REGULATION OF STEROIDOGENESIS IN THE HUMAN FETAL ADRENAL

人类胎儿肾上腺中类固醇生成的调节

• 批准号: 6240849
• 负责人: EVAN R SIMPSON
• 金额: $ 18.32万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6240849
• 关键词: RNase protection assay; SDS polyacrylamide gel electrophoresis; adrenal glands; birth; cytochrome P450; embryo /fetus; embryo /fetus membrane; enzyme biosynthesis; enzyme linked immunosorbent assay; gel mobility shift assay; gene expression; genetic regulatory element; histochemistry /cytochemistry; hormone regulation /control mechanism; human fetus tissue; human tissue; in situ hybridization; messenger RNA; northern blottings; organ culture; parathyroid hormone related protein; placenta; polymerase chain reaction; protein kinase; radioimmunoassay; sheep; steroid biosynthesis; steroid hormone biosynthesis; transfection; western blottings

The long-range goal of this project is to define the molecular mechanisms that regulate the expression of the enzymes required for the synthesis of progesterone and estradiol-17beta (E20 in the human and sheep placenta and fetal adrenal. These steroidal secretions are important in effecting the maintenance of uterine quiescence; but the rates of secretion or bioaction must be altered to initiate the preparatory and active phases of labor. The molecular basis of regulation of 3beta-hydroxysteroid dehydrogenase/delta 5->4-isomerase (3beta-HSD) type I in human trophoblast will be determined: (i) The levels of 3beta-HSD MRNA and protein in cultured trophoblast, choriocarcinoma, chorion laeve, and amnion cells will be evaluated after treatments with agonists of protein kinases A and C, and Ca2+-dependent signal transduction; (ii) with selected placental-derived growth factors [insulin-like growth factors (IGFs) I and II, and transforming growth of factor-betas(TGF-betas)]. The nature of the cis- regulatory and trans-acting elements in the 5'-untranscribed flanking sequence, which account for the high expression of this gene in trophoblast, will be established using transfection assays of chimeric gene constructs and gel-shift assays. The level of 3 beta-HSD expression in human fetal adrenal (HFA) is low compared with that in placenta throughout most of pregnancy; but increased of the adrenal enzyme must occur near the time of parturition. The HFA 3beta-HSD is postulated to be the product of a gene distinct from that which encodes the placental (type I) isoform and regulated separately. The action of ACTH, TGF-betas, parathyroid hormone- related protein (PTH-rP), IGFs, and E2 on the level of MRNA encoding HFA 3beta-HSD and the cellular content and activity of this enzyme will be defined. By immunohistochemistry and in situ hybridization, the cell types that express 3beta-HSD and 17alpha-hydroxylase cytochrome P450 (P45017alpha) in the ovine placentome during dexamethasone-induced parturition will be determined. The in vitro action of potential regulators (e.g., IGF-1/II, TGF-betas, glucocorticosteroids) of 3beta-HSD and P450alpha expression in ovine trophoblast cells will be evaluated. Glucocorticosteroid-induced expression of P45017alpha in ovine placenta near the time of parturition may involve alternative, tissue-specific promoters. Unique, untranslated exonic sequences, obtained from amplification of full-length placental and adrenal CDNAS, will be sought to identify placental- and adrenal-specific P45017alpha transcripts and promoters in the CYP17 gene; by means of "foot-printing" studies, the role of putative steroid enhancer and TGF-beta-inhibitory sequences in the 5'- flanking sequence will be investigated. We will determine whether the enhanced placental expression of this gene involves a trans-acting factor that interacts with a steroid enhancer-like consensus sequence. We will ascertain if differences in 5'-regulatory elements in the human an sheep CYP17 genes account for differences in placental P45017alpha expression.

该项目的长期目标是确定分子机制 调节合成所需的酶的表达 孕酮和雌二醇-17 β（E20在人类和绵羊胎盘中， 胎儿肾上腺。 这些甾体分泌物在影响 维持子宫静止;但分泌或生物作用的速率 必须改变以启动分娩的准备和主动阶段。 3 β-羟基类固醇调节的分子基础 人滋养层中的脱氢酶/δ 5->4-异构酶（3 β-HSD）I型 将测定：（i）在20天内， 培养的滋养层、绒毛膜癌、绒毛膜层和羊膜细胞将 在用蛋白激酶A和C激动剂治疗后进行评价，和 Ca 2+依赖性信号转导;（ii）选择胎盘来源的 生长因子[胰岛素样生长因子（IGF）I和II，和 转化因子-β（TGF-β）的生长]。 顺- 调控和反式作用元件在5 '-非转录侧翼 序列，这解释了该基因在大肠杆菌中的高表达。 滋养层细胞，将使用嵌合基因的转染测定来建立 构建体和凝胶迁移测定。 3 β-HSD表达水平在 人胎儿肾上腺（HFA）含量始终低于胎盘 大多数怀孕;但增加肾上腺酶必须发生在附近的 分娩时间。 HFA 3 β-HSD被假定为以下物质的产物： 与编码胎盘（I型）同种型的基因不同的基因， 单独监管。 促肾上腺皮质激素，转化生长因子β，甲状旁腺激素的作用- 在编码HFA的mRNA水平上的PTH-rP、IGF和E2 3 β-HSD和细胞内容物和这种酶的活性将是 定义了 通过免疫组织化学和原位杂交， 其表达3 β-HSD和17 α-羟化酶细胞色素P450 （P45017 α）在地塞米松诱导的 分娩将被确定。 电位的体外作用 调节器（例如，IGF-1/II、TGF-β、糖皮质激素） 和P450 α在绵羊滋养层细胞中的表达。 糖皮质激素对绵羊胎盘P45017 α表达的影响 分娩时可能涉及替代性，组织特异性 发起人。 独特的，未翻译的外显子序列，从 全长胎盘和肾上腺CDNAS的扩增，将寻求 鉴定胎盘和肾上腺特异性P45017 α转录物， CYP 17基因启动子;通过“足迹”研究， 在5 '端的推定类固醇增强子和TGF-β抑制序列， 将研究侧翼序列。 我们将决定是否 该基因的胎盘表达增强涉及反式作用因子 与类固醇增强子样共有序列相互作用。 我们将 确定人和羊的5 '调控元件是否存在差异 CYP 17基因解释了胎盘P45017 α表达的差异。