FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2107008249> ?p ?o ?g. }

• W2107008249 endingPage "671" @default.
• W2107008249 startingPage "661" @default.
• W2107008249 abstract "Interferon (IFN)-γ plays crucial roles in regulating both innate and adaptive immunity. The existence of IFN-γ receptor 1 (IFNGR1) molecules on the cell surface is a prerequisite to the initiation of IFN-γ signaling; low expression of IFNGR1 leads to a functional blockade of IFN-γ signaling. However, the molecular mechanisms by which IFNGR1 expression is controlled are unclear. In the present study, we demonstrated that IFNGR1 expression was reduced or lost in breast cancer. Heterogeneous IFNGR1 immunoreactivity appeared to be associated with the morphological heterogeneity of breast cancer, and loss of IFNGR1 expression was predominantly observed in poorly differentiated areas. We identified the functional activating protein (AP)-2 and specificity protein (SP)-1 sites within the IFNGR1 promoter. Ectopic expression of AP-2α drastically repressed the expression of IFNGR1 and hindered IFN-γ signaling, whereas AP-2α gene silencing elevated IFNGR1 levels. Overexpression of SP-1 effectively antagonized the repressive effects of AP-2α. Simultaneous recruitment of both transcription factors to the AP-2 and SP-1 motifs, respectively, in the IFNGR1 promoter was demonstrated, implying that AP-2α and SP-1 may synergistically modulate IFNGR1 transcription. Moreover, AP-2α overexpression in AP-2–deficient SW480 cells remarkably inhibited Stat1 phosphorylation and the anti-proliferative effects of IFN-γ, whereas knockdown of the AP-2α expression dramatically enhanced the sensitivities of HeLa cells highly expressing AP-2 to IFN-γ, indicating that dysregulation of AP-2α expression is associated with impaired IFN-γ actions in cancer cells. Interferon (IFN)-γ plays crucial roles in regulating both innate and adaptive immunity. The existence of IFN-γ receptor 1 (IFNGR1) molecules on the cell surface is a prerequisite to the initiation of IFN-γ signaling; low expression of IFNGR1 leads to a functional blockade of IFN-γ signaling. However, the molecular mechanisms by which IFNGR1 expression is controlled are unclear. In the present study, we demonstrated that IFNGR1 expression was reduced or lost in breast cancer. Heterogeneous IFNGR1 immunoreactivity appeared to be associated with the morphological heterogeneity of breast cancer, and loss of IFNGR1 expression was predominantly observed in poorly differentiated areas. We identified the functional activating protein (AP)-2 and specificity protein (SP)-1 sites within the IFNGR1 promoter. Ectopic expression of AP-2α drastically repressed the expression of IFNGR1 and hindered IFN-γ signaling, whereas AP-2α gene silencing elevated IFNGR1 levels. Overexpression of SP-1 effectively antagonized the repressive effects of AP-2α. Simultaneous recruitment of both transcription factors to the AP-2 and SP-1 motifs, respectively, in the IFNGR1 promoter was demonstrated, implying that AP-2α and SP-1 may synergistically modulate IFNGR1 transcription. Moreover, AP-2α overexpression in AP-2–deficient SW480 cells remarkably inhibited Stat1 phosphorylation and the anti-proliferative effects of IFN-γ, whereas knockdown of the AP-2α expression dramatically enhanced the sensitivities of HeLa cells highly expressing AP-2 to IFN-γ, indicating that dysregulation of AP-2α expression is associated with impaired IFN-γ actions in cancer cells. Interferon (IFN)-γ is a key proinflammatory cytokine and plays crucial roles in regulating both innate and adaptive immunity.1Schroder K. Hertzog P.J. Ravasi T. Hume D.A. Interferon-gamma: an overview of signals, mechanisms and functions.J Leukoc Biol. 2004; 75: 163-189Crossref PubMed Scopus (3033) Google Scholar, 2Boehm U. Klamp T. Groot M. Howard J.C. Cellular responses to interferon-gamma.Annu Rev Immunol. 1997; 15: 749-795Crossref PubMed Scopus (2519) Google Scholar The findings that IFN-γ protects the host against the growth of chemically induced, spontaneously arising, and transplantable tumors have generated great interests in the obligate activities of IFN-γ in cancer immunoediting, which refers to the dual anti-tumor/tumor-sculpting actions of IFN-γ and the immune system.3Blankenstein T. Qin Z. The role of IFN-gamma in tumor transplantation immunity and inhibition of chemical carcinogenesis.Curr Opin Immunol. 