|
1
|
Wang Y, Hoeppner LH, Angom RS, Wang E, Dutta S, Doeppler HR, Wang F, Shen T, Scarisbrick IA, Guha S, Storz P, Bhattacharya R, Mukhopadhyay D. Protein kinase D up-regulates transcription of VEGF receptor-2 in endothelial cells by suppressing nuclear localization of the transcription factor AP2β. J Biol Chem 2019; 294:15759-15767. [PMID: 31492751 PMCID: PMC6816101 DOI: 10.1074/jbc.ra119.010152] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2019] [Revised: 08/19/2019] [Indexed: 01/29/2023] Open
Abstract
Vascular endothelial growth factor A (VEGF) signals primarily through its cognate receptor VEGF receptor-2 (VEGFR-2) to control vasculogenesis and angiogenesis, key physiological processes in cardiovascular disease and cancer. In human umbilical vein endothelial cells (HUVECs), knockdown of protein kinase D-1 (PKD1) or PKD2 down-regulates VEGFR-2 expression and inhibits VEGF-induced cell proliferation and migration. However, how PKD regulates VEGF signaling is unclear. Previous bioinformatics analyses have identified binding sites for the transcription factor activating enhancer-binding protein 2 (AP2) in the VEGFR-2 promoter. Using ChIP analyses, here we found that PKD knockdown in HUVECs increases binding of AP2β to the VEGFR-2 promoter. Luciferase reporter assays with serial deletions of AP2-binding sites within the VEGFR-2 promoter revealed that its transcriptional activity negatively correlates with the number of these sites. Next we demonstrated that AP2β up-regulation decreases VEGFR-2 expression and that loss of AP2β enhances VEGFR-2 expression in HUVECs. In vivo experiments confirmed increased VEGFR-2 immunostaining in the spinal cord of AP2β knockout mouse embryos. Mechanistically, we observed that PKD phosphorylates AP2β at Ser258 and Ser277 and suppresses its nuclear accumulation. Inhibition of PKD activity with a pan-PKD inhibitor increased AP2β nuclear localization, and overexpression of both WT and constitutively active PKD1 or PKD2 reduced AP2β nuclear localization through a Ser258- and Ser277-dependent mechanism. Furthermore, substitution of Ser277 in AP2β increased its binding to the VEGFR-2 promoter. Our findings uncover evidence of a molecular pathway that regulates VEGFR-2 expression, insights that may shed light on the etiology of diseases associated with aberrant VEGF/VEGFR signaling.
Collapse
Affiliation(s)
- Ying Wang
- Department of Biochemistry and Molecular Biology, College of Medicine and Science, Mayo Clinic, Jacksonville, Florida 32224
| | - Luke H Hoeppner
- Department of Biochemistry and Molecular Biology, College of Medicine and Science, Mayo Clinic, Rochester, Minnesota 55905
| | - Ramcharan Singh Angom
- Department of Biochemistry and Molecular Biology, College of Medicine and Science, Mayo Clinic, Jacksonville, Florida 32224
| | - Enfeng Wang
- Department of Biochemistry and Molecular Biology, College of Medicine and Science, Mayo Clinic, Jacksonville, Florida 32224
| | - Shamit Dutta
- Department of Biochemistry and Molecular Biology, College of Medicine and Science, Mayo Clinic, Jacksonville, Florida 32224
| | - Heike R Doeppler
- Department of Cancer Biology, College of Medicine and Science, Mayo Clinic, Jacksonville, Florida 32224
| | - Fei Wang
- Department of Biochemistry and Molecular Biology, College of Medicine and Science, Mayo Clinic, Jacksonville, Florida 32224
- Department of Neurosurgery, Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010050, China
| | - Tao Shen
- Department of Biochemistry and Molecular Biology, College of Medicine and Science, Mayo Clinic, Jacksonville, Florida 32224
- Department of Colorectal Surgery, Third Affiliated Hospital of Kunming Medical University, Kunming 650221, China
| | - Isobel A Scarisbrick
- Department of Physical Medicine and Rehabilitation, College of Medicine and Science, Mayo Clinic, Rochester, Minnesota 55905
| | - Sushovan Guha
- University of Arizona College of Medicine, Phoenix, Arizona 85004
| | - Peter Storz
- Department of Cancer Biology, College of Medicine and Science, Mayo Clinic, Jacksonville, Florida 32224
| | - Resham Bhattacharya
- Department of Biochemistry and Molecular Biology, College of Medicine and Science, Mayo Clinic, Rochester, Minnesota 55905
| | - Debabrata Mukhopadhyay
- Department of Biochemistry and Molecular Biology, College of Medicine and Science, Mayo Clinic, Jacksonville, Florida 32224
| |
Collapse
|
|
2
|
Pfister NT, Fomin V, Regunath K, Zhou JY, Zhou W, Silwal-Pandit L, Freed-Pastor WA, Laptenko O, Neo SP, Bargonetti J, Hoque M, Tian B, Gunaratne J, Engebraaten O, Manley JL, Børresen-Dale AL, Neilsen PM, Prives C. Mutant p53 cooperates with the SWI/SNF chromatin remodeling complex to regulate VEGFR2 in breast cancer cells. Genes Dev 2015; 29:1298-315. [PMID: 26080815 PMCID: PMC4495400 DOI: 10.1101/gad.263202.115] [Citation(s) in RCA: 111] [Impact Index Per Article: 11.1] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2015] [Accepted: 05/26/2015] [Indexed: 01/15/2023]
Abstract
In this study, Pfister et al. identified a new mutant p53 target gene, VEGFR2, and demonstrated that mutant p53 stimulates expression of VEGFR2 by cooperating with the SWI/SNF chromatin remodeling complex to superactivate the VEGFR2 gene. They also show that >50% of all mutant p53-regulated gene expression is mediated by SWI/SNF, providing insight into the observation that mutant p53 alters the expression of many genes. Mutant p53 impacts the expression of numerous genes at the level of transcription to mediate oncogenesis. We identified vascular endothelial growth factor receptor 2 (VEGFR2), the primary functional VEGF receptor that mediates endothelial cell vascularization, as a mutant p53 transcriptional target in multiple breast cancer cell lines. Up-regulation of VEGFR2 mediates the role of mutant p53 in increasing cellular growth in two-dimensional (2D) and three-dimensional (3D) culture conditions. Mutant p53 binds near the VEGFR2 promoter transcriptional start site and plays a role in maintaining an open conformation at that location. Relatedly, mutant p53 interacts with the SWI/SNF complex, which is required for remodeling the VEGFR2 promoter. By both querying individual genes regulated by mutant p53 and performing RNA sequencing, the results indicate that >40% of all mutant p53-regulated gene expression is mediated by SWI/SNF. We surmise that mutant p53 impacts transcription of VEGFR2 as well as myriad other genes by promoter remodeling through interaction with and likely regulation of the SWI/SNF chromatin remodeling complex. Therefore, not only might mutant p53-expressing tumors be susceptible to anti VEGF therapies, impacting SWI/SNF tumor suppressor function in mutant p53 tumors may also have therapeutic potential.
Collapse
Affiliation(s)
- Neil T Pfister
- Department of Biological Sciences, Columbia University, New York, New York 10027, USA
| | - Vitalay Fomin
- Department of Biological Sciences, Columbia University, New York, New York 10027, USA
| | - Kausik Regunath
- Department of Biological Sciences, Columbia University, New York, New York 10027, USA
| | - Jeffrey Y Zhou
- Department of Biological Sciences, Columbia University, New York, New York 10027, USA
| | - Wen Zhou
- Department of Biological Sciences, Columbia University, New York, New York 10027, USA
| | - Laxmi Silwal-Pandit
- Department of Genetics, Institute for Cancer Research, Oslo University Hospital, The Norwegian Radiumhospital, 0310 Oslo, Norway; The K.G. Jebsen Center for Breast Cancer Research, Faculty of Medicine, Institute for Clinical Medicine, University of Oslo, 0450 Oslo, Norway
| | - William A Freed-Pastor
- Department of Biological Sciences, Columbia University, New York, New York 10027, USA; Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114, USA
| | - Oleg Laptenko
- Department of Biological Sciences, Columbia University, New York, New York 10027, USA
| | - Suat Peng Neo
- Quantitative Proteomics Group, Institute of Molecular and Cell Biology, Agency for Science, Technology, and Research, Singapore S138673
| | - Jill Bargonetti
- Department of Biological Sciences, Hunter College, City University of New York, New York, New York 10065, USA
| | - Mainul Hoque
- Department of Microbiology, Biochemistry, and Molecular Genetics, Rutgers New Jersey Medical School, Newark, New Jersey 07103, USA
| | - Bin Tian
- Department of Microbiology, Biochemistry, and Molecular Genetics, Rutgers New Jersey Medical School, Newark, New Jersey 07103, USA
| | - Jayantha Gunaratne
- Quantitative Proteomics Group, Institute of Molecular and Cell Biology, Agency for Science, Technology, and Research, Singapore S138673; Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597
| | - Olav Engebraaten
- The K.G. Jebsen Center for Breast Cancer Research, Faculty of Medicine, Institute for Clinical Medicine, University of Oslo, 0450 Oslo, Norway; Department of Oncology, Oslo University Hospital, 0424 Oslo, Norway
| | - James L Manley
- Department of Biological Sciences, Columbia University, New York, New York 10027, USA
| | - Anne-Lise Børresen-Dale
- Department of Genetics, Institute for Cancer Research, Oslo University Hospital, The Norwegian Radiumhospital, 0310 Oslo, Norway; The K.G. Jebsen Center for Breast Cancer Research, Faculty of Medicine, Institute for Clinical Medicine, University of Oslo, 0450 Oslo, Norway
| | - Paul M Neilsen
- Swinburne University of Technology, Kuching 93350, Sarawak, Malaysia
| | - Carol Prives
- Department of Biological Sciences, Columbia University, New York, New York 10027, USA
| |
Collapse
|
|
3
|
Donovan K, Alekseev O, Qi X, Cho W, Azizkhan-Clifford J. O-GlcNAc modification of transcription factor Sp1 mediates hyperglycemia-induced VEGF-A upregulation in retinal cells. Invest Ophthalmol Vis Sci 2014; 55:7862-73. [PMID: 25352121 DOI: 10.1167/iovs.14-14048] [Citation(s) in RCA: 47] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
PURPOSE Proangiogenic protein VEGF-A contributes significantly to retinal lesions and neovascularization in diabetic retinopathy (DR). In preclinical DR, hyperglycemia can upregulate VEGF-A in retinal cells. The VEGF-A promoter is responsive to the transcription factor specificity protein 1 (Sp1). The O-GlcNAc modification is driven by glucose concentration and has a profound effect on Sp1 activity. This study investigated the effects of hyperglycemia on Sp1-mediated expression of VEGF-A in the retinal endothelium and pigment epithelium. METHODS Hyperglycemia-exposed ARPE-19 (human retinal pigment epithelial cells) and TR-iBRB (rat retinal microendothelial cells) were assayed for levels of VEGF-A by qRT-PCR, Western blot, and ELISA. Small molecule inhibitors of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) were used to manipulate O-GlcNAc levels. Vascular endothelial growth factor-A protein and transcript were measured in cells depleted of OGT or Sp1 by shRNA. The proximal VEGF-A promoter was analyzed for glucose sensitivity by luciferase assay. Chromatin immunoprecipitation (ChIP) was used to assess Sp1 occupancy on the VEGF-A promoter. RESULTS Hyperglycemia increased VEGF-A promoter activity and upregulated VEGF-A transcript and protein. Elevation of O-GlcNAc by OGA inhibitors was sufficient to increase VEGF-A. O-GlcNAc transferase inhibition abrogated glucose-driven VEGF-A. Cellular depletion of OGT or Sp1 by shRNA significantly abrogated glucose-induced changes in VEGF-A. ChIP analysis showed that hyperglycemia significantly increased binding of Sp1 to the VEGF-A promoter. CONCLUSIONS Hyperglycemia-driven VEGF-A production is mediated by elevated O-GlcNAc modification of the Sp1 transcription factor. This mechanism may be significant in the pathogenesis of preclinical DR through VEGF-A upregulation.
