Regulation of the Human P-selectin Promoter by Bcl-3 and Specific Homodimeric Members of the NF- (cid:107) B/Rel Family*

P-selectin, an adhesion receptor for leukocytes, is con- stitutively expressed by megakaryocytes and endothelial cells. Synthesis of P-selectin is also increased by some inflammatory mediators. We characterized a previously identified (cid:107) B site ( (cid:50) 218 GGGGGTGACCCC (cid:50) 207 ) in the promoter of the human P-selectin gene. The (cid:107) B site was unique in that it bound constitutive nuclear protein complexes containing p50 or p52, but not inducible nuclear protein complexes containing p65. Furthermore, the element bound recombinant p50 or p52 homodimers, but not p65 homodimers. Methylation interference anal- ysis indicated that p50 or p52 homodimers contacted the guanines at positions (cid:50) 218 to (cid:50) 214 on the coding strand and at (cid:50) 210 to (cid:50) 207 on the noncoding strand. Changes in the three central residues at (cid:50) 213 to (cid:50) 211 altered binding specificity for members of the NF- (cid:107) B/Rel family. Mutations that eliminated binding to NF- (cid:107) B/Rel proteins reduced by (cid:59) 40% the expression of a reporter gene driven by the P-selectin promoter in transfected bovine aortic endothelial cells. Overexpression of p52 enhanced P-selectin promoter activity, and co-overexpression of Bcl-3 further induced promoter activity in a (cid:107) B site-de-pendent manner. In contrast, overexpression of

Trafficking of leukocytes into inflammatory sites requires the regulated expression of adhesion molecules on activated endothelial cells (1,2). Inflammatory mediators such as interleukin-1 (IL-1), 1 TNF-␣, and LPS induce human umbilical vein endothelial cells (HUVEC) to transcribe mRNAs encoding the adhesion molecules E-selectin, VCAM-1, and ICAM-1. Transcription increases within 1-2 h and persists for 6 -72 h, de-pending on the stimulus and the gene (3,4). During active mRNA transcription, newly synthesized protein appears on the cell surface. In contrast to these adhesion proteins, P-selectin is constitutively synthesized by megakaryocytes (the precursors of platelets) and endothelial cells, where it is packaged into the membranes of secretory granules (5). Upon stimulation of these cells by agonists such as thrombin, P-selectin is rapidly redistributed to the plasma membrane. In vivo, LPS and TNF-␣ markedly increase P-selectin transcripts and protein in endothelial cells of multiple tissues (6 -9). These agonists also augment P-selectin mRNA levels in cultured murine, rat, and bovine endothelial cells (7,9,10). However, LPS and TNF-␣ do not increase P-selectin transcripts in cultured HUVEC, 2 suggesting that the transcriptional regulation of P-selectin differs from that of E-selectin, VCAM-1, and ICAM-1.
We have previously isolated and conducted a preliminary analysis of the 5Ј-flanking region of the human P-selectin gene (11). Transcription of the gene is initiated at multiple sites, consistent with the lack of a canonical TATA box in the promoter region. The sequence from Ϫ249 to Ϫ13 relative to the translational start site confers tissue-specific expression of a reporter gene in cultured bovine aortic endothelial cells (BAEC). Serial deletions of this sequence revealed at least three positive regulatory regions; a GATA element in the region from Ϫ197 to Ϫ147 was demonstrated to be functional. The region from Ϫ249 to Ϫ197 contains at least three potential elements, including a putative recognition site for NF-B/Rel transcription factors.
