Intracellular Signaling In Endocrine Cells

内分泌细胞的细胞内信号传导

基本信息

批准号：
10913217
负责人：
STANKO S. STOJILKOVIC
金额：
$ 133.67万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10913217
关键词：
Address Adult Affect Age Anterior Anterior Pituitary Gland Astrocytes Blood Vessels Calcium Calcium Signaling Cell Differentiation process Cell Proliferation Regulation Cell membrane Cell physiology Cells Code Collaborations Communities Complement Complex Coupling Data Delayed Puberty Development Embryo Endocrine Enzyme Inhibition Enzymes Event Exhibits Exocytosis Female Fiber Follicle Stimulating Hormone G-Protein-Coupled Receptors GNRH1 gene Gene Expression Generations Genes Genetic Transcription Genetic Variation Golgi Apparatus Gonadal structure Gonadotropin Hormone Releasing Hormone Gonadotropin-Releasing Hormone Receptor Gonadotropins Heterogeneity Hormone Receptor Hormones Hypothalamic structure Infertility Inositol Investigation Ion Channel Ion Channel Gating KISS1 gene Knock-out Knockout Mice Lactation Ligands Lipids Lobe Luteinizing Hormone Maintenance Membrane Potentials Mus National Institute of Child Health and Human Development Neonatal Neuroendocrine Cell Neurons Neurosecretory Systems Ovarian Ovulation P2X-receptor PIK3CG gene PIK4CB gene PTPRN gene Pathway interactions Pattern Pericytes Periodicals Phosphatidylinositols Phospholipase C Phosphotransferases Physiological Pituitary Gland Pituitary Hormones Play Population Process Prolactin Protein Tyrosine Phosphatase Proteins Puberty Rattus Receptor Gene Recombinants Reproduction Research Personnel Research Project Grants Residual state Role Sampling Signal Transduction Sister Specificity Stem cell pluripotency Steroid biosynthesis Structure Structure of nucleus infundibularis hypothalami System Third ventricle structure Tissues Transgenic Organisms Vascular Endothelial Cell Ventricular Work cell type conditional knockout density experimental study hypothalamic pituitary gonadal axis immunoreactivity inorganic phosphate lactotroph male mouse model organ growth pharmacologic phosphoinositide-3,4,5-triphosphate phosphoinositide-3,4-bisphosphate postnatal postnatal development postnatal period preservation receptor regulated endocrine secretory protein 18 reproductive function reproductive organ reproductive tract sex sexual dimorphism single-cell RNA sequencing stem cell niche stem cells stem-like cell transcriptome transcriptomics voltage voltage gated channel

项目摘要

We continue investigations on genes expressed in mammalian pituitary cells and their role in pituitary cell signaling and function. Our experiments on transcriptome profiles of secretory and nonsecretory cell types using single cell RNA sequencing (scRNAseq) of freshly dispersed pituitary cells revealed the presence of six hormone-producing cell types melanotrophs, corticotrophs, gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs. We also identified four non-hormonal cell types folliculostellate cells (FSCs), pituicytes, and vascular endothelial cells and pericytes. We recently summarized scRNAseq and immunohistofluorescence analyses