FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2074582794> ?p ?o ?g. }

• W2074582794 endingPage "24714" @default.
• W2074582794 startingPage "24705" @default.
• W2074582794 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 α (hERα) 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 ERα 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-κB binds to this region. An inhibition of NF-κB activity by parthenolide significantly increased the transcriptional activity of the F-promoter. Increasing NF-κB expression by tumor necrosis factor-α reduced the expression of ERα, indicating that the NF-κB pathway inhibits expression of ERα in human cardiomyocytes. Finally, 17β-estradiol induced the transcriptional activity of hERα promoters A, B, C, and F. In conclusion, inflammatory stimuli suppress hERα expression via activation and subsequent binding of NF-κB to the ERα F-promoter, and 17β-estradiol/hERα may antagonize the inhibitory effect of NF-κB. This suggests interplay between estrogen/estrogen receptors and the pro-hypertrophic and inflammatory responses to NF-κB. 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 α (hERα) 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 ERα 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-κB binds to this region. An inhibition of NF-κB activity by parthenolide significantly increased the transcriptional activity of the F-promoter. Increasing NF-κB expression by tumor necrosis factor-α reduced the expression of ERα, indicating that the NF-κB pathway inhibits expression of ERα in human cardiomyocytes. Finally, 17β-estradiol induced the transcriptional activity of hERα promoters A, B, C, and F. In conclusion, inflammatory stimuli suppress hERα expression via activation and subsequent binding of NF-κB to the ERα F-promoter, and 17β-estradiol/hERα may antagonize the inhibitory effect of NF-κB. This suggests interplay between estrogen/estrogen receptors and the pro-hypertrophic and inflammatory responses to NF-κB. Estrogens play an important role in mammal normal physiological functions and also in the pathology of several diseases (1Mendelsohn M.E. Karas R.H. Science. 2005; 308: 1583-1587Crossref PubMed Scopus (846) Google Scholar). One important target organ for estrogen action is the cardiovascular system. Estrogen exerts its effects mainly through its cognate receptors, estrogen receptor α (ERα) 3The abbreviations used are:ERestrogen receptorhERhuman ERTNFtumor necrosis factorE217β-estradiolLVleft ventricle5′-UTR5′-untranslated regionNF-κBnuclear factor-κBSOEsplicing overlap extension5′-RACE5′-rapid amplification of cDNA endsFWforwardRVreverse. and estrogen receptor beta (ERβ), members of the nuclear hormone receptor superfamily of ligand activated transcription factors (2Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (5999) Google Scholar). ERs have been identified in both vascular endothelial and smooth muscle cells of blood vessel walls as well as in cardiac fibroblasts and myocytes, in humans, and rodents (3Karas R.H. Patterson B.L. Mendelsohn M.E. Circulation. 1994; 89: 1943-1950Crossref PubMed Scopus (446) Google Scholar, 4Venkov C.D. Rankin A.B. Vaughan D.E. Circulation. 1996; 94: 727-733Crossref PubMed Scopus (300) Google Scholar, 5Nordmeyer J. Eder S. Mahmoodzadeh S. Martus P. Fielitz J. Bass J. Bethke N. Zurbrügg H.R. Pregla R. Hetzer R. Regitz-Zagrosek V. Circulation. 2004; 110: 3270-3275Crossref PubMed Scopus (101) Google Scholar, 6Grohé C. Kahlert S. Löbbert K. Stimpel M. Karas R.H. Vetter H. Neyses L. FEBS Lett. 1997; 416: 107-112Crossref PubMed Scopus (317) Google Scholar, 7Mahmoodzadeh S. Eder S. Nordmeyer J. Ehler E. Huber O. Martus P. Weiske J. Pregla R. Hetzer R. Regitz-Zagrosek V. FASEB J. 2006; 20: 926-934Crossref PubMed Scopus (106) Google Scholar, 8Ropero A.B. Eghbali M. Minosyan T.Y. Tang G. Toro L. Stefani E. J. Mol. Cell. Cardiol. 2006; 41: 496-510Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). These receptors have been found to mediate the effects of 17β-estradiol (E2) on the cardiovascular system, e.g. rapid vasodilatation, reduction of vessel walls responses to injury, decreasing the development of atherosclerosis, and preventing apoptosis in cardiac myocytes in heart failure (9Mendelsohn M.E. Karas R.H. N. Engl. J. Med. 1999; 340: 1801-1811Crossref PubMed Scopus (2464) Google Scholar, 10Simoncini T. Genazzani A.R. Liao J.K. Circulation. 