Androgen Receptor (AR) modulation through and cAMP signaling in prostate epithelium

目的为了理解细胞内信号过程的区隔和串扰的分子机制，目前有很大的兴趣来理解基本的见解。在这方面，cAMP信号通路提供了重要的范例。CAMP-磷酸二酯酶(PDE‘s)是细胞内cAMP信号的基础。这个项目的重点是了解和开发特定的PDE在男性前列腺上皮中的功能作用。特别是定义cAMP信号系统和雄激素受体(AR)之间的相互作用，雄激素受体(AR)的作用是正常前列腺发育和功能所必需的。雄激素受体信号及其由cAMP、前列腺发育和正常前列腺功能调节需要雄激素信号和作用(1)。男性雄激素，特别是睾丸睾酮及其更活跃的代谢物双氢睾酮(DHT)，通过核激素受体雄激素受体(AR)在前列腺中发挥作用。雄性器官与性分化和成熟有关的发育和生理强烈依赖于雄激素和AR的活性。110 kDa的AR是核受体超家族中的一员，在与各种亲脂配体(类固醇、维甲酸等)结合后被激活。配基结合促进AR共调节因子的结合，然后蛋白质复合体移位到细胞核中。DNA转录是通过AR复合体与靶基因启动子区域的雄激素反应元件(ARES)结合而诱导的。AR转录活性的调节似乎受到许多共调节因子和磷酸化事件的影响。已有研究表明，cAMP水平的升高可以以非配体依赖的方式诱导AR活性。这被认为是通过激活PKA来调节的。然而，还没有进行分析来确定是否可以通过最近发现的涉及cAMP GTP交换因子EPAC的cAMP信号系统来进行额外的调节。然而，已经在AR(Ser81/94/650)上发现了几个潜在的PKA磷酸化位点，并推测PKA的磷酸化为AR转录的非配体激活提供了一种机制。因此，需要确定可能控制这些位点上PKA磷酸化的参数，例如PDE的调节、AKAPs和PKA亚型的参与及其功能意义。然而，也有人提出，cAMP与AR的串扰可能(另外)发生在CREB(cAMP反应元件结合蛋白)的水平。该模型提示，PKA通过CREB Ser133的磷酸化间接影响AR的兴奋。这种PKA的磷酸化促进CREB与靶候选基因中的cAMP反应元件结合，并招募更多的共激活子，从而增强靶基因的转录。在PSA基因5‘上游调控区存在一个可能的cAMP反应位点(cAMP Response Site，CRE)，这使得CREB可能成为介导PKA效应的候选基因。因此，PKA可以在两个地方管理AR的运作，AR本身和CREB。尽管多年来一直表明，cAMP-PKA途径的激活增强了AR的转录活性，但其确切机制尚未被揭示。这种作用既可以通过cAMP-PKA直接磷酸化AR来实现，也可以通过间接的PKA磷酸化来实现。工作计划本研究旨在确定cAMP-PKA对AR信号的调制。这些研究将对一个重要的生物系统中的阵营划分和串扰提供新的理解。这些使用最先进技术的多学科研究将为参与其中的学生提供一流的培训。

Aim There is currently a great interest in understanding the molecular mechanism that underpin compartmentalisation and cross-talk of intracellular signalling processes in order to appreciate fundamental insights. In this, the cAMP signalling pathway has provided important paradigms. cAMP-phosphodiesterases (PDE's) underpin compartmentalised cAMP signalling in cells. This project focuses on understanding and exploiting the functional role of specific PDE's in male prostate epithelium. In particular defining interactions between the cAMP signalling system and that of the androgen receptor (AR), whose action is required for normal prostate development and function. Androgen Receptor signalling and its modulation by cAMP Prostate development and normal prostate function requires androgen signaling and action (1). The male androgens, in particular the testicular testosterone and its more active metabolite dihydrotestosterone (DHT), exert their action in the prostate through a nuclear hormone receptor, the Androgen Receptor (AR). The development and physiology of the male organism regarding sexual differentiation and maturation is strongly dependent on androgen and AR activity. The 110-kDa AR is a member of the super-family of nuclear receptors, and is activated upon binding of various lipophilic ligands (steroids, retinoids, etc). Ligand binding promotes the association of AR co-regulators, and the protein complex then translocates into the cell nucleus. DNA transcription is induced by binding of AR complexes to AREs (Androgen Responsive Elements) in the promotor region of target genes. The modulation of transcriptional activity of the AR appears to be influenced by a number of co-regulators and phosphorylation events. It has been suggested that AR activity can be induced in a ligand-independent manner by the elevation of cAMP levels. This has been presumed to be mediated via the activation of PKA. However, no analysis has yet been done to determine whether regulation can additionally occur through the recently discovered cAMP signalling system involving the cAMP GTP exchange factor, EPAC. Nevertheless, several potential PKA phosphorylation sites have been identified on the AR (Ser81/94/650) and phosphorylation by PKA has been speculated to provide a mechanism for ligand-independent activation of AR transcription. Thus work is needed to define the parameters that may control PKA phosphorylation at these sites, such as regulation by PDEs and the involvement of AKAPs and PKA isoforms as well as their functional significance. However, it has also been proposed that cAMP cross-talk with the AR may occur (additionally) at the level of CREB (cAMP responsive element-binding protein). This model suggests an indirect effect of PKA on AR stimulation by the phosphorylation of Ser133 of CREB. Such PKA phosphorylation promotes CREB binding to cAMP responsive elements in target candidate genes and the recruitment of additional co-activators, thereby enhancing target gene transcription. The presence of a putative cAMP responsive site (CRE) at the 5'-upstream regulatory region in the PSA gene makes CREB a conceivable candidate for mediating PKA effects. Thus PKA may regulate AR functioning at two localities, the AR itself and also CREB. Although it has been shown consistently over the years that the activation of cAMP-PKA pathway enhances AR transcriptional activity, the exact mechanism for this has not been shown. This effect could be mediated through either direct phosphorylation of the AR by cAMP-PKA, or mediated through indirect PKA phosphorylation effects. Workplan This study aims to identify the modulation of AR signalling by cAMP-PKA. These investigations will provide novel understanding about cAMP compartmentalisation and cross-talk in an important biological system. These multi-disciplinary studies using 'state-of-the-art' technologies will provide a first-rate training for the student involved.