Analysis of estrogen receptor alpha-Sp1 interactions in breast cancer cells by fluorescence resonance energy transfer.

Estrogen-dependent regulation of several genes associated with cell cycle progression, proliferation, and nucleotide metabolism in breast cancer cells is associated with interactions of estrogen receptor (ER)alpha/Sp1 with GC-rich promoter elements. This study investigates ligand-dependent interactions of ERalpha and Sp1 in MCF-7 breast cancer cells using fluorescence resonance energy transfer (FRET). Chimeric ERalpha and Sp1 proteins fused to cyan fluorescent protein or yellow fluorescent protein were transfected into MCF-7 cells, and a FRET signal was induced after treatment with 17beta-estradiol, 4'-hydroxytamoxifen, or ICI 182,780. Induction of FRET by these ERalpha agonists/antagonists was paralleled by their activation of gene expression in cells transfected with a construct (pSp1(3)) containing three tandem Sp1 binding sites linked to a luciferase reporter gene. In contrast, interactions between ERalpha and Sp1DeltaDBD [a DNA binding domain (DBD) deletion mutant of Sp1] are not observed, and this is consistent with the critical role of the C-terminal DBD of Sp1 for interaction with ERalpha. Results of the FRET assay are consistent with in vitro studies on ERalpha/Sp1 interactions and transactivation, and confirm that ERalpha and Sp1 interact in living breast cancer cells.

[1]  A. Nardulli,et al.  Estrogen receptor α and Sp1 regulate progesterone receptor gene expression , 2003, Molecular and Cellular Endocrinology.

[2]  P. Webb,et al.  Tamoxifen activation of the estrogen receptor/AP-1 pathway: potential origin for the cell-specific estrogen-like effects of antiestrogens. , 1995, Molecular endocrinology.

[3]  S. Safe,et al.  Transcriptional activation of rat creatine kinase B by 17β‐estradiol in MCF‐7 cells involves an estrogen responsive element and GC‐rich sites , 2001, Journal of cellular biochemistry.

[4]  J. Olefsky,et al.  Nuclear Receptor Minireview Series* , 2001, The Journal of Biological Chemistry.

[5]  R. Pepperkok,et al.  Spectral imaging and linear un‐mixing enables improved FRET efficiency with a novel GFP2–YFP FRET pair , 2002, FEBS letters.

[6]  Brad Saville,et al.  Estrogen regulation of vascular endothelial growth factor gene expression in ZR-75 breast cancer cells through interaction of estrogen receptor α and SP proteins , 2004, Oncogene.

[7]  B. Katzenellenbogen,et al.  The estrogen receptor enhances AP-1 activity by two distinct mechanisms with different requirements for receptor transactivation functions. , 1999, Molecular endocrinology.

[8]  Horst Wallrabe,et al.  Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy. , 2003, Methods.

[9]  John Calvin Reed,et al.  Mechanisms of Transcriptional Activation of bcl-2Gene Expression by 17β-Estradiol in Breast Cancer Cells* , 1999, The Journal of Biological Chemistry.

[10]  J. Ellenberg,et al.  Cyclic, proteasome-mediated turnover of unliganded and liganded ERalpha on responsive promoters is an integral feature of estrogen signaling. , 2003, Molecular cell.

[11]  Carolyn L. Smith,et al.  Subnuclear Trafficking of Estrogen Receptor-α and Steroid Receptor Coactivator-1 , 2000 .

[12]  K. Horwitz,et al.  Progesterone Regulates Transcription of the p21 WAF1 Cyclindependent Kinase Inhibitor Gene through Sp1 and CBP/p300* , 1998, The Journal of Biological Chemistry.

[13]  V. Giguère,et al.  Isoform-selective interactions between estrogen receptors and steroid receptor coactivators promoted by estradiol and ErbB-2 signaling in living cells. , 2003, Molecular endocrinology.

[14]  E. Simpson,et al.  Molecular mechanism for cooperation between Sp1 and steroidogenic factor-1 (SF-1) to regulate bovine CYP11A gene expression , 1999, Molecular and Cellular Endocrinology.

[15]  Hong Wang,et al.  Yeast Two-hybrid System Demonstrates That Estrogen Receptor Dimerization Is Ligand-dependent in Vivo(*) , 1995, The Journal of Biological Chemistry.

[16]  S. Safe,et al.  Estrogen Induces Adenosine Deaminase Gene Expression in MCF-7 Human Breast Cancer Cells: Role of Estrogen Receptor-Sp1 Interactions* *This work was supported by the National Institutes of Health (Grant CA-76636), the Welch Foundation, and the Texas Agricultural Experiment Station. , 1999, Endocrinology.

