©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of the Human P-selectin Promoter by Bcl-3 and Specific Homodimeric Members of the NF-B/Rel Family (*)

(Received for publication, May 5, 1995)

Junliang Pan Rodger P. McEver (§)

From the Departments of Medicine and Biochemistry & Molecular Biology, W. K. Warren Medical Research Institute, University of Oklahoma, Health Sciences Center, and the Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

P-selectin, an adhesion receptor for leukocytes, is constitutively expressed by megakaryocytes and endothelial cells. Synthesis of P-selectin is also increased by some inflammatory mediators. We characterized a previously identified kappaB site (GGGGGTGACCCC) in the promoter of the human P-selectin gene. The kappaB 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 analysis indicated that p50 or p52 homodimers contacted the guanines at positions -218 to -214 on the coding strand and at -210 to -207 on the noncoding strand. Changes in the three central residues at -213 to -211 altered binding specificity for members of the NF-kappaB/Rel family. Mutations that eliminated binding to NF-kappaB/Rel proteins reduced by 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 kappaB site-dependent manner. In contrast, overexpression of p50 repressed promoter activity; this repression was prevented by co-overexpression of Bcl-3. Similar phenomena were observed with reporter gene constructs driven by two tandem P-selectin kappaB sequences linked to the SV40 minimal promoter. These data suggest that Bcl-3 differentially regulates the effects of p50 and p52 homodimers bound to the kappaB site of the P-selectin promoter. This site may be a prototype for kappaB elements in other genes that bind specifically to p50 and/or p52 homodimers.


INTRODUCTION

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-alpha, 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, depending 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-alpha markedly increase P-selectin transcripts and protein in endothelial cells of multiple tissues (6, 7, 8, 9) . These agonists also augment P-selectin mRNA levels in cultured murine, rat, and bovine endothelial cells(7, 9, 10) . However, LPS and TNF-alpha 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 5`-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 -249 to -13 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 -197 to -147 was demonstrated to be functional. The region from -249 to -197 contains at least three potential elements, including a putative recognition site for NF-kappaB/Rel transcription factors.

The DNA-binding forms of NF-kappaB/Rel transcription factors are homodimers or heterodimers that bind to kappaB 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 IkappaBalpha and related proteins. Upon cellular stimulation, IkappaBalpha is degraded; the dimeric complexes then migrate to the nucleus where they function as potent activators of many genes with kappaB 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-kappaB1) and p100 (NF-kappaB2), 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 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 IkappaBalpha, 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 kappaB elements that bind only to these proteins.

In this paper we characterize the properties of the kappaB element in the human P-selectin promoter. We find that this kappaB site has the unique property of binding only to p50 and p52 homodimers. Interactions of Bcl-3, p52 homodimers, and the kappaB element augment transcription. In contrast, interactions of p50 homodimers with the kappaB 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 kappaB sites specific for these homodimers.


EXPERIMENTAL PROCEDURES

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 pmkappaB309LUC, which contained two base changes in the kappaB site (GGGGGTGACCCC to GCCGGTGACCCC) was constructed by an overlap extension polymerase chain reaction protocol(38) . Plasmids p2xkappaBSV40 and p2xmkappaBSV40 were constructed through direct ligation of double-stranded oligonucleotides encoding two wild-type or mutated P-selectin kappaB sites into the MluI and XhoI sites of the pGL2-promoter vector. These oligonucleotides were: 5`-cgcgGAAGGGGGTGACCCCTTGCCGAAGGGGGTGACCCCTTGCC-3` and 3`-CTTCCCCCACTGGGGAACGGCTTCCCCCACTGGGGAACGGagct-5` for the wild-type P-selectin kappaB sites, and 5`-cgcgGAAGGGGGTGAATAGTTGCCGAAGGGGGTGAATAGTTGCC-3` and 3`-CTTCCCCCACTTATCAACGGCTTCCCCCACTTATCAACGGagct-5`, 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 µg/dish of cells by adding an appropriate amount of a control plasmid without an insert (pRc/CMV, Invitrogen). Equal volumes of 30 µg of test plasmids and 50 µg 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-phenol-chloroform extraction(40) , and Northern blot analysis was performed as described previously(41) .


RESULTS

The Sequence from -232 to -192 in the 5`-Flanking Region of the P-selectin Gene Forms Five DNA-Nuclear Protein Complexes

We previously showed that deletion of the sequence from -249 to -197 in the 5`-flanking region of the human P-selectin gene decreased expression of a reporter gene in transfected BAEC by 40%(11) . This region contains putative recognition elements for ETS proteins, certain zinc finger proteins induced by phorbol esters, and members of the NF-kappaB/Rel family of proteins. To determine whether nuclear proteins bound to this region, we synthesized a 41-base pair double-stranded oligonucleotide encompassing the sequence -232 to -192, 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.


Figure 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 5`-flanking sequences of the P-selectin gene, as described previously(11) . The sequence deleted in the second construct includes a kappaB element (overlined) that is aligned with the sequence of the kappaB 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.



The kappaB Element in the P-selectin Promoter Binds Homodimers Containing p50 or p52 but Not Homodimers or Heterodimers Containing p65

The putative kappaB element in Seq I