Molecule-1 Promoter the Platelet Endothelial Cell Adhesion B Site In

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

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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 Sańchez-Elsner,* Eduardo Munõz,† Angel Corbı´,* and Carmelo Bernabeú*

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 expression 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 Fisiologıá e Inmunologıá, 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 Comisioń Interministerial de Ciencia proteasome. Then, the activated NF- B dimer translocates to the y Tecnologıá (CICYT-SAF97-0034 to C.B. and CICYT-SAF98-0068 to A.C.) and nucleus and regulates gene transcription. The NF-␬B activation Comunidad Autońoma 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

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. GTGAGGGGATTAAACCCTGC-3Ј) were designed. At the same time, Because PECAM-1 expression is up-regulated upon treatment of Downloaded from oligodeoxynucleotides L (5Ј-GATCCAAGCTTGTCGACCC-3Ј) and R (5Ј-GATCTCAGACTCGGTACCC-3Ј), corresponding to the polylinker monocytic cell lines with phorbol esters (16, 22, 23), which are flanking regions of plasmid pXP2, were also synthesized and used as prim- strong NF-␬B activators, we assayed the effect of NF-␬B modu- ers. Using the pCD31-0.44-LUC construct as a template, single PCRs with lators on the PECAM-1 cell-surface expression and gene promoter A and R, as well as B and L, primers were performed. The resulting am- activity. PDTC has been described as a potent inhibitor of NF-␬B plification products were used as templates, in the presence of primers L and R, to carry out a third recombinant PCR. The recombinant product was activation (28). Thus, U937 cells were treated with PMA, in the

cloned in PCRII TOPO (Invitrogen, San Diego, CA), released upon EcoRI presence of different doses of PDTC, and expression of PECAM-1 http://www.jimmunol.org/ digestion, and subcloned into the SmaI site of pXP2, resulting in the at the cell surface was measured by flow cytometry (Fig. 1A). As pCD31-0.44-Mut-LUC construct. Plasmids were sequenced to confirm the previously described (16), when U937 cells were treated with mutated sequence. Plasmids pSEAP, which contains the alkaline phosphatase gene driven PMA, the expression of PECAM-1 increased 2.5-fold over the by the SV40 enhancer and early promoter (Clontech, Palo Alto, CA), and basal level. Importantly, addition of PDTC at 10–70 ␮M inhibited pCMV-␤gal, which contains the ␤-galactosidase gene driven by the CMV the PMA-dependent induction up to eight times. At the highest enhancer and promoter (Clontech), were used to normalize transfection concentration, PDTC lowered the PECAM-1 expression below the efficiencies. Plasmid pRc/CMV-p65 contains the gene encoding the p65 ␬ subunit of NF-␬B driven by the CMV promoter (25). Plasmid pKBF-Luc basal levels observed in untreated cells, suggesting that NF- Bis contains three repeats of the NF-␬B consensus elements present in the involved in basal expression of PECAM-1. As a control, the via- by guest on September 26, 2021 H-2Kb gene upstream of the herpes simplex thymidine kinase gene (26). bility of PDTC-treated cells was measured and found unaffected Transfections with respect to that of untreated cells (data not shown). Moreover, the effect of PDTC on PECAM-1 expression is not a general in- U937 (human promonocytic), K-562 (human erythropoietic), and Raw hibitory phenomenon, as PDTC has been reported to up-regulate 264.7 (mouse macrophage) cell lines were cultured in RPMI 1640, sup- ␤ plemented with 10% heat-inactivated FCS, 2 mM L-glutamine, in a 5% the expression of 2 integrins (CD11a-c/CD18) in U937 cells (29).

