A PR-Induced Autocrine/Paracrine Pathway in Female Brain

卵巢类固醇激素，雌激素(E)和孕酮(P)，通过激活同源核受体(Era、PR亚型a和b)对发育、生殖和衰老产生深远的影响。除了在女性下丘脑腹内侧核(VMN)合成PR外，E还引起局部微电路的变化，这一效应需要cAMP活性和感受性。由于cAMP增加了其他组织中构成性腺苷环化酶激活多肽(PACAP)的合成和释放，以与PACAP受体局部结合，本研究的目的是确定E诱导的PACAP是否在VMN微电路中介导了一个新的自分泌/旁分泌跨突触环路，这是启动P促进感受性所必需的。由于缺乏对表达PR亚型的细胞谱系的了解，PR的生物学作用的研究一直受到阻碍，因此将研究选择性消融PRA(PRAKO)和PRB(PRBKO)的小鼠。本项目的具体目标是：1)确定E和P及其同源受体[ERA、PRA、PRB]是否调节VMN微循环中垂体腺苷酸环化酶激活多肽的合成和释放，最终实现感受性；2)研究VMN中单个PR表达细胞和相邻的非PR表达细胞介导异构型PR易化行为的基因表达谱(分子指纹)；3)确定类固醇诱导的机制(S)及其主要成分(膜结合受体的VMN表达模式[PAC1、VPAC、VPAC2])；PACAP介导异构体特异性PR易化行为的信号通路[cAMP，MAPK]。将使用成熟的程序[原位杂交、印迹分析、免疫组织化学、类固醇受体依赖的行为]和新技术[实时RT-PCR、单细胞表达谱(分子指纹)、微阵列、激光捕获单个细胞]。通过监测单个VMN细胞中与激素状态和行为相关的PACAP活性、细胞内信号转导和基因表达诱导的特定类固醇受体(PRA和PRB)的协调变化，将识别对细胞表达的遗传变异性的生物学后果的独特见解。这将提供对产生类固醇受体依赖性疾病病因的潜在细胞和分子机制的洞察，并应有助于开发新的治疗策略。

Ovarian steroid hormones, estrogen (E) and progesterone (P), exert profound influence on development, reproduction, and aging through activation of cognate nuclear receptors (ERa, PR isoforms a and b). In addition to synthesis of PR in female hypothalamic ventromedial nucleus (VMN), E induces changes in local microcircuitry, an effect that requires cAMP activity and for receptivity. Since cAMP increases synthesis and release of constitutive pituitary adenylyl cyclase-activating polypeptide (PACAP) in other tissue for local binding to PACAP receptors, the goal of this proposal is to determine whether E-induced PACAP mediates a novel autocrine/paracrine trans-synaptic loop within VMN microcircuitry required for onset of P-facilitated receptivity. Since studies on the biological role of PR have been hampered by a lack of knowledge of the cell-lineages that express PR isoforms, mice with selective ablation of PRa (PRAKO) and PRb (PRBKO) will be studied. The specific aims of this project are: 1) To determine whether E and P and their cognate receptors [ERa, PRa, PRb] regulate the synthesis and release of pituitary adenylyl cyclase-activating polypeptide (PACAP) in the microcircuitry of the VMN ultimately for receptivity, 2) To characterize the gene expression profile ('molecular fingerprints') of individual PR-expressing and adjacent nonPR- expressing cells in the VMN that mediate isoform-specific PR- facilitated behavior, 3) To identify the steroid-induced mechanism(s) and its major components (VMN expression pattern of membrane-bound receptors [PAC1, VPAC, VPAC2]; signaling pathway [cAMP, MAPK] by which PACAP mediates isoform-specific PR- facilitated behavior. Well-established procedures [in situ hybridization, blot analysis, immunohistochemistry, steroid receptor-dependent behavioral] and new technologies [real-time RT-PCR, single cell expression profiling (molecular fingerprinting), microarrays, Laser capture of individual cells] will be used. By monitoring coordinate changes in PACAP activity, intracellular signaling, and gene expression induced specific steroid receptors (PRa and PRb) in the individual VMN cells that correlate with hormone status and behavior, unique insight into the biological consequences of expressed genetic variability of cells will be identified. This will furnish insight into the underlying cellular and molecular mechanisms that produce steroid receptor-dependent disease etiologies and should contribute to the development of new therapeutic strategies.