of pituitary cells of adult female rats with a focus on transcriptomic profiles of nonhormonal cell types. Samples obtained from whole pituitaries and separated anterior and posterior lobe cells contained all expected pituitary resident cell types and lobe-specific vascular cell subpopulations. FSCs and pituicytes expressed S100B, ALDOC, EAAT1, ALDH1A1, and VIM genes and proteins, as well as other astroglial marker genes, some common and some cell type specific. We also found that the SOX2 gene and protein were expressed in 15% of pituitary cells, including FSCs, pituicytes, and a fraction of hormone-producing cells, arguing against its stem cell-specificity. FSCs comprised two Sox2-expressing subclusters; FSC1 contained more cells but lower genetic diversity, while FSC2 contained proliferative cells, shared genes with hormone-producing cells, and expressed genes consistent with stem cell niche formation, regulation of cell proliferation and stem cell pluripotency, including the Hippo and Wnt pathways. FSC1 cells were randomly distributed in the anterior and intermediate lobes, while FSC2 cells were localized exclusively in cells of the marginal zone between the anterior and intermediate lobes. These data indicate the identity of these cells as specialized anterior pituitary-specific astroglia, with FSC1 representing differentiated cells with transcriptomes consistent with classical FSC roles and FSC2 exhibiting additional stem cell-like features. All pituitary hormone-producing cells expressed common genes related to secretory functions, such as sister genes coding for regulated endocrine-specific protein 18, Resp18, and the protein tyrosine phosphatase receptor genes Ptprn and Ptprn2, as well as Chga, Chgb, Scg2, Snap25, and Uchl1. Unlike cell type-specific hormone and hormone receptor genes, the roles of these common genes are not well characterized. Our recent studies confirmed that simultaneous knockout of the neuroendocrine marker genes Ptprn and Ptprn2 causes infertility in female mice while males are fertile. To elucidate the mechanism of the sex-specific roles of Ptprn and Ptprn2 in mouse reproduction, we further analyzed the effects of their double knockout (DKO) on the hypothalamic-pituitary-gonadal axis. In DKO females, delayed puberty and lack of ovulation were observed, complemented by changes in ovarian gene expression and steroidogenesis. In contrast, testicular gene expression, steroidogenesis, and reproductive organs development were not significantly affected in DKO males. However, in both sexes, pituitary luteinizing hormone (LH) beta gene expression and LH levels were reduced, as well as follicle-stimulating hormone beta gene and gonadotropin-releasing hormone (GnRH) gene, while the calcium-mobilizing and LH secretory actions of GnRH were preserved. Hypothalamic Gnrh1 and Kiss1 gene expression was also reduced in DKO females and males. In parallel, a significant decrease in the density of immunoreactive GnRH and kisspeptin fibers was detected in the hypothalamic arcuate nucleus of DKO females and males. The female-specific