CO2 atmosphere at 37°C. U937 and Raw 264.7 cells, but not K-562 cells, To determine whether PMA and PDTC were altering PECAM-1 express PECAM-1. Transfection of U937, K562 (106), or Raw 264.7 (2 ϫ expression at the transcriptional level, U937 cells were transfected 5 10 ) cells was conducted using Superfect (Qiagen, Chatsworth, CA) and 1 with the pCD31-1.42-LUC reporter construct. PMA addition ␮g of the appropriate PECAM-1 promoter-pXP2 luciferase vector. Lucif- erase relative units were determined in a TD20/20 luminometer (Promega, caused a 2-fold increase in the PECAM-1 promoter activity (Fig. Madison, WI). When required, cells were treated with TNF-␣ at 50 or 100 1B). In addition, PDTC abrogated the PMA-induced increase in ng/ml, with pyrrolidine dithiocarbamate (PDTC) (10–150 ␮M), or with the promoter activity and, when used at 30 ␮M, even reduced the PMA (40 ng/ml) for the times indicated, 24 h after transfection. Stable basal level of promoter activity observed in untreated cells (Fig. transfectants of the PECAM-1 promoter were obtained by electroporation of U937 cells as described (16). Briefly, plasmids pCD31-1.42-LUC and 1B). Therefore, both PMA and PDTC directly affect the activity of pBSpacAp, encoding the puromycin resistance gene, were cotransfected, the PECAM-1 promoter. and puromycin-resistant cells were isolated and maintained in culture at 0.2 Subsequently, we analyzed the effect of TNF-␣, the strongest ␮ g/ml of antibiotic. inductor of NF-␬B (19), on the transcription of PECAM-1. To that Flow cytometry end, U-937 cells stably transfected with the PECAM-1 promoter were treated with TNF-␣ and the promoter activity was analyzed Flow cytometric analyses were performed with an Epics-CS (Coulter Ci- ␣ entifica, Madrid, Spain) using log amplifiers. U937 cells were treated with (Fig. 2). Addition of TNF- resulted in a 2.5-fold induction of the PMA or PDTC as indicated and incubated with HC1/6 (anti-PECAM-1) PECAM-1 promoter activity at 12 h after treatment. Taken to- mAb (21) for 30 min at 4°C. After two washes with PBS containing 0.1% gether, these experiments support the involvement of NF-␬Binthe BSA, FITC-labeled F(abЈ) rabbit anti-mouse IgG (Dakopatts, Copenha- 2 transcription of the PECAM-1 gene. gen, Denmark) was added, and incubation proceeded for an additional 30-min period at 4°C. Finally, cells were washed twice, and their fluores- Genomic sequence analysis of the 1.55-kb upstream region of cence was estimated. the PECAM-1 promoter revealed two possible consensus sites for NF-␬B (16). One of them was found at positions Ϫ409/Ϫ418 EMSA (GGGGTTCTCC) within an AluI element, and the second one was Nuclear extracts from treated or untreated U937 or Raw 264.7 cells were located at ϩ110/ϩ120 (GAGGAATCCCC) within a BglII/NotI obtained as described (27). Probes consisted of either the 185-bp BglII/NotI restriction fragment (Fig. 3A). This latter fragment was used as a fragment of PECAM-1, end labeled by Klenow with 10 ␮Ci of [␣-32P]dCTP, or the oligonucleotides, containing NF-␬B consensus ϩ110/ probe for binding experiments with nuclear extracts from PMA- ϩ120 (wild type (WT) and mutant) and Ϫ409/Ϫ418 (WT), labeled by treated or untreated U937 cells (Fig. 3B). Two specific bands could 1374 NF-␬B ROLE IN PECAM-1 TRANSCRIPTION

FIGURE 2. Effect of TNF-␣ on the PECAM-1 promoter activity. U-937 Downloaded from cells stably transfected with the PECAM-1 reporter pCD31-1.42-LUC were incubated either in the absence or in the presence of TNF-␣ 100 ng/ml for the times indicated. Cells were lysed and luciferase activity was measured using a luminometer. For comparative purposes, the activity of PECAM-1 transfectants in the absence of treatment was given the arbitrary value of 100. As a negative control, the luciferase activity of cells trans-

fected with the empty pXP2 vector (MOCK) was included. The mean of http://www.jimmunol.org/ three different experiments is shown.

