REGULATION OF STEROIDOGENESIS IN THE HUMAN FETAL ADRENAL

这个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 人和绵羊胎盘中的孕酮和雌二醇-17β(E20)和 胎儿肾上腺。这些类固醇分泌物在影响 维持子宫静止；但分泌或生物作用的速度 必须改变，以启动准备和活跃期的分娩。 3β-羟基类固醇调节的分子基础 人滋养层细胞中脱氢酶/β-5-和4-异构酶(3beta-HSD)的研究 将测定：(1)3β-HSD的mRNA和蛋白水平 培养的滋养层细胞、绒毛膜癌、绒毛膜和羊膜细胞将 在用蛋白激酶A和C激动剂治疗后进行评估，以及 依赖于CA2的信号转导；(Ii)与选定的胎盘来源 生长因子[胰岛素样生长因子I和II，以及 转化生长因子-β(转化生长因子-β)]。独联体的性质-- 5‘-非转录侧翼中的调控和反式作用元件 序列，这解释了该基因在 将通过嵌合基因的转染实验建立滋养层细胞 构建和凝胶移位分析。3β-HSD在大鼠体内的表达水平 人胎儿肾上腺(HFA)始终低于胎盘。 大多数妊娠；但肾上腺酶的增加必须发生在 分娩时间。HFA 3beta-HSD被认为是以下产品 一种不同于编码胎盘(I型)亚型的基因 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- 甲状旁腺素相关蛋白(PTH-RP)、胰岛素样生长因子(IGFS)和雌二醇(E_2)在HFA编码基因水平的表达 3β-HSD和这种酶的细胞含量和活性将是 已定义。通过免疫组织化学和原位杂交检测细胞类型 表达3β-HSD和17α-羟基酶细胞色素P450 (P45017α)在地塞米松诱导的绵羊胎盘中的表达 分娩情况将会确定。电势的体外作用 3β-HSD的调节因子(如IGF-1/II、转化生长因子-β1、糖皮质激素) 和P450α在绵羊滋养层细胞中的表达将被评估。 糖皮质激素诱导绵羊胎盘P45017α的表达 临近分娩的时间可能涉及到组织特异性的替代 推动者。唯一的、未翻译的外显子序列，从 全长胎盘和肾上腺cDNA的扩增，将寻求 鉴定胎盘和肾上腺特异的P45017α转录本和 CYP17基因中的启动子；通过“足迹”研究，角色 可能的类固醇增强子和转化生长因子-β抑制序列在5‘- 将对侧翼序列进行调查。我们将确定是否 该基因在胎盘中的表达增强涉及反式作用因子 它与类固醇增强剂类似的共识序列相互作用。我们会 确定人类和绵羊的5‘-调控元件是否存在差异 细胞色素P45017基因是胎盘P45017α表达差异的原因。

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.