kisspeptin immunoreactivity in the rostral periventricular region of the third ventricle was also reduced in DKO females, but not in DKO males. These data indicate a critical role of Ptprn and Ptprn2 in kisspeptin-GnRH neuronal function and sexual dimorphism in the threshold levels of GnRH required to preserve reproductive functions. Ongoing experiments on this topic are focuses on the physiological status of anterior pituitary corticotrophs and intermediate lobe-located melanotrophs of DKO mice. In collaboration with the group of Dr. Balla at the NICHD, we are also studying the functions of three phosphoinositides, PI4P, PI(4,5)P2, and PI(3,4,5)P3, in cellular signaling and exocytosis, focusing on hormone-producing pituitary cells. PI(4,5)P2, acting as a substrate for phospholipase C, plays a key role in the control of pituitary cell functions, including hormone synthesis and secretion. PI(4,5)P2 also acts as a substrate for class I PI3-kinases, leading to the generation of two intracellular messengers, PI(3,4,5)P3 and PI(3,4)P2, which act through their intracellular effectors, including Akt. PI(4,5)P2 can also influence the release of pituitary hormones acting as an intact lipid to regulate ion channel gating and concomitant calcium signaling, as well as the exocytic pathway. Recent findings also showed the expression of several PI lipid kinases, including Pi4ka, Pi4kb, Pi4k2a, Pi4k2b, Pip5k1a, Pip5k1c, and Pik3ca, as well as Pikfyve and Pip4k2c, in pituitary lactotrophs, which are responsible for the secretion of prolactin (PRL), a hormone controlling lactation. Using a pharmacological approach to specifically inhibit these enzymes we show that PI4P made in the plasma membrane by PI4KA is critical for exocytosis without affecting the calcium signals that trigger secretion. Our experiments also indicate that inhibition of the PI4KB enzyme that generates PI4P in the Golgi is dispensable for the exocytic step. These experiments revealed a key role of PI4KA-derived PI4P in the plasma membrane in calcium-secretion coupling in pituitary lactotrophs downstream of voltage-gated and PI(4,5)P2-dependent calcium signaling. The ongoing study on this topic is focused on the role of PI4KA in gonadotroph function by knocking out this enzyme in cells expressing the GnRH receptor. Knockout mice were infertile, reflecting underdeveloped gonads and reproductive tracts, and lack of puberty. The number and distribution of hypothalamic GnRH neurons and Gnrh1 expression in postnatal knockouts were not affected, while Kiss1/kisspeptin expression was increased. Knockout of PI4KA also did not alter embryonic establishment and neonatal development and function of the gonadotroph population. However, during the postnatal period, there was a progressive loss of expression of gonadotroph-specific genes, including Fshb and Lhb, accompanied by low synthesis of gonadotropins, but not of other pituitary lineage-specific genes and their hormones. The postnatal gonadotroph population also progressively declined, reaching approximately 25% of that observed in controls at 100 days of age. In these residual gonadotrophs, GnRH-dependent calcium signaling, and calcium-dependent membrane potential changes were lost, but intracellular administration of inositol-1,4,5-trisphosphate rescued this signaling. These results indicate a key role for PI4KA in the postnatal development and maintenance of a functional gonadotroph population.