gonucleotides (Fig. 3C). The probe containing the NF-␬B site at ϩ110/ϩ120 showed speciﬁc bands in U937 and Raw 264.7 cells. By contrast, the oligonucleotide containing the NF-␬B site at Ϫ409/Ϫ418 did not show any binding when used as a probe, nor competed the binding of the ϩ110/ϩ120 site. Therefore, further

studies were focused only on the NF-␬B motif at ϩ110/ϩ120, as by guest on September 26, 2021 a bona fide NF-␬B site. As expected, both PMA and TNF-␣ increased the two specific NF-␬B bands (Figs. 3, B and D). Also, PDTC inhibited the NF-␬B complex formation and prevented its increase due to either PMA or TNF-␣ (Fig. 3D). Identification of the specific complexes p50/p65 or p50/p50 was made by Abs against p50 and p65. Addition of anti-p65 resulted in inhibition of the upper complex associated with a supershift effect, whereas the presence of anti-p50 Abs led to inhibition of both complexes and FIGURE 1. Effect of the NF-␬B inhibitor PDTC on the PMA-induced to a supershift effect (Fig. 3D). These results suggest that the upper PECAM-1 expression. A, Cell-surface expression of PECAM-1. U937 cells band is formed by p65/p50 heterodimers, while the lower complex were incubated either in the absence or in the presence of PMA (10ng/ml) is formed by p50 homodimers. Overall, these findings support the or PDTC (10–70 ␮M) for 2 days, as indicated. Then, cells were stained for involvement of NF-␬B on the PECAM-1 transcription. indirect immunofluorescence using the mAb HC1/6 (anti-PECAM-1) and analyzed by flow cytometry. Values of mean fluorescence intensity are Involvement of NF-␬B site at ϩ110 in PECAM-1 expression indicated in the upper right corner. B, U937 cells were transiently trans- ␬ ϩ fected with the PECAM-1 promoter construct pCD31-1.42-LUC. After To test whether the NF- B site at 110 was involved in the tran- 24 h, cells were treated in the presence or in the absence of PMA (40 scriptional activity of PECAM-1, its consensus sequence was mu- ng/ml) or PDTC (10–30 ␮M), as indicated. Transcriptional activity was tated in the context of the pCD31-0.44-LUC plasmid. Then, wild- measured using the luciferase reporter assay. Correction for transfection type and mutant constructs were used to transfect Raw 264.7 cells efficiency was made by cotransfection with a ␤-galactosidase expression as these cells constitutively express high levels of TNF-␣ (30) and vector. For comparative purposes, the activity of the PECAM-1 construct are likely to have constitutively active NF-␬B. As shown in Fig. in the absence of treatment was given the arbitrary value of 100. As a 4A, the mutant construct displayed a clear reduction (seven times) negative control, the promoter-less vector pXP2 was used. The mean of of its promoter activity respect to the wild-type control. Moreover, three different experiments is shown. we found that addition of the NF-␬B inhibitor PDTC decreased the promoter activity of the wild-type construct in a dose-dependent manner (Fig. 4B). Therefore, the NF-␬B element at ϩ110 directly be detected, which were competed by a consensus NF-␬B oligo- contributes to the activity of the PECAM-1 promoter in Raw 246.7 nucleotide, while the complexes remained unaltered in the pres- cells. ence of heat shock protein factor or AP-1 recognition sequences. To confirm the transcriptional involvement of the NF-␬B site at Both putative NF-␬B sites at Ϫ409/Ϫ418 and ϩ110/ϩ120 were ϩ110, EMSA experiments using the wild-type and mutated ver- also studied in binding experiments using their corresponding oli- sions of the NF-␬B oligonucleotide were conducted in Raw 264.7 The Journal of Immunology 1375 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. Characterization of a NF-␬B consensus site in PECAM-1 promoter. A, Diagram of the 1.42-Kb SmaI/PstI fragment of the PECAM-1 promoter containing the NF-␬B site at ϩ102. Numbers at sites of restriction enzymes refer to the transcription start site (ϩ1) indicated by an arrow. The 185-bp BglII/NotI fragment (ϩ47/ϩ232) and the synthetic oligonucleotide ϩ102/ϩ126 were used as probes in the EMSA experiments corresponding to B–D. B, EMSA with PMA-treated or untreated U937 nuclear extracts. The 185-bp BglII/NotI fragment used as a probe was end labeled with [␣-32P]dCTP. The presence of competitors (ϫ100-fold excess) heat shock protein factor, AP-1, and NF-␬B (NF) consensus oligonucleotides, and the 185-bp BglII/NotI fragment (F) is indicated. Arrows point to the NF-␬B complexes, identified according to E. C, EMSA using probes containing the Ϫ409/Ϫ418 and ϩ110/ϩ120 NF-␬B consensus sites. Nuclear extracts from U937 (U) or Raw 264.7 (R) cells were incubated with oligonucletides Ϫ422/Ϫ404 (Ϫ404) or ϩ103/ϩ124 (ϩ102) as indicated. D, Specific blocking of NF-␬B mobilization by PDTC. Nuclear extracts from cells treated with different combinations of TNF-␣, PMA, or PDTC were used as indicated. EMSA experiments were performed using as a probe the oligonucleotide ϩ110/ϩ120 containing a consensus NF-␬B site. E, Identification of NF-␬B/Rel subunits present in the protein-DNA complexes of PECAM-1 promoter. Untreated or TNF-␣-treated U937 cells were used for EMSA experiments using as a probe the oligonucleotide ϩ110/ϩ120 containing a consensus NF-␬B site. The presence of competitor unlabeled oligonucleotide and Abs to p65 and p50 is indicated. The arrows indicate the original NF-␬B complexes whose electrophoretic mobility is modified by the presence of Abs. 1376 NF-␬B ROLE IN PECAM-1 TRANSCRIPTION Downloaded from

