Estradiol down regulates expression of vasoactive intestinal polypeptide receptor type-1 in breast cancer cell lines
ABSTRACT 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.
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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 and (125)I-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.Journal of Biological Chemistry 05/2002; 277(16):13488-93. DOI:10.1074/jbc.M107922200 · 4.57 Impact Factor
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ABSTRACT: The role of steroid hormones in regulating vaginal smooth muscle contractility was investigated. Rabbits were kept intact or ovariectomized. After 2 weeks, animals were continuously infused with vehicle or supraphysiological levels of testosterone (100 microg/day), or estradiol (200 microg/day), for an additional 2 weeks. The distal vaginal tissue was used to assess contractility in organ baths and changes in tissue structure were assessed by histology. Ovariectomized animals infused with vehicle exhibited significant atrophy of the muscularis and decreased epithelial height, resulting in thinning of the vaginal wall. Estradiol infusion increased epithelial height, comparable to that of intact animals, but only partially restored the muscularis layer. In contrast, testosterone infusion completely restored the muscularis layer, but only partially restored the epithelial height. In vaginal tissue strips contracted with norepinephrine and treated with bretylium, electrical field stimulation (EFS) caused frequency-dependent relaxation that was slightly attenuated with vehicle, significantly inhibited with estradiol and significantly enhanced with testosterone. VIP-induced relaxation was slightly attenuated in tissues from vehicle and estradiol-infused groups, but was enhanced in tissues from testosterone-infused animals. Contraction elicited by EFS or exogenous norepinephrine was not significantly altered with ovariectomy or steroid hormone infusion when data were normalized to potassium contraction. However, the tissue from testosterone-infused animals developed significantly greater contractile force to norepinephrine. These observations suggest that steroid hormones may be important regulators of vaginal tissue structure and contractility.International Journal of Impotence Research 03/2004; 16(1):43-50. DOI:10.1038/sj.ijir.3901138 · 1.76 Impact Factor