Identification of a Functional NF-κB Site in the Platelet Endothelial Cell Adhesion Molecule-1 Promoter This information is current as Luisa M. Botella, Amaya Puig-Kröger, Nuria Almendro, of September 26, 2021. Tilman Sánchez-Elsner, Eduardo Muñoz, Angel Corbí and Carmelo Bernabéu J Immunol 2000; 164:1372-1378; ; doi: 10.4049/jimmunol.164.3.1372 http://www.jimmunol.org/content/164/3/1372 Downloaded from References This article cites 35 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/164/3/1372.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Identification of a Functional NF-␬B Site in the Platelet Endothelial Cell Adhesion Molecule-1 Promoter1 Luisa M. Botella,2,3* Amaya Puig-Kro¨ger,3* Nuria Almendro,* Tilman Sa´nchez-Elsner,* Eduardo Mun˜oz,† Angel Corbı´,* and Carmelo Bernabe´u* Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a type I transmembrane adhesion protein of 130 kDa that belongs to a subgroup of the Ig gene superfamily, characterized by the presence of immunoreceptor tyrosine-based inhibitory motifs. PECAM-1 is expressed in circulating platelets, monocytes, neutrophils, a selective subgroup of T cells, and in endothelial cells, where it is preferentially located at intercellular junctions and participates in leukocyte transmigratory processes. The identifi- cation of two consensus NF-␬B sites within the PECAM-1 promoter led us to analyze their possible involvement in the PECAM-1 expression regulated by inflammatory stimuli. We found that surface expression and promoter activity of PECAM-1 in myeloid cells are regulated by modulators of NF-␬B, including TNF-␣, PMA, and pyrrolidine dithiocarbamate. Mobility shifts assays Downloaded from identified a specific NF-␬B-binding element at ؉110/؉120, whose mutation abolished the basal promoter activity of PECAM-1 and decreased NF-␬B-dependent responses of the PECAM-1 gene promoter. Furthermore, cotransfection experiments with an ex- pression vector encoding the p65 subunit of NF-␬B showed transactivation of the PECAM-1 promoter. These results demonstrate that NF-␬B can regulate the transcriptional activity of PECAM-1. The Journal of Immunology, 2000, 164: 1372–1378. nflammation is a physiological process triggered in organ- can inhibit protein tyrosine kinase-dependent signals transduced http://www.jimmunol.org/ isms by infectious agents, foreign bodies, or injuries and by the TCR (9). PECAM-1 is expressed in circulating platelets, I characterized by pain and fever (1). It is associated with an monocytes, neutrophils, and a selective subgroup of T cells. In outlet of leukocytes, migrating toward the inflammatory focus endothelial cells, PECAM-1 is preferentially located at intercellu- where different adhesion molecules allow the interaction between lar junctions and participates in leukocyte transmigratory pro- immune cells and endothelium. These adhesion molecules belong cesses. PECAM-1 mediates homotypic adhesion among endothe- to the mucin, selectin, cadherin, integrin, and Ig superfamily (2–4). lial cells, as well as monocyte/neutrophil adhesion to endothelium The inflammatory focus releases cytokines such as TNF-␣, IL-1, through homophilic interactions. It also interacts in a heterophilic IL-8, or IFN-␥, which induce cell-surface expression of adhesion ␣ ␤ way with ligands such as v 3, CD38, and Plasmodium falcipa- by guest on September 26, 2021 molecules and activate endothelial cells of neighboring vessels. rum-infected erythrocytes (7, 10–13). PECAM-1 is encoded by a Leukocytes leave the circulation at sites of local inflammation 65-kb gene allocated in the long arm of chromosome 17 (14, 15), through a series of sequential steps classified as rolling, activation, and the region driving its transcription has been identified as a tight adhesion, transmigration, and passage across the vascular TATA-less promoter containing relevant EGR-1 and GATA-2 cis- basement membrane. regulatory elements (16, 17). In addition, two consensus sites for 4 Platelet endothelial cell adhesion molecule-1 (PECAM-1), also NF-␬B were identified at Ϫ409 (GGGGTTCTCC) and at ϩ110 known as CD31, has been implicated as a critical mediator of (GAGGAATCCCC) (16), although their functional relevance is transendothelial migration (5, 6). This is a type I transmembrane not known. This family of transcription factors regulates the tran- adhesion protein of 130 kDa, which belongs to a subgroup of the scription of adhesion molecules such as E-selectin, VCAM-1, and Ig superfamily, characterized by