The DNA-binding forms of NF-B/Rel transcription factors are homodimers or heterodimers that bind to B elements in many genes involved in inflammation, acute phase responses, cell proliferation, and differentiation (12)(13)(14)(15)(16). The family members share a Rel homology domain and can be divided into two groups. The first group includes Rel A (p65), Rel (c-Rel), Rel B, v-Rel, and the Drosophila proteins Dorsal and Dif. Members of this group share an acidic transactivation domain. Homodimers or heterodimers containing at least one of these molecules are retained in the cytoplasm by complex formation with IB␣ and related proteins. Upon cellular stimulation, IB␣ is degraded; the dimeric complexes then migrate to the nucleus where they function as potent activators of many genes with B elements, including those for E-selectin, VCAM-1, and ICAM-1 (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). The second group includes the precursor proteins p105 (NF-B1) and p100 (NF-B2), which are proteolytically cleaved to the mature p50 and p52 proteins, respectively. Most studies indicate that homodimers containing p50 or p52 have little or no ability to transactivate gene expression (14), although there are some dissenting reports (28,29). Homodimers of p50 are present constitutively in the nucleus of * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed: Dept. of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104. Tel.: 405-271-6480; Fax: 405-271-3137. 1 The abbreviations used are: IL-1, interleukin-1; BAEC, bovine aortic endothelial cells; HUVEC, human umbilical vein endothelial cells; ICAM-1, intercellular adhesion molecule-1; LPS, lipopolysaccharide; TNF-␣, tumor necrosis factor ␣; VCAM-1, vascular cell adhesion molecule-1. some cells, where they may prevent binding of inducible complexes containing members of the first group of proteins such as p65 (30). Bcl-3, a protein structurally related to IB␣, dissociates bound p50 homodimers from DNA, allowing heterodimers containing p65 to bind and transactivate (31). In contrast, Bcl-3 forms a ternary complex with p52 homodimers on DNA, resulting in transactivation (32,33). There is disagreement as to whether Bcl-3 can form ternary complexes with p50 homodimers on DNA that transactivate (32,33). One difficulty in interpreting the roles of p50 and p52 homodimers in gene expression has been the inability to identify B elements that bind only to these proteins.
In this paper we characterize the properties of the B element in the human P-selectin promoter. We find that this B site has the unique property of binding only to p50 and p52 homodimers. Interactions of Bcl-3, p52 homodimers, and the B element augment transcription. In contrast, interactions of p50 homodimers with the B element repress transcription; however, repression is prevented by co-expression of Bcl-3. These data suggest that differential interactions of Bcl-3 with p50 and p52 homodimers regulate the constitutive and inducible expression of the P-selectin gene, and perhaps other genes with B sites specific for these homodimers.

Cells, Proteins, Antibodies, and Expression Plasmids-CHRF-288
human megakaryocytic cells were maintained in Fisher's medium supplemented with 20% horse serum. BAEC, HUVEC, HL-60 cells, HEL cells, and Jurkat cells were cultured as described previously (11). Recombinant p50 and p49 (a variant form of p52) were obtained from Promega. Purified, bacterially expressed p50 (amino acids 1-503) and p65 were gifts from Dr. Craig Rosen (34). Antibodies against p50, p52, and c-Rel were obtained from Santa Cruz. The expression plasmid encoding Bcl-3, driven by the Rc/CMV promoter, was a gift from Dr. Timothy McKeithan (35). The expression plasmid encoding p52, driven by the adenovirus major late promoter, was a gift from Dr. Riccardo Dalla-Favera (36). The expression plasmid p50XbaI, encoding amino acids 1-503 of p50 under the control of the Rc/CMV promoter, was provided by Dr. Rosen (34). An expression plasmid encoding amino acids 1-401 of p50 under the control of the same promoter was constructed by polymerase chain reaction.
Gel Mobility Shift Assay-Gel mobility shift assays were performed as described previously (11), except that KCl was used at a final concentration of 100 mM. In some experiments, nuclear extracts were preincubated with antibodies at room temperature for 45 min before the addition of labeled probe.
Methylation Interference Assay-Methylation interference analysis was performed as described previously (37). Aliquots of the Seq I oligonucleotide were end-labeled on each strand. The labeled oligonucleotides were partially methylated and then incubated with recombinant p50 or p52 homodimers. Bound and free DNA were separated by native 4% polyacrylamide gel electrophoresis, eluted from the gel, and cleaved with piperidine. The cleaved products were electrophoresed in 12.5% polyacrylamide, 7.5 M urea gels and subjected to autoradiography.