Collapse
Affiliation(s)
- Kelly Donovan
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States
| | - Oleg Alekseev
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States
| | - Xin Qi
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States Department of Ophthalmology, The Second Xiangya Hospital of Central South University, Changsha, Hunan, China
| | - William Cho
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States
| | - Jane Azizkhan-Clifford
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States
| |
Collapse
|
|
4
|
Murakami M, Nguyen LT, Hatanaka K, Schachterle W, Chen PY, Zhuang ZW, Black BL, Simons M. FGF-dependent regulation of VEGF receptor 2 expression in mice. J Clin Invest 2011; 121:2668-78. [PMID: 21633168 DOI: 10.1172/jci44762] [Citation(s) in RCA: 144] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2010] [Accepted: 04/13/2011] [Indexed: 01/19/2023] Open
Abstract
Numerous studies have suggested a link between the angiogenic FGF and VEGF signaling pathways; however, the nature of this link has not been established. To evaluate this relationship, we investigated VEGF signaling in ECs with disrupted FGF signaling in vitro and in vivo. ECs lacking FGF signaling became unresponsive to VEGF, caused by downregulation of VEGF receptor 2 (VEGFR2) expression after reduced Vegfr2 enhancer activation. FGF mediated VEGFR2 expression via activation of Erk1/2. Transcriptional analysis revealed that Ets transcription factors controlled VEGFR2 expression in an FGF- and Erk1/2-dependent manner. Mice with defective FGF signaling exhibited loss of vascular integrity and reduced vascular morphogenesis. Thus, basal FGF stimulation of the endothelium is required for maintenance of VEGFR2 expression and the ability to respond to VEGF stimulation and accounts for the hierarchic control of vascular formation by FGFs and VEGF.
Collapse
Affiliation(s)
- Masahiro Murakami
- Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA
| | | | | | | | | | | | | | | |
Collapse
|
|
5
|
Domingues I, Rino J, Demmers JAA, de Lanerolle P, Santos SCR. VEGFR2 translocates to the nucleus to regulate its own transcription. PLoS One 2011; 6:e25668. [PMID: 21980525 PMCID: PMC3182252 DOI: 10.1371/journal.pone.0025668] [Citation(s) in RCA: 78] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2011] [Accepted: 09/09/2011] [Indexed: 01/01/2023] Open
Abstract
Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) is the major mediator of the angiogenic effects of VEGF. In addition to its well known role as a membrane receptor that activates multiple signaling pathways, VEGFR2 also has a nuclear localization. However, what VEGFR2 does in the nucleus is still unknown. In the present report we show that, in endothelial cells, nuclear VEGFR2 interacts with several nuclear proteins, including the Sp1, a transcription factor that has been implicated in the regulation of genes needed for angiogenesis. By in vivo chromatin immunoprecipitation (ChIP) assays, we found that VEGFR2 binds to the Sp1-responsive region of the VEGFR2 proximal promoter. These results were confirmed by EMSA assays, using the same region of the VEGFR2 promoter. Importantly, we show that the VEGFR2 DNA binding is directly linked to the transcriptional activation of the VEGFR2 promoter. By reporter assays, we found that the region between -300/-116 relative to the transcription start site is essential to confer VEGFR2-dependent transcriptional activity. It was previously described that nuclear translocation of the VEGFR2 is dependent on its activation by VEGF. In agreement, we observed that the binding of VEGFR2 to DNA requires VEGF activation, being blocked by Bevacizumab and Sunitinib, two anti-angiogenic agents that inhibit VEGFR2 activation. Our findings demonstrate a new mechanism by which VEGFR2 activates its own promoter that could be involved in amplifying the angiogenic response.
Collapse
Affiliation(s)
- Inês Domingues
- Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisbon, Portugal
| | - José Rino
- Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisbon, Portugal
| | - Jeroen A. A. Demmers
- Proteomics Center, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Primal de Lanerolle
- Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois, United States of America
| | | |
Collapse
|
|
6
|
Guo S, Colbert LS, Fuller M, Zhang Y, Gonzalez-Perez RR. Vascular endothelial growth factor receptor-2 in breast cancer. BIOCHIMICA ET BIOPHYSICA ACTA 2010; 1806:108-21. [PMID: 20462514 PMCID: PMC2885515 DOI: 10.1016/j.bbcan.2010.04.004] [Citation(s) in RCA: 115] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 01/28/2010] [Revised: 04/16/2010] [Accepted: 04/21/2010] [Indexed: 12/31/2022]
Abstract
Investigations over the last decade have established the essential role of growth factors and their receptors during angiogenesis and carcinogenesis. The vascular endothelial growth factor receptor (VEGFR) family in mammals contains three members, VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR-3 (Flt-4), which are transmembrane tyrosine kinase receptors that regulate the formation of blood and lymphatic vessels. In the early 1990s, the above VEGFR was structurally characterized by cDNA cloning. Among these three receptors, VEGFR-2 is generally recognized to have a principal role in mediating VEGF-induced responses. VEGFR-2 is considered as the earliest marker for endothelial cell development. Importantly, VEGFR-2 directly regulates tumor angiogenesis. Therefore, several inhibitors of VEGFR-2 have been developed and many of them are now in clinical trials. In addition to targeting endothelial cells, the VEGF/VEGFR-2 system works as an essential autocrine/paracrine process for cancer cell proliferation and survival. Recent studies mark the continuous and increased interest in this related, but distinct, function of VEGF/VEGFR-2 in cancer cells: the autocrine/paracrine loop. Several mechanisms regulate VEGFR-2 levels and modulate its role in tumor angiogenesis and physiologic functions, i.e.: cellular localization/trafficking, regulation of cis-elements of promoter, epigenetic regulation and signaling from Notch, cytokines/growth factors and estrogen, etc. In this review, we will focus on updated information regarding VEGFR-2 research with respect to the molecular mechanisms of VEGFR-2 regulation in human breast cancer. Investigations in the activation, function, and regulation of VEGFR-2 in breast cancer will allow the development of new pharmacological strategies aimed at directly targeting cancer cell proliferation and survival.
Collapse
Affiliation(s)
- Shanchun Guo
- Microbiology, Biochemistry & Immunology, Morehouse School of Medicine, Atlanta, GA 30310
| | - Laronna S. Colbert
- Clinical Medicine, Hematology/Oncology Section, Morehouse School of Medicine, Atlanta, GA 30310
| | - Miles Fuller
- Microbiology, Biochemistry & Immunology, Morehouse School of Medicine, Atlanta, GA 30310
| | - Yuanyuan Zhang
- Microbiology, Biochemistry & Immunology, Morehouse School of Medicine, Atlanta, GA 30310
| | | |
Collapse
|
|
7
|
PRH/Hhex controls cell survival through coordinate transcriptional regulation of vascular endothelial growth factor signaling. Mol Cell Biol 2010; 30:2120-34. [PMID: 20176809 DOI: 10.1128/mcb.01511-09] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The proline-rich homeodomain protein (PRH) plays multiple roles in the control of gene expression during embryonic development and in the adult. Vascular endothelial growth factor (VEGF) is a mitogen that stimulates cell proliferation and survival via cell surface receptors including VEGFR-1 and VEGFR-2. VEGF signaling is of critical importance in angiogenesis and hematopoiesis and is elevated in many tumors. Here we show that PRH binds directly to the promoter regions of the Vegf, Vegfr-1, and Vegfr-2 genes and that in each case PRH represses transcription. We demonstrate that overexpression or knockdown of PRH directly impinges on the survival of both leukemic and tumor cells and that the modulation of VEGF and VEGF receptor signaling by PRH mediates these effects. Our findings demonstrate that PRH is a key regulator of the VEGF signaling pathway and describe a mechanism whereby PRH plays an important role in tumorigenesis and leukemogenesis.
Collapse
|
|
8
|
Le Bras A, Soncin F. [Genes that make the endothelial identity]. JOURNAL DE LA SOCIETE DE BIOLOGIE 2009; 203:125-41. [PMID: 19527626 DOI: 10.1051/jbio/2009016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/14/2022]
Abstract
The endothelium is a tissue with a distinct identity due to the specific expression of molecular markers by endothelial cells. Further, the endothelium displays a structural heterogeneity illustrated by the expression of specific markers in arteries and in veins. Here, we present a review of the transcriptional and epigenetic mechanisms regulating the expression of the main markers of endothelial cells in man and mouse, demonstrating that there is no common and unique mechanism of specific expression of genes in these cells.