[17]  Lucia Altucci,et al.  Nuclear receptors in cell life and death , 2001, Trends in Endocrinology & Metabolism.

[18]  T. Hunter,et al.  Dimerization of Receptor Protein-Tyrosine Phosphatase alpha in living cells , 2001, BMC Cell Biology.

[19]  T. Sugawara,et al.  Sp1 and SF-1 interact and cooperate in the regulation of human steroidogenic acute regulatory protein gene expression. , 2000, Endocrinology.

[20]  R. Heyman,et al.  Effect of the retinoid X receptor-selective ligand LGD1069 on mammary carcinoma after tamoxifen failure. , 1999, Journal of the National Cancer Institute.

[21]  K. Korach,et al.  The Multifaceted Mechanisms of Estradiol and Estrogen Receptor Signaling* , 2001, The Journal of Biological Chemistry.

[22]  Richard N. Day,et al.  Fluorescence resonance energy transfer microscopy of localized protein interactions in the living cell nucleus. , 2001, Methods.

[23]  A. Sugawara,et al.  Transcription Suppression of Thromboxane Receptor Gene by Peroxisome Proliferator-activated Receptor-γ via an Interaction with Sp1 in Vascular Smooth Muscle Cells* , 2002, The Journal of Biological Chemistry.

[24]  M. Husmann,et al.  Nuclear receptors modulate the interaction of Sp1 and GC-rich DNA via ternary complex formation. , 2000, The Biochemical journal.

[25]  Eva Enmark,et al.  Ligand-, Cell-, and Estrogen Receptor Subtype (α/β)-dependent Activation at GC-rich (Sp1) Promoter Elements* , 2000, The Journal of Biological Chemistry.

[26]  J. Adelman,et al.  Determinants contributing to estrogen-regulated expression of SK3. , 2003, Biochemical and biophysical research communications.

[27]  J. Gustafsson,et al.  Biological Role of Estrogen and Estrogen Receptors , 2002, Critical reviews in biochemistry and molecular biology.

[28]  E. Wilson,et al.  Androgen suppression of GnRH-stimulated rat LHbeta gene transcription occurs through Sp1 sites in the distal GnRH-responsive promoter region. , 2001, Molecular endocrinology.

[29]  Jingwen Liu,et al.  Requirement of Sp1 and Estrogen Receptor α Interaction in 17β-Estradiol-Mediated Transcriptional Activation of the Low Density Lipoprotein Receptor Gene Expression. , 2001, Endocrinology.

[30]  P. Silver,et al.  Mapping interactions between nuclear transport factors in living cells reveals pathways through the nuclear pore complex. , 2000, Molecular cell.

[31]  S. Safe,et al.  Transcriptional Activation of Thymidylate Synthase by 17β-Estradiol in MCF-7 Human Breast Cancer Cells. , 2000, Endocrinology.

[32]  P. Chambon,et al.  The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer , 1988, Cell.

[33]  Heike Brand,et al.  Estrogen Receptor-α Directs Ordered, Cyclical, and Combinatorial Recruitment of Cofactors on a Natural Target Promoter , 2003, Cell.

[34]  S. Safe,et al.  Transcriptional activation of E2F1 gene expression by 17beta-estradiol in MCF-7 cells is regulated by NF-Y-Sp1/estrogen receptor interactions. , 1999, Molecular endocrinology.

[35]  M. Tsai,et al.  COUP-TF Upregulates NGFI-A Gene Expression through an Sp1 Binding Site , 1999, Molecular and Cellular Biology.

[36]  P. Devreotes,et al.  Receptor-Mediated Activation of Heterotrimeric G-Proteins in Living Cells , 2001, Science.

[37]  B. Katzenellenbogen,et al.  Selective recognition of distinct classes of coactivators by a ligand-inducible activation domain. , 2004, Molecular cell.

[38]  B. Carlsson,et al.  Equilibrium hormone binding to human estrogen receptors in highly diluted cell extracts is non-cooperative and has a K d of approximately 10 pM , 1994, The Journal of Steroid Biochemistry and Molecular Biology.

[39]  R. Tsien,et al.  Ligand-dependent interactions of coactivators steroid receptor coactivator-1 and peroxisome proliferator-activated receptor binding protein with nuclear hormone receptors can be imaged in live cells and are required for transcription. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[40]  S. Safe,et al.  Functional synergy between the transcription factor Sp1 and the estrogen receptor. , 1997, Molecular endocrinology.

