Nuclear factor-kappaB regulates estrogen receptor-alpha transcription in the human heart.

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.

Identification and Functional Characterization of Cis-Regulatory Elements Controlling Expression of the Porcine ADRB2 Gene.

The beta-2 adrenergic receptor (beta-2 AR) modulates metabolic processes in skeletal muscle, liver, and adipose tissue in response to catecholamine stimulation. We showed previously that expression of the porcine beta-2 AR gene (ADRB2) is affected by cis-regulatory polymorphisms. These are most likely responsible for the association of ADRB2 with economically relevant muscle-related traits in pigs. The present study focused on characterization of promoter elements involved in basal transcriptional regulation of the porcine ADRB2 in different cell types to aid identification of its cis-regulatory polymorphisms. Based on in silico analysis, luciferase reporter gene assays and gel shift assays were performed using COS-7, HepG2, C2C12, and 3T3-L1 cells. Deletion mapping of the 5´ flanking region (-1324 to +33) of ADRB2 revealed the region between -307 and -269 to be the minimal promoter, including regulatory elements essential for the basal transcriptional activity in all four tested cell types. Directly upstream (-400 to -323) we identified an important enhancer element required for maximal promoter activity. In silico analysis and gel shift assays revealed that this GC-rich element harbors two evolutionarily conserved binding sites of Sp1, a constitutive transcriptional activator. Significant transcriptional activation of the porcine ADRB2 promoter was demonstrated by overexpression of Sp1. Our results demonstrate, for the first time, an important role of Sp1 and of the responsive enhancer element in the regulation of ADRB2 expression. Polymorphisms located in this domain of the porcine ADRB2 promoter represent candidate causal cis-regulatory variants.

A substitution in the ligand binding domain of the porcine glucocorticoid receptor affects activity of the adrenal gland.

Glucocorticoids produced in the adrenal cortex under the control of the hypothalamic-pituitary axis play a vital role in the maintenance of basal and stress-related homeostasis and influence health and well-being. To identify loci affecting regulation of the hypothalamic-pituitary-adrenal (HPA) axis in the pig we performed a genome-wide association study for two parameters of acute and long-term adrenal activity: plasma cortisol level and adrenal weight. We detected a major quantitative trait locus at the position of the glucocorticoid receptor gene (NR3C1) - a key regulator of HPA axis activity. To determine the causal variant(s), we resequenced the coding region of NR3C1 and found three missense single nucleotide polymorphisms (SNPs). SNP c.1829C>T, leading to a p.Ala610Val substitution in the ligand binding domain, showed large (about 0.6× and 1.2× phenotypic standard deviations for cortisol level and adrenal weight, respectively), and highly significant (2.1E-39≤log10(1/p)≤1.7E+0) negative effects on both traits. We were able to replicate the association in three commercial pig populations with different breed origins. We analyzed effects of the p.Ala610Val substitution on glucocorticoid-induced transcriptional activity of porcine glucocorticoid receptor (GR) in vitro and determined that the substitution introduced by SNP c.1829C>T increased sensitivity of GR by about two-fold. Finally, we found that non-coding polymorphisms in linkage disequilibrium with SNP c.1829C>T have only a minor effect on the expression of NR3C1 in tissues related to the HPA axis. Our findings provide compelling evidence that SNP c.1829C>T in porcine NR3C1 is a gain-of-function mutation with a major effect on the activity of the adrenal gland. Pigs carrying this SNP could provide a new animal model to study neurobiological and physiological consequences of genetically based GR hypersensitivity and adrenal hypofunction.

17beta-Estradiol inhibits matrix metalloproteinase-2 transcription via MAP kinase in fibroblasts.

AIMS: Female sex and sex hormones contribute to cardiac remodelling. 17beta-estradiol (E2) is involved in the modulation of extracellular matrix composition and function. Here, we analysed the effect of E2 on matrix metalloproteinase (MMP)-2 gene expression and studied the underlying molecular mechanisms in rat cardiac fibroblasts and in a human fibroblast cell line. METHODS AND RESULTS: In adult rat cardiac fibroblasts, E2 significantly decreased MMP-2 gene expression in an estrogen receptor (ER)-dependent manner. Transient transfection experiments of human MMP-2 (hMMP-2) promoter deletion constructs in a human fibroblast cell line revealed a regulatory region between -324 and -260 bp that is involved in E2/ERalpha-mediated repression of hMMP-2 gene transcription. Electrophoretic mobility shift assays (EMSA) and supershift analysis demonstrated the binding of transcription factor Elk-1 within this promoter region. Elk-1 was phosphorylated by E2 via the mitogen-activated protein kinase (MAPK) signalling pathway as shown by western blotting. Treatment of cells with the MAPK inhibitor PD98059 blocked the E2-dependent repression of hMMP-2 promoter activity as well as the endogenous MMP-2 mRNA levels in both human fibroblast cells and rat cardiac fibroblasts. CONCLUSION: E2 inhibits MMP-2 expression via the ER and the MAPK pathway in rat cardiac fibroblasts and in a human fibroblast cell line. These mechanisms may contribute to sex-specific differences in fibrotic processes that are observed in human heart and other diseases.