ABSTRACT
AIMS: 17ß-Oestradiol (E2) and its receptors (ERα and ERß) are important regulators of physiological and pathological processes in the cardiovascular system. ER act in concert with other regulatory factors mediating oestrogenic effects. However, the underlying mechanisms modulating ER transcriptional activity are not fully elucidated. To gain better understanding of E2-induced ERα action in the human heart, we aimed to identify and functionally analyse interaction partners of ERα. METHODS AND RESULTS: Using yeast two-hybrid assays with a human heart cDNA library, we identified atrial natriuretic peptide precursor A (NPPA), a well-known cardiac hypertrophy marker, as a novel ERα interaction partner interacting in an E2-dependent manner. Mutation analyses and immunofluorescence data indicated that the LXXLL motif within NPPA is necessary for its E2-induced interaction with ERα, its action as a co-repressor of ERα, and its translocation into the nucleus of human and rat cardiomyocytes. Expression analysis and chromatin immunoprecipitation assays in a human left ventricular cardiomyocyte cell line, AC16, showed that NPPA interacts with E2/ERα, suppressing the transcriptional activity of ERα on E2-target genes, such as NPPA, connexin43, αactinin-2, nuclear factor of activated T-cells, and collagens I and III. CONCLUSION: We characterize for the first time an E2-regulated interaction of NPPA with ERα in cardiomyocytes, that may be crucial in physiological and/or pathological cardiac processes, thereby representing a potential therapeutic target.
Subject(s)
Atrial Natriuretic Factor/metabolism , Estradiol/pharmacology , Estrogen Receptor alpha/agonists , Myocytes, Cardiac/drug effects , Active Transport, Cell Nucleus , Animals , Animals, Newborn , Atrial Natriuretic Factor/genetics , Cell Line , Chromatin Immunoprecipitation , Estrogen Receptor alpha/deficiency , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Female , Fluorescent Antibody Technique , Gene Expression Regulation , Humans , Mice , Mice, Knockout , Mutagenesis, Site-Directed , Mutation , Myocytes, Cardiac/metabolism , Natriuretic Peptide, C-Type/genetics , Natriuretic Peptide, C-Type/metabolism , Promoter Regions, Genetic , Protein Binding , Protein Interaction Domains and Motifs , Protein Interaction Mapping , Protein Precursors/genetics , Protein Precursors/metabolism , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Transcription, Genetic , Transfection , Two-Hybrid System TechniquesABSTRACT
Estrogen receptor (ER)-mediated effects have been associated with the modulation of myocardial hypertrophy in animal models and in humans, but the regulation of ER expression in the human heart has not yet been analyzed. In various cell lines and tissues, multiple human estrogen receptor alpha (hERalpha) mRNA isoforms are transcribed from distinct promoters and differ in their 5'-untranslated regions. Using PCR-based strategies, we show that in the human heart the ERalpha mRNA is transcribed from multiple promoters, namely, A, B, C, and F, of which the F-promoter is most frequently used variant. Transient transfection reporter assays in a human cardiac myocyte cell line (AC16) with F-promoter deletion constructs demonstrated a negative regulatory region within this promoter. Site-directed mutagenesis and electrophoretic mobility shift assays indicated that NF-kappaB binds to this region. An inhibition of NF-kappaB activity by parthenolide significantly increased the transcriptional activity of the F-promoter. Increasing NF-kappaB expression by tumor necrosis factor-alpha reduced the expression of ERalpha, indicating that the NF-kappaB pathway inhibits expression of ERalpha in human cardiomyocytes. Finally, 17beta-estradiol induced the transcriptional activity of hERalpha promoters A, B, C, and F. In conclusion, inflammatory stimuli suppress hERalpha expression via activation and subsequent binding of NF-kappaB to the ERalpha F-promoter, and 17beta-estradiol/hERalpha may antagonize the inhibitory effect of NF-kappaB. This suggests interplay between estrogen/estrogen receptors and the pro-hypertrophic and inflammatory responses to NF-kappaB.