[41]  S. Safe,et al.  Cell context-dependent differences in the induction of E2F-1 gene expression by 17 beta-estradiol in MCF-7 and ZR-75 cells. , 2003, Endocrinology.

[42]  H. Imataka,et al.  Trans-activation functions of the Sp-related nuclear factor, basic transcription element-binding protein, and progesterone receptor in endometrial epithelial cells. , 1999, Endocrinology.

[43]  R. Tsien,et al.  Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin , 1997, Nature.

[44]  B. O’Malley,et al.  FRAP reveals that mobility of oestrogen receptor-α is ligand- and proteasome-dependent , 2000, Nature Cell Biology.

[45]  S. Kojima,et al.  Transactivation via RAR / RXR-Sp 1 Interaction : Characterization of Binding Between Sp 1 and GC Box Motif , 2001 .

[46]  S. Safe,et al.  Transcriptional Activation of Deoxyribonucleic Acid Polymerase α Gene Expression in MCF-7 Cells by 17β-Estradiol. , 2001, Endocrinology.

[47]  Horst Wallrabe,et al.  Confocal FRET microscopy to measure clustering of ligand-receptor complexes in endocytic membranes. , 2003, Biophysical journal.

[48]  S. Safe,et al.  Nuclear receptor-mediated transactivation through interaction with Sp proteins. , 2004, Progress in nucleic acid research and molecular biology.

[49]  S. Safe,et al.  Estrogen Regulation of Cyclin D1 Gene Expression in ZR-75 Breast Cancer Cells Involves Multiple Enhancer Elements* , 2001, The Journal of Biological Chemistry.

[50]  G. Jenster,et al.  Androgen induction of cyclin-dependent kinase inhibitor p21 gene: role of androgen receptor and transcription factor Sp1 complex. , 2000, Molecular endocrinology.

[51]  S. Safe,et al.  Estrogen-induced retinoic acid receptor alpha 1 gene expression: role of estrogen receptor-Sp1 complex. , 1998, Molecular endocrinology.

[52]  K. Umesono,et al.  The nuclear receptor superfamily: The second decade , 1995, Cell.

[53]  Brad Saville,et al.  Domains of Estrogen Receptor α (ERα) Required for ERα/Sp1-Mediated Activation of GC-Rich Promoters by Estrogens and Antiestrogens in Breast Cancer Cells , 2003 .

[54]  W. Hung,et al.  Vitamin D3 receptor/Sp1 complex is required for the induction of p27Kip1 expression by vitamin D3 , 2004, Oncogene.

[55]  J. Katzenellenbogen,et al.  Estrogen receptor dimerization: ligand binding regulates dimer affinity and dimer dissociation rate. , 2002, Molecular endocrinology.

[56]  J. Baxter,et al.  Ligand-selective interactions of ER detected in living cells by fluorescence resonance energy transfer. , 2002, Molecular endocrinology.

[57]  Hiroshi Yamamoto,et al.  The Receptor for Advanced Glycation End Products Is Induced by the Glycation Products Themselves and Tumor Necrosis Factor-α through Nuclear Factor-κB, and by 17β-Estradiol through Sp-1 in Human Vascular Endothelial Cells* , 2000, The Journal of Biological Chemistry.

[58]  S. Kojima,et al.  HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Physical Interaction Between Retinoic Acid Receptor and Sp1: Mechanism for Induction of Urokinase by Retinoic Acid , 2016 .

[59]  D. Aunis,et al.  COUP-TF and Sp1 Interact and Cooperate in the Transcriptional Activation of the Human Immunodeficiency Virus Type 1 Long Terminal Repeat in Human Microglial Cells* , 1997, The Journal of Biological Chemistry.

[60]  B. Herman,et al.  Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. , 1998, Biophysical journal.

[61]  S. Safe,et al.  Estrogen receptor/Sp1 complexes are required for induction of cad gene expression by 17beta-estradiol in breast cancer cells. , 2003, Endocrinology.

[62]  S. Safe,et al.  Transcriptional Activation of Insulin-Like Growth Factor-Binding Protein-4 by 17β-Estradiol in MCF-7 Cells: Role of Estrogen Receptor-Sp1 Complexes. , 1999, Endocrinology.

[63]  A. Periasamy,et al.  Fluorescence resonance energy transfer microscopy: a mini review. , 2001, Journal of biomedical optics.

[64]  W. Kraus,et al.  Mediator and p300/CBP-Steroid Receptor Coactivator Complexes Have Distinct Roles, but Function Synergistically, during Estrogen Receptor α-Dependent Transcription with Chromatin Templates , 2003, Molecular and Cellular Biology.