the presence of immunoreceptor ICAM-1 (18). Thus, structural and functional similarities of tyrosine-based inhibitory motifs (7, 8). Accordingly, PECAM-1 PECAM-1 and these adhesion molecules support the involvement of NF-␬B in the transcription of PECAM-1. The NF-␬B/Rel family of transcription factors is composed of five distinct DNA-binding subunits called p50, p52, p65 (RelA), *Centro de Investigaciones Biolo´gicas, Consejo Superior de Investigaciones Cientı´- ficas, Madrid, Spain; and †Departamento de Fisiologı´a e Inmunologı´a, Facultad de c-Rel, and Rel-B (19–21). The different family members can Medicina, Universidad de Co´rdoba, Co´rdoba, Spain associate in various homo- or heterodimers through a highly con- Received for publication August 6, 1999. Accepted for publication November 10, served N-terminal 300-aa region known as the rel homology 1999. domain. Inactive NF-␬B bound to the inhibitory protein I-␬Bis The costs of publication of this article were defrayed in part by the payment of page present in the cytoplasm and released upon phosphorylation of charges. This article must therefore be hereby marked advertisement in accordance ␬ with 18 U.S.C. Section 1734 solely to indicate this fact. I- B that regulates its ubiquitin-dependent degradation by the 26S ␬ 1 This work has been supported by grants from Comisio´n Interministerial de Ciencia proteasome. Then, the activated NF- B dimer translocates to the y Tecnologı´a (CICYT-SAF97-0034 to C.B. and CICYT-SAF98-0068 to A.C.) and nucleus and regulates gene transcription. The NF-␬B activation Comunidad Auto´noma de Madrid (fellowship to T.S.-E.). process can be triggered by physiological stimuli such as viral or 2 Address correspondence and reprint requests to Dr. Luisa M. Botella, Centro de bacterial infections, as well as inflammatory cytokines as TNF-␣ Investigaciones Biolo´gicas, Consejo Superior de Investigaciones Cientı´ficas, Vela´zquez 144, 28006 Madrid, Spain. E-mail address: [email protected] or IL-1. In addition, phorbol esters can be used in vitro as non- ␬ 3 L.M.B. and A.P.-K. contributed equally to this work. physiological activators of NF- B. Previous studies have demon- 4 Abbreviations used in this paper: PECAM-1, platelet endothelial cell adhesion mol- strated that PECAM-1 expression is up-regulated upon treatment ecule-1; PDTC, pyrrolidine dithiocarbamate. of monocytic cell lines with phorbol esters (16, 22, 23), a finding Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 1373 compatible with the involvement of NF-␬B on PECAM-1 expres- polynucleotide kinase. These oligonucleotides were WT, ϩ103GCAGG ␬ ϩ GAGGAATCCCCTCACAϩ124; mutated (MUT), ϩ103GCAGGGTTTA sion. This and the existence of two consensus NF- B sites at 110 ϩ Ϫ Ϫ Ϫ ATCCCCTCACA 124; and WT, 422TACAGGGGTTCTCCACCA 404. and 409 within the promoter region led us to assess the possible ␮ ␮ ␬ Binding reaction was performed with 10 g of nuclear extracts, 2.5 gof involvement of NF- B on PECAM-1 expression. Here, we have poly (dI-dC) in a buffer containing 70 mM KCl, 5 mM MgCl , 0.1 mM characterized the NF-␬B site at ϩ110 as a functional motif in- 2 ZnCl2, 0.5 mM DTT, 0.05% Nonidet P-40, 10% glycerol, and 20 mM volved in PECAM-1 transcription. HEPES, pH 7.5, on ice for 1 h. An amount of 2 ng of labeled probe (105 cpm) was added to the reaction mixture. Samples were electrophoresed on Materials and Methods a 7.5% polyacrylamide gel in 0.5ϫ TBE at 175 V for 3 h. For competition experiments a 100-fold excess of cold oligonucleotides were incubated in Plasmids the reaction mixture. The pXP2 vector contains the promoterless firefly luciferase gene (24). Reporter plasmids pCD31-0.22 LUC, pCD31-0.44 LUC, pCD31-0.66 LUC, pCD31-0.98 LUC, and pCD31-1.42 LUC, containing different frag- ments of the PECAM-1 promoter inserted into the pXP2 vector, were Results previously described (16). Mutagenesis of the ϩ110/ϩ120 NF-␬B site of Transcriptional regulation of PECAM-1 by NF-␬B PECAM-1 sequence was made by recombinant PCR from construct PECAM-1, as other adhesion molecules, is involved in the recruit- pCD31-0.44-LUC. NF-␬B consensus sequence GCAGGGAGGAATC CCC (ϩ110/ϩ120) was changed to GCAGGGTTTAATCCC, where the ment of neutrophils and leukocytes toward the inflammatory foci. mutated bases are underlined. For this purpose, complementary oligode- Structural characterization of the PECAM-1 promoter region re- oxynucleotides A (5Ј- GCAGGGTTTAATCCCCTCAC-3Ј)andB(5Ј-CT vealed consensus sites for nuclear factors of the NF-␬B family.