Construction of Chimeric Luciferase Expression Vectors-Plasmid p309LUC was described previously (11) and plasmid pGL2-promoter was from Promega. Plasmid pmB309LUC, which contained two base changes in the B site (GGGGGTGACCCC to GCCGGTGACCCC) was constructed by an overlap extension polymerase chain reaction protocol (38). Plasmids p2xBSV40 and p2xmBSV40 were constructed through direct ligation of double-stranded oligonucleotides encoding two wildtype or mutated P-selectin B sites into the MluI and XhoI sites of the pGL2-promoter vector. These oligonucleotides were: 5Ј-cgcgGAAGGG-GGTGACCCCTTGCCGAAGGGGGTGACCCCTTGCC-3Ј and 3Ј-CTTC-CCCCACTGGGGAACGGCTTCCCCCACTGGGGAACGGagct-5Ј for the wild-type P-selectin B sites, and 5Ј-cgcgGAAGGGGGTGAATAGTT-GCCGAAGGGGGTGAATAGTTGCC-3Ј and 3Ј-CTTCCCCCACTTAT-CAACGGCTTCCCCCACTTATCAACGGagct-5Ј, for the mutant. These complementary oligonucleotides were annealed and phosphorylated (39), and then used for ligation. The fidelity of all constructs was verified by dideoxynucleotide sequencing.
Transfection and Luciferase Assay-Preparation of plasmids, transfections, and luciferase assays were performed as described previously (11), with the following minor modifications. For co-transfections, the total amount of DNA was kept constant at 10 g/dish of cells by adding an appropriate amount of a control plasmid without an insert (pRc/ CMV, Invitrogen). Equal volumes of 30 g of test plasmids and 50 g of Lipofectin reagent (Life Technologies, Inc.), each diluted in 3.75 ml of OptiMEM medium (Life Technologies, Inc.), were incubated for 20 min. The resulting transfection mixture was divided into three parts, and each part was then added to cells in a separate 60-mm dish. After incubation for 7-8 h at 37°C, the transfection medium was replaced by complete medium for an additional 36 h and the cells were then harvested for luciferase assays.
Northern Blot Analysis-Total RNA was prepared from CHRF-288 cells, HEL cells, and HUVEC by acid guanidium thiocyanate-phenolchloroform extraction (40), and Northern blot analysis was performed as described previously (41).

RESULTS
The Sequence from Ϫ232 to Ϫ192 in the 5Ј-Flanking Region of the P-selectin Gene Forms Five DNA-Nuclear Protein Complexes-We previously showed that deletion of the sequence from Ϫ249 to Ϫ197 in the 5Ј-flanking region of the human P-selectin gene decreased expression of a reporter gene in transfected BAEC by ϳ40% (11). This region contains putative recognition elements for ETS proteins, certain zinc finger proteins induced by phorbol esters, and members of the NF-B/Rel family of proteins. To determine whether nuclear proteins bound to this region, we synthesized a 41-base pair doublestranded oligonucleotide encompassing the sequence Ϫ232 to Ϫ192, which contained all the putative regulatory elements (Fig. 1A). The labeled oligonucleotide, termed Seq I, formed five complexes with nuclear extracts from the megakaryocytic cell line CHRF-288 (Fig. 1B) and from all other cells tested (BAEC, HEL cells, Hy.EA926 hybrid endothelial cells, and Jurkat cells, data not shown). Formation of all complexes was prevented by addition of a 100-fold excess of unlabeled Seq I probe, but not by an unlabeled probe containing an unrelated GATA element (11) (Fig. 1B). Complex II was resolved into separate complexes, termed IIa and IIb, only after electrophoresis for longer periods. Complexes III and IV were not consistently formed by all nuclear extracts from a given cell type.
The B Element in the P-selectin Promoter Binds Homodimers Containing p50 or p52 but Not Homodimers or Heterodimers Containing p65-The putative B element in Seq I is very similar to a B element in the murine H-2K b gene that binds constitutive p50 and p52 homodimers as well as inducible p50/p65 heterodimers (42)(43)(44) (Fig. 1A). As shown in Fig. 2, a 50-to 200-fold excess of an unlabeled oligonucleotide encompassing the H2-K b sequence prevented Seq I from forming complex II, but not the other complexes, with nuclear extracts. We next synthesized a shorter oligonucleotide, termed Seq B, that contained only the sequence from Ϫ222 to Ϫ200 immediately surrounding the putative B element (Fig. 1A). Seq B, when labeled, formed only complex II, and complex formation was specifically inhibited by addition of unlabeled Seq I, Seq B, or the H2-K b probe (Fig. 2). These results indicate that Seq B contains a B element that is functionally related to the element in the H2-K b promoter.
