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.
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