Abstract
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.
Highlights
Estrogen receptor (ER)␣ expression has been detected in several tissues with considerably different expression levels among these tissues [12]
ER␣ Gene Is Regulated by the F-promoter Variant in the Human Heart—To identify the alternative 5Ј-untranslated regions (5Ј-UTRs) usage in ER␣ transcripts in the human heart, we performed nested 5Ј-rapid amplification of cDNA ends (5Ј-RACE), as described under “Experimental Procedures.”
The existence of these three alternatives 5Ј-UTRs points to the presence of three alternative promoters of ER␣ in the human heart. This experiment suggests that the F-variant is the predominant promoter form of the ER␣ gene in the human myocardium, as a majority of the 5Ј-RACE clones were initiated by the promoter variant F
Summary
ER␣ expression has been detected in several tissues with considerably different expression levels among these tissues [12]. To identify the regulatory elements controlling the expression of the ER␣ gene in the human heart, the activity of 1.2kilobase pair F-promoter (full-length) and the deletion F-promoter fragments were investigated by luciferase reporter assay in AC16 cells.
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