Estradiol down regulates expression of vasoactive intestinal polypeptide receptor type-1 in breast cancer cell lines
Department of Clinical Biochemistry, Bispebjerg Hospital, University of Copenhagen NV, Bispebjerg Bakke 23, Dk-2400, Copenhagen, Denmark. Molecular and Cellular Endocrinology
(Impact Factor: 4.41).
03/2001; 172(1-2):203-11. DOI: 10.1016/S0303-7207(00)00341-5
Three breast carcinoma cell lines were tested for 17beta-estradiol (E(2)) mediated regulation of vasoactive intestinal polypeptide receptor type-1 (VPAC(1)) expression. In all three, E(2) was found to down-regulate the mRNA level. We studied T47D cells in more details and found a 25 and 70% decrease in the VPAC(1) mRNA level upon 7 and 48 h of E(2) treatment, respectively. The number of vasoactive intestinal polypeptide (VIP) binding sites was reduced 66% upon treatment with E(2) for 72 h. After cycloheximide pretreatment, the E(2) mediated mRNA reduction was attenuated from 50% to 25% after 24 h suggesting the effect to be at least partly independent of protein synthesis. Experiments with the transcriptional inhibitor actinomycin D showed that E(2) did not influence the VPAC(1) mRNA half-life while nuclear run-on experiments indicated that E(2) decreased the VPAC(1) transcription rate. Two antiestrogens: ICI 182780 (ICI) and 4-hydroxy-tamoxifen (4-OHT) mediated a concentration dependent inhibition of E(2)'s effect on the mRNA level. Transient transfection with reporter-gene constructs containing various portions of the VPAC(1) 5'-flanking sequence revealed the most proximal 100 bp to be essential for the basal transcriptional activity. However, E(2) did not influence the expression of the reporter gene using up to 3250 bp of the VPAC(1) 5'-flariking region.
Available from: Finn Gyntelberg
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ABSTRACT: VIP and PACAP are two prominent neuropeptides that share two common G protein-coupled receptors, VPAC1 and VPAC2, while PACAP has an additional specific receptor, PAC1. This article reviews the present knowledge regarding various aspects of VPAC receptors including: 1) receptor specificity toward natural VIP-related peptides and pharmacology of synthetic agonists or antagonists; 2) genomic organization and chromosomal localization; 3) signaling and established or putative interactions with G proteins or accessory proteins such as RAMPs or PDZ-containing proteins; 4) molecular basis of ligand-receptor interaction as determined by site-directed mutagenesis, construction of receptor chimeras, and structural modeling; 5) constitutively active receptor mutants; 6) short-term (desensitization, internalization, phosphorylation) and long-term (transcription) regulations and transgenic models; 7) receptor polymorphisms.
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ABSTRACT: Vasoactive intestinal peptide and its G-protein-coupled receptors, VPAC-1 and VPAC-2, are highly expressed in the immune system
and modulate diverse T cell functions. The human VPAC-1 5′-flanking region (1.4 kb) contains four high affinity Ikaros (IK)
consensus sequences. Ikaros native protein from T cell nuclear extracts and IK-1 and IK-2 recombinant proteins recognized
an IK high affinity binding motif in the VPAC-1 promoter in electrophoretic mobility shift assays by a sequence-specific mechanism,
and anti-IK antibodies supershifted this complex. Stable NIH-3T3 clones overexpressing IK-1 or IK-2 isoforms were generated
to investigate Ikaros regulation of endogenous VPAC-1 expression as assessed by quantifying VPAC-1 mRNA and protein. By traditional
and fluorometric-based kinetic reverse transcription-PCR and125I-labeled vasoactive intestinal peptide binding, both IK-1 and IK-2 suppressed endogenous VPAC-1 expression in NIH-3T3 clones
by a range of 50–93%. When a series of nested deletions of the VPAC-1 luciferase reporter construct were transiently transfected
into IK-2 clones there was up to a 41% decrease in transcriptional activity compared with vector control. Two major IK-2 binding
domains also were identified at −1076 to −623 bp and at −222 to −35 bp, respectively. As both Ikaros and its novel target
VPAC-1 are highly expressed in T cells, this system may be a dominant determinant of the VPAC-1 expression in immune responses.
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