2003; 15: 148-154Crossref PubMed Scopus (117) Google Scholar, 4Bui J.D. Schreiber R.D. Cancer immunosurveillance, immunoediting and inflammation: independent or interdependent processes?.Curr Opin Immunol. 2007; 19: 203-208Crossref PubMed Scopus (248) Google Scholar, 5Dighe A.S. Richards E. Old L.J. Schreiber R.D. Enhanced in vivo growth and resistance to rejection of tumor cells expressing dominant negative IFN gamma receptors.Immunity. 1994; 1: 447-456Abstract Full Text PDF PubMed Scopus (511) Google Scholar, 6Dunn G.P. Ikeda H. Bruce A.T. Koebel C. Uppaluri R. Bui J. Chan R. Diamond M. White J.M. Sheehan K.C. Schreiber R.D. Interferon-gamma and cancer immunoediting.Immunol Res. 2005; 32: 231-245Crossref PubMed Scopus (119) Google Scholar Several lines of evidence indicate that IFN-γ acts as a central coordinator in all phases of the cancer-immunoediting process.1Schroder K. Hertzog P.J. Ravasi T. Hume D.A. Interferon-gamma: an overview of signals, mechanisms and functions.J Leukoc Biol. 2004; 75: 163-189Crossref PubMed Scopus (3033) Google Scholar, 7Dunn G.P. Old L.J. Schreiber R.D. The immunobiology of cancer immunosurveillance and immunoediting.Immunity. 2004; 21: 137-148Abstract Full Text Full Text PDF PubMed Scopus (2204) Google Scholar, 8Kaplan D.H. Shankaran V. Dighe A.S. Stockert E. Aguet M. Old L.J. Schreiber R.D. Demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice.Proc Natl Acad Sci U S A. 1998; 95: 7556-7561Crossref PubMed Scopus (1180) Google Scholar Experimental investigations show that IFN-γ exerts its anti-tumor effects by regulating various biological programs, including activating natural killer cells, natural killer T cells, and macrophages; driving CD4+ type 1 helper T-cell development; promoting the major histocompatibility complex class I pathway of antigen processing and presentation in tumor cells; inhibiting the generation and/or activation of CD4+CD25+ regulatory T cells; and directly inhibiting tumor cell growth, inducing cellular apoptosis, and blocking neovascularization.6Dunn G.P. Ikeda H. Bruce A.T. Koebel C. Uppaluri R. Bui J. Chan R. Diamond M. White J.M. Sheehan K.C. Schreiber R.D. Interferon-gamma and cancer immunoediting.Immunol Res. 2005; 32: 231-245Crossref PubMed Scopus (119) Google Scholar, 9Xu X. Fu X.Y. Plate J. Chong A.S. IFN-gamma induces cell growth inhibition by Fas-mediated apoptosis: requirement of STAT1 protein for up-regulation of Fas and FasL expression.Cancer Res. 1998; 58: 2832-2837PubMed Google Scholar, 10Mumberg D. Monach P.A. Wanderling S. Philip M. Toledano A.Y. Schreiber R.D. Schreiber H. CD4(+) T cells eliminate MHC class II-negative cancer cells in vivo by indirect effects of IFN-gamma.Proc Natl Acad Sci U S A. 1999; 96: 8633-8638Crossref PubMed Scopus (314) Google Scholar, 11Nishikawa H. Kato T. Tawara I. Ikeda H. Kuribayashi K. Allen P.M. Schreiber R.D. Old L.J. Shiku H. IFN-gamma controls the generation/activation of CD4+ CD25+ regulatory T cells in antitumor immune response.J Immunol. 2005; 175: 4433-4440PubMed Google Scholar, 12Beatty G. Paterson Y. IFN-gamma-dependent inhibition of tumor angiogenesis by tumor-infiltrating CD4+ T cells requires tumor responsiveness to IFN-gamma.J Immunol. 2001; 166: 2276-2282PubMed Google Scholar IFN-γ signaling is triggered by an engagement of its heterodimeric receptor (IFNGR), which is ubiquitously expressed on virtually all normal cell surfaces.13Bach E.A. Aguet M. Schreiber R.D. The IFN gamma receptor: a paradigm for cytokine receptor signaling.Annu Rev Immunol. 1997; 15: 563-591Crossref PubMed Scopus (883) Google Scholar IFNGR consists of two ligand-binding IFNGR1 chains and two signal-transducing IFNGR2 chains.13Bach E.A. Aguet M. Schreiber R.D. The IFN gamma receptor: a paradigm for cytokine receptor signaling.Annu Rev Immunol. 1997; 15: 563-591Crossref PubMed Scopus (883) Google Scholar Binding of IFN-γ to IFNGR1 initiates autophosphorylation and trans-phosphorylation and activation of the Janus-activated kinases (JAKs), which, in turn, phosphorylate the intracellular domain of IFNGR1, leading to the recruitment of Stat1 proteins.2Boehm U. Klamp T. Groot M. Howard J.C. Cellular responses to interferon-gamma.Annu Rev Immunol. 