To determine whether Seq B bound constitutive p50 or p52 homodimers as well as inducible NF-B dimers containing p65, we first performed gel shift assays with nuclear extracts from BAEC treated with or without phorbol myristate acetate, which induces degradation of IB␣ and release of p50/p65 complexes into the nucleus (Fig. 3A). The labeled H2-K b probe formed two complexes. The faster migrating complex, found in both unstimulated and stimulated cells, corresponded to constitutively expressed p50 or p52 homodimers. The slower moving complex represented p50/p65 heterodimers that were more abundant in extracts from stimulated cells (43). The labeled Seq B also formed the faster moving complex, but not the slower moving complex. Furthermore, unlabeled Seq B pre-vented the labeled H2-K b probe from forming the faster moving complex, but not the slower moving complex (Fig. 3B). These results suggest that the P-selectin B site, unlike the H2-K b B element, interacts with p50 or p52 homodimers but not inducible dimers containing p65. In other experiments, Seq B failed to form slower moving complexes with nuclear extracts from HUVEC treated with LPS for 2, 4, or 24 h, or from BAEC treated with TNF-␣ for 2 or 4 h (data not shown). Because dimers containing c-Rel also migrate to the nuclei of stimulated HUVEC (45), this result suggests that Seq B does not bind inducible dimers containing c-Rel.
To confirm the differential specificities of the Seq B and H2-K b probes, we performed gel shift assays with purified, recombinant p50, p52, and p65; each of these proteins forms homodimers when expressed in bacteria (34). Seq B formed complexes with p50 and p52 homodimers, but not with p65 homodimers, whereas the H2-K b probe formed complexes with all three proteins (Fig. 4A). To determine whether complexes IIa and IIb represented interactions of Seq I with p52 and p50 homodimers, we preincubated nuclear extracts or purified p50 or p52 with specific antibodies prior to gel shift analysis. To resolve complex IIa from complex IIb, electrophoresis was performed for a longer period such that the faster migrating com-plexes III and IV exited the gel. Antibodies to p50 supershifted complex IIb to a slower migrating position, and antibodies to p52 supershifted complex IIa (Fig. 4B). In contrast, antibodies to c-Rel had no effect on either complex. None of the antibodies affected formation of complex I. Collectively, these data indicate that the P-selectin B element interacts with constitutively expressed nuclear complexes consisting of p50 or p52 homodimers, but not inducible nuclear complexes containing p65.
Characterization of the Nucleotides in the P-selectin B Sequence That Contact p50 and p52 Homodimers-We used methylation interference analysis to characterize the sites on the P-selectin B element that bind p50 and p52 homodimers. As shown in Fig. 5, methylation of five guanines at Ϫ218 to FIG. 1. Nuclear proteins bind to the sequence from ؊232 to ؊192 in the 5-flanking region of the P-selectin gene. A, at the top are shown the relative expression levels of two reporter genes driven by 5Ј-flanking sequences of the P-selectin gene, as described previously (11). The sequence deleted in the second construct includes a B element (overlined) that is aligned with the sequence of the B site of the H2-K b promoter. The three central residues in the H2-K b element that differ from those in the P-selectin element are italicized. The coding strands of the double-stranded Seq I, Seq B, and H2-K b oligonucleotides used as probes and competitors in gel mobility shift experiments are shown. B, nuclear extracts from CHRF-288 cells were incubated with a labeled Seq I probe in the presence or absence of a 100-fold excess of the unlabeled Seq I probe or an unlabeled probe containing an unrelated GATA element (11). The positions of the specific DNA-protein complexes are indicated. Ϫ214 in the coding strand and four guanines at Ϫ210 to Ϫ207 in the non-coding strand suppressed binding to p52 and p50. Methylation of the guanine at Ϫ212 on the coding strand also prevented binding to both proteins, although the effect was more pronounced for p52.