FIGURE 5. Analysis by EMSA using as probes the wild-type and the mutated NF-␬B oligonucleotides. A, Sequences of wild-type and mutated oligonucleotides, used as probes, are shown. B, Different amounts of nuclear extracts (5 or 10 ␮g of total protein) from Raw 264.7 cells were

incubated with the wild-type (wt) or the mutated (mut) oligonucleotides http://www.jimmunol.org/ (ϩ110/ϩ120) as probes. In each sample, 105 cpm were used. Binding reactions were loaded in a 7.5% polyacrylamide gel and electrophoresed. The presence of 100 times excess competitor is indicated.

of PECAM-1 displayed a reduction (2.5 times) of its promoter activity in respect to the wild-type plasmid (Fig. 6). In addition, the activity of the wild-type promoter was increased by TNF-␣ in a FIGURE 4. Role of the NF-␬B site at ϩ110 on the PECAM-1 promoter dose-dependent manner, whereas that of the mutated version was by guest on September 26, 2021 activity of Raw cells. A, Effect of mutation of the NF-␬B site at ϩ110 on either unaffected or inhibited (Fig. 6). the PECAM-1 promoter activity. Raw 264.7 cells were transiently trans- To further demonstrate the specific involvement of members of fected with the wild-type pCD31-0.44-LUC plasmid (WT) or the corre- the NF-␬B family in the PECAM-1 promoter activity, transacti- sponding mutant construct (MUT), and, 24 h later, the luciferase activity vation experiments were conducted in Raw 264.7 cells using an was measured. Correction for transfection efficiency was made by cotrans- expression vector for the p65 subunit (Fig. 7). Coexpression of the fection with an alkaline phosphatase expression vector. The mean of three different experiments is shown. As a negative control, the promoter-less vector pXP2 was used. B, Effect of PDTC on the promoter activity of PECAM-1. Raw 264.7 cells were transiently transfected with the pCD31- 0.44-LUC plasmid in the presence of the indicated concentrations of PDTC, and luciferase activity was measured after 24 h of treatment. Cor- rection for transfection efficiency was made by cotransfection with an alkaline phosphatase expression vector. The mean of three different experiments is shown. cells (Fig. 5, A and B). When the wild-type oligonucleotide ϩ110/ ϩ120 was labeled as a probe, two specific bands corresponding to the NF-␬B transcription factor appeared (Fig. 5B). The identification of these bands was made with Abs specific for p50 and p65, respectively (data not shown). However, when the mutated oligonucleotide was used as a probe, no specific complexes could be detected. Taken together, these results support the involvement of the NF-␬Batϩ110 in the promoter activity of PECAM-1. FIGURE 6. Effect of TNF-␣ on the activity of the PECAM-1 promoter mutated at the NF-␬B site ϩ110/ϩ120. K-562 cells were transiently trans- The p65 subunit of NF-␬B modulates the promoter activity of fected with the wild-type pCD31-0.44-LUC construct (Ⅺ) or the corre- PECAM-1 sponding mutant construct (o) in the presence of the indicated concentra- ␣ ␬ ϩ tions of TNF- . Luciferase activity was measured after 24 h of treatment. The transcriptional involvement of the NF- B site at 110 in the Correction for transfection efficiency was made by cotransfection with an promoter activity of PECAM-1 was also assessed by treatment alkaline phosphatase expression vector. For comparative purposes, the pro- with exogenous TNF-␣ of erythropoietic K562 cells because this moter activity of the wild-type pCD31-0.44-LUC construct in the absence cell lineage does not express TNF-␣. Similarly to results obtained of TNF-␣ was given the arbitrary value of 100. The mean of four different with Raw 264.7 cells (Fig. 4A), in K562 cells the mutant construct experiments is shown. The Journal of Immunology 1377