1997; 15: 749-795Crossref PubMed Scopus (2519) Google Scholar The phosphorylated Stat1 proteins, forming reciprocal homodimers, dissociate from the receptor complex, translocate to the nucleus, and regulate the transcription of IFN-γ–inducible genes.14Decker T. Kovarik P. Meinke A. GAS elements: a few nucleotides with a major impact on cytokine-induced gene expression.J Interferon Cytokine Res. 1997; 17: 121-134Crossref PubMed Scopus (344) Google Scholar Studies6Dunn G.P. Ikeda H. Bruce A.T. Koebel C. Uppaluri R. Bui J. Chan R. Diamond M. White J.M. Sheehan K.C. Schreiber R.D. Interferon-gamma and cancer immunoediting.Immunol Res. 2005; 32: 231-245Crossref PubMed Scopus (119) Google Scholar, 8Kaplan D.H. Shankaran V. Dighe A.S. Stockert E. Aguet M. Old L.J. Schreiber R.D. Demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice.Proc Natl Acad Sci U S A. 1998; 95: 7556-7561Crossref PubMed Scopus (1180) Google Scholar using mice with the genetic deficiencies of the components involved in the IFN-γ signaling pathway have demonstrated that IFNGR1, JAK1, JAK2, and Stat1 are unequivocally required for IFN-γ–induced biological responses. Investigations on human tumor cells have revealed that the resistance of a variety of tumor cell lines to IFN-γ is owing to the defects in IFNGR-mediated signaling cascades, including loss of IFNGR1 expression, underexpression of JAK1 and Stat1, and production of abnormal JAK2.3Blankenstein T. Qin Z. The role of IFN-gamma in tumor transplantation immunity and inhibition of chemical carcinogenesis.Curr Opin Immunol. 2003; 15: 148-154Crossref PubMed Scopus (117) Google Scholar, 4Bui J.D. Schreiber R.D. Cancer immunosurveillance, immunoediting and inflammation: independent or interdependent processes?.Curr Opin Immunol. 2007; 19: 203-208Crossref PubMed Scopus (248) Google Scholar, 6Dunn G.P. Ikeda H. Bruce A.T. Koebel C. Uppaluri R. Bui J. Chan R. Diamond M. White J.M. Sheehan K.C. Schreiber R.D. Interferon-gamma and cancer immunoediting.Immunol Res. 2005; 32: 231-245Crossref PubMed Scopus (119) Google Scholar, 15Dunn G.P. Koebel C.M. Schreiber R.D. Interferons, immunity and cancer immunoediting.Nat Rev Immunol. 2006; 6: 836-848Crossref PubMed Scopus (1221) Google Scholar Clinical studies have underpinned the importance of IFNGR1 expression in esophageal and ovarian cancers. The reduced expression of IFNGR1 was tightly associated with clinicopathologic features of esophageal cancer16Wang Y. Liu D. Chen P. Koeffler H.P. Tong X. Xie D. Negative feedback regulation of IFN-gamma pathway by IFN regulatory factor 2 in esophageal cancers.Cancer Res. 2008; 68: 1136-1143Crossref PubMed Scopus (41) Google Scholar and prognosis of ovarian cancers.17Duncan T.J. Rolland P. Deen S. Scott I.V. Liu D.T. Spendlove I. Durrant L.G. Loss of IFN gamma receptor is an independent prognostic factor in ovarian cancer.Clin Cancer Res. 2007; 13: 4139-4145Crossref PubMed Scopus (39) Google Scholar However, the molecular mechanisms of IFNGR1 down-regulation are poorly understood. The expression of IFNGR1 is usually in surplus and can be further up-regulated at the transcription level in certain settings. Proinflammatory cytokines, such as tumor necrosis factor-α, IL-1, IL-6, and IFN-γ, stimulate the expression of IFNGR1 mRNA and protein.13Bach E.A. Aguet M. Schreiber R.D. The IFN gamma receptor: a paradigm for cytokine receptor signaling.Annu Rev Immunol. 1997; 15: 563-591Crossref PubMed Scopus (883) Google Scholar, 18Shirey K.A. Jung J.Y. Maeder G.S. Carlin J.M. Upregulation of IFN-gamma receptor expression by proinflammatory cytokines influences IDO activation in epithelial cells.J Interferon Cytokine Res. 2006; 26: 53-62Crossref PubMed Scopus (51) Google Scholar The transcriptional activation of IFNGR1 was postulated to depend on the transcription factor NF-κB.18Shirey K.A. Jung J.Y. Maeder G.S. Carlin J.M. Upregulation of IFN-gamma receptor expression by proinflammatory cytokines influences IDO activation in epithelial cells.J Interferon Cytokine Res. 2006; 26: 53-62Crossref PubMed Scopus (51) Google Scholar, 19Shirey K.A. Jung J.Y. Carlin J.M. Up-regulation of gamma interferon receptor expression due to Chlamydia-toll-like receptor interaction does not enhance signal transducer and activator of transcription 1 signaling.Infect Immun. 