2002; 105: 1368-1373Crossref PubMed Scopus (106) Google Scholar, 11Kim J.K. Pedram A. Razandi M. Levin E.R. J. Biol. Chem. 2006; 281: 6760-6767Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Our recent studies in patients with aortic stenosis and dilated cardiomyopathy showed that the expression of the ERα gene is regulated in a disease-dependent manner (5Nordmeyer J. Eder S. Mahmoodzadeh S. Martus P. Fielitz J. Bass J. Bethke N. Zurbrügg H.R. Pregla R. Hetzer R. Regitz-Zagrosek V. Circulation. 2004; 110: 3270-3275Crossref PubMed Scopus (101) Google Scholar, 7Mahmoodzadeh S. Eder S. Nordmeyer J. Ehler E. Huber O. Martus P. Weiske J. Pregla R. Hetzer R. Regitz-Zagrosek V. FASEB J. 2006; 20: 926-934Crossref PubMed Scopus (106) Google Scholar). However, the mechanisms involved in the regulation of ERα gene expression in the human myocardium have not been addressed to date. estrogen receptor human ER tumor necrosis factor 17β-estradiol left ventricle 5′-untranslated region nuclear factor-κB splicing overlap extension 5′-rapid amplification of cDNA ends forward reverse. ERα expression has been detected in several tissues with considerably different expression levels among these tissues (12Flouriot G. Griffin C. Kenealy M. Sonntag-Buck V. Gannon F. Mol. Endocrinol. 1998; 12: 1939-1954Crossref PubMed Google Scholar). The transcription of the ERα gene plays an important role in regulating the expression of ERα in a cell- and tissue-specific manner (13Shupnik M.A. Gordon M.S. Chin W.W. Mol. Endocrinol. 1989; 3: 660-665Crossref PubMed Scopus (169) Google Scholar, 14Cho H.S. Ng P.A. Katzenellenbogen B.S. Mol. Endocrinol. 1991; 5: 1323-1330Crossref PubMed Scopus (56) Google Scholar, 15Freyschuss B. Sahlin L. Masironi B. Eriksson H. J. Endocrinol. 1994; 142: 285-298Crossref PubMed Scopus (48) Google Scholar, 16Denger S. Reid G. Brand H. Kos M. Gannon F. Mol. Cell. Endocrinol. 2001; 178: 155-160Crossref PubMed Scopus (43) Google Scholar). The human ERα mRNA is transcribed from at least seven different promoters with unique 5′-untranslated regions (5′-UTRs) (A, B, C, D, E, F, and T) (17Kos M. Reid G. Denger S. Gannon F. Mol. Endocrinol. 2001; 15: 2057-2063Crossref PubMed Scopus (225) Google Scholar, 18Okuda Y. Hirata S. Watanabe N. Shoda T. Kato J. Hoshi K. Endocr. J. 2003; 50: 97-104Crossref PubMed Scopus (20) Google Scholar). All these ERα transcripts initiate at cap sites upstream of exon 1 and utilize a splice acceptor site at nucleotide +163 in the originally identified exon 1 (19Thompson D.A. McPherson L.A. Carmeci C. deConinck E.C. Weigel R.J. J. Steroid Biochem. Mol. Biol. 1997; 62: 143-153Crossref PubMed Scopus (42) Google Scholar). These multiple promoters are utilized in a cell and tissue type-specific manner (20Grandien K. Mol. Cell. Endocrinol. 1996; 116: 207-212Crossref PubMed Scopus (93) Google Scholar). For example the predominant promoter variants utilized for the expression of the ERα gene are A and C promoters in the endometrium, C and F promoters in ovaries, and only F promoter variant in osteoblasts (12Flouriot G. Griffin C. Kenealy M. Sonntag-Buck V. Gannon F. Mol. Endocrinol. 1998; 12: 1939-1954Crossref PubMed Google Scholar, 21Lambertini E. Penolazzi L. Giordano S. Del Senno L. Piva R. Biochem. J. 2003; 372: 831-839Crossref PubMed Scopus (24) Google Scholar). In addition to the differential promoter usage, it appears that there are a variety of cell/tissue-specific factors that interact with these various ERα promoters with trans-activating (AP1, ERBF-1, AP2) or trans-repressing functions, which also affect the regulation of the transcription of the ERα gene in a cell- and tissue- specific manner (22Schuur E.R. McPherson L.A. Yang G.P. Weigel R.J. J. Biol. Chem. 2001; 276: 15519-15526Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 23Tang Z. Treilleux I. Brown M. Mol. Cell. Biol. 1997; 17: 1274-1280Crossref PubMed Scopus (60) Google Scholar, 24Tanimoto K. Eguchi H. Yoshida T. Hajiro-Nakanishi K. Hayashi S. Nucleic Acids Res. 1999; 27: 903-909Crossref PubMed Scopus (41) Google Scholar). Furthermore, it has been shown that E2 differentially regulates the levels of ERα in a cell type- and tissue type-specific manner. Although E2 down-regulates the level of ERα gene expression in MCF7 cells, it leads to an increase of ERα mRNA levels in other cell lines such as FEM-19 and ZR-75 and in tissues such as liver (12Flouriot G. Griffin C. Kenealy M. Sonntag-Buck V. Gannon F. Mol. Endocrinol. 