macrophages (16, 22, 23). The results here presented suggest that part of this regulation in macrophages is mediated through the NF-␬B factor, active at the ϩ110/ϩ120 site of the PECAM-1 promoter used in this study. Interestingly enough, mobilization of NF-␬B in monocytes/macrophages can be readily achieved by a variety of stimuli, including cell adherence to the substratum, or the presence of TNF-␣ or bacterial LPS (19), suggesting a functional versatility of PECAM-1. The involvement of NF-␬Bin PECAM-1 transcription was demonstrated based upon several lines of evidence: 1) surface expression and promoter activity of PECAM-1 are regulated by modulators of NF-␬B, including TNF-␣, PMA, and PDTC; 2) cotransfection experiments with a PECAM-1 promoter-derived reporter construct and an expression vector encoding the p65 subunit of NF-␬B show transactivation of the PECAM-1 promoter; 3) EMSA experiments demonstrate that the NF-␬B site at ϩ110/ϩ120 of the PECAM-1 promoter binds FIGURE 7. Effect of p65 overexpression on the PECAM-1 promoter NF-␬B transcription factor; and 4) mutation of the NF-␬B site at activity. Raw 264.7 cells were transiently transfected with the pCD31-0.22- ϩ110/ϩ120 abolishes the basal promoter activity of PECAM-1

LUC, pCD31-0.44-LUC (both wild-type and mutant versions), pCD31- and inhibits the TNF-␣-dependent induction of the promoter. Downloaded from 0.98-LUC, or pCD31-1.42-LUC constructs, either in the absence or in the A major secretory product of macrophages is TNF-␣, which in ␬ presence of the plasmid encoding the p65 subunit of NF- B, as indicated. turn stimulates mononuclear phagocytes to secrete other cytokines After 24 h, the luciferase activity was measured. Correction for transfection contributing to the leukocyte adhesion and to activate transcription efﬁciency was made by cotransfection with an alkaline phosphatase ex- of surface receptors, leading to recruitment of neutrophils, mono- pression vector. The mean of three different experiments is shown. As a positive control for the transactivation effect of p65, the plasmid KBF-1 cytes, and lymphocytes to the inﬂammatory sites. One way for