2006; 74: 6877-6884Crossref PubMed Scopus (10) Google Scholar Specificity protein (SP)-1 may regulate 12-otetradecanoylphorbol-13-acetate (TPA)–induced transcription of the IFNGR1 gene through binding to TPA response element within IFNGR1 promoter.20Sakamoto S. Taniguchi T. Identification of a phorbol ester-responsive element in the interferon-gamma receptor 1 chain gene.J Biol Chem. 2001; 276: 37237-37241Crossref PubMed Scopus (20) Google Scholar How the expression of IFNGR1 is controlled, either positively or negatively, is basically unknown. In the present study, we demonstrate that IFNGR1 expression is reduced or lost in breast cancer tissues. Our data prove that AP-2α represses the expression of IFNGR1, hinders IFN-γ signaling, and attenuates the sensitivities of tumor cells to IFN-γ stimulation. All human tumor cell lines used are from American Type Culture Collection, including breast cancer cell lines MCF-7, SKBR3, T47D, MDA-453, MDA-231, and MDA-435S; cervical carcinoma cell line HeLa; embryonic kidney cell line transformed with SV-40 large T-antigen 293T; hepatoblastoma cell line HepG2; colon carcinoma cell line SW480; lymphoblastic T-cell line Jurkat; Burkitt's lymphoma cell line Raji; and myeloid leukemic cell line U937. Before the treatment with TPA (0.2 μmol/L), isoproterenol (10 μmol/L), forskolin (10 μmol/L) (Sigma-Aldrich, St. Louis, MO), and tumor necrosis factor-α (10 ng/mL) (PeproTech, Rocky Hill, NJ), the cells were starved for 1 hour. For the treatment with IFN-γ (20 ng/mL) and IFN-β (10 ng/mL) (PeproTech), the cells were routinely cultured. A 770-bp IFNGR1 promoter fragment (nucleotide −770 to −1 relative to the translation initiation site) was amplified by PCR from HeLa cell genomic DNA using the primers P1 and P2 (Table 1) and inserted into the plasmid pGL3-Basic (Promega, Madison, WI) designated as p770. A series of 5′-deletions were produced by PCR using p770 as a template with the distinct 5′ primers P3 to P5 and a common 3′ primer P2 (Table 1). The products were cloned into pGL3-Basic to generate p550, p260, and p160. The plasmids containing the site-directed mutations in IFNGR1 promoter, p770/AP2M, p770/SP1M, and p770/AP2M/SP1M, were constructed by overlap extension PCR with the primers P6 to P9 (Table 1). The IFN-γ–inducible class II transactivator (CIITA) IV promoter21Dong Y. Rohn W.M. Benveniste E.N. IFN-gamma regulation of the type IV class II transactivator promoter in astrocytes.J Immunol. 1999; 162: 4731-4739PubMed Google Scholar was amplified from Raji cell genomic DNA using P10 and P11 and also cloned into pGL3-Basic. SP-1 cDNA (Origene Technologies, Inc., Rockville, MD) was PCR amplified with the primers P12 and P13 (Table 1) and cloned into the plasmid pXJ40-HA (pXJ40-HA/SP-1). The plasmids pAP-1-Luc and pNF-κB-Luc were generous gifts from Dr. Xuemin Zhang (National Center of Biomedical Analysis, Beijing, China). The plasmids pCMV-myc/AP-2α and pCREB-Luc were provided by Dr. Yingli Zhong (College of Life Science, Hunan Normal University, Changsha, China) and Dr. Jiyan Zhang (Beijing Institute of Basic Medical Sciences, Beijing), respectively. The small-interfering RNAs (siRNAs) targeting AP-2α (Table 2) were synthesized by Ribio Biotech (Guangzhou, China).Table 1The Primers UsedApplication designationPrimersConstruction of p770P15′-CCCCTCGAGGGTGAGATCATTAGACATTCGCATG-3′ (XhoI)P25′-CCCAAGCTTCTGCTACCGACGGTCGCTG-3′ (HindIII)Construction of p550, p260, and p160P35′-CCCCTCGAGGTCAAGCCGATTTGATTTGGG-3′ (XhoI)P45′-CCCCTCGAGTCAAATTCCTCCCACACCCAG-3′ (XhoI)P55′-CCCCTCGAGCGCCTCCTGCGGCTTCCCGGACTTG-3′ (XhoI)Construction of p770/AP2M, p770/SP1M, and p770/AP2M/SP1MP65′-CCCAAGCTTGCTGCTACCGACGGTCGCTGGCTCCAACCCCGAGCGCCTG-3′P75′-GAGCGCCTGCTTTCCCAGCCCAGC-3′P85′-CCCCGCCCACGCCCTGGTAGTGACTCCTGCCGACGCCGGCAC-3′P95′-GTGCCGGCGTCGGCAGGAGTCACTACCAGGGCGTGGGCGGGG-3′Construction of HA-SP1P105′-CCCAAGCTTATG AGCGACCAAG ATCACTCCAT-3′ (HindIII)P115′-CGGGGTACCTCAGAAGCCATTGCCACTGATATTA-3′ (KpnI)RT-PCR for IFNGR1P125′-TCCTCAGTGCCTACACCAACTAATG-3′P135′-CTGGATCTCACTTCCGTTCATTCTC-3′RT-PCR for GAPDHP145′-ACCCAGAAGACTGTGGATGG-3′P155′-CAGTGAGCTTCCCGTTCAG-3′ChIP-PCR for IFNGR1 promoterP1′5′-CGCCTCCTGCGGCTTCCCGGACTTG-3′P2′5′-GCCGACGCCGGCACAGAC-3′P3′5′-GTCTGTGCCGGCGTCGGC-3′P4′5′-TATTGTCACACCGACGTCACCT-3′ChIP-PCR for ICAM-1 promoterP5′5′-AGGATGACCCTCTCGGCC-3′P6′5′-TGCTGCAGTTATTTCCGGACT-3′Real-time RT-PCR for