Collapse
Affiliation(s)
- Alexandra Le Bras
- Institut de Biologie de Lille, CNRS UMR8161, Equipe Labellisée Ligue Nationale contre le Cancer 2008, Université de Lille I, Université de Lille II, Institut Pasteur de Lille, 1, rue Calmette, 59021 Lille Cedex, France
| | | |
Collapse
|
|
9
|
Higgins KJ, Liu S, Abdelrahim M, Vanderlaag K, Liu X, Porter W, Metz R, Safe S. Vascular endothelial growth factor receptor-2 expression is down-regulated by 17beta-estradiol in MCF-7 breast cancer cells by estrogen receptor alpha/Sp proteins. Mol Endocrinol 2008; 22:388-402. [PMID: 18006642 PMCID: PMC2234589 DOI: 10.1210/me.2007-0319] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2007] [Accepted: 11/05/2007] [Indexed: 02/08/2023] Open
Abstract
17beta-Estradiol (E2) induces and represses gene expression in breast cancer cells; however, the mechanisms of gene repression are not well understood. In this study, we show that E2 decreases vascular endothelial growth factor receptor 2 (VEGFR2) mRNA levels in MCF-7 cells, and this gene was used as a model for investigating pathways associated with E2-dependent gene repression. Deletion analysis of the VEGFR2 promoter indicates that the proximal GC-rich motifs at -58 and -44 are critical for the E2-dependent decreased response in MCF-7 cells. Mutation or deletion of these GC-rich elements results in loss of hormone responsiveness and shows that the -60 to -37 region of the VEGFR2 promoter is critical for both basal and hormone-dependent decreased VEGFR2 expression in MCF-7 cells. Western blot, immunofluorescent staining, RNA interference, and EMSAs support a role for Sp proteins in hormone-dependent down-regulation of VEGFR2 in MCF-7 cells, primarily through estrogen receptor (ER)alpha/Sp1 and ERalpha/Sp3 interactions with the VEGFR2 promoter. Using chromatin immuno-precipitation and transient transfection/RNA interference assays we show that the ERalpha/Sp protein-promoter interactions are accompanied by recruitment of the co-repressors SMRT (silencing mediator of retinoid and thyroid hormone receptor) and NCoR (nuclear receptor corepressor) to the promoter and that SMRT and NCoR knockdown reverse E2-mediated down-regulation of VEGFR2 expression in MCF-7 cells. This study illustrates that both SMRT and NCoR are involved in E2-dependent repression of VEGFR2 in MCF-7 cells.
Collapse
Affiliation(s)
- Kelly J Higgins
- Department of Veterinary Physiology and Pharmacology, Texas A&M University, 4466 TAMU, College Station, TX 77843-4466, USA
| | | | | | | | | | | | | | | |
Collapse
|
|
10
|
Li BJ, Zhang C, Yi YX, Hao Y, Liu XP, Ou QJ. Vascular damage and anti-angiogenic effects of tumor vessel-targeted adenovirus-mediated herpes simplex virus thymidine kinase gene. World J Gastroenterol 2007; 13:4006-10. [PMID: 17663519 PMCID: PMC4171177 DOI: 10.3748/wjg.v13.i29.4006] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To explore the therapeutic efficacy and mechanism of herpes simplex virus-thymidine kinase (HSV-tk) targeting angiogenesis against hepatocellular carcinoma in vivio and in vitro.
METHODS: Recombinant adenovirus containing kinase domain insert with receptor (KDR) or cytomegalovirus (CMV) promoter-controlled HSV-tk gene (AdKDR-tk and AdCMV-tk) was constructed using pAdeasy system. The expression of KDR antigen in human umbilical venous endothelial cells (HUVEC) and HepG2 was detected with histological analysis of cells. The virus was used to infect HUVEC and HepG2. Following administration of ganciclovir (GCV), the survival rate of gene-transfected HUVEC and HepG2 was evaluated by MTT method. To develop hepatocarcinomas in 32 Balb/C mice with HepG2 cells, the mice were divided into four groups: ganciclovir group (I), Ad group (II), AdCMV-tk group (III) and AdKDR-tk group (IV). Then selective administration of recombinant adenovirus or Ad via the intratumorial was given to all rats. Ganciclovir (GCV) was given at a dose of 100 mg·kg-1·d-1 (ip) started on the following day and lasted 10 d. Microvessel density (MVD) of tumor in all the treated animals were examined by the immunohistochemical methods and tumor burden was evaluated 10 d before and after the last GCV dose.
RESULTS: Immunocytochemical staining indicated the expression of KDR antigen in HUVEC. Under adenovirus infection index of 100, with increasing GCV concentration from 0 up to 50 mg/L, the survival rate of AdKDR-tk-transfected HUVEC and HepG2 decreased from 100% to (28.94 ± 5.67)% and (75.45 ± 2.91)% at proper order, respectively (P < 0.01), while the survival rate of AdCMV-tk-transfected HUVEC and HepG2 declined from 100% to (17.56 ± 2.48)% and (23.15 ± 5.72)%, respectively (P > 0.05). Compared with groupI, there was a decrease of tumor weight by 14.7% in group III and by 23.6% in group IV. And there was a distinct difference between group III and IV (P < 0.05). The median MVD for all groups was 37.4 ± 8.6, 30.6 ± 7.8, 27.6 ± 7.1, and 10.7 ± 4.1 (microvessels/mm2) in groupI, II, III and IV, respectively. And there was a marked difference between group III and II (P < 0.05), IV and II (P < 0.01), and IV and III (P < 0.01).
CONCLUSION: KDR promoter-HSV-tk gene may effectually restrain the growth of tumor via targeting angiogenesis for hepatocellular carcinoma with treatment of GCV.
Collapse
Affiliation(s)
- Bao-Jin Li
- Department of Hepatobillary and Pancreatic Surgery, Peking University Shenzhen Hospital, Shenzhen PKU-HKUST Medical Center, No. 1120, Lianhua Road, Shenzhen 518036, Guangdong Province, China.
| | | | | | | | | | | |
Collapse
|
|
11
|
Brockington A, Wokke B, Nixon H, Hartley J, Shaw PJ. Screening of the transcriptional regulatory regions of vascular endothelial growth factor receptor 2 (VEGFR2) in amyotrophic lateral sclerosis. BMC MEDICAL GENETICS 2007; 8:23. [PMID: 17456229 PMCID: PMC1868706 DOI: 10.1186/1471-2350-8-23] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/14/2006] [Accepted: 04/24/2007] [Indexed: 11/10/2022]
Abstract
BACKGROUND Vascular endothelial growth factor (VEGF) has neurotrophic activity which is mediated by its main agonist receptor, VEGFR2. Dysregulation of VEGF causes motor neurone degeneration in a mouse model of amyotrophic lateral sclerosis (ALS), and expression of VEGFR2 is reduced in motor neurones and spinal cord of patients with ALS. METHODS We have screened the promoter region and 4 exonic regions of functional significance of the VEGFR2 gene in a UK population of patients with ALS, for mutations and polymorphisms that may affect expression or function of this VEGF receptor. RESULTS No mutations were identified in the VEGFR2 gene. We found no association between polymorphisms in the regulatory regions of the VEGFR2 gene and ALS. CONCLUSION Mechanisms other than genetic variation may downregulate expression or function of the VEGFR2 receptor in patients with ALS.
Collapse
Affiliation(s)
- Alice Brockington
- Academic Neurology Unit, University of Sheffield, E Floor, Medical School, Beech Hill Road, Sheffield S10 2RX, UK
| | - Beatrijs Wokke
- Leiden University Medical Centre, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
| | - Hannah Nixon
- Academic Neurology Unit, University of Sheffield, E Floor, Medical School, Beech Hill Road, Sheffield S10 2RX, UK
| | - Judith Hartley
- Academic Neurology Unit, University of Sheffield, E Floor, Medical School, Beech Hill Road, Sheffield S10 2RX, UK
| | - Pamela J Shaw
- Academic Neurology Unit, University of Sheffield, E Floor, Medical School, Beech Hill Road, Sheffield S10 2RX, UK
| |
Collapse
|
|
12
|
Abdelrahim M, Baker CH, Abbruzzese JL, Sheikh-Hamad D, Liu S, Cho SD, Yoon K, Safe S. Regulation of vascular endothelial growth factor receptor-1 expression by specificity proteins 1, 3, and 4 in pancreatic cancer cells. Cancer Res 2007; 67:3286-3294. [PMID: 17409437 DOI: 10.1158/0008-5472.can-06-3831] [Citation(s) in RCA: 87] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Vascular endothelial growth factor receptor-1 (VEGFR1) is expressed in cancer cell lines and tumors and, in pancreatic and colon cancer cells, activation of VEGFR1 is linked to increased tumor migration and invasiveness. Tolfenamic acid, a nonsteroidal anti-inflammatory drug, decreases Sp protein expression in Panc-1 and L3.6pl pancreatic cancer cells, and this was accompanied by decreased VEGFR1 protein and mRNA and decreased luciferase activity on cells transfected with constructs (pVEGFR1) containing VEGFR1 promoter inserts. Comparable results were obtained in pancreatic cancer cells transfected with small inhibitory RNAs for Sp1, Sp3, and Sp4 and all three proteins bound to GC-rich elements in the VEGFR1 promoter. These results show that VEGFR1 is regulated by Sp proteins and that treatment with tolfenamic acid decreases expression of this critical angiogenic factor. Moreover, in vitro studies in Panc-1 cells show that activation of VEGFR1 by VEGFB to increase mitogen-activated protein kinase 1/2 phosphorylation and cell migration on collagen-coated plates is also inhibited by tolfenamic acid. Thus, targeted degradation of Sp proteins is highly effective for inhibiting VEGFR1 and associated angiogenic responses in pancreatic cancer.
Collapse
Affiliation(s)
- Maen Abdelrahim
- Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas, USA
| | | | | | | | | | | | | | | |
Collapse
|
|
13
|
Adenovirus-mediated herpes simplex virus thymidine kinase gene transfer driver by KDR promoter in treatment of experimental human hepatocelLular carcinoma in nude mice. Chin J Cancer Res 2007. [DOI: 10.1007/s11670-007-0022-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022] Open
|
|
14
|
Higgins KJ, Liu S, Abdelrahim M, Yoon K, Vanderlaag K, Porter W, Metz RP, Safe S. Vascular endothelial growth factor receptor-2 expression is induced by 17beta-estradiol in ZR-75 breast cancer cells by estrogen receptor alpha/Sp proteins. Endocrinology 2006; 147:3285-3295. [PMID: 16574784 DOI: 10.1210/en.2006-0081] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Vascular endothelial growth factor receptor-2 kinase insert domain receptor (VEGFR2/KDR) is critical for angiogenesis, and VEGFR2 mRNA and protein are expressed in ZR-75 breast cancer cells and induced by 17beta-estradiol (E2). Deletion analysis of the VEGFR2 promoter indicates that the proximal GC-rich region is required for both basal and hormone-induced transactivation, and mutation of one or both of the GC-rich motifs at -58 and -44 results in loss of transactivation. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that Sp1, Sp3, and Sp4 proteins bind the GC-rich region of the VEGFR2 promoter. Results of the chromatin immunoprecipitation assay also demonstrate that ERalpha is constitutively bound to the VEGFR2 promoter and that these interactions are not enhanced after treatment with E2, whereas ERalpha binding to the region of the pS2 promoter containing an estrogen-responsive element is enhanced by E2. RNA interference studies show that hormone-induced activation of the VEGFR2 promoter constructs requires Sp3 and Sp4 but not Sp1, demonstrating that hormonal activation of VEGFR2 involves a nonclassical mechanism in which ERalpha/Sp3 and ERalpha/Sp4 complexes activate GC-rich sites where Sp proteins but not ERalpha bind DNA. These results show for the first time that Sp3 and Sp4 cooperatively interact with ERalpha to activate VEGFR2 and are in contrast to previous results showing that several hormone-responsive genes are activated by ERalpha/Sp1 in breast cancer cell lines.