我们继续研究在哺乳动物垂体细胞中表达的基因及其在垂体细胞信号传导和功能中的作用。 Our experiments on transcriptome profiles of secretory and nonsecretory cell types using single cell RNA sequencing (scRNAseq) of freshly dispersed pituitary cells revealed the presence of six hormone-producing cell types melanotrophs, corticotrophs, gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs.我们还鉴定了四种非激素细胞类型的卵泡细胞（FSC），皮尿素和血管内皮细胞和周细胞。我们最近总结了成年雌性大鼠的垂体细胞的SCRNASEQ和免疫晶状体荧光分析，重点是非激素细胞类型的转录组。从整个垂体中获得的样品并分离前叶和后叶细胞，其中包含所有预期的垂体居民细胞类型和叶特异性血管细胞亚群。 FSC和Pituicytes表示S100B，ALDOC，EAAT1，ALDH1A1和VIM基因和蛋白质以及其他星形胶质标记基因，有些是常见和某些细胞类型的特异性。我们还发现，Sox2基因和蛋白质在15％的垂体细胞中表达，包括FSC，pituicytes和一小部分产生激素的细胞，反对其干细胞特异性。 FSC包括两个表达Sox2的子截面。 FSC1包含更多的细胞但较低的遗传多样性，而FSC2包含增殖细胞，与产生激素的细胞共享基因，并表达与干细胞形成，细胞增殖和干细胞多能性的调节，包括河马和WNT途径。 FSC1细胞随机分布在前叶和中间叶中，而FSC2细胞仅位于前叶和中间叶之间边缘区的细胞中。这些数据表明这些细胞的身份是专门的垂体特异性星形胶质细胞，其中FSC1代表具有与经典FSC角色一致的转录组和FSC2一致的分化细胞，而FSC2表现出其他类似干细胞的特征。所有垂体激素产生的细胞都表达了与分泌功能相关的共同基因，例如编码调节内分泌特异性蛋白18，RESS18和蛋白酪氨酸磷酸酶受体基因PTPRN和PTPRN2的姐妹基因，以及CHGA，CHGA，CHGB，CHGB，CHGB，SCG2，SNAP25和UCHL1。与细胞类型特异性激素和激素受体基因不同，这些常见基因的作用没有很好地表征。我们最近的研究证实，同时敲除神经内分泌标记基因PTPRN和PTPRN2导致雌性小鼠的不孕症，而雄性肥沃。为了阐明PTPRN和PTPRN2在小鼠繁殖中的性别特异性作用的机制，我们进一步分析了它们的双基因敲除（DKO）对下丘脑 - 垂体 - 垂体轴轴的影响。在DKO女性中，观察到延迟的青春期和缺乏排卵，并与卵巢基因表达和类固醇发生的变化相辅相成。相反，在DKO雄性中，睾丸基因表达，类固醇生成和生殖器官发育没有显着影响。然而，在男女中，垂体黄体激素（LH）β基因表达和LH水平降低，以及卵泡刺激的激素β基因和促性腺激素释放激素（GNRH）的基因，而钙模化和LH分泌作用是Gnrh的钙化和LH分泌作用。在DKO女性和男性中，下丘脑GNRH1和KISS1基因表达也降低了。同时，在DKO雌性和雄性的下丘脑弧形核中检测到免疫反应性GNRH和Kisspeptin纤维的密度显着降低。在DKO雌性中，在第三个心室的脑室周围区域中，女性特异性的亲吻蛋白免疫反应性也降低，但在DKO雄性中也没有降低。这些数据表明PTPRN和PTPRN2在Kisspeptin-GNRH神经元功能和性二态性中的关键作用在保留生殖功能所需的GnRH阈值中。关于该主题的持续实验侧重于垂体前肌营养物的生理状态和DKO小鼠的中间叶中间叶片的黑色素芽孢杆菌。在NICHD与Balla博士的小组合作，我们还研究了三种磷酸肌醇，PI4P，PI（4,5）P2和PI（3,4,5）P3的功能，在细胞信号传导和胞吞作用中的功能，专注于激素产生的垂体细胞。 PI（4,5）P2充当磷脂酶C的底物，在控制垂体细胞功能（包括激素合成和分泌）中起关键作用。 PI（4,5）P2还充当I类PI3-激酶的底物，从而导致两个细胞内信使生成Pi（3,4,5）P3和PI（3,4）P2，它们通过其细胞内效应子（包括Akt）起作用。 PI（4,5）P2还可以影响垂体激素的释放，该激素可作为调节离子通道门控和伴随钙信号的完整脂质，以及外生途径。 Recent findings also showed the expression of several PI lipid kinases, including Pi4ka, Pi4kb, Pi4k2a, Pi4k2b, Pip5k1a, Pip5k1c, and Pik3ca, as well as Pikfyve and Pip4k2c, in pituitary lactotrophs, which are responsible for the secretion of prolactin (PRL), a hormone controlling lactation.使用药理学方法特异性抑制这些酶，我们表明，PI4KA在质膜中制造的PI4P对于胞吐作用至关重要，而不会影响触发分泌的钙信号。我们的实验还表明，在高尔基体中生成PI4P的PI4KB酶是外囊性步骤可分配的。这些实验揭示了PI4KA衍生的PI4P在钙 - 分泌耦合中的质膜膜中的关键作用，在电压门控和PI（4,5）P2依赖性钙信号传导下下游的垂体乳腺营养中。正在进行的有关该主题的研究集中在PI4KA在促性腺营养功能中的作用，通过在表达GNRH受体的细胞中删除这种酶。敲除小鼠是不育的，反映了欠发达的性腺和生殖道，缺乏青春期。下丘脑GNRH神经元和GNRH1表达在产后敲除中的数量和分布没有影响，而KISS1/Kisspeptin表达则增加。 PI4KA的敲除也没有改变促性腺激素种群的胚胎建立和新生儿的发展和功能。然而，在产后，促性腺营养特异性基因（包括FSHB和LHB）的表达逐渐丧失，伴随着促性腺激素的低合成，但没有其他垂体谱系特异性基因及其激素。产后促性腺营养的种群也逐渐下降，大约达到了100天大的对照中观察到的25％。在这些残留的促性腺营养物中，GNRH依赖性钙信号传导和钙依赖性的膜电位变化丢失了，但细胞内肌内抗磷酸1,4,5-三磷酸磷酸盐的施用挽救了这种信号传导。这些结果表明，PI4KA在产后发育和维持功能性促性腺营养群中的关键作用。