AP-2P165′-CCGTGTCCCTGTCCAAGT-3′P175′-TTCCGCCACCGTGACCTT-3′Real-time RT-PCR for IFNGR1P185′-CTTAGCCTGGTATTCATC-3′P195′-CTCTTCACAGACCACCTC-3′Restriction enzyme recognition sites are underlined; restriction enzymes are noted in parentheses.GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin; ICAM, intercellular adhesion molecule 1. Open table in a new tab Table 25′-Biotinylated Double-Stranded Oligonucleotides and siRNA SequencesProbesSequencesAP-2α siRNA 15′-CACGGACAACAACGCCAAAdTdT-3′ 25′-GGAAGAUCUUUAAGAGAAAdTdT-3′NC siRNA5′-ACGUGCACCGUUCGGAGAAdTdT-3′Oligo AP-2Biotin-5′-GTGCTGGGCTGGTCCCGCAGGCGCTCGGGGTTG-3′5′-CAACCCCGAGCGCCTGCGGGACCAGCCCAGCAC-3′ SP-1Biotin-5′-CCCACGCCCTGGTCCC GCCTCCTGCCGACGCCG-3′5′-CGGCGTCGGCAGGAGGCGGGACCAGGGCGTGGG-3′ NSBiotin-5′-ATTTTTAACAAAAGTTCATGATACTCAGAGAATT-3′5′-AATTCTCTGAGTATCATGAACTTTTGTTAAAAAT-3′The AP-2 and SP1 binding sites are underlined.NS, nonspecific. Open table in a new tab Restriction enzyme recognition sites are underlined; restriction enzymes are noted in parentheses. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin; ICAM, intercellular adhesion molecule 1. The AP-2 and SP1 binding sites are underlined. NS, nonspecific. A total of 2 × 104 to 5 × 104 MCF-7, 293T, HepG2, and SW480 cells were transfected with 200 ng of each reporter construct, 20 ng of pRL-TK vector (Promega), and 0.1 to 0.5 μg of the plasmid expressing AP-2α or SP-1 or the empty plasmid using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). A total of 1 × 106 Raji, Jurkat, and U937 cells were transfected with 500 ng of p770, 50 ng of pRL-TK, and 1 μg of the effector plasmid using Cell Line Nuclefeofector Kit V (Amaxa, Cologne, Germany). The luciferase activities were assayed with a dual luciferase assay kit (Promega). The assay was repeated twice, independently. The whole cell lysates were prepared, and protein concentration of the lysates was determined by a BCA Protein Assay Kit (Pierce, Rockford, IL). The protein samples were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membranes were probed with the primary antibodies against Myc tag (9 E10, number SC-40; Santa Cruz, Santa Cruz, CA), AP-2α (H-79, number SC-8975; Santa Cruz), SP-1 (H-225, number SC-14027; Santa Cruz), IFNGR1 (c-20, number SC-700; Santa Cruz), and p-Stat1 (Tyr701, number SC-7988; Santa Cruz), followed by washing and incubating with horseradish peroxidase–conjugated secondary antibodies (Zhongshan Golden Bridge, Beijing, China). Equal loading was verified by detection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with a specific antibody (kc-5G4; Kangchen, Shanghai, China). Bands were visualized by the Enhanced Chemiluminescence System (Amersham Pharmacia Biotech, Newcastle upon Tyne, UK). For coimmunoprecipitation analysis, SW480 cells transfected with the plasmid expressing Myc-tagged AP-2α were harvested and lysed in ice-cold lysis buffer. After clarification by centrifugation, the supernatants were incubated with the antibodies against Myc or SP-1 in the presence of 40 μL of protein A/G PLUS-Agarose (Santa Cruz) at 4°C for 4 hours. Immunoprecipitated samples were then blotted with the indicated antibodies. The experiments were performed in duplicate. The level of IFNGR1 mRNA was detected by RT-PCR with a pair of primers, P12 and P13 (Table 1). Amplification of the GAPDH gene with the primers P14 and P15 (Table 1) was used as an internal control. The intensity of bands was quantified using LabWorks Image Acquisition and Analysis software (UVP Mini Darkroom, Upland, CA). The experiment was conducted in duplicate. Cell surface expression of IFNGR1 was detected by flow cytometry using a BD Biosciences FACSCalibur (San Jose, CA). A chromatin immunoprecipitation (ChIP) assay was performed by using the SimpleChIPTM Enzymatic Chromatin IP Kit (Cell Signaling Technology Inc., Beverly, MA). The AP-2α/SP-1/DNA complexes were precipitated by either anti-Myc or anti-SP-1 antibody or by rabbit IgG as the negative control. The precipitated DNA was amplified by PCR using primers P1′ and P3′ (Table 1) specific for a 109-bp region flanking the SP-1 element, P2′ and P4′ for an 85-bp region spanning the AP-2 element, and P1′ and P4′ for a 160-bp region harboring both elements, or P5′ and P6′ for a 52-bp fragment spanning the known AP-2 element in the intercellular adhesion molecular 1 promoter as