Collapse
Affiliation(s)
- Kelly J Higgins
- Department of Biochemistry, Texas A&M University, College Station, 77843-4466, USA
| | | | | | | | | | | | | | | |
Collapse
|
|
15
|
Higgins KJ, Abdelrahim M, Liu S, Yoon K, Safe S. Regulation of vascular endothelial growth factor receptor-2 expression in pancreatic cancer cells by Sp proteins. Biochem Biophys Res Commun 2006; 345:292-301. [PMID: 16678129 DOI: 10.1016/j.bbrc.2006.04.111] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2006] [Accepted: 04/18/2006] [Indexed: 11/19/2022]
Abstract
Vascular endothelial growth factor receptor-2 (VEGFR2/KDR) is an important mediator of angiogenesis, and VEGFR2 mRNA is expressed in several pancreatic cancer cell lines. Deletion analysis of the VEGFR2 promoter in Panc-1, AsPC-1, and MiaPaCa-2 pancreatic cancer cells shows that the proximal region of the promoter is primarily responsible for VEGFR2 expression, and two GC-rich sites at -58 and -44 are critical elements in all three cell lines. Panc-1, AsPC-1, and MiaPaCa-2 cells also express Sp1, Sp3, and Sp4 proteins which bind to the GC-rich region of the VEGFR2 promoter in electrophoretic mobility shift and chromatin immunoprecipitation assays. RNA interference with small inhibitory RNAs for Sp1, Sp3, and Sp4 decreases VEGFR2 mRNA and reporter gene activity in transfection assays, confirming that VEGFR2 expression in pancreatic cancer cells is regulated by Sp proteins. These results suggest that VEGFR2 cannot only be targeted by receptor tyrosine kinase inhibitors but also by drugs that downregulate Sp proteins or block Sp-dependent transactivation.
Collapse
Affiliation(s)
- Kelly J Higgins
- Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, USA
| | | | | | | | | |
Collapse
|
|
16
|
Bhattacharya R, Senbanerjee S, Lin Z, Mir S, Hamik A, Wang P, Mukherjee P, Mukhopadhyay D, Jain MK. Inhibition of vascular permeability factor/vascular endothelial growth factor-mediated angiogenesis by the Kruppel-like factor KLF2. J Biol Chem 2005; 280:28848-51. [PMID: 15980434 DOI: 10.1074/jbc.c500200200] [Citation(s) in RCA: 138] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
The Kruppel-like factor KLF2 was recently identified as a novel regulator of endothelial pro-inflammatory and pro-thrombotic function. Here it is shown that overexpression of KLF2 potently inhibits vascular permeability factor/vascular endothelial growth factor (VEGF-A)-mediated angiogenesis and tissue edema in the nude ear mouse model of angiogenesis. In vitro, KLF2 expression retards VEGF-mediated calcium flux, proliferation and induction of pro-inflammatory factors in endothelial cells. This effect is due to a potent inhibition of VEGFR2/KDR expression and promoter activity. These observations identify KLF2 as a regulator of VEGFR2/KDR and provide a foundation for novel approaches to regulate angiogenesis.
Collapse
Affiliation(s)
- Resham Bhattacharya
- Program in Cardiovascular Transcriptional Biology, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA
| | | | | | | | | | | | | | | | | |
Collapse
|
|
17
|
Wu J, Iwata F, Grass JA, Osborne CS, Elnitski L, Fraser P, Ohneda O, Yamamoto M, Bresnick EH. Molecular determinants of NOTCH4 transcription in vascular endothelium. Mol Cell Biol 2005; 25:1458-74. [PMID: 15684396 PMCID: PMC548019 DOI: 10.1128/mcb.25.4.1458-1474.2005] [Citation(s) in RCA: 58] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
The process whereby the primitive vascular network develops into the mature vasculature, known as angiogenic vascular remodeling, is controlled by the Notch signaling pathway. Of the two mammalian Notch receptors expressed in vascular endothelium, Notch1 is broadly expressed in diverse cell types, whereas Notch4 is preferentially expressed in endothelial cells. As mechanisms that confer Notch4 expression were unknown, we investigated how NOTCH4 transcription is regulated in human endothelial cells and in transgenic mice. The NOTCH4 promoter and the 5' portion of NOTCH4 assembled into an endothelial cell-specific histone modification pattern. Analysis of NOTCH4 primary transcripts in human umbilical vein endothelial cells by RNA fluorescence in situ hybridization revealed that 36% of the cells transcribed one or both NOTCH4 alleles. The NOTCH4 promoter was sufficient to confer endothelial cell-specific transcription in transfection assays, but intron 1 or upstream sequences were required for expression in the vasculature of transgenic mouse embryos. Cell-type-specific activator protein 1 (AP-1) complexes occupied NOTCH4 chromatin and conferred endothelial cell-specific transcription. Vascular angiogenic factors activated AP-1 and reprogrammed the endogenous NOTCH4 gene in HeLa cells from a repressed to a transcriptionally active state. These results reveal an AP-1-Notch4 pathway, which we propose to be crucial for transducing angiogenic signals and to be deregulated upon aberrant signal transduction in cancer.
Collapse
MESH Headings
- 5' Flanking Region/genetics
- Alleles
- Animals
- Cells, Cultured
- Embryo, Mammalian/cytology
- Embryo, Mammalian/metabolism
- Endothelial Cells/metabolism
- HeLa Cells
- Histones/genetics
- Histones/metabolism
- Humans
- In Situ Hybridization, Fluorescence
- Introns/genetics
- Mice
- Mice, Transgenic
- Promoter Regions, Genetic
- Proto-Oncogene Proteins/genetics
- Proto-Oncogene Proteins/metabolism
- Receptor, Notch1
- Receptor, Notch4
- Receptors, Cell Surface/genetics
- Receptors, Cell Surface/metabolism
- Receptors, Notch
- Transcription Factor AP-1/genetics
- Transcription Factor AP-1/metabolism
- Transcription Factors/genetics
- Transcription Factors/metabolism
- Transcription, Genetic/genetics
Collapse
Affiliation(s)
- Jing Wu
- Molecular and Cellular Pharmacology Program, Department of Pharmacology, University of Wisconsin Medical School, 1300 University Ave., 383 Medical Sciences Center, Madison, WI 53706, USA.
| | | | | | | | | | | | | | | | | |
Collapse
|
|
18
|
Seki T, Hong KH, Yun J, Kim SJ, Oh SP. Isolation of a Regulatory Region of Activin Receptor-Like Kinase 1 Gene Sufficient for Arterial Endothelium-Specific Expression. Circ Res 2004; 94:e72-7. [PMID: 15059937 DOI: 10.1161/01.res.0000127048.81744.31] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Activin receptor-like kinase 1 (Acvrl1; Alk1) is a type I receptor for transforming growth factor-beta (TGF-beta). ALK1 plays a pivotal role in vascular development and is involved in the development of hereditary hemorrhagic telangiectasia 2 (HHT2), a dominantly inherited vascular disorder, and pulmonary hypertension. We have previously shown that Alk1 is expressed predominantly in arterial endothelial cells (ECs). Despite recent discoveries of a number of artery-specific genes, the regulatory elements of these genes have not been characterized. To investigate the cis-acting elements essential for the artery-specific Alk1 expression, we have generated a series of transgenic constructs with various lengths and regions of Alk1 genomic fragments connected to a LacZ reporter gene, and analyzed the reporter gene expression in transgenic mice. We found that a 9.2-kb genomic fragment, which includes 2.7-kb promoter region and the entire intron 2, is sufficient to drive arterial endothelium-specific expression. The defined regulatory region, as well as the transgenic mouse lines, would be invaluable resources in studying the mechanisms underlying angiogenesis, arteriogenesis, and vascular disorders, such as HHT and pulmonary hypertension. The full text of this article is available online at http://circres.ahajournals.org.
Collapse
MESH Headings
- Activin Receptors, Type I/biosynthesis
- Activin Receptors, Type I/genetics
- Activin Receptors, Type I/physiology
- Activin Receptors, Type II
- Animals
- Arteries/cytology
- Arteries/embryology
- Arteries/growth & development
- Arteries/metabolism
- Binding Sites
- Consensus Sequence
- Endothelium, Vascular/metabolism
- Exons/genetics
- Female
- Gene Expression Regulation, Developmental
- Genes, Reporter
- Humans
- Introns/genetics
- Lac Operon
- Male
- Mice
- Mice, Transgenic
- Neovascularization, Physiologic/genetics
- Organ Specificity
- Promoter Regions, Genetic/genetics
- Regulatory Sequences, Nucleic Acid
- Sequence Alignment
- Sequence Homology
- Skin/injuries
- Species Specificity
- Transcription Factors/metabolism
- Wound Healing/genetics
Collapse
Affiliation(s)
- Tsugio Seki
- Department of Physiology and Functional Genomics, University of Florida,Gainesville, Fla 32610, USA
| | | | | | | | | |
Collapse
|
|
19
|
Wu Y, Moser M, Bautch VL, Patterson C. HoxB5 is an upstream transcriptional switch for differentiation of the vascular endothelium from precursor cells. Mol Cell Biol 2003; 23:5680-91. [PMID: 12897140 PMCID: PMC166331 DOI: 10.1128/mcb.23.16.5680-5691.2003] [Citation(s) in RCA: 67] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Endothelial cells differentiate from mesoderm-derived precursors to initiate the earliest events in vascular development. Although the signaling events that regulate the successive steps of vascular development are known in some detail, the transcriptional processes that regulate the first steps in vasculogenesis are not well defined. We have studied the regulatory mechanisms of flk1 expression as a model to understand the upstream events in endothelial cell differentiation, since flk1 is the earliest marker of endothelial precursors. Using a variety of biochemical approaches, we identified a cis-acting element in the first intron of the flk1 gene that is required for endothelium-dependent expression in transgenic reporter gene assays. Using the yeast one-hybrid system, we identified HoxB5 as the transcription factor that binds this cis-acting element, the HoxB5-binding element (HBE). HoxB5 mRNA colocalized with flk1 expression in differentiating embryoid bodies, and HoxB5 potently transactivated the flk1 promoter in an HBE-dependent fashion in transient-transfection assays. Overexpression of HoxB5 led to expansion of flk1(+) angioblasts in differentiating embryoid bodies and increased the number of PECAM (platelet-endothelial cell adhesion molecule)-positive primitive blood vessels. HoxB5 is necessary and sufficient to activate the cell-intrinsic events that regulate the differentiation of angioblasts and mature endothelial cells from their mesoderm-derived precursors.