The three core nucleotides at Ϫ213 to Ϫ211 in the P-selectin B element differ from those in the H2-K b element, suggesting that they are important for recognition specificity. We used gel shift assays to test the effects of some changes in the core sequence of the B element of Seq I. Substitution of the G at Ϫ212 with C or A preserved recognition specificity for p50 and p52 homodimers, whereas substitution to T also conferred binding to p50/p65 heterodimers (data not shown). No clear rules for recognition specificity emerged from this limited survey. In conjunction with the results from methylation interference analysis, however, the data indicate that p50 and p52 homodimers bind specifically to four symmetrical guanines on each strand. These guanines are separated by three core nucleotides that participate in recognition specificity. The B Element Is Required for Optimal Constitutive Expression of the P-selectin Gene-To determine whether the B element was required for optimal constitutive expression of Pselectin, we changed two guanines at Ϫ217 and Ϫ216 to cytosines (Fig. 6A). These mutations specifically affected the binding site for NF-B proteins; a Seq I oligonucleotide containing the mutations did not form complex II but did form other protein complexes with nuclear extracts from CHRF-288 cells (Fig. 6B). The mutant oligonucleotide also failed to bind purified p50 or p52 dimers (Fig. 6C). The same two mutations were then introduced into a luciferase reporter gene driven by the P-selectin 5Ј-flanking sequence from Ϫ309 to Ϫ13. When transfected into BAEC, the mutant construct was expressed at levels ϳ40% lower than those of the wild-type sequence (Fig.  6D). The reduction in expression was similar to that produced by deletion of the region from Ϫ309 to Ϫ197 (11). These data indicate that a functional B element is required for optimal constitutive expression of the P-selectin gene in BAEC.

Function of the P-selectin B Element Is Differentially Regulated by Interactions of p50 and p52 Homodimers with Bcl-3-
Interactions of p50 and p52 homodimers with Bcl-3 have been reported to stimulate or repress B-dependent gene expression (14). The previously studied genes have B elements that bind both p50 and p52 homodimers as well as heterodimers containing p65. In contrast, the B site in the P-selectin promoter binds only p50 and p52 homodimers. Therefore, we asked whether p50, p52, and Bcl-3 regulate B-dependent expression of the P-selectin gene. BAEC were co-transfected with plasmids encoding various combinations of these proteins with a luciferase reporter gene driven by the P-selectin promoter, containing either a wild-type or mutated B element.
Co-expression of p52 augmented luciferase expression driven by the wild-type promoter, but not the promoter with the mutated B element (Fig. 7A). Co-expression of increasing amounts of Bcl-3 with a constant amount of p52 further increased luciferase expression by the wild-type, but not the FIG. 4. The P-selectin B element binds p50 and p52 homodimers but not p65 homodimers. A, the labeled Seq B and H2-K b probes were incubated with purified recombinant homodimers containing p50, p52, or p65. B, the labeled Seq I probe was incubated with nuclear extracts from CHRF-288 cells or with purified p50 or p52, in the presence or absence of the indicated antibodies. The DNA-protein mixtures were electrophoresed for a longer period to resolve complexes IIa and IIb. Antibodies to p50 supershifted complex IIb, and antibodies to p52 supershifted complex IIa.

FIG. 5. Methylation interference analysis of the nucleotides in the P-selectin B element that contact p50 and p52 homodimers.
A, aliquots of the Seq I oligonucleotide were end-labeled on each strand. The labeled oligonucleotides were partially methylated and then incubated with purified p50 or p52 homodimers. Bound and free DNA were separated by electrophoresis in native 4% polyacrylamide gels, eluted, and cleaved with piperidine. The cleaved products were resolved in 12.5% polyacrylamide, 7 M urea gels and subjected to autoradiography. B, sequence of the P-selectin B element. The filled symbols indicate guanines that, when methylated, blocked binding of the oligonucleotide to p50 or p52. The open symbols indicate guanines that, when methylated, partially inhibited binding of the oligonucleotide to p50 or p52. mutant, promoter. The expression observed without co-transfection of p52 or Bcl-3 may reflect the basal functions of the endogenously expressed proteins (42,44,46). Similar stimulatory effects were observed with a reporter gene driven by an SV40 minimal promoter linked to two copies of wild-type Seq B, but not Seq B containing a mutated B element (Fig. 7B). The mutated Seq B oligonucleotide also failed to interact with p50 or p52 in gel shift assays (data not shown).
In sharp contrast, co-expression of p50 repressed luciferase expression driven by the wild-type P-selectin promoter (Fig.  8A). However, co-expression of increasing amounts of Bcl-3 prevented the inhibitory effects of p50 on reporter gene expression. Similar effects were observed with the reporter gene containing two copies of wild-type Seq B linked to the SV40 minimal promoter (Fig. 8B). The p50 construct in these experiments encompassed residues 1-503, whereas the p50 protein generated by proteolysis in intact cells may span only the first 400 amino acids (33). It has been suggested that only homodimers containing the larger form of p50 repress B-dependent gene expression (33). However, we found that a p50 construct encoding residues 1-401 had similar inhibitory effects on P-selectin reporter gene expression (data not shown). These data indicate that the function of the B element in the Pselectin promoter is differentially regulated by interactions of Bcl-3 with p52 and p50 homodimers.