␣ http://www.jimmunol.org/ containing multiple repeats of NF-␬B consensus elements was used. The TNF- to increase transcription of its target genes is through mo- pXP2 vector was used as a negative control. bilization of transcription factors such as those of the NF-␬B family (19, 20). Monocytes, rather undifferentiated cells, circulate in the blood stream and can be recruited to inﬂammatory foci, dif- ferentiate into macrophages, and be activated by cytokines to re- p65 subunit resulted in a 4.5-fold increase of the transcriptional spond against foreign Ags, to eliminate tumor cells, and to induce activity displayed by the pCD31-0.44 LUC wild-type PECAM-1 proliferation and migration of endothelial cells. NF-␬B is involved promoter construct, whereas the activity in the mutant construct in these processes by coordinately controlling gene expression of was not signiﬁcantly increased. In addition, PECAM-1 promoter ILs, adhesion molecules, and cytokines expressed by monocytes/

constructs containing (pCD31-0.44-LUC and pCD31-1.42-LUC) by guest on September 26, 2021 macrophages (19). Therefore, a possible induction of PECAM-1 or not (pCD31-0.22-LUC and pCD31-0.98-LUC) the NF-␬B site transcription through NF-␬B activation can be interpreted as an at ϩ110 were analyzed. Only constructs pCD31-0.44-LUC and adaptative response of the organism to meet inflammation. In this pCD31-1.42-LUC containing the NF-␬B site at ϩ110 could be sense, PECAM-1 participates in neutrophil recruitment at inflam- transactivated at similar levels with the p65 subunit. It is worth matory sites (5, 6) and is down-regulated after leukocyte extrav- noting that the unresponsive pCD31-0.98-LUC construct contains asation (31). To our knowledge, the effect of TNF-␣ on PECAM-1 the putative NF-␬B site at Ϫ409. This is in agreement with the lack expression by myeloid cells had not been reported to date. How- of specific NF-␬B complexes when using the oligonucleotide ever, three reports have analyzed the combined action of TNF-␣ Ϫ422/Ϫ404 in EMSA studies (Fig. 3C) and suggests that this is a and IFN-␥ on endothelial cells (32–34). Thus, it has been shown nonfunctional motif. As a control of the experiment, the KBF-1 that this cytokine combination leads to a redistribution of the promoter construct, containing three NF-␬B responsive elements, PECAM-1 Ag on human endothelial cells (32). However, while showed a 5-fold transcription stimulation upon transfection with some authors find a reduction in the levels of PECAM-1 (33, 34), the p65 subunit. These results demonstrate that the p65 subunit of others do not find alterations in the levels of PECAM-1 transcrip- the NF-␬B complex takes part in the transcriptional regulation of tion or surface expression (32). These contradictory reports might PECAM-1. reflect the existence of soluble forms of PECAM-1, generated by alternative splicing (7), which likely show a transcriptional regu- Discussion lation different from the membrane-bound PECAM-1. As all these The NF-␬B family of transcription factors has been reported to be studies were conducted using the combined action of TNF-␣ and involved in the control of several important physiological pro- IFN-␥ in endothelial cells, it is not possible to compare these re- cesses, including inflammation, cell cycle regulation, and HIV vi- sults with the single TNF-␣ treatments on myeloid cells analyzed ral replication (19–21). Thus, NF-␬B regulates transcription of in this report. In addition, it can be argued that the inducible ex- cytokines (e.g., TNF-␣, IL-1, or IFN-␥), and cell-surface receptors pression of PECAM-1 detected in myeloid cells should have a (e.g., ICAM-1, VCAM-1, E-selectin, or CD69), involved in the different type of regulation than the constitutive expression found inflammatory response. Here, we report the transcriptional regu- in endothelial cells. lation of the cell adhesion molecule PECAM-1 by NF-␬Binmy- It is worth noting that NF-␬B was initially identified as an ac- eloid cells. tivator of the Ig ␬ light chain (35). Since then, an increasing num- PECAM-1 is a TATA-less gene, which is a characteristic of ber of genes, belonging to the Ig superfamily, have been reported constitutively expressed genes, containing multiple transcription to be regulated by NF-␬B, including the TCR ␣ and ␤, MHC class initiation sites (16, 17). Endothelial cells express constitutively I and II, VCAM-1, ICAM-1, or MadCAM (18, 19, 36). Among high levels of PECAM-1, while its expression is regulated in these, the list of members of the CAM subfamily is now increased monocytes, during the process of maturation and differentiation to with the identification of a functional NF-␬B site in the PECAM-1 1378 NF-␬B ROLE IN PECAM-1 TRANSCRIPTION

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