The effect of AIP mutations on the apoptotic RET pathway in pituitary adenomas

垂体腺瘤是位于大脑底部的内分泌腺--垂体腺的良性肿瘤。如果腺瘤在这里发展，它可能会引起问题，因为它的大小，它可能会损害周围的关键结构(如视神经)，或由于过量的激素释放。最近，一种名为AIP的基因突变被发现，该基因易导致儿童或青年发病的腺瘤，通常会导致一种称为巨人症的毁灭性疾病。尽管AIP蛋白存在于身体的每个细胞中，但具有生殖系AIP突变的患者的一个独特特征是，他们只会患上垂体腺瘤，最常见的是来自垂体腺的生长激素(GH)分泌细胞。我们研究的总体目标是确定与AIP相关的肿瘤发展的机制。基于我们的初步数据，这项建议旨在阐明RET蛋白及其途径成员在AIP相关肿瘤发生机制中的作用。RET蛋白是细胞生长和死亡的重要调节因子。在过去的几年里，研究发现，干细胞在人的一生中不断提供新的分化的内分泌细胞，并且有一种控制机制，即凋亡，移除细胞来维持正常脑下垂体中的细胞数量。在脑下垂体中，RET主要在生长激素细胞中表达，并参与了这种凋亡过程。细胞凋亡的RET途径负责细胞的受控死亡，为生长激素细胞中垂体细胞的更新提供了第一条生理途径。该项目将探索破坏凋亡的RET途径在人类AIP相关肿瘤发生中的作用。我们将使用生长激素细胞系来研究这些途径，以及来自大鼠和小鼠脑垂体腺的细胞，因为它们比细胞系更像生理上的生长激素分泌细胞。我们将用突变的AIP转导细胞，或者我们将敲除细胞自身的AIP(从而使细胞缺乏AIP)，并将研究由此导致的RET途径成员如RET、Caspase 3活性、PKCDelta、CREB、JNK、P53和Pit-1蛋白的变化。我们还将探索AIP作为热休克蛋白90的合作伙伴，是否在RET的生物发生、运输或降解中发挥‘伴侣’作用。此外，我们还将研究在手术中从有AIP突变的患者身上取出的垂体瘤样本，并将数据与没有AIP突变的人类腺瘤样本进行比较。我们已经建立了一个在脑下垂体不表达AIP的小鼠模型，我们将利用这一模型来研究出生前和出生后脑下垂体的发育，包括青春期，那时携带AIP突变的人通常会发展为腺瘤。在项目的最后部分，我们将把含有突变型或野生型自然产生的AIP的病毒颗粒(wt-AIP)注射到新生和青春期动物的脑垂体中，并研究它们对脑垂体和身体生长的影响。我们希望识别AIP在RET调节的增殖和细胞死亡途径中的作用将有助于为患者寻找新的治疗靶点。

Pituitary adenomas are benign tumours of the pituitary, an endocrine gland located at the base of the brain. If an adenoma develops here it can cause problems either because of its size, where it can damage the surrounding crucial structures (e.g. optic nerve), or due to excess hormone release. Recently mutations in a gene called AIP have been identified which predisposes to childhood or young-adult onset adenomas often leading to a devastating condition called gigantism. Despite the AIP protein being present in every cell of the body, a unique feature of patients with germline AIP mutations is that they only develop pituitary adenoma, most commonly from the growth hormone (GH)-secreting cells of the pituitary gland. The overall aim of our study is to identify the mechanism by which AIP-related tumours develop. Based on our preliminary data this proposal aims to clarify the role of the RET protein, an important regulator of cell growth and death, and its pathway members in the mechanism of the AIP-related tumorigenesis. Over the last few years it has been revealed that stem cells constantly provide new differentiated endocrine cells throughout a person's life and there is a control mechanism, apoptosis, removing cells to maintain the number of cells in the normal pituitary gland. In the pituitary gland RET is predominantly expressed in the growth hormone cells and contributes to this apoptotic process. The apoptotic RET pathway, responsible for controlled cell death, provides the first physiological pathway contributing to pituitary cell turnover in GH cells. This project will explore the possibility that disruption of the apoptotic RET pathway contributes to AIP-related tumorigenesis in humans. We will use a somatotroph cell line to study these pathways as well as cells from rat and mouse pituitary glands as they resemble physiological growth hormone-secreting cells better than the cell line. We will transfect the cells with mutant AIP or we will knock down the cell's own AIP (therefore making the cell deficient in AIP) and will study resulting changes in RET pathway members such RET, Caspase 3 activity, PKCdelta, CREB, JNK, p53 and Pit-1 proteins. We will also explore if AIP, as a partner to the heat-shock protein 90, plays a 'chaperone' role in RET biogenesis, trafficking or degradation. In addition, we will study pituitary tumour samples removed during surgery from patients with AIP mutations and compare data to human adenoma samples without AIP mutation. We have created a mouse model which does not express AIP in the pituitary gland and we will use this to study the development of the pituitary gland both before and after birth including at puberty, when humans with AIP mutations often develop adenomas. In the final part of the project we will microinject virus particles containing mutant or wild type, naturally occurring AIP (wt-AIP), into the pituitary gland of newborn and pubescent animals and will study their impact on pituitary gland and body growth.We hope that identifying the role of AIP in the RET-regulated proliferation and cell death pathways will help to identify new therapeutic targets for patients.