1998; 12: 1939-1954Crossref PubMed Google Scholar, 25Clayton S.J. May F.E. Westley B.R. Mol. Cell. Endocrinol. 1997; 128: 57-68Crossref PubMed Scopus (30) Google Scholar, 26Donaghue C. Westley B.R. May F.E. Mol. Endocrinol. 1999; 13: 1934-1950PubMed Google Scholar). These findings suggested that the differential regulation of ERα gene expression by E2 in part is due to different promoter usage and/or transcription factors present within a cell (12Flouriot G. Griffin C. Kenealy M. Sonntag-Buck V. Gannon F. Mol. Endocrinol. 1998; 12: 1939-1954Crossref PubMed Google Scholar, 26Donaghue C. Westley B.R. May F.E. Mol. Endocrinol. 1999; 13: 1934-1950PubMed Google Scholar). To understand the molecular mechanisms controlling ERα gene expression in the human heart, we first report the characterization of the ERα promoter variants in the human left ventricular (LV) tissue and subsequently examine the molecular mechanism involved in the regulation of the most frequently utilized promoter variant. Finally, we study the effect of E2 and ERα itself on the transcriptional activity of the identified human ERα promoters. Human LV myocardial samples used in this study were composed of tissue samples of non-used donor hearts with originally normal systolic cardiac function, no history of cardiac disease, and normal postmortem histology. However, they did not qualify for transplantation at the time of organ harvesting because of functional reasons. All subjects were Caucasian. The study followed the rules of the Declaration of Helsinki. Total RNA from LV tissue of human hearts was isolated using the guanidinium isothiocyanate based method (RNAzolB, Friendswood) as previously described (5Nordmeyer J. Eder S. Mahmoodzadeh S. Martus P. Fielitz J. Bass J. Bethke N. Zurbrügg H.R. Pregla R. Hetzer R. Regitz-Zagrosek V. Circulation. 2004; 110: 3270-3275Crossref PubMed Scopus (101) Google Scholar). To determine the 5′-UTRs of the ERα transcript in the human myocardium, 5′-rapid amplification of cDNA ends (5′-RACE) was performed using a GenRacerTM kit according to the manufacturer's instructions (Invitrogen). The template for 5′-RACE was total RNA isolated from LV tissue of 5 human hearts (3 females and 2 males; age 55.8 ± 10.8). To increase the specificity and product yield of 5′-RACE, nested PCR was then performed using another internal gene-specific primer and geneRacer-nested primer. First strand synthesis of hERα cDNAs was carried out from isolated total RNA using a gene-specific primer, RV4 oligonucleotide, located in exon 2. Subsequently, for the amplification of cDNAs, we performed, first, hot-start PCR followed by nested PCR using the GenRacerTM 5′-primer and GenRacerTM 5′-nested primer as forward primer and the gene-specific primer RV1, RV2, RV3, and RV4 located in exon 1 or exon 2 of the ERα gene as the reverse primer (for primer sequences see supplemental Table 1). The PCR reactions were carried out under standard conditions. The 5′-RACE PCR products were subcloned into pCR®4-TOPO® vector using a TA cloning kit (Invitrogen) for subsequent DNA sequence analysis. Total RNA isolated from 14 human LV samples (7 females and 7 males; age: 50.9 ± 12) was used as the template for reverse transcriptase-PCR. cDNA was synthesized from 500 ng of total RNA from each sample using a random primer and a high capacity cDNA reverse transcription kit according to standard protocol (Applied Biosystems). PCRs were then carried out according to standard protocol using the following sense and antisense primers specific for each 5′-UTR variant of the hERα gene: A-variant, FW/RV; B-variant, FW/RV; C-variant, FW/RV; D-variant, FW/RV; E-variant, FW/RV; F-variant, FW/RV (for the primer sequences, see supplemental Table 1). The resulting PCR products were analyzed in 1% agarose gels stained with ethidium bromide. Semiquantitative PCR was performed on a cDNA pool generated from the RNA of the same 14 human LV samples using primers specific for 5′-UTR A-, B-, C-, and F-variants according to standard protocols. PCR reactions were stopped after 28, 30, 32, 35, 38, and 40 cycles of amplification. The amplification of human β-actin gene was used as a reference gene for semiquantitative comparison. Equal aliquots of each PCR reaction were electrophoresed on a 1% agarose gel stained with ethidium bromide. Human genomic DNA was prepared from peripheral blood samples from healthy volunteers (n = 3) by using QIAamp DNA blood kit (Qiagen) according to the manufacturer's instructions. To generate the reporter construct containing the 5′-flanking region of the hERα F-variant, the sequence of the 5′-UTR F-variant and a part of coding exon 1 of the hERα gene (from +55 to +359 bp, relative to transcription start site; accession number U68068/AJ002562) (17Kos M. Reid G. Denger S. Gannon F. Mol. Endocrinol. 2001; 15: 2057-2063Crossref PubMed Scopus (225) Google Scholar) was fused to the −1,218/+83-bp fragment of hERα promoter F sequence (from −118,358 to −117,140 bp; upstream of the originally described transcription start site (17Kos M. Reid G. Denger S. Gannon F. Mol. Endocrinol. 2001; 15: 2057-2063Crossref PubMed Scopus (225) Google Scholar)) using a splicing overlap extension method (SOE-PCR). The fragment +55/+359 bp, amplified with primer pairs FW-C1/RV-D1, was generated using human ERα cDNA as template, and the fragment −1218/+83 bp (relative to the transcription start site of F-variant), amplified with primer pairs FW-A1/RV-B1, was generated using human genomic DNA as template (see Fig. 1, also see supplemental Table 1). The primer RV-B1 was the reverse complement to the primer FW-C1. Amplified fragments were cloned into pCR®4-TOPO® and subsequently used as template for SOE-PCR amplification with primer pairs MluI site-linked FW-A1 and XhoI site-linked RV-D1. The resulting SOE-PCR fragment (referred herein and thereafter as full-length fragment F: −1218/+359 bp) was subcloned into a pCR®4-TOPO® vector using a TA cloning kit (Invitrogen). This sequence was then used as a template to prepare a series of deletion ERα F-variant DNA fragments (−910/+359 bp, −457/+359 bp, −910/−487 bp, and −910/−9 bp) by PCR (for primer binding sites see Fig. 1, FW-A6/RV-D1, FW-A5/RV-D1, FW-A6/RV-G4, FW-A6/RV-G1). Additionally, to generate reporter constructs containing the 5′-flanking region of hERα A-, hERα B-, and hERα C-transcript (−1019/+260 bp; −1303/−175 bp; −3215/−1859 bp respectively, relative to the originally identified transcription start site) (17Kos M. Reid G. Denger S. Gannon F. Mol. Endocrinol. 2001; 15: 2057-2063Crossref PubMed Scopus (225) Google Scholar), we performed PCR as described above (for the primer sequence see supplemental Table 1). The resulting sequences referred herein as to promoter variant A-, B-, and C- were then subcloned into the pCR®4-TOPO® vector. All constructs were verified by restriction site digestion and sequence analysis. Thereafter, luciferase reporter constructs were generated by using restriction sites MluI and XhoI; the resulting fragments were gel-purified and subcloned into promoterless pGl2-basic vector (Promega). The resulting luciferase reporter constructs are referred to as: A-promoter-pGL2, B-promoter-pGL2, C-promoter-pGL2, and F-promoter −pGL2. The different F-promoter constructs are as follows: −1218/+359-pGL2, −910/+359-pGL2, −457/+359-pGL2, −910/−487-pGL2, −910/−9-pGL2. QuikChange® site-directed mutagenesis kit (Stratagene) was used for generating mutants of potential transcription factor nuclear factor-κB (NF-κB) binding sites within the hERα F-promoter. The −910/−9-pGL2 reporter construct was used as a wild type construct. PCR oligonucleotide primer pairs used for generating mutants are listed in supplemental Table 1. The mutation was confirmed by sequencing. Deletion constructs are referred to as M1 (−910/−9)-pGL2 and M2 (−910/−9)-pGL2. AC16 cells (human cardiomyocyte cell line) (27Davidson M.M. Nesti C. Palenzuela L. Walker W.F. Hernandez E. Protas L. Hirano M. Isaac N.D. J. Mol. Cell. Cardiol. 