Cells Expressing P-selectin Also Transcribe mRNAs for Bcl-3 and the Precursors of p50 and p52-Endothelial cells and megakaryocytes constitutively synthesize P-selectin in vivo (47). Northern blot analysis of RNA from HUVEC and the megakaryocytic HEL and CHRF-228 cell lines, which also con-stitutively express P-selectin (41), identified transcripts for NF-B1 (p105), NF-B2 (p100), and Bcl-3 (Fig. 9). The presence of these transcripts is consistent with a role of Bcl-3, p50, and p52 in regulating the expression of P-selectin in megakaryocytes and endothelial cells. DISCUSSION We defined a unique B site in the P-selectin promoter that recognized homodimers containing p50 and p52, but not homodimers or heterodimers containing p65. Interactions of Bcl-3 with p50 and p52 homodimers differentially regulated the activity of the B site. The gene for P-selectin may be a prototype for other genes whose expression may be regulated by B sites that do not bind inducible heterodimeric NF-B complexes.
Inducible NF-B complexes containing p65 activate gene transcription in response to a variety of inflammatory signals (14). These complexes are normally sequestered in the cytoplasm by IB␣. Upon cellular stimulation, IB␣ is phosphoryl-FIG. 6. A mutation in the P-selectin B element eliminates binding to p50 and p52 homodimers and decreases constitutive promoter activity. A, sequence of wild-type and mutant Seq I probes used in gel shift studies. B, the wild-type or mutant Seq I probes were incubated with nuclear extracts from CHRF-288 cells. The mutant probe failed to form complex II. C, the wild-type or mutant Seq I probes were incubated with purified recombinant p50 or p52 homodimers. D, the same mutations were introduced into a luciferase reporter gene driven by the P-selectin 5Ј-flanking sequence from Ϫ309 to Ϫ13. The mutant and wild-type reporter genes were transfected into BAEC, and the luciferase activities were measured. The activities of the wild-type promoter gene were normalized to 100%. The data represent the mean Ϯ S.D. of three independent experiments. Triplicate transfections were performed in each experiment. ated and degraded, releasing the p65-containing heterodimers to the nucleus. In contrast, p50 and p52 homodimers are constitutively expressed in the nucleus, at least in some cells (14). Most reports suggest that p50 homodimers do not transactivate gene expression (14). Instead, it has been proposed that p50 homodimers serve as repressors of gene activation by competing with p50/p65 heterodimers for binding to B elements (30,31). For example, p50 homodimers in nuclear extracts of unstimulated T cells bind the B element in the IL-2 gene (30). Upon antigenic stimulation, fewer p50 homodimers bind to the element, whereas p50/p65 heterodimers that have moved to the nucleus then bind. Activation of the IL-2 gene is correlated with the change in binding profiles. Notably, inhibitors of protein synthesis prevent the loss of binding of p50 homodimers as well as activation of the IL-2 gene, suggesting that a newly synthesized protein sequesters p50 homodimers in the nucleus. This protein might correspond to Bcl-3, which is inducibly expressed in some cells (48) and dissociates p50 homodimers from bound DNA (31). In contrast, ternary complexes of Bcl-3 and p52 homodimers may activate expression of some genes (32,33). For example, the B site in the H2-K b promoter is required for constitutive gene expression, and expression is correlated with binding of nuclear p52 homodimers to the B element (42,44).