Collapse
Affiliation(s)
- Yaxu Wu
- Carolina Cardiovascular Biology Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA
| | | | | | | |
Collapse
|
|
20
|
Rance JB, Follows GA, Cockerill PN, Bonifer C, Lane DA, Simmonds RE. Regulation of the human endothelial cell protein C receptor gene promoter by multiple Sp1 binding sites. Blood 2003; 101:4393-401. [PMID: 12560236 DOI: 10.1182/blood-2002-05-1570] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The human endothelial cell protein C receptor (hEPCR) is normally expressed by the endothelium of large blood vessels, but the molecular basis for its in vivo specificity is uncertain. In this study, DNaseI hypersensitive site mapping demonstrated the presence of a hypersensitive site in the 5' flanking region of the hEPCR gene in endothelial cells and certain transformed cells (HeLa and U937) known to express hEPCR in vitro. Conversely, this site was only weakly hypersensitive in HepG2 cells, cells which do not express hEPCR mRNA. Functional analysis of this 5' flanking region by in vivo dimethylsulfate footprinting in cultured endothelial cells identified multiple regions, containing high and low homology consensus Sp1 binding sequences, that were protected from methylation in endothelial cells. These sequences were not protected in HepG2 cells. Reporter gene analysis of this region in endothelial cells demonstrated the presence of promoter activity conferred by the proximal 572 bp but failed to identify a functional TATA-box. This promoter was inactive in HepG2 cells. Electrophoresis mobility shift assays using endothelial cell nuclear extracts identified Sp1 family proteins binding to sites that were protected during footprinting. Sp1 sites were identified in regions at -368, -232, -226, -201, -146, and -102 bp relative to the translation start site. With the exception of the site at -102 bp, each identified Sp1 binding site made a positive contribution to reporter gene expression, although no individual site was critically important. We conclude that transcription factor binding to Sp1 binding sites in the 5' flanking region is critical for normal hEPCR gene expression in endothelial cells.
Collapse
Affiliation(s)
- James B Rance
- Department of Haematology, Imperial College London-Faculty of Medicine, Hammersmith Hospital Campus, London, United Kingdom.
| | | | | | | | | | | |
Collapse
|
|
21
|
Urbich C, Stein M, Reisinger K, Kaufmann R, Dimmeler S, Gille J. Fluid shear stress-induced transcriptional activation of the vascular endothelial growth factor receptor-2 gene requires Sp1-dependent DNA binding. FEBS Lett 2003; 535:87-93. [PMID: 12560084 DOI: 10.1016/s0014-5793(02)03879-6] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Hemodynamic forces play a fundamental role in the regulation of endothelial cell survival. As signaling via the vascular endothelial growth factor (VEGF) receptor-2 pathway has been previously demonstrated to impact endothelial cell survival, we hypothesized that laminar shear stress may facilitate survival in part by inducing VEGF receptor-2 expression. This study shows a time- and dose-dependent upregulation of endothelial VEGF receptor-2 expression by fluid shear stress in microvascular and large-vessel derived endothelial cells. A functional analysis of the 5'-regulatory region of the VEGF receptor-2 promoter localized the shear stress-response element to a sequence between bp -60 and -37 that encompasses two adjacent consensus Sp1 transcription factor binding sites. Constitutive and shear stress-inducible Sp1-dependent complexes are bound to this element, indicating that fluid shear stress-induced transcriptional activation of the VEGF receptor-2 gene requires Sp1-dependent DNA binding. Together, these results suggest that biomechanical stimulation may lead to endothelial cell survival by upregulating VEGF receptor-2 expression.
Collapse
Affiliation(s)
- Carmen Urbich
- Molecular Cardiology, Department of Internal Medicine IV, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany
| | | | | | | | | | | |
Collapse
|
|
22
|
Abumiya T, Sasaguri T, Taba Y, Miwa Y, Miyagi M. Shear stress induces expression of vascular endothelial growth factor receptor Flk-1/KDR through the CT-rich Sp1 binding site. Arterioscler Thromb Vasc Biol 2002; 22:907-13. [PMID: 12067897 DOI: 10.1161/01.atv.0000018300.43492.83] [Citation(s) in RCA: 42] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Fluid shear stress is 1 of the major factors that control gene expression in vascular endothelial cells. We investigated the role of shear stress in the regulation of the expression of fetal liver kinase-1/kinase domain region (Flk-1/KDR), a vascular endothelial growth factor receptor, by using human umbilical vein endothelial cells. Laminar shear stress (15 dyne/cm2) elevated Flk-1/KDR mRNA levels by approximately 3-fold for 8 hours, and the expression was upregulated within the range of 5 to 40 dyne/cm2. Deletion analysis of the 5'-flanking region of the Flk-1/KDR gene promoter by use of a luciferase reporter vector revealed that a shear stress-responsive element resided in the sequence between -94 and -31 bp, which contained putative nuclear factor-kappaB, activator protein-2, and GC-rich Sp1 and CT-rich Sp1 binding sites. Electrophoretic mobility shift assay demonstrated that nuclear extract was bound to the GC-rich Sp1 sites and the CT-rich Sp1 site with a similar pattern. However, shear stress enhanced the DNA-protein interactions only on the CT-rich Sp1 site but not on the GC-rich Sp1 sites. A 3-bp mutation in the CT-rich Sp1 site eliminated the response to shear stress in electrophoretic mobility shift assay and luciferase reporter assay. These results suggest that shear stress induces Flk-1/KDR expression through the CT-rich Sp1 binding site.
Collapse
MESH Headings
- Binding Sites/genetics
- Binding Sites/physiology
- Cells, Cultured
- Cytosine/physiology
- Electrophoretic Mobility Shift Assay/methods
- Endothelial Growth Factors/metabolism
- Endothelium, Vascular/chemistry
- Endothelium, Vascular/cytology
- Endothelium, Vascular/enzymology
- Endothelium, Vascular/metabolism
- Humans
- Lymphokines/metabolism
- Mutagenesis, Site-Directed/genetics
- Mutagenesis, Site-Directed/physiology
- Promoter Regions, Genetic/genetics
- Promoter Regions, Genetic/physiology
- Protein Kinases/genetics
- Protein Kinases/metabolism
- Receptor Protein-Tyrosine Kinases/biosynthesis
- Receptors, Growth Factor/biosynthesis
- Receptors, Mitogen/biosynthesis
- Receptors, Vascular Endothelial Growth Factor
- Rheology
- Stress, Mechanical
- Thymine/physiology
- Umbilical Veins/cytology
- Vascular Endothelial Growth Factor A
- Vascular Endothelial Growth Factors
Collapse
Affiliation(s)
- Takeo Abumiya
- National Cardiovascular Center Research Institute, Osaka, Japan.
| | | | | | | | | |
Collapse
|
|
23
|
Nawrocki AR, Goldring CE, Kostadinova RM, Frey FJ, Frey BM. In vivo footprinting of the human 11beta-hydroxysteroid dehydrogenase type 2 promoter: evidence for cell-specific regulation by Sp1 and Sp3. J Biol Chem 2002; 277:14647-56. [PMID: 11850421 DOI: 10.1074/jbc.m111549200] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
11beta-Hydroxysteroid dehydrogenase type 2 is selectively expressed in aldosterone target tissues, where it confers aldosterone selectivity for the mineralocorticoid receptor by inactivating 11beta-hydroxyglucocorticoids with a high affinity for the mineralocorticoid receptor. The present investigation aimed to elucidate the mechanisms accounting for the rigorous control of the HSD11B2 gene in humans. Using dimethyl sulfate in vivo footprinting via ligation-mediated PCR, we identified potentially important regions for HSD11B2 regulation in human cell lines: two GC-rich regions in the first exon (I and II) and two upstream elements (III and IV). The footprints suggest a correlation between the extent of in vivo protein occupancy at three of these regions (I, II, and III) and the rate of HSD11B2 transcription in cells with high (SW620), intermediate (HCD, MCF-7, and HK-2), or low HSD11B2 mRNA levels (SUT). Moreover, gel shift assays with nuclear extracts from these cell lines revealed that decreased HSD11B2 expression is related to a decreased binding activity with oligonucleotides containing the putative regulatory elements. Antibody supershifts identified the majority of the components of the binding complexes as the transcription factors Sp1 and Sp3. Finally, transient transfections with various deletion mutant reporters define positive regulatory elements that might account for basal and selective expression of 11beta-hydroxysteroid dehydrogenase type 2.
Collapse
Affiliation(s)
- Andrea R Nawrocki
- Division of Nephrology and Hypertension, Department of Internal Medicine, University Hospital of Berne, CH-3010 Berne, Switzerland.
| | | | | | | | | |
Collapse
|
|
24
|
Aitsebaomo J, Kingsley-Kallesen ML, Wu Y, Quertermous T, Patterson C. Vezf1/DB1 is an endothelial cell-specific transcription factor that regulates expression of the endothelin-1 promoter. J Biol Chem 2001; 276:39197-205. [PMID: 11504723 DOI: 10.1074/jbc.m105166200] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Coordinated gene regulation within the vascular endothelium is required for normal cardiovascular patterning during development and for vascular homeostasis during adulthood, yet little is known about the mechanisms that regulate endothelial transcriptional events. Vascular endothelial zinc finger 1 (Vezf1)/DB1 is a recently identified zinc finger-containing protein that is expressed specifically within endothelial cells during development. In this report, we demonstrate that Vezf1/DB1 is a nuclear localizing protein that potently and specifically activates transcription mediated by the human endothelin-1 promoter, in a Tax-independent manner, in transient transfection assays. Using a combination of deletion mutagenesis and electrophoretic mobility shift assays, a novel Vezf1/DB1-responsive element was localized to a 6-base pair (bp) motif, ACCCCC, located 47 bp upstream of the endothelin-1 transcription start site. Recombinant Vezf1/DB1 also bound to this sequence, and a 2-bp mutation in this element abolished Vezf1/DB1 responsiveness by the endothelin-1 promoter. Vezf1/DB1 could be identified with a specific antibody in nuclear complexes from endothelial cells that bound to this element. Regulation of endothelin-1 promoter activity by Vezf1/DB1 provides a mechanism for endothelin-1 expression in the vascular endothelium during development and to maintain vascular tone; Vezf1/DB1 itself is a candidate transcription factor for modifying endothelial cell phenotypes in order to appropriately assemble and maintain the cardiovascular system.
Collapse
Affiliation(s)
- J Aitsebaomo
- Program in Molecular Cardiology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7075, USA
| | | | | | | | | |
Collapse
|
|
25
|
Abstract
Epilysin (MMP-28) is a recently cloned member of the matrix metalloproteinase family (Lohi et al., J. Biol. Chem. 276 (2001) 10134). It is expressed at highest levels in the skin by basal and suprabasal keratinocytes, and in testis by developing germ cells. To characterize the epilysin promoter, we isolated a 3.0 kb fragment of human genomic DNA containing 5'-flanking sequence of the epilysin gene, and a corresponding 660 bp fragment from the mouse. The 5'-flanking sequences contain no typical TATA-boxes or CCAAT sequences close to the translation initiation sites. RNase protection assay revealed that two transcription start sites are utilized in the human epilysin gene, situated 210 and 230 bp upstream from the translation start site. The promoter contains a GT-box, situated 300 bp upstream from the translation start site, with homology to the consensus binding site for transcription factors of the Sp family. This site is perfectly conserved between the human and mouse promoters. For reporter gene assays a series of constructs with fragments of increasing length of the epilysin promoter were coupled to the firefly luciferase gene. Reporter gene assays indicated that deletion or mutation of the GT-box dramatically reduces the transcriptional activity both in keratinocytes and in spermatogonia. Gel mobility shift assays showed that several nuclear proteins bind specifically to this sequence. Supershift assays with antibodies specific for members of the Sp family identified Sp1 and Sp3 as components of these protein/DNA complexes and hence as possible regulators of the epilysin gene. Our results indicate that the epilysin promoter has distinctive structural and functional features, which may control the unique expression and regulation patterns of the epilysin gene.
