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      Cloning and Characterization of the Human Lung Endothelial-Cell-Specific Molecule-1 Promoter

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          Abstract

          Endothelial-cell-specific molecule-1 (ESM-1) is a cysteine-rich protein that is expressed primarily in endothelial cells of the lung, kidney and gut. In the present study, we have cloned and sequenced 3,888 bp of the 5′ flanking region of the human ESM-1 gene. The full-length promoter directed high-level expression of the luciferase reporter gene in bovine lung microvascular endothelial cells and bovine aortic endothelial cells, but not in nonendothelial cell types. In 5′ deletion analyses, a region spanning –81 to +58 was shown to contain information for endothelial-cell-specific expression. Mutational analysis in transient transfection assays uncovered an important role for an Ets-binding motif located between –77 and –74 and a cAMP-response-element (CRE)-like motif located between –68 and –62 in mediating high-level expression in endothelial cells. A second Ets site (–63 to –60) as well as a novel 6-bp palindromic sequence (–58 to –53) were found to inhibit expression. In DNase footprint analyses, both the Ets-binding motifs were protected specifically in endothelial cells, while the CRE-like element and palindrome were protected in endothelial and nonendothelial cells alike. Taken together, these results provide an important foundation for studying endothelial-cell-subtype-specific gene regulation.

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          Most cited references 15

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          ESM-1 is a novel human endothelial cell-specific molecule expressed in lung and regulated by cytokines.

          We here report the identification of a novel human endothelial cell-specific molecule (called ESM-1) cloned from a human umbilical vein endothelial cell (HUVEC) cDNA library. Constitutive ESM-1 gene expression (as demonstrated by Northern blot and reverse transcription-polymerase chain reaction analysis) was found in HUVECs but not in the other human cell lines tested. The cDNA sequence contains an open reading frame of 552 nucleotides and a 1398-nucleotide 3'-untranslated region including several domains involved in mRNA instability and five putative polyadenylation consensus sequences. The deduced 184-amino acid sequence defines a cysteine-rich protein with a functional NH2-terminal hydrophobic signal sequence. Searches in several data bases confirmed the unique identity of this sequence. A rabbit immune serum raised against the 14-kDa COOH-terminal peptide of ESM-1 immunoprecipitated a 20-kDa protein only in ESM-1-transfected COS cells. Immunoblotting and immunoprecipitation of HUVEC lysates revealed a specific 20-kDa band corresponding to ESM-1. In addition, constitutive ESM-1 gene expression was shown to be tissue-restricted to the human lung. Southern blot analysis suggests that a single gene encodes ESM-1. A time-dependent up-regulation of ESM-1 mRNA was seen after addition of tumor necrosis factor alpha (TNFalpha) or interleukin (IL)-1beta but not with IL-4 or interferon gamma (IFNgamma) alone. In addition, when IFNgamma was combined with TNFalpha, IFNgamma inhibited the TNFalpha-induced increase of ESM-1 mRNA level. These data suggest that ESM-1 may have potent implications in the areas of vascular cell biology and human lung physiology.
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            Single-copy transgenic mice with chosen-site integration.

            We describe a general way of introducing transgenes into the mouse germ line for comparing different sequences without the complications of variation in copy number and insertion site. The method uses homologous recombination in embryonic stem (ES) cells to generate mice having a single copy of a transgene integrated into a chosen location in the genome. To test the method, a single copy murine bcl-2 cDNA driven by either a chicken beta-actin promoter or a human beta-actin promoter has been inserted immediately 5' to the X-linked hypoxanthine phosphoribosyltransferase locus by a directly selectable homologous recombination event. The level of expression of the targeted bcl-2 transgene in ES cells is identical in independently isolated homologous recombinants having the same promoter yet varies between the different promoters. In contrast, the expression of bcl-2 transgenes having the same (chicken beta-actin) promoter varies drastically when they are independently integrated at random insertion sites. Both promoters direct broad expression of the single-copy transgene in mice derived from the respective targeted ES cells. In vitro and in vivo, the human beta-actin promoter consistently directed a higher level of transgene expression than the chicken beta-actin promoter.
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              Uniform vascular-endothelial-cell-specific gene expression in both embryonic and adult transgenic mice.

              TIE2 is a vascular endothelial-specific receptor tyrosine kinase essential for the regulation of vascular network formation and remodeling. Previously, we have shown that the 1.2-kb 5' flanking region of the TIE2 promoter is capable of directing beta-galactosidase reporter gene expression specifically into a subset of endothelial cells (ECs) of transgenic mouse embryos. However, transgene activity was restricted to early embryonic stages and not detectable in adult mice. Herein we describe the identification and characterization of an autonomous endothelial-specific enhancer in the first intron of the mouse TIE2 gene. Furthermore, combination of the TIE2 promoter with an intron fragment containing this enhancer allows it to target reporter gene expression specifically and uniformly to virtually all vascular ECs throughout embryogenesis and adulthood. To our knowledge, this is the first time that an in vivo expression system has been assembled by which heterologous genes can be targeted exclusively to the ECs of the entire vasculature. This should be a valuable tool to address the function of genes during physiological and pathological processes of vascular ECs in vivo. Furthermore, we were able to identify a short region critical for enhancer function in vivo that contains putative binding sites for Ets-like transcription factors. This should, therefore, allow us to determine the molecular mechanisms underlying the vascular-EC-specific expression of the TIE2 gene.
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                Author and article information

                Journal
                JVR
                J Vasc Res
                10.1159/issn.1018-1172
                Journal of Vascular Research
                S. Karger AG
                1018-1172
                1423-0135
                2002
                April 2002
                10 May 2002
                : 39
                : 2
                : 148-159
                Affiliations
                aDepartment of Medicine, Beth Israel Deaconess Medical Center, Boston, Mass., USA; bInstitut National de la Santé et de la Recherche Médicale, Unité 416, Institut Pasteur de Lille, France
                Article
                57763 J Vasc Res 2002;39:148–159
                10.1159/000057763
                12011586
                © 2002 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 10, References: 34, Pages: 12
                Categories
                Research Paper

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