A difficulty in interpreting previous studies of p50 and p52 homodimers is that the B elements of genes encoding proteins such as IL-2 and H2-K b also bind p65-containing heterodimers. Furthermore, variable levels of such heterodimers have been found in cells in the absence of overt stimulation (14). Thus, it has not been clear whether p50 and p52 homodimers regulate gene expression directly, or function indirectly by affecting binding of p65-containing heterodimers to B elements. Because the B site in the P-selectin gene did not bind p65, the role of the interactions of Bcl-3 with p50 and p52 homodimers could be more clearly assessed. Mutations of the B element that abolished binding to p50 and p52 homodimers reduced gene expression directed by the P-selectin promoter in transfected BAEC. Co-expression of p52 and Bcl-3 augmented expression in a concentration-dependent manner. In contrast, co-expression of p50 repressed expression, but this repression was prevented by co-expression of Bcl-3. These data suggest that p50 and p52 homodimers compete for the P-selectin B site. In this model, binding of Bcl-3 to DNA-bound p52 homodimers activates gene expression, whereas binding of Bcl-3 to DNA-bound p50 homodimers results in their dissociation from DNA, allowing p52 homodimers to bind. The model predicts that the constitutive expression of P-selectin is partially regulated by the relative amounts of p50, p52, and Bcl-3 in megakaryocytes and endothelial cells; basal levels of mRNA encoding all three proteins were detected in the cultured endothelial cells and megakaryocytic cell lines that we examined. The function of the B site may also be regulated by inflammatory stimuli. LPS increases transcripts for the precursors of p50 and p52 in cultured HUVEC, although the relative amounts of these transcripts were not quantified (22,49). Mitogen stimulation increases transcripts for Bcl-3 in peripheral blood mononuclear cells (48). Phosphorylation of Bcl-3 may also affect its activity (33,46).
Regulation of P-selectin expression probably requires cooperative interactions of proteins binding to the B site with proteins binding to other elements in the promoter/enhancer. Mutation of the B element reduced but did not eliminate constitutive expression in BAEC. A GATA element downstream of the B site was previously demonstrated to be required for optimal P-selectin expression, and several other putative regulatory elements in the promoter have been identified (11). An oligonucleotide encoding Seq I, which spanned the area immediately surrounding the B site, formed several other complexes with nuclear proteins. These and other proteins may positively or negatively regulate binding of p50 or FIG. 8. Interactions of p50 with the B element inhibit P-selectin promoter activity, but Bcl-3 prevents this inhibition. BAEC were transfected with a reporter gene (7 g) containing the P-selectin promoter with the wild-type or mutated B element (panel A) or with two copies of Seq B containing the wild-type or mutated B element linked to the SV40 minimal promoter (panel B). The cells were cotransfected with the indicated amounts of expression plasmids encoding p50 and/or Bcl FIG. 9. Cells expressing P-selectin transcribe mRNAs for Bcl-3 and the precursors of p50 and p52. Northern blots of total RNA from HUVEC and the megakaryocytic HEL and CHRF-288 cell lines were probed with 32 P-labeled cDNAs encoding p50, p52, and Bcl-3. p52 homodimers to the B site, and may affect the ability of Bcl-3 to activate gene expression.
Methylation interference analysis indicated that p50 and p52 homodimers contacted four adjacent guanines on each half-site of the P-selectin B element, consistent with a preference for these homodimers to bind symmetrical half-sites (42,50). The B elements in the P-selectin and H2-K b genes are similar, except that the three residues separating the two halfsites differ. These three residues contribute to recognition specificity, since the H2-K b element also binds p50/p65, and certain substitutions of these residues in the P-selectin element conferred binding to p50/65 as well as to p50 and p52 homodimers. The sequences of the P-selectin B element and the altered versions that retained specificity for p50 and p52 homodimers were not identified by random amplification of sequences by polymerase chain reaction (50). We hypothesize that other genes have B elements that bind only p50 and/or p52 homodimers. One candidate is the gene encoding Bcl-3, which has two putative B elements in its 5Ј-flanking region (48). The sequence of one of these elements, GGGGACACCCC, is similar to that of the P-selectin B element, and might have similar recognition specificity. If so, expression of the Bcl-3 gene could be positively autoregulated by its protein product. Bcl-3 could dissociate "repressive" p50 homodimers from the B element and/or form activating complexes with p52 homodimers bound to the B element.
Expression of the gene for P-selectin is clearly regulated differently than that of the genes encoding the endothelial adhesion receptors E-selectin, VCAM-1, and ICAM-1. Transcriptional induction of the latter genes by LPS, IL-1, and TNF-␣ requires binding of p65-containing NF-B dimers to B elements present in each promoter/enhancer (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). P-selectin is constitutively synthesized and packaged in secretory granules of megakaryocytes/platelets and endothelial cells. However, increased synthesis may account for the observed surface expression of P-selectin on endothelial cells overlying atherosclerotic plaques (51) and at sites of chronic or allergic inflammation (52,53). In these areas, one or more of the other adhesion proteins are not expressed. Understanding the mechanisms underlying differential expression of these molecules may provide insight into their roles in various inflammatory and thrombotic conditions.