a positive control.22Grether-Beck S. Olaizola-Horn S. Schmitt H. Grewe M. Jahnke A. Johnson J.P. Briviba K. Sies H. Krutmann J. Activation of transcription factor AP-2 mediates UVA radiation- and singlet oxygen-induced expression of the human intercellular adhesion molecule 1 gene.Proc Natl Acad Sci U S A. 1996; 93: 14586-14591Crossref PubMed Scopus (195) Google Scholar The biotinylated oligonucleotides (Table 2) corresponding to the IFNGR1 promoter region spanning the AP-2 or SP-1 site were synthesized by AuGCT Biotechnology (Beijing). The biotinylated irrelevant oligonucleotides (Table 2) were used as the control. The DNA/nuclear protein complexes were precipitated by anti-AP-2α, anti-Myc, and anti-SP-1 or rabbit IgG using Dynabeads M-280 (Invitrogen) and detected by using Western blot analysis with the antibodies against AP-2α and SP-1. A TMA containing cores from 50 breast cancer and adjacent normal tissues was obtained from US Biomax (Rockville, MD). Twenty invasive ductal carcinoma tissues and five normal breast tissues (from mamma accessoria) were obtained from Donghua Hospital, Dongguan, Guangdong, China, with the informed consent of patients and approval for the experiments from Donghua Hospital. Immunohistochemistry (IHC) staining was performed as previously described,23Shi M. Liu D. Duan H. Qian L. Wang L. Niu L. Zhang H. Yong Z. Gong Z. Song L. Yu M. Hu M. Xia Q. Shen B. Guo N. The β2-adrenergic receptor and Her2 comprise a positive feedback loop in human breast cancer cells.Breast Cancer Res Treat. 2011; 125: 351-362Crossref PubMed Scopus (100) Google Scholar using the rabbit polyclonal antibodies against IFNGR1 (ab61179; Abcam, Cambridge, UK) and AP-2α (number 3208; Abcam). Staining was assessed microscopically by two independent pathologists (M.H. and T.Y.W). The immunostaining was scored as strong (3 and 4+), weak (1 and 2+) or negative (0), according to the rate of labeled tumor cells and staining intensity, respectively. A Real-Time PCR-Based TissueScan Breast Cancer Panel, consisting of cDNAs obtained from 48 samples covering four disease stages of breast invasive ductal carcinoma and normal tissues, with all of the clinical information associated with each of these samples on the OriGene web site, was obtained from OriGene Technologies (Rockville, MD). Reactions were amplified using the β-actin primers provided by the manufacturer and the primers for human AP-2α (P16 and P17) and IFNGR1 (P18 and P19). SW480 cells were transfected with either the plasmid expressing AP-2α or control plasmid, and HeLa cells were transfected with the siRNA specific for AP-2α or a control siRNA. After 24 hours, the transfected cells were trypsinized, seeded in 96-well plates to a density of 2.5 × 103 per well, and then treated with 5, 25, or 50 ng/mL of IFN-γ for 72 hours. Cell viability was assessed by measurement of cellular ATP levels using the ATPlite Luminescence Assay kit (PerkinElmer, Waltham, MA). All samples were assayed in triplicate, and the data were reported as the mean ± SE. This experiment was performed in duplicate. For comparisons among the groups in the experiments, an analysis of variance test was performed. For the breast cancer tissues array analysis, a Mann-Whitney test was used. A Pearson correlation was used for the correlation analysis between IFNGR1 and AP-2α expression. P < 0.05 was considered statistically significant. Among 88 tumor cores (two cores from each case), 43.2% was reduced (22 cores) or completely lacking (18 cores) of IFNGR1 staining (Figure 1A; see also Supplemental Table S1 at http://ajp.amjpathol.org), which was strongly positive in pericancerous tissues. Of 20 infiltrating ductal carcinoma samples, IFNGR1 expression was down-regulated in 10 with pronounced heterogeneity in staining intensity, including three cases with complete loss of IFNGR1 immunoreactivity, compared with normal breast tissues. Interestingly, in the same tissue section, IFNGR1 expression was strong in well-differentiated areas but weak or absent in poorly differentiated areas (Figure 1B). In some cases, well and poorly differentiated areas were close together, exhibiting a remarkably differential IFNGR1 staining (Figure 1B). Loss of IFNGR1 could be observed mainly in the cells with distinct heteromorphism. To investigate the molecular mechanisms of IFNGR1 down-regulation, we searched for consensus motifs of transcription factors by computational sequence analysis. The results revealed several potential sites, including a putative AP-2 site (CCCGCAGGCG), which was presumed to be an NF-κB site located at −43 to −34 in a previous study,24Juliger S. Bongartz M. Luty A.J. Kremsner P.G. Kun J.F. Functional analysis of a promoter variant of the gene encoding the interferon-gamma receptor chain I.Immunogenetics. 