2005; 39: 133-147Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) were grown in Dulbecco's modified Eagle's medium/F-12 (InvitrogenTM) supplemented with 12.5% fetal bovine serum (PAA Laboratories), penicillin/streptomycin (100 units/ml, 100 μg/ml; PPA), and amphotericin B (0.25 μg/ml, InvitrogenTM) at 37 °C in 5% CO2. For stimulation experiments with E2 (10−8 mol/liter, Sigma), cells were cultured in phenol red-free Dulbecco's modified Eagle's medium/F-12 supplemented with 2.5% charcoal stripped fetal bovine serum (CS-FBS, Biochrom AG), penicillin/streptomycin (100 units/ml, 100 μg/ml), and amphotericin B (0.25 μg/ml) at 37 °C in 5% CO2 for 48 h. AC16 cells were treated with parthenolide (10 μmol/liter, Biomol) for 6 h and with ICI 182,780 (10−5 mol/liter, Tocris) 30 min before starting the E2 treatment. For stimulation experiments with TNFα, AC16 cells were cultured in normal medium with TNFα (10 ng/ml, R&D system) for 15 and 30 min. For the transient expression analysis of hERα promoter constructs, ∼1.5 × 105 cells/well were plated onto 6-well plates. After 24 h of incubation, promoter-luciferase reporter construct (1 μg) and the internal reference Renilla luciferase reporter plasmid phRL-TK vector (10 ng, Promega) were transfected to each well using FuGENE® 6 reagent according to the manufacturer's recommendations (Roche Diagnostics). For co-transfection experiments, 1 μg of each pSG-hERα66 vector (HEGO-vector, kindly donated by Dr. P. Chambon) or appropriate empty vector was used. After treatments, cell extracts were prepared, and Firefly and Renilla luciferase activities were sequentially measured using the Dual-GloTM-Luciferase assay system (Promega) following the manufacturer's instructions in a multilabel counter Victor3TM (PerkinElmer Life Sciences). Variations in transfection efficiency were normalized to Renilla luciferase activity. All transfections were carried out in triplicate for each construct and performed independently at least three times. Transfection results were averaged and are expressed as the mean ± S.E. Nuclear proteins from cultured (stimulated or non-stimulated) AC16 cells were extracted from cells grown in 100-mm culture plates. The AC16 cell pellets were resuspended in Nonidet P-40 containing saccharose buffer (for all buffers see supplemental Table 2). After centrifugation, the pellet was gently resuspended in a low salt buffer before the same volume of high salt buffer was gradually added in small aliquots to the cells. Afterward, the samples were incubated for 45 min at 4 °C on a rotating wheel. After centrifugation, the supernatant (nuclear proteins) was collected and stored at −80 °C. The protein concentration of nuclear extracts was determined by BCA protein assay kit (Pierce). Five μg of nuclear protein or 50 μg of whole cell extract isolated from AC16 cells was separated by SDS-polyacrylamide gel electrophoresis and electrotransferred onto nitrocellulose membranes. The membranes were immunoblotted overnight with antibodies against anti-NF-κB p50 (1:500; H-119, Santa Cruz) or anti-ERα (1:300, G-20, Sc-544; Santa Cruz) followed by incubation for 1 h with horseradish peroxidase-conjugated donkey anti-rabbit antibody (1:10,000, Dianova). Nuclear-specific protein TFIID (TBP, N-12, Santa Cruz) or anti-glyceraldehyde-3-phosphate dehydrogenase antibody (Chemicon) was used for normalization. Immunoreactive bands were visualized with a chemiluminescent detection kit (ECLTM, GE Healthcare), and the density of protein bands were quantified by Alpha Ease FCTM software (Version 3.1.2, Alpha Innotech Corp.). AC16 cells were grown on eight-chamber culture slides (BD Bioscience) at a density of 30,000 cells/well. The cells were treated with or without NF-κB inhibitor, parthenolide (10 μmol/liter), for 6 h. Cells were fixed with 3% buffered formaldehyde (20 min), permeabilized with 0.1% Triton X-100/phosphate-buffered saline (PBS, 4 min), blocked with 1% bovine serum albumin/PBS (1 h), and then stained overnight with rabbit anti-NF-κB p50 polyclonal antibody (1:100, H-119, Santa Cruz) and mouse anti-ERα monoclonal antibody (1:50, ab2746, Abcam). Subsequently the cells were incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody (1:100, Jackson ImmunoResearch Laboratories) and Cy-3 conjugated goat F(ab′)2 Fragment anti-mouse secondary antibody (1:100, Jackson ImmunoResearch Laboratories) for 1 h. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole for 10 min. Subsequently, slides were mounted with Vectashield mounting medium for fluorescence (H-1000, Vectashield, Vector Laboratories). Confocal images were acquired using a Leica TCS-SPE spectral laser scanning microscope, and images were processed by Leica Application Suite AF software (Version 1.8.0). For electrophoretic mobility shift assays, 5 μg of nuclear extracts were incubated for 1 h at room temperature with 2 μg poly(dI-dC) and 60,000 cpm radiolabeled oligonucleotide (5′-AACCTCATTAATCGGTAACAAGAAGTGCAGAGCGGGCT-3′, containing the putative binding site for NF-κB (Fig. 1), adjusted to 20 μl with a 5× binding buffer (for the buffer, see supplemental Table 2). For competition experiments, unlabeled oligonucleotides were added in a 100-fold molar excess to the reaction mixture before the addition of radiolabeled probe. For supershift assays, increasing amounts of antibody against NF-κB p50 (H-119, Santa Cruz) was added 30 min at 4 °C before the addition of the 32P-labeled probe. Each reaction was loaded on a native 5% polyacrylamide gel and run at 150 V for ∼2 h. After electrophoresis, gels were dried, exposed to imaging plates at −20 °C for up to 1 week, and visualized by autoradiography and quantified using phosphorimaging (GE Healthcare). All graphic representations and statistical analysis were accomplished using SPSS Program for windows (Version 13; SPSS, Inc.). Statistical comparisons between unpaired groups were performed using the Mann-Whitney test. The data are expressed as the means ± S.E. A p value <0.05 was regarded as significant. To identify the alternative 5′-UTR usage in ERα transcripts in the human heart, we performed nested 5′-RACE, as described under “Experimental Procedures.” Sequence analysis of 41 positive clones demonstrated that 85.4% of these clones contained the 5′-UTR F-variant, 12.2% contained the C-variant, and 2.4% contained the B-variant. The existence of these three alternatives 5′-UTRs points to the presence of three alternative promoters of ERα in the human heart. Furthermore, this experiment suggests that the F-variant is the predominant promoter form of the ERα gene in the human myocardium, as a majority of the 5′-RACE clones were initiated by the promoter variant F (herein designated as F-promoter). To confirm the results obtained from 5′-RACE, we measured the relative abundance of the ERα transcripts containing different variants of the 5′-UTR by semiquantitative PCR. As shown in Fig. 2, the F-transcript exhibited the greatest abundance followed by C, B, and A transcripts. Additionally, 5′-UTR-specific PCR revealed that the transcript variants A, B, C, and F were present in all LV samples (data not shown). The 5′-UTR variants D and E were not detected in any tested sample. These findings suggest that the F-promoter is the most frequently utilized promoter in the basal transcription of the ERα gene in the human myocardium. To identify the regulatory elements controlling the expression of the ERα gene in the human heart, the activity of 1.2-kilobase pair F-promoter (full-length) and the deletion F-promoter fragments were investigated by luciferase reporter assay in AC16 cells. The full-length luciferase reporter construct (−1218/+359-pGL2) showed ∼4-fold promoter activity in comparison with the promoterless construct pGl2-basic (Fig. 3). Deletion of the region from −1218 to −911 bp to yield −910/+359-pGL2 decreased the promoter activity. These findings suggest that the region from −1218 to −910 bp contains an enhancer element(s) and/or the region from −910 to +359 bp contains a strong negative cis-acting element(s). To determine the region responsible for lowering the promoter activity, we generated two expression