Collapse
Affiliation(s)
- S A Illman
- Department of Pathology, Haartman Institute and Biomedicum Helsinki, University of Helsinki and Helsinki University Hospital, FIN-00014 Helsinki, Finland
| | | | | |
Collapse
|
|
26
|
Thompson LJ, Jiang J, Madamanchi N, Runge MS, Patterson C. PTP-epsilon, a tyrosine phosphatase expressed in endothelium, negatively regulates endothelial cell proliferation. Am J Physiol Heart Circ Physiol 2001; 281:H396-403. [PMID: 11406508 DOI: 10.1152/ajpheart.2001.281.1.h396] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
The vascular endothelium is a dynamic interface between the blood vessel and circulating factors and, as such, plays a critical role in vascular events like inflammation, angiogenesis, and hemostasis. Whereas specific protein tyrosine kinases have been identified in these processes, less is known about their protein tyrosine phosphatase (PTP) counterparts. We utilized a RT-PCR/differential hybridization assay to identify PTP-epsilon as a highly abundant endothelial cell PTP. PTP-epsilon mRNA expression is growth factor responsive, suggesting a role for this enzyme in endothelial cell proliferation. Overexpression of PTP-epsilon decreases proliferation by 60% in human umbilical vein endothelial cells (HUVEC) but not in smooth muscle cells or fibroblasts. In contrast, overexpression of PTP-epsilon (D284A), a catalytically inactive mutant, has no significant effect on HUVEC proliferation. These data provide the first functional characterization of PTP-epsilon in endothelial cells and identify a novel pathway that negatively regulates endothelial cell growth. Such a pathway may have important implications in vascular development and angiogenesis.
Collapse
Affiliation(s)
- L J Thompson
- Sealy Center for Molecular Cardiology, University of Texas Medical Branch, Galveston, Texas 77555, USA
| | | | | | | | | |
Collapse
|
|
27
|
Novikov DK, Kamps ME. Characterization of the Promoter Region of the Human Peroxisomal Multifunctional Enzyme Type 2 Gene. Biochem Biophys Res Commun 2001; 284:226-31. [PMID: 11374894 DOI: 10.1006/bbrc.2001.4949] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Peroxisomal multifunctional enzyme type 2 (perMFE-2) catalyzes conversion of (24E)-3alpha,7alpha, 12alpha-trihydroxy-5beta-cholest-24-enoyl-CoA to (24-keto)-3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoyl-CoA, which are physiological intermediates in cholic acid synthesis. In contrast to long chain fatty acid oxidizing enzymes clofibrate does not induce peroxisomal enzymes metabolizing bile acid intermediates. We proposed the existence of PPAR-independent regulation of cholesterol side chain oxidation in the process of bile acid synthesis. In the present study, we characterized the promoter region of the human perMFE-2 gene. The promoter contains the Sp1/AP2 binding site (-151/-142) within 197 base pairs upstream of the translation start site. Mutation of the Sp1/AP2 binding site decreases the promoter activity. Analysis by the luciferase assay revealed that the activity of the promoter region is strong in HepG2 and HeLa cell lines, although the activity in HepG2 cells was five- to sixfold higher than that in HeLa cells. Transient transfection assays have confirmed that AP2alpha and AP2gamma were able to transactivate the perMFE-2 promoter/luciferase chimeric gene. Cotransfections with Sp1 expression plasmid decreased the promoter activity. We suggest that perMFE-2 promoter activity is the result of both the abundance of AP2 and Sp1 family members and their relative ratios.
Collapse
Affiliation(s)
- D K Novikov
- Biocenter Oulu and Department of Biochemistry, University of Oulu, Oulu, FIN-90570, Finland.
| | | |
Collapse
|
|
28
|
Minami T, Rosenberg RD, Aird WC. Transforming growth factor-beta 1-mediated inhibition of the flk-1/KDR gene is mediated by a 5'-untranslated region palindromic GATA site. J Biol Chem 2001; 276:5395-402. [PMID: 11098056 DOI: 10.1074/jbc.m008798200] [Citation(s) in RCA: 58] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The angiogenic effects of vascular endothelial growth factor are mediated predominantly by the FLK-1/KDR receptor. An understanding of the transcriptional control mechanisms underlying flk-1/KDR expression should provide insight into the molecular basis of angiogenesis. In this study, we show that transforming growth factor-beta(1) (TGF-beta(1)) down-regulates expression of the endogenous flk-1/KDR gene in endothelial cells. In transient transfection assays, this effect was mapped to a palindromic GATA site in the 5'-untranslated region. In electrophoretic mobility shift assays, the palindromic GATA site was shown to bind to two molecules of GATA protein. Moreover, DNA-GATA interactions were inhibited by TGF-beta(1). Finally, in cotransfection assays, transactivation of the flk-1/KDR promoter by GATA-1 or GATA-2 was attenuated in TGF-beta(1)-treated cells. Taken together, these results suggest that the TGF-beta-1-mediated inhibition of the flk-1/KDR gene is mediated by a 5'-untranslated region palindromic GATA site.
Collapse
Affiliation(s)
- T Minami
- Department of Molecular Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA
| | | | | |
Collapse
|
|
29
|
Role of SCL/Tal-1, GATA, and Ets transcription factor binding sites for the regulation of Flk-1 expression during murine vascular development. Blood 2000. [DOI: 10.1182/blood.v96.9.3078] [Citation(s) in RCA: 112] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
AbstractThe receptor tyrosine kinase Flk-1 is essential for embryonic blood vessel development and for tumor angiogenesis. To identify upstream transcriptional regulators of Flk-1, the gene regulatory elements that mediate endothelium-specific expression in mouse embryos were characterized. By mutational analysis, binding sites for SCL/Tal-1, GATA, and Ets transcription factors located in theFlk-1 enhancer were identified as critical elements for the endothelium-specific Flk-1 gene expression in transgenic mice. c-Ets1, a transcription factor that is coexpressed withFlk-1 during embryonic development and tumor angiogenesis, activated the Flk-1 promoter via 2 binding sites. One of these sites was required for Flk-1 promoter function in the embryonic vasculature. These results provide the first evidence that SCL/Tal-1, GATA, and Ets transcription factors act upstream ofFlk-1 in a combinatorial fashion to determine embryonic blood vessel formation and are key regulators not only of the hematopoietic program, but also of vascular development.
Collapse
|
|
30
|
Role of SCL/Tal-1, GATA, and Ets transcription factor binding sites for the regulation of Flk-1 expression during murine vascular development. Blood 2000. [DOI: 10.1182/blood.v96.9.3078.h8003078_3078_3085] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The receptor tyrosine kinase Flk-1 is essential for embryonic blood vessel development and for tumor angiogenesis. To identify upstream transcriptional regulators of Flk-1, the gene regulatory elements that mediate endothelium-specific expression in mouse embryos were characterized. By mutational analysis, binding sites for SCL/Tal-1, GATA, and Ets transcription factors located in theFlk-1 enhancer were identified as critical elements for the endothelium-specific Flk-1 gene expression in transgenic mice. c-Ets1, a transcription factor that is coexpressed withFlk-1 during embryonic development and tumor angiogenesis, activated the Flk-1 promoter via 2 binding sites. One of these sites was required for Flk-1 promoter function in the embryonic vasculature. These results provide the first evidence that SCL/Tal-1, GATA, and Ets transcription factors act upstream ofFlk-1 in a combinatorial fashion to determine embryonic blood vessel formation and are key regulators not only of the hematopoietic program, but also of vascular development.
Collapse
|
|
31
|
Taranenko N, Krause DS. Regulation of CD34 transcription by Sp1 requires sites upstream and downstream of the transcription start site. Exp Hematol 2000; 28:974-84. [PMID: 10989198 DOI: 10.1016/s0301-472x(00)00492-6] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
CD34 is a cell surface glycoprotein expressed on hematopoietic stem and progenitor cells, but not on fully differentiated cells in the peripheral blood. To better understand the molecular regulation of early hematopoiesis, we are elucidating the mechanisms of CD34 transcriptional regulation. By deletion analysis we identify a 39-bp element in the proximal region of murine CD34 promoter that is critical for promoter activity. Electromobility shift assays indicate that nuclear proteins of hematopoietic cells bind to this domain; however, the presence of this binding activity does not correlate directly with CD34 expression.Using methylation interference, the DNA binding site for this activity was localized to four guanine residues within the GGGGTCGG sequence from -48 to -54 bp. When the four contact guanines were mutated, both protein binding and promoter activity were abolished. Although this sequence does not contain a standard consensus for Sp1, this transcription factor binds specifically to the 39-bp region and stimulates promoter activity in both hematopoietic cells and in Sp1 null Drosophila S2 cells. In addition, Ku binds to this domain in a sequence-specific manner. Activation of the CD34 promoter by Sp1 requires the presence of a binding domain at -48 bp as well as the 5' untranslated region, which also binds Sp1.A functional interaction between regulatory regions upstream and downstream of the transcription start site is required for CD34 gene expression.
Collapse
Affiliation(s)
- N Taranenko
- Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA
| | | |
Collapse
|
|
32
|
Modlich U, Pugh CW, Bicknell R. Increasing endothelial cell specific expression by the use of heterologous hypoxic and cytokine-inducible enhancers. Gene Ther 2000; 7:896-902. [PMID: 10845728 DOI: 10.1038/sj.gt.3301177] [Citation(s) in RCA: 44] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
One of the current challenges in gene therapy is to construct a vector that will target specific tissues. Targeting expression to endothelium is of particular interest in the treatment of several pathologies. We have shown previously that defined regions of the E-selectin and KDR promoters confer endothelial cell specific expression following retroviral delivery. However, the levels of expression were low. In an attempt to increase expression but to preserve the tissue specificity we have examined hypoxic and cytokine-inducible enhancer elements in combination with the KDR and E-selectin promoters. Both enhancers should be active in the tumour environment, boosting expression and giving additional specificity of gene expression in the tumour endothelium. The hypoxia response element (HRE) of the murine phosphoglycerate kinase-1 (PGK-1) promoter was used as a hypoxic enhancer and the tandem-binding site for NFKB from the murine vascular cell adhesion molecule-1 (VCAM-1) promoter as a cytokine-inducible enhancer. The HRE conferred hypoxia inducibility to the KDR and E-selectin promoters. Endothelial specificity of expression was retained with the KDR but not the E-selectin promoter. The NFKB-binding site conferred responsiveness to TNF-alpha to the KDR promoter, however the level of induction was less than that achieved with the HRE. Retrovirus combining both enhancer elements transferred inducibility by hypoxia and TNF-alpha, and reached the highest expression levels upon stimulation. These results confirm that heterologous enhancer elements may operate on a single endothelial cell specific promoter. These findings make the use of inducible enhancers a promising strategy for increasing tissue specific gene expression.