2003; 54: 675-680PubMed Google Scholar an AP-1 site (TGAGTCA) at −83 to −76, and a CREB site (TGACGGAA) at −91 to −84 nucleotides upstream of the ATG codon (Figure 2A). To test the functional significance of these cis-acting regulatory elements, a 770-bp of IFNGR1 5′-flaking region was cloned into a luciferase reporter plasmid designated p770. 293T cells were transiently cotransfected with p770 and an internal control plasmid, pRL-TK. The roles of AP-1, CREB, and NF-κB elements in the transcriptional activation of IFNGR1 promoter were excluded by our data (see Supplemental Figure S1 at http://ajp.amjpathol.org). Notably, the sequence features of the NF-κB site, which were previously described,24Juliger S. Bongartz M. Luty A.J. Kremsner P.G. Kun J.F. Functional analysis of a promoter variant of the gene encoding the interferon-gamma receptor chain I.Immunogenetics. 2003; 54: 675-680PubMed Google Scholar are highly homologous to the AP-2 consensus core sequences, existing at equivalent position of mouse, rat, and Xenopus IFNGR1 promoters (Figure 2B). Therefore, we inspected the effects of AP-2α on IFNGR1 promoter activation by the expression of AP-2α in several cell lines, including the epithelial-derived cell lines HepG2, MCF-7, and SW480, and the nonepithelial cell lines Raji and Jurkat. Surprisingly, IFNGR1 promoter activities were strikingly suppressed in all cell lines tested (Figure 2C). AP-2 recognizes and binds to GC-rich DNA sequences of its target genes, mediating both the activation and repression of regulation. To further localize the proximal region of IFNGR1 promoter and define the functional significance of AP-2α, the plasmids containing a series of truncated promoter fragments were generated (Figure 2D). The transactivation activities of the fragments were estimated in 293T cells transiently transfected with an AP-2α–expressing plasmid. The deletion of the IFNGR1 5′-flanking region progressively decreased the luciferase activities; nevertheless, the plasmid p160 still retained significant promoter activity, suggesting that the −160-bp region played a key role in the basal expression of the IFNGR1 gene. Notably, AP-2α overexpression prominently repressed the transcriptional activities of all promoter fragments (Figure 2D). To further testify about the role of the AP-2 element, we performed a site-directed mutagenesis analysis for the AP-2 site by altering the highly conserved nucleotides and generated the plasmid p770/AP2M. The mutagenesis resulted in a remarkable increase of IFNGR1 promoter activities by 76.6% in 293T and 49.0% in HeLa cells. The inhibition of the basal transcription of IFNGR1 by AP-2α was also reversed (from 84.1% to 68.8% in 293T and from 59% to 23.8% in HeLa cells; Figure 2E), indicating that the AP-2 element is important in transcriptional regulation of IFNGR1. To testify about the impact of AP-2α on the expression of IFNGR1, HepG2, MCF-7, SW480, Raji, and Jurkat cells were transiently transfected with pCMV-myc/AP-2α. The effect of AP-2α on the expression of IFNGR1 mRNA was evaluated by semiquantitative RT-PCR. Figure 3A showed that the IFNGR1 mRNA level was significantly decreased in the transfected cells. Moreover, the ectopic expression of AP-2α resulted in strong inhibition of IFNGR1 protein expression in a dose-dependent manner (Figure 3B) and also caused diminished IFNGR1 molecules on the cellular surface, as analyze" @default.
• W2107008249 created "2016-06-24" @default.
• W2107008249 creator A5020955864 @default.
• W2107008249 creator A5026467025 @default.
• W2107008249 creator A5031067040 @default.
• W2107008249 creator A5038796794 @default.
• W2107008249 creator A5047622083 @default.
• W2107008249 creator A5051318920 @default.
• W2107008249 creator A5057741794 @default.
• W2107008249 creator A5060514130 @default.
• W2107008249 creator A5065435488 @default.
