result in parathyroid atrophy. Moreover, using well-established murine models of primary hyperparathyroidism, we expect that timed deletion of Gcm2 in hyperplastic or adenomatous parathyroid glands will result in decreased survival of parathyroid cells and regression of hyperparathyroidism. The reagents and mouse models are in hand, and the techniques have been established as feasible in the applicants' labs. The approach is innovative because it utilizes novel mouse models, applies new techniques such as enhanced yeast 1-hybrid screening to uncover genes that regulate Gcm2 expression, and uses RNA-seq to develop a comprehensive catalog of the Gcm2-dependent transcriptosome. The proposed research is significant because it is expected to advance our understanding of parathyroid cell biology, and ultimately, to identify molecular targets that will allow development of new medical treatments for primary and tertiary hyperparathyroidism in humans.