Collapse
Affiliation(s)
- U Modlich
- Molecular Angiogenesis Laboratory, Imperial Cancer Research Fund, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, UK
| | | | | |
Collapse
|
|
33
|
Martin SG, Murray JC. Gene-transfer systems for human endothelial cells. stewart.martin@nottingham.ac.uk. Adv Drug Deliv Rev 2000; 41:223-33. [PMID: 10699317 DOI: 10.1016/s0169-409x(99)00068-x] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
By virtue of its location and importance in a number of pathophysiological processes the endothelium represents an attractive target tissue for gene-transfer and gene-therapy strategies. Although it is important to maximise gene-transfer to endothelial cells in such strategies primary human endothelial cells have proven to be rather intransigent to a variety of transfection techniques both in vitro and in vivo. We report on the variety of techniques in current use, revealing their strengths and weaknesses, indicate the steps that should ideally be taken to optimise expression and discuss the usefulness and future directions for viral mediated transduction.
Collapse
Affiliation(s)
- S G Martin
- University of Nottingham, Laboratory of Molecular Oncology, Cancer Research Campaign Department of Clinical Oncology, City Hospital, Nottingham, UK
| | | |
Collapse
|
|
34
|
Moreira AL, Friedlander DR, Shif B, Kaplan G, Zagzag D. Thalidomide and a thalidomide analogue inhibit endothelial cell proliferation in vitro. J Neurooncol 1999; 43:109-14. [PMID: 10533722 DOI: 10.1023/a:1006202700039] [Citation(s) in RCA: 66] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Angiogenesis is a crucial process in inflammatory reactions as well as in tumor implantation and growth. Tumors with high rates of invasion and recurrence such as gliomas, are specially dependent on neovascularization. This suggests that inhibition of angiogenesis might reduce the growth of these tumors. Thalidomide has been previously shown to inhibit angiogenesis induced by basic fibroblast growth factor in vivo, using the rabbit corneal micropocket assay. Therefore, the effect of thalidomide and a thalidomide analogue (cc-1069) on the proliferation in vitro of endothelial and glioma cells was tested. We observed a decrease in endothelial cell proliferation in cultures treated with thalidomide or the thalidomide analogue cc-1069. The analogue inhibited endothelial cell proliferation more efficiently than thalidomide. The inhibition occurred in association with a marked decrease in the activity of the nuclear factor SP1 and a moderate inhibition of NF-kappaB activation in nuclear extracts of endothelial cells. The drugs did not impair cell viability. There was no effect of thalidomide or the thalidomide analogue on the proliferation of the glioma cell line (U251) in vitro.
Collapse
Affiliation(s)
- A L Moreira
- Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, NY, USA
| | | | | | | | | |
Collapse
|
|
35
|
Sibinga NE, Wang H, Perrella MA, Endege WO, Patterson C, Yoshizumi M, Haber E, Lee ME. Interferon-gamma-mediated inhibition of cyclin A gene transcription is independent of individual cis-acting elements in the cyclin A promoter. J Biol Chem 1999; 274:12139-46. [PMID: 10207041 DOI: 10.1074/jbc.274.17.12139] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Interferons (IFNs) affect cellular functions by altering gene expression. The eukaryotic cell cycle is governed in part by the periodic transcription of cyclin genes, whose protein products associate with and positively regulate the cyclin-dependent kinases. To understand better the growth inhibitory effect of IFN-gamma on vascular smooth muscle cells (VSMCs), we compared the expression and activity of G1 and S phase cyclins in control and IFN-gamma-treated VSMCs. IFN-gamma treatment did not inhibit the G1 cyclins but did decrease cyclin A protein, mRNA, and associated kinase activity by 85, 90, and 90%, respectively. Nuclear run-on and mRNA stability determinations indicated that this decrease was the result of transcriptional inhibition. To investigate the molecular basis of this inhibition, we examined protein-DNA interactions involving the cyclin A promoter. Electromobility shift assays showed little change with IFN-gamma treatment in the binding of nuclear proteins to isolated ATF, NF-Y, and CDE elements. In vivo genomic footprinting indicated that IFN-gamma treatment changed the occupancy of chromosomal NF-Y and CDE sites slightly and did not affect occupancy of the ATF site. In a previous study of transforming growth factor-beta1-mediated inhibition of the cyclin A promoter, we mapped the inhibitory effect to the ATF site; in the present study of IFN-gamma treatment, functional analysis by transient transfection showed that inhibition of the cyclin A promoter persisted despite mutation of the ATF, NF-Y, or CDE elements. We hypothesize that IFN-gamma inhibits cyclin A transcription by modifying co-activators or general transcription factors within the complex that drives transcription of the cyclin A gene.
Collapse
Affiliation(s)
- N E Sibinga
- Cardiovascular Biology Laboratory, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
| | | | | | | | | | | | | | | |
Collapse
|
|
36
|
|
|
37
|
Iljin K, Dube A, Kontusaari S, Korhonen J, Lahtinen I, Oettgen P, Alitalo K. Role of ets factors in the activity and endothelial cell specificity of the mouse Tie gene promoter. FASEB J 1999; 13:377-86. [PMID: 9973326 DOI: 10.1096/fasebj.13.2.377] [Citation(s) in RCA: 75] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
The Tie gene encodes an endothelial cell receptor tyrosine kinase necessary for normal vascular development. The Tie gene promoter targets expression of heterologous genes specifically to endothelial cells in transgenic mice. Here we have characterized the promoter sequences critical for endothelial cell-specific activity in cultured cells and transgenic mice. Progressive deletions and site-directed mutations of the promoter showed that the critical endothelial cell-specific elements are an octamer transcription factor binding site and several Ets binding sites located in two clusters within 300 bp upstream of the major transcription initiation site. Among members of the Ets transcription factor family tested, NERF-2 (a novel transcription factor related to the ets factor ELF-1), which is expressed in endothelial cells, and ETS2 showed the strongest transactivation of the Tie promoter; ETS1 gave lower levels of stimulation and the other Ets factors gave little or no transactivation. Furthermore, the Tie promoter directed the production of high amounts of human growth hormone into the circulation of transgenic mice. The secreted amounts correlated with transgene copy number, being relatively insensitive to the effects of the transgene integration site. These properties suggest that Tie promoter activity is controlled by endothelial cell Ets factors and that it has potential for use in vectors for endothelial cell-specific gene expression.
Collapse
Affiliation(s)
- K Iljin
- Molecular/Cancer Biology Laboratory, Haartman Institute and Department of Biomedicine, University of Helsinki, Helsinki, Finland
| | | | | | | | | | | | | |
Collapse
|
|
38
|
Wu Y, Patterson C. The human KDR/flk-1 gene contains a functional initiator element that is bound and transactivated by TFII-I. J Biol Chem 1999; 274:3207-14. [PMID: 9915861 DOI: 10.1074/jbc.274.5.3207] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
KDR/flk-1, the receptor for vascular endothelial growth factor, is required for normal vascular development. KDR/flk-1 is a TATA-less gene, containing four upstream Sp1 sites and a single transcription start site, although analysis of the start site sequence discloses only weak similarities with the consensus initiator element (Inr) sequence. In vitro transcription assays, however, demonstrate that the region from -10 to +10 relative to the start site contains Inr activity that is orientation- and position-dependent, and mutagenesis of the KDR/flk-1 Inr reduces promoter activity to 28% of the wild-type promoter in transient transfection assays. Gel shift assays confirm that nuclear proteins specifically bind the Inr, and competition experiments demonstrate that TFII-I, a multifunctional Inr-binding nuclear protein, is a component of these DNA-protein complexes. TFII-I transactivates the wild-type KDR/flk-1 promoter, but not a promoter containing a mutated Inr, in transient transfection assays. Immunodepletion of TFII-I from nuclear extracts prior to in vitro transcription assays abolishes transcription from the KDR/flk-1 Inr, an effect that can be rescued by adding back purified TFII-I, reflecting the importance of TFII-I in KDR/flk-1 Inr activity. These experiments demonstrate that the KDR/flk-1 gene contains a functional Inr that is bound by TFII-I and that both the functional Inr and TFII-I activity are essential for transcription.
Collapse
Affiliation(s)
- Y Wu
- University of Texas Medical Branch, Division of Cardiology and Sealy Center for Molecular Cardiology, Galveston, Texas 77555-1064, USA
| | | |
Collapse
|
|
39
|
Huang YS, Chuang DT. Mechanism for basal expression of rat mitochondrial branched-chain-2-oxo-acid dehydrogenase kinase [corrected]. Biochem J 1998; 334 ( Pt 3):713-22. [PMID: 9729481 PMCID: PMC1219742 DOI: 10.1042/bj3340713] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The rat branched-chain-2-oxo-acid dehydrogenase (BCOD) kinase mRNA is transcribed from a TATA-less promoter that has GC-rich sequences and two putative Sp1 binding sites near the transcription start site. We demonstrated previously that the 5' region of the kinase gene, base pairs -128 to +264, contained promoter activity when assayed using luciferase as a reporter (Huang and Chuang (1996) Biochem. J. 313, 603-609). To define DNA elements required for efficient expression of the kinase gene, nested deletion constructs of the above promoter region fused with a luciferase reporter gene were transfected into cultured H4IIE (hepatoma) and NRK-52E (kidney) cells. The results showed that the region between nucleotides -58 and +21 was indispensable for the kinase basal promoter activity. Methylation-interference and mutagenesis-promoter assays identified nucleotides -50 to -40 (ACAACTCCCA) as cis-acting DNA sequences that are required for nuclear protein binding and efficient promoter activity. Gel-supershift analysis with anti-Sp1 antibody suggested that the nuclear protein capable of binding to the -58 oligonucleotide (bp -58 to -34) was immunologically related to the Sp1 protein. The -58 oligonucleotide formed a DNA-protein complex with recombinant Sp1 protein with an affinity approximately ten-fold lower than that of the consensus Sp1 oligonucleotide. Co-transfection of the Sp1 expression plasmid and the -58 promoter construct into Drosophila Schneider cells revealed that Sp1 contributed to the kinase basal promoter activity by binding to the non-consensus site in the -58 region. Deletion of two consensus Sp1 binding sites (bases -150 to -140 and bases +29 to +38) in the kinase gene did not affect the basal promoter activity. Therefore binding of Sp1 or Sp1-like proteins to the above single non-consensus Sp1 sequence in the -58 region plays a major role of transactivating basal expression of the BCOD kinase.