• W2107008249 creator A5077713155 @default.
• W2107008249 date "2012-02-01" @default.
• W2107008249 modified "2023-10-03" @default.
• W2107008249 title "Modulation of IFN-γ Receptor 1 Expression by AP-2α Influences IFN-γ Sensitivity of Cancer Cells" @default.
• W2107008249 cites W1585344925 @default.
• W2107008249 cites W1637209605 @default.
• W2107008249 cites W1860599747 @default.
• W2107008249 cites W1969591502 @default.
• W2107008249 cites W1970569661 @default.
• W2107008249 cites W1976121925 @default.
• W2107008249 cites W1991293039 @default.
• W2107008249 cites W1996663731 @default.
• W2107008249 cites W1998837866 @default.
• W2107008249 cites W2004433270 @default.
• W2107008249 cites W2013958910 @default.
• W2107008249 cites W2019214729 @default.
• W2107008249 cites W2023030470 @default.
• W2107008249 cites W2024050621 @default.
• W2107008249 cites W2025723633 @default.
• W2107008249 cites W2026318599 @default.
• W2107008249 cites W2030954212 @default.
• W2107008249 cites W2035932824 @default.
• W2107008249 cites W2038634209 @default.
• W2107008249 cites W2041233862 @default.
• W2107008249 cites W2042687451 @default.
• W2107008249 cites W2052313621 @default.
• W2107008249 cites W2052363929 @default.
• W2107008249 cites W2053220974 @default.
• W2107008249 cites W2062889582 @default.
• W2107008249 cites W2076954365 @default.
• W2107008249 cites W2084545547 @default.
• W2107008249 cites W2086739969 @default.
• W2107008249 cites W2105358835 @default.
• W2107008249 cites W2106169455 @default.
• W2107008249 cites W2134768631 @default.
• W2107008249 cites W2145084871 @default.
• W2107008249 cites W2147147369 @default.
• W2107008249 cites W2153398168 @default.
• W2107008249 cites W2158153035 @default.
• W2107008249 cites W2159830797 @default.
• W2107008249 cites W2164151058 @default.
• W2107008249 cites W2167017590 @default.
• W2107008249 cites W4313376818 @default.
• W2107008249 doi "https://doi.org/10.1016/j.ajpath.2011.10.040" @default.
• W2107008249 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/22182699" @default.
• W2107008249 hasPublicationYear "2012" @default.
• W2107008249 type Work @default.
• W2107008249 sameAs 2107008249 @default.
• W2107008249 citedByCount "18" @default.
• W2107008249 countsByYear W21070082492013 @default.
• W2107008249 countsByYear W21070082492014 @default.
• W2107008249 countsByYear W21070082492015 @default.
• W2107008249 countsByYear W21070082492016 @default.
• W2107008249 countsByYear W21070082492017 @default.
• W2107008249 countsByYear W21070082492018 @default.
• W2107008249 countsByYear W21070082492019 @default.
• W2107008249 countsByYear W21070082492020 @default.
• W2107008249 countsByYear W21070082492021 @default.
• W2107008249 countsByYear W21070082492022 @default.
• W2107008249 countsByYear W21070082492023 @default.
• W2107008249 crossrefType "journal-article" @default.
• W2107008249 hasAuthorship W2107008249A5020955864 @default.
• W2107008249 hasAuthorship W2107008249A5026467025 @default.
• W2107008249 hasAuthorship W2107008249A5031067040 @default.
• W2107008249 hasAuthorship W2107008249A5038796794 @default.
• W2107008249 hasAuthorship W2107008249A5047622083 @default.
• W2107008249 hasAuthorship W2107008249A5051318920 @default.
• W2107008249 hasAuthorship W2107008249A5057741794 @default.
• W2107008249 hasAuthorship W2107008249A5060514130 @default.
• W2107008249 hasAuthorship W2107008249A5065435488 @default.
• W2107008249 hasAuthorship W2107008249A5077713155 @default.
• W2107008249 hasBestOaLocation W21070082491 @default.
• W2107008249 hasConcept C121608353 @default.
• W2107008249 hasConcept C170493617 @default.
• W2107008249 hasConcept C203014093 @default.
• W2107008249 hasConcept C502942594 @default.
• W2107008249 hasConcept C54355233 @default.
• W2107008249 hasConcept C86803240 @default.
• W2107008249 hasConcept C96232424 @default.
• W2107008249 hasConceptScore W2107008249C121608353 @default.
• W2107008249 hasConceptScore W2107008249C170493617 @default.
• W2107008249 hasConceptScore W2107008249C203014093 @default.
• W2107008249 hasConceptScore W2107008249C502942594 @default.
• W2107008249 hasConceptScore W2107008249C54355233 @default.
• W2107008249 hasConceptScore W2107008249C86803240 @default.
• W2107008249 hasConceptScore W2107008249C96232424 @default.
• W2107008249 hasIssue "2" @default.

• next