constructs, −910/−487-pGL2 and −457/+359-pGL2. Interestingly, both expression constructs showed a significant increase of luciferase activity, 6- and 12-fold, respectively (Fig. 3). Because the region from −486 to −458 bp is not present in both of these constructs, we therefore speculated that this region and most likely the adjacent sequences (from −490 to −440 bp) contain a negative cis-acting element(s) critical for the basal F-promoter activity in AC16 cells (Fig. 3, hatched column). Computer-assisted analysis (MatInspector 7.4.3./06, TESS (TRANSFAC Version 6.0) and Alibaba2.1) of the sequence from −490 to −440 bp showed several potential transcription factor binding sites, including NF-κB among others (Fig. 1). The functional significance of the NF-κB binding site to the hERα F-promoter was first investigated by site-directed mutagenesis. Mutation within the NF-κB binding sites (M2 (−910/−9)-pGL2) resulted in a significant increase of basal F-promoter activity in AC16 cells (Fig. 4). In contrast, no significant changes in luciferase activity were observed when the second putative NF-κB binding site, located downstream of the identified regulatory region, was mutated (M1 (−910/−9-pGL2)-pGL2). This experiment suggests that the NF-κB binding site located within the region −490 to −440 bp mediates the inhibition of the basal activity of hERα F-promoter. To confirm whether the NF-κB transcription factor binds within the region −490 to −440 bp, we performed electrophoretic mobility shift/supershift assays using nuclear extracts prepared from AC16 cells and synthetic oligonucleotides containing the NF-κB binding site. Three different DNA-protein complexes were formed (Fig. 5). These shifted bands could be competed by 100-fold molar excesses of the unlabeled oligonucleotide (Fig. 5). The addition of antibody against NF-κB p50 resulted in a supershifted band demonstrating the binding of the p50 subunit of the NF-κB transcription factor to its consensus sequence (Fig. 5). Taken together, the transcription factor NF-κB (p50) interacts with the ERα F-promoter. Most likely, NF-κB functions as a suppressor in the transcriptional regulation of the ERα gene in the human heart. In further experiments, we confirmed the inhibitory effect of NF-κB on the expression of ERα gene. The AC16 cells were transiently transfected with the −910/−9-pGL2 expression construct and treated with parthenolide, a well known inhibitor of NF-κB activation (28Hehner S.P. Heinrich M. Bork P.M. Vogt M. Ratter F. Lehmann V. Schulze-Osthoff K. Dröge W. Schmitz M.L. J. Biol. Chem. 1998; 273: 1288-1297Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). Parthenolide blocks the NF-κB activation by stabilizing its inhibitor IκB, resulting in cytoplasmic retention of NF-κB. The incubation of AC16 cells with parthenolide led to a significant increase of hERα F-promoter activity in comparison with vehicle-treated cells (Fig. 6A). We conclude that NF-κB binding reduces the transcriptional activation of the hERα promoter. Furthermore, the amount of NF-κB p50 was significantly decreased in the nuclear extract of AC16 cells treated with parthenolide (Fig. 6B). Thus, the inhibition of translocation of NF-κB into the nucleus leads to an increase of hERα F-promoter activity in AC16 cells. To characterize more extensively the inhibitory role of NF-κB in the regulation of hERα gene, we examined the effects of an inhibition of NF-κB on the h" @default.
• W2074582794 created "2016-06-24" @default.
• W2074582794 creator A5002573739 @default.
• W2074582794 creator A5019742604 @default.
• W2074582794 creator A5031326148 @default.
• W2074582794 creator A5040192098 @default.
• W2074582794 creator A5057574658 @default.
• W2074582794 creator A5058149904 @default.
• W2074582794 creator A5062581990 @default.
• W2074582794 creator A5072077160 @default.
• W2074582794 date "2009-09-01" @default.
• W2074582794 modified "2023-10-05" @default.
• W2074582794 title "Nuclear Factor-κB Regulates Estrogen Receptor-α Transcription in the Human Heart" @default.
• W2074582794 cites W1520650580 @default.
• W2074582794 cites W187180120 @default.
• W2074582794 cites W1964808756 @default.