Collapse
Affiliation(s)
- Y S Huang
- Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9038, USA
| | | |
Collapse
|
|
40
|
Giraudo E, Primo L, Audero E, Gerber HP, Koolwijk P, Soker S, Klagsbrun M, Ferrara N, Bussolino F. Tumor necrosis factor-alpha regulates expression of vascular endothelial growth factor receptor-2 and of its co-receptor neuropilin-1 in human vascular endothelial cells. J Biol Chem 1998; 273:22128-35. [PMID: 9705358 DOI: 10.1074/jbc.273.34.22128] [Citation(s) in RCA: 201] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Tumor necrosis factor-alpha (TNF-alpha) modulates gene expression in endothelial cells and is angiogenic in vivo. TNF-alpha does not activate in vitro migration and proliferation of endothelium, and its angiogenic activity is elicited by synthesis of direct angiogenic inducers or of proteases. Here, we show that TNF-alpha up-regulates in a dose- and time-dependent manner the expression and the function of vascular endothelial growth factor receptor-2 (VEGFR-2) as well as the expression of its co-receptor neuropilin-1 in human endothelium. As inferred by nuclear run-on assay and transient expression of VEGFR-2 promoter-based reporter gene construct, the cytokine increased the transcription of the VEGFR-2 gene. Mithramycin, an inhibitor of binding of nuclear transcription factor Sp1 to the promoter consensus sequence, blocked activation of VEGFR-2, suggesting that the up-regulation of the receptor required Sp1 binding sites. TNF-alpha increased the cellular amounts of VEGFR-2 protein and tripled the high affinity 125I-VEGF-A165 capacity without affecting the Kd of ligand-receptor interaction. As a consequence, TNF-alpha enhanced the migration and the wound healing triggered by VEGF-A165. Since VEGFR-2 mediates angiogenic signals in endothelium, our data indicate that its up-regulation is another mechanism by which TNF-alpha is angiogenic and may provide insight into the mechanism of neovascularization as occurs in TNF-alpha-mediated pathological settings.
Collapse
Affiliation(s)
- E Giraudo
- Vascular Biology Laboratory, Department of Genetics, Biology and Biochemistry, Medical School, University of Torino, Torino, 10126 Italy
| | | | | | | | | | | | | | | | | |
Collapse
|
|
41
|
Hata Y, Duh E, Zhang K, Robinson GS, Aiello LP. Transcription factors Sp1 and Sp3 alter vascular endothelial growth factor receptor expression through a novel recognition sequence. J Biol Chem 1998; 273:19294-303. [PMID: 9668119 DOI: 10.1074/jbc.273.30.19294] [Citation(s) in RCA: 104] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Kinase domain receptor (KDR) is a high affinity, endothelial cell-specific, autophosphorylating tyrosine kinase receptor for vascular endothelial growth factor. This transcriptionally regulated receptor is a critical mediator of endothelial cell (EC) growth and vascular development. In this study, we identify a DNA element modulating KDR promoter activity and evaluate the nuclear binding proteins accounting for a portion of the cell-type specificity of the region. KDR promoter luciferase activity was retained within -85/+296 and was 10-30-fold higher in EC than non-EC. Electrophoretic mobility shift assays demonstrated specific nuclear protein binding to -85/-64, and single point mutations suggested important binding nucleotides between -79/-68 with five critical bases between -74/-70 (5'-CTCCT-3'). DNA-protein complexes were displaced by Sp1 consensus sequence oligodeoxynucleotides and supershifted by Sp1- and Sp3-specific antibodies. Sp1 and Sp3 protein in EC nuclear extracts bound the -79/-68 region even when all surrounding classic Sp1 recognition sites were removed. Sp1 protein in nuclear extracts was 4-24-fold higher in EC than non-EC, whereas Sp3 was 3-7-fold higher. Sp1/Sp3 ratios in EC were 2-10-fold higher. Overexpression of Sp1 protein increased KDR promoter activity 3-fold in both EC and non-EC, whereas simultaneous co-expression of Sp3 attenuated this response. An Sp1 consensus sequence cis element "decoy" reduced EC KDR promoter activity and mRNA expression by 85 and 69%, respectively. An antisense phosphorothioate oligodeoxynucleotide to Sp1 inhibited Sp1 and KDR protein expression by 66 and 68%, respectively, without changing Sp3 protein expression. These data illustrate that Sp1 and Sp3 modulate KDR promoter activity through a novel recognition binding sequence. However, since Sp1-mediated promoter activation is attenuated by Sp3, endothelial selective KDR promoter activity may be partially regulated by variations in the Sp1/Sp3 ratio.
Collapse
Affiliation(s)
- Y Hata
- Research Division, Joslin Diabetes Center, Boston, Massachusetts 02215, USA
| | | | | | | | | |
Collapse
|
|
42
|
Cowan PJ, Tsang D, Pedic CM, Abbott LR, Shinkel TA, d'Apice AJ, Pearse MJ. The human ICAM-2 promoter is endothelial cell-specific in vitro and in vivo and contains critical Sp1 and GATA binding sites. J Biol Chem 1998; 273:11737-44. [PMID: 9565596 DOI: 10.1074/jbc.273.19.11737] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The expression of intercellular adhesion molecule 2 (ICAM-2) in adult tissues is restricted to vascular endothelial cells and megakaryocytes. We have previously shown that the endothelial-specific in vivo activity of the human ICAM-2 promoter is contained within a small (0.33-kilobase (kbp)) 5'-flanking region of the gene. Here we describe the in vitro characterization of this region. The ICAM-2 promoter is TATA-less, and transcription in endothelial cells initiates at four sites. Reporter gene expression directed by the promoter was 125-fold greater than vector alone in bovine aortic endothelial cells but less than 2-fold vector alone in non-endothelial (COS) cells, confirming that specificity in vivo was paralleled in vitro. The addition of 2.7 kbp of 5'-flanking region to the 0.33-kbp fragment had no effect on promoter activity or specificity. The mutation of an Sp1 motif centered at base pair -194 or an eight-base pair palindrome at -268 each reduced promoter activity by 70%. Mutation of GATA motifs at -145 and -53 reduced promoter activity by 78 and 61%, respectively. Specific binding of bovine aortic endothelial cells nuclear proteins to the Sp1 and GATA sites was demonstrated by gel shift analysis. Promoter activity in COS cells was transactivated 3-4-fold by overexpression of GATA-2. The results presented here suggest that transcription from the ICAM-2 promoter in endothelial cells is regulated by the interplay of several positive-acting factors and provide the basis for further analysis of endothelial-specific gene expression.
Collapse
Affiliation(s)
- P J Cowan
- Immunology Research Center, St Vincent's Hospital, Fitzroy 3065, Victoria, Australia
| | | | | | | | | | | | | |
Collapse
|
|
43
|
Affiliation(s)
- L Y Yin
- Division of Cardiology, University of Texas Medical Branch at Galveston, 301 University Boulevard, 9.138 Medical Research Building, Galveston, Texas 77555-1064, USA
| | | | | | | |
Collapse
|
|
44
|
Gory S, Dalmon J, Prandini MH, Kortulewski T, de Launoit Y, Huber P. Requirement of a GT box (Sp1 site) and two Ets binding sites for vascular endothelial cadherin gene transcription. J Biol Chem 1998; 273:6750-5. [PMID: 9506975 DOI: 10.1074/jbc.273.12.6750] [Citation(s) in RCA: 94] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Vascular endothelial cadherin (VE cadherin) gene encodes a Ca2+-dependent cell adhesion molecule required for the organization of interendothelial junctions. This gene is exclusively and constitutively expressed in endothelial cells. Previous data with transgenic mice revealed that the transcriptional regulatory elements present within a -2486/+24 DNA fragment of mouse VE cadherin gene mimic the tissue-specific activity of the endogenous promoter. In this study, we analyzed elements implicated in the function of the proximal regulatory region. Electrophoretic mobility shift assay identified a GT-rich sequence (positions -49/-39) interacting with factors related to the Sp1 family. Point mutations abolished the binding of nuclear proteins in vitro and drastically diminished the activity of the promoter in transient transfection assay. Supershift assays with antibodies against proteins of the Sp1 family revealed that Sp1 and Sp3 interact with this region of the VE cadherin promoter. Furthermore, two GGAA motifs, located at positions -93/-90 and -109/-106, were shown to interact with nuclear factors. Site-directed mutagenesis of these sequences demonstrated that these Ets binding sites are essential for promoter activity. In vitro binding assays in the presence of various antisera suggest that Erg is one of the proteins interacting with the -109/-106 site.
Collapse
Affiliation(s)
- S Gory
- CEA, Laboratoire de Transgénèse et Différenciation Cellulaire, Département de Biologie Moléculaire et Structurale, 17 rue des Martyrs, 38054 Grenoble, France
| | | | | | | | | | | |
Collapse
|
|
45
|
Chang BB, Persengiev SP, de Diego JG, Sacristan MP, Martin-Zanca D, Kilpatrick DL. Proximal promoter sequences mediate cell-specific and elevated expression of the favorable prognosis marker TrkA in human neuroblastoma cells. J Biol Chem 1998; 273:39-44. [PMID: 9417044 DOI: 10.1074/jbc.273.1.39] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
The nerve growth factor receptor, TrkA, has a critical role in the survival, differentiation, and function of neurons in the peripheral and central nervous systems. Recent studies have demonstrated a strong correlation between abundant expression of TrkA and a favorable prognosis of the pediatric tumor, neuroblastoma. This correlation suggests that TrkA may actively promote growth arrest and differentiation of neuroblastoma tumor cells and may be an important therapeutic target in the treatment of this disease. In the present study, we have examined the mechanistic basis for TrkA gene expression in human neuroblastoma cells. Northern blotting and nuclear run-on analyses demonstrated that transcription is a primary determinant of both cell-specific and variable expression of the TrkA gene in neuroblastoma cell lines that express it to different degrees. Cell-specific and variable transcription in neuroblastoma cells was recapitulated by transient transfection of TrkA promoter-luciferase reporter constructs, and regulatory sequences mediating these processes were localized to a 138-base pair region lying just upstream of the transcription initiation region. This neuroblastoma regulatory region formed multiple DNA-protein complexes in gel shift assays that were highly enriched in neuroblastoma cells exhibiting abundant TrkA expression. Thus, TrkA-positive neuroblastoma cells are distinguished by differential expression of putative transcription factors that ultimately may serve as targets for up-regulating TrkA expression in tumors with poor prognosis.
Collapse
Affiliation(s)
- B B Chang
- Physiology Department, University of Massachusetts Medical Center, Worcester, Massachusetts 01655, USA
| | | | | | | | | | | |
Collapse
|