Autoregulation of human relaxin-2 gene expression critically involves relaxin and glucocorticoid receptor binding to glucocorticoid response half-sites in the relaxin-2 promoter.
Relaxin peptides act in brain, reproductive and cardiovascular systems, kidneys, and connective tissue through different G protein-coupled receptors. We reported that human relaxin-2 and porcine relaxin are both agonists at the human glucocorticoid receptor (GR). Here, we investigated the possible auto-regulation of relaxin-2 gene expression via recently discovered GR-binding sites in the relaxin-2 promoter. We found that porcine relaxin increased the secretion of human relaxin-like immunoreactivity in HeLa and THP-1 cells. Silencing of GR gene expression completely abolished this effect whereas transfection of wild-type GR into naturally GR-devoid HT-29 cells established relaxin sensitivity. Relaxin was shown to stimulate CAT expression driven by different deletion constructs of the 5'-flanking region of the relaxin-2 promoter. In chromatin immunoprecipitation assays, we detected both GR and relaxin binding to the relaxin-2 promoter. Gel shift assays indicated binding of relaxin-activated GR to half-GREs located between 160 and 200 bp upstream of transcription start but not to the GRE at -900 bp. Relaxin bound to human GR and displaced established GR agonists. Immunofluorescence experiments visualized nuclear co-localization of relaxin and GR in response to relaxin. In conclusion, we have identified a positive auto-regulatory loop of human relaxin-2 expression which involves GR and relaxin/GR binding to half-GREs in the relaxin-2 promoter.
Dschietzig
Thomas
T
Department of Cardiology and Angiology, Charité, University Medicine Berlin (Campus Mitte), Berlin, Germany. thomas.dschietzig@charite.de
Bartsch
Cornelia
C
Wessler
Silja
S
Baumann
Gert
G
Stangl
Karl
K
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D003907:Dexamethasone / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000187:drug effects*; D005938:Glucocorticoids / Q000494:pharmacology; D019073:HT29 Cells; D006367:HeLa Cells; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D012065:Relaxin / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology* / Q000502:physiology*; D013552:Swine
D005938:Glucocorticoids; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D012065:Relaxin