Gonadal Receptors/mechanisms Of Action Of Peptide Hormon

1) LH受体：黄体生成素受体（luteinizing hormone receptor， LHR）是生殖功能必需的G蛋白偶联受体，主要存在于性腺细胞的质膜中。它介导促性腺激素信号传导并触发参与性腺成熟和功能的细胞内反应，以及调节甾体生成和配子发生。LHR基因也在一些非性腺组织中表达，包括子宫和胎盘，其功能尚未确定。我们之前证明了核孤儿受体（EAR2, EAR3/COUP-TFI，抑制性；TR4，刺激性）可以调节Sp1/ sp3驱动的人LHR的TATA-less 176 bp启动子转录。目前的研究探讨了培养的人绒毛膜癌细胞中组蛋白去乙酰化酶对人LHR的调节作用。组蛋白去乙酰化酶（HDCs）和乙酰化酶修饰染色质结构是调控真核基因转录的重要机制。Trichostatin A （TSA）抑制HDAC可使hLHR增加40倍，并显著提高内源性hLHR mRNA水平（高达11倍）。这些研究表明组蛋白去乙酰化在这些细胞中引起了显著的抑制。乙酰化组蛋白（H3, H4）在tsa处理的细胞中积累，CHIP实验显示其主要与LHR启动子相关。此外，TSA显著增强了RNA Pol II对启动子的招募。在基础启动子活性必需的两个位点中，只有一个位点被确定为TSA效应的关键位点（Sp1-1， ATG +1的-79/-71），但Sp1/Sp3与该位点的结合保持不变。一个多蛋白复合物（Sp1/Sp3, HDC1,2, RbAp48, Sin3A）通过与Sp1和Sp3的相互作用被招募到启动子中。HDAC1和HDAC2通过RbAp48直接停靠在sp1结合的DNA上或间接停靠在Sp3结合的DNA上，而mSin3A（抑制剂）与HDACs相互作用。共转染研究表明，HDAC1和hdac 2能有效抑制hLHR基因转录，而mSin3A能增强HDAC1的抑制作用。LHR基因的表达受到其基因启动子去乙酰化的强直性抑制。通过未知的信号输入，对该基因的抑制控制进行调控，可能在性腺细胞分化、生长和发育的诱导和周期性变化过程中提供功能控制。

1) The LH receptor: The luteinizing hormone receptor (LHR) is an essential G protein-coupled receptor for reproductive function and is predominantly located in the plasma membrane of gonadal cells. It mediates gonadotropin signaling and triggers intracellular responses that participate in gonadal maturation and function, as well as in the regulation of steroidogenesis and gametogenesis. The LHR gene is also expressed in several non-gonadal tissues, including the uterus and placenta, where its functions have not been determined. We previously demonstrated regulation of the Sp1/Sp3-driven transcription of the TATA-less 176 bp promoter of the human LHR by nuclear orphan receptors (EAR2, EAR3/COUP-TFI, inhibitory; TR4, stimulatory). Current studies investigated the regulation of the human LHR by histone deacetylases in human choriocarcinoma cells in culture. Modification of chromatin structure by histone deacetylases (HDCs) and acetylases are important mechanisms in the modulation of eukaryotic gene transcription. Inhibition of HDAC by Trichostatin A (TSA) increased hLHR by 40-fold, and markedly elevated endogenous hLHR mRNA levels (up to 11-fold). These studies demonstrated that histone deacetylation caused significant repression in these cells. Acetylated histones (H3, H4) accumulated in TSA-treated cells, and were shown by CHIP assay to be predominantly associated with LHR promoter. Also, TSA significantly enhanced the recruitment of RNA Pol II to the promoter. Only one of the two sites essential for basal promoter activity was identified as critical for the TSA effect (Sp1-1,-79/-71 from ATG +1), but the binding of Sp1/Sp3 to the site remained unchanged. A multiprotein complex (Sp1/Sp3, HDC1,2, RbAp48, Sin3A) was recruited to the promoter via interaction with Sp1 and Sp3. HDAC1 and HDAC2 were docked directly to Sp1-bound DNA and indirectly to Sp3 bound DNA through RbAp48 while mSin3A (inhibitor) interacted with HDACs. Cotransfection studies revealed that the HDACs 1 and 2 potently repressed hLHR gene transcription and mSin3A potentiated the inhibition by HDAC1. Expression of the LHR gene is subject to tonic repression by deacetylation of its gene promoter. The regulated derepression of such inhibitory control of this gene, through as yet unidentified signal imputs, may provide functional control during the induction and cyclical changes in the differentiation, growth and development of gonadal cells. 2) Prolactin receptors: Prolactin is a polypeptide hormone with highly diversified biological actions. Its actions are mediated by a single transmembrane domain receptor (PRLR) which is widely distributed and exists in several forms (long, intermediate and short inhibitory forms of the stimulatory action of the long form). Prolactin could contribute to tumor biogenesis through its receptor-mediated actions. Adipose cells are the major source of estrogen in postmenopausal women and could exert paracrine control of prolactin and PRLR synthesis in epithelial cells of the breast. To determine the mechanism(s) underlying human PRLR gene expression we defined the 5 prime exonic gene structure and initiated studies on promoter identification and regulation. Of the three promoters utilized for transcription of non-coding exons-1 in the rat (PI,PII and PIII), and two in the mouse (PII and PIII) only one, PIII is shared with the human (PIII). The generic hPIII promoter directs transcription of hE1/3 exon-1 Five additional non-coding human exons-1 (hE1N1-5) were revealed by 5-prime RACE of hPRLR mRNAs from several tissues and breast cancer cells. Resolution of the complete genomic structure of the hPRLR revealed that hPRLR transcription is controlled by multiple promoters that utilize distinct mechanism to regulate expression (hPIII, generic; hPIN1-5, human specific). Sp1/Sp3 are the major transcription factors that participate in hPIII promoter activation (80%) while C/EBPbeta can minimally support activity when Sp1 is mutated (20%) in T47D breast cancer cells. Estradiol significantly increased the expression of both hPRLR mRNA transcripts hE1/3 generic and hE1N1 assessed by quantitative competitive RTPCR in these cells. In transfected T47D cells estradiol activated the hPIII promoter in a dose-dependent manner. This effect of estradiol was markedly inhibited by the specific estrogen receptor antagonist, ICI 182,780. The lack of a formal ERE in these promoters suggests that the effect of estradiol is mediated through association of the activated ER with relevant DNA binding transfactor(s). These findings support the role of estradiol in the regulation of hPRLR expression in human breast cancer cell lines. Since prolactin could influence the growth of mammary tumors, blockade of its stimulatory action on neoplastic cells could provide novel therapeutic applications based on suppression of the expression of the prolactin receptor through its multiple promoters, and inhibition of its cellular actions by short receptors isoforms.