• W2074582794 cites W1966323574 @default.
• W2074582794 cites W1969882888 @default.
• W2074582794 cites W1970479761 @default.
• W2074582794 cites W1975751793 @default.
• W2074582794 cites W1977909041 @default.
• W2074582794 cites W1978847352 @default.
• W2074582794 cites W2002809517 @default.
• W2074582794 cites W2006862162 @default.
• W2074582794 cites W2007705378 @default.
• W2074582794 cites W2008914962 @default.
• W2074582794 cites W2009816222 @default.
• W2074582794 cites W2014252953 @default.
• W2074582794 cites W2032006984 @default.
• W2074582794 cites W2033436442 @default.
• W2074582794 cites W2034008568 @default.
• W2074582794 cites W2040995356 @default.
• W2074582794 cites W2046335503 @default.
• W2074582794 cites W2049158573 @default.
• W2074582794 cites W2050312701 @default.
• W2074582794 cites W2050903498 @default.
• W2074582794 cites W2051135818 @default.
• W2074582794 cites W2052490065 @default.
• W2074582794 cites W2054465223 @default.
• W2074582794 cites W2056034929 @default.
• W2074582794 cites W2057324918 @default.
• W2074582794 cites W2059477274 @default.
• W2074582794 cites W2064904470 @default.
• W2074582794 cites W2077038454 @default.
• W2074582794 cites W2086908859 @default.
• W2074582794 cites W2098881788 @default.
• W2074582794 cites W2101566761 @default.
• W2074582794 cites W2102691726 @default.
• W2074582794 cites W2108452673 @default.
• W2074582794 cites W2114299563 @default.
• W2074582794 cites W2120065100 @default.
• W2074582794 cites W2121514085 @default.
• W2074582794 cites W2122530165 @default.
• W2074582794 cites W2128291744 @default.
• W2074582794 cites W2131577319 @default.
• W2074582794 cites W2134043187 @default.
• W2074582794 cites W2139204764 @default.
• W2074582794 cites W2154080287 @default.
• W2074582794 cites W2155598737 @default.
• W2074582794 cites W2172256092 @default.
• W2074582794 cites W2615633554 @default.
• W2074582794 cites W4232678241 @default.
• W2074582794 doi "https://doi.org/10.1074/jbc.m109.000463" @default.
• W2074582794 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2757174" @default.
• W2074582794 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19584059" @default.
• W2074582794 hasPublicationYear "2009" @default.
• W2074582794 type Work @default.
• W2074582794 sameAs 2074582794 @default.
• W2074582794 citedByCount "26" @default.
• W2074582794 countsByYear W20745827942012 @default.
• W2074582794 countsByYear W20745827942013 @default.
• W2074582794 countsByYear W20745827942014 @default.
• W2074582794 countsByYear W20745827942015 @default.
• W2074582794 countsByYear W20745827942016 @default.
• W2074582794 countsByYear W20745827942017 @default.
• W2074582794 countsByYear W20745827942018 @default.
• W2074582794 countsByYear W20745827942019 @default.
• W2074582794 countsByYear W20745827942020 @default.
• W2074582794 countsByYear W20745827942021 @default.
• W2074582794 countsByYear W20745827942022 @default.
• W2074582794 countsByYear W20745827942023 @default.
• W2074582794 crossrefType "journal-article" @default.
• W2074582794 hasAuthorship W2074582794A5002573739 @default.
• W2074582794 hasAuthorship W2074582794A5019742604 @default.
• W2074582794 hasAuthorship W2074582794A5031326148 @default.
• W2074582794 hasAuthorship W2074582794A5040192098 @default.
• W2074582794 hasAuthorship W2074582794A5057574658 @default.
• W2074582794 hasAuthorship W2074582794A5058149904 @default.
• W2074582794 hasAuthorship W2074582794A5062581990 @default.
• W2074582794 hasAuthorship W2074582794A5072077160 @default.
• W2074582794 hasBestOaLocation W20745827941 @default.
• W2074582794 hasConcept C104317684 @default.
• W2074582794 hasConcept C121608353 @default.
• W2074582794 hasConcept C126322002 @default.
• W2074582794 hasConcept C134018914 @default.
• W2074582794 hasConcept C170493617 @default.
• W2074582794 hasConcept C172313692 @default.
• W2074582794 hasConcept C185592680 @default.

• next