Molecular interactions between glucocorticoid and catecholamine signaling pathways  Yi-Tang Tseng, PhD, Rajan Wadhawan, MD, Joan P. Stabila, BS, Bethany G. McGonnigal, BS, James F. Padbury, MD  Journal of Allergy and Clinical Immunology  Volume 110, Issue 6, Pages S247-S254 (December 2002) DOI: 10.1067/mai.2002.129946 Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 1 Schematic of ovine β1AR 5′ flanking region. Regulatory elements including GRE half-sites (white squares) , functional inverted cyclic adenosine monophosphate response element (black triangle) , and activator protein-1 site (white circle) are indicated relative to translation start site. The consensus GRE half-site (TGTTCT at −1244 from translation start site, in bold ) and an overlapping consensus Myc/Max-binding site (CACGTG, underlined) are also indicated (black square) . ( From Tseng YT, Stabila J, Nguyen TT, et al. A novel glucocorticoid regulatory unit mediates the hormone responsiveness of the β1-adrenergic receptor gene. Mol Cell Endocrinol 2001;181:165-78. With permission. ) Journal of Allergy and Clinical Immunology 2002 110, S247-S254DOI: (10.1067/mai.2002.129946) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 2 Detailed sequence identified by deoxyribonuclease I footprinting assay [under bracket] and downstream sequence from the GRU. HD element containing TAATTA within the footprint region is indicated by arrows . Consensus Myc/Max-binding site (E-box) is underlined; overlapping consensus GRE half-site is shown in bold. ( From Tseng YT, Stabila J, Nguyen TT, McGonnigal BG, Waschek JA, Padbury JF. A novel glucocorticoid regulatory unit mediates the hormone responsiveness of the β1-adrenergic receptor gene. Mol Cell Endocrinol 2001;181:165-78. With permission. ) Journal of Allergy and Clinical Immunology 2002 110, S247-S254DOI: (10.1067/mai.2002.129946) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 3 GRU confers glucocorticoid responsiveness in primary cardiomyocytes. Transient transfection of the GRU and 3 mutation constructs subcloned into the pGL2C vector was performed in 3-day-old rat primary cardiomyocytes as described by Tseng.19 Luciferase activities (in relative light units, RLU ) are normalized to the cotransfected RSV-CAT activity and are expressed as mean ± SEM from 4 independent experiments. Data are presented as x -fold increase of each dexamethasone-treated construct relative to its individual control condition. Mut-A, Mutation of HD region with E-box sequence intact; Mut-B, mutation of both E-box and GRE half-site; Mut-C, mutation of entire GRU element; *P < .01. ( From Tseng YT, Stabila J, Nguyen TT, et al. A novel glucocorticoid regulatory unit mediates the hormone responsiveness of the β1-adrenergic receptor gene. Mol Cell Endocrinol 2001;181:165-78. With permission. ) Journal of Allergy and Clinical Immunology 2002 110, S247-S254DOI: (10.1067/mai.2002.129946) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 4 GR as part of the GRU-protein interaction complex. EMSA of the labeled GRU and TAT GRE to SK-N-MC cell nuclear extracts and partially purified GR were performed as described by Tseng.19 Cold competitors (100-fold molar excess) were included as indicated (lane 3) . Arrows indicate two major complexes formed. Arrowheads indicate supershifts by anti-GR150-175 antibody (lanes 4, 6). Lane 1 indicates labeled GRU in the absence of nuclear protein. ( From Tseng YT, Stabila J, Nguyen TT, et al. A novel glucocorticoid regulatory unit mediates the hormone responsiveness of the β1-adrenergic receptor gene. Mol Cell Endocrinol 2001;181:165-78. With permission. ) Journal of Allergy and Clinical Immunology 2002 110, S247-S254DOI: (10.1067/mai.2002.129946) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 5 Sequences of the GRU are critical for DNA-protein interaction. EMSA of labeled GRU and Mut-C to SK-N-MC cell nuclear extracts and partially purified human GR. Mut-C mutant has both HD region and E-box/GRE half-site replaced with nonsense sequence. Arrows indicate two major complexes formed. Lanes 1 and 5 indicate individual probe in the absence of nuclear protein. ( From Tseng YT, Stabila J, Nguyen TT, et al. A novel glucocorticoid regulatory unit mediates the hormone responsiveness of the β1-adrenergic receptor gene. Mol Cell Endocrinol 2001;181:165-78. With permission. ) Journal of Allergy and Clinical Immunology 2002 110, S247-S254DOI: (10.1067/mai.2002.129946) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 6 A, EMSA in SK-N-MC cell nuclear extracts, with labeled GRU and shorter probes representing each element or combination of two elements in the GRU. Arrows indicate two major complexes formed by the GRU. Lane 1 indicates labeled GRU in the absence of nuclear protein. B, Competition of labeled GRU binding to SK-N-MC cell nuclear extracts by 200-fold molar excess of cold GRU and individual elements or combined elements as indicated. Lane 1 indicates binding in the absence of competitor. EB , E-box. ( From Tseng YT, Stabila J, Nguyen TT, McGonnigal BG, Waschek JA, Padbury JF. A novel glucocorticoid regulatory unit mediates the hormone responsiveness of the β1-adrenergic receptor gene. Mol Cell Endocrinol 2001;181:165-78. With permission. ) Journal of Allergy and Clinical Immunology 2002 110, S247-S254DOI: (10.1067/mai.2002.129946) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 7 Competition of GRU binding to SK-N-MC cell nuclear extracts by cold GRU (lane 3), consensus Myc/Max-binding oligonucleotide (lane 4), or mutated Myc/Max-binding oligonucleotide (lane 5). Lane 1 indicates labeled GRU in the absence of nuclear protein; lane 2 shows binding without competitors. Arrows indicate two major complexes formed by the GRU. Right panel shows competition of labeled consensus Myc/Max-binding oligonucleotide binding to SK-N-MC cell nuclear extracts by 100-fold molar excess of the cold probe (lane 9), the GRU (lane 8), or a mutated (CACGTG to CACGGA) Myc/Max-binding oligonucleotide (lane 10). Lane 6 indicates labeled Myc/Max-binding oligonucleotide in the absence of nuclear protein; lane 7 shows binding without competitors. (From Tseng YT, Stabila J, Nguyen TT, et al. A novel glucocorticoid regulatory unit mediates the hormone responsiveness of the β1-adrenergic receptor gene. Mol Cell Endocrinol 2001;181:165-78. With permission.) Journal of Allergy and Clinical Immunology 2002 110, S247-S254DOI: (10.1067/mai.2002.129946) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 8 Glucocorticoid responsiveness of the β1AR is greatly reduced by cotransfection of a plasmid expressing c-myc gene in the antisense orientation. In 3-day-old rat primary cardiomyocytes, a 2.3-kb full-length β1AR promoter construct subcloned into the pGL2B vector was transiently cotransfected with a c-myc antisense-expressing vector (0.15 μg) or with an empty vector (pcDNA3). Cardiomyocytes were treated with ethanol (control) or 100 nmol/L dexamethasone for 24 hours. Luciferase activities (in relative light units, RLU ) are normalized to cotransfected RSV-CAT activity (cpm) and are expressed as mean ± SEM from 4 independent experiments. Data are presented as percentage activity of control. (From Tseng YT, Stabila J, Nguyen TT, et al. A novel glucocorticoid regulatory unit mediates the hormone responsiveness of the β1-adrenergic receptor gene. Mol Cell Endocrinol 2001;181:165-78. With permission.) Journal of Allergy and Clinical Immunology 2002 110, S247-S254DOI: (10.1067/mai.2002.129946) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 9 Developmental expression of proteins interacting with the GRU. EMSA was performed with labeled GRU to nuclear extracts prepared from 18-day-old rat embryonic (E18), 3-day postnatal (P3), and adult (AD) hearts. Two major complexes formed in E18/P3 are indicated by arrows . ( From Tseng YT, Stabila J, Nguyen TT, et al. A novel glucocorticoid regulatory unit mediates the hormone responsiveness of the β1-adrenergic receptor gene. Mol Cell Endocrinol 2001;181:165-78. With permission. ) Journal of Allergy and Clinical Immunology 2002 110, S247-S254DOI: (10.1067/mai.2002.129946) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 10 Model of a novel GRU mediating glucocorticoid responsiveness of the β1AR gene. GRU is composed of 3 elements: liganded GR, which is required for hormone responsiveness; an HD region, which may interact with an unknown GRU accessory protein (GRUAP) ; and an E-box/GRE half-site, which is critical for DNA-protein interaction and forms complexes with Myc/Max family proteins. Two-headed arrow indicates that binding of both Myc/Max and GRUAP to GRU is required. ( From Tseng YT, Stabila J, Nguyen TT, et al. A novel glucocorticoid regulatory unit mediates the hormone responsiveness of the β1-adrenergic receptor gene. Mol Cell Endocrinol 2001;181:165-78. With permission. ) Journal of Allergy and Clinical Immunology 2002 110, S247-S254DOI: (10.1067/mai.2002.129946) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 11 Run-on assays were performed on nuclei isolated from hearts of 19-day-old embryonic (E19) and 15-day postnatal (P15) rats. Nuclei were incubated with 32P-radiolabeled uridine triphosphate at room temperature for 30 minutes. Labeled mRNA were then hybridized to nylon membrane with cDNA of interest. cDNA immobilized onto membranes included β1AR, c-myc, β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and α-myosin heavy chain (α-MHC). Membranes were washed after hybridization for 72 hours, and radioactivity was quantified by phosphorimaging. ( Data from Wadhawan R, Tseng YT, Stabila J, McGonnigal B, Sarkar S, Rubin L, et al. The Role of c-myc in regulation of β1AR transcription rate during the developmental transition in cardiac growth. Pediatr Res 2002;51:32A. ) Journal of Allergy and Clinical Immunology 2002 110, S247-S254DOI: (10.1067/mai.2002.129946) Copyright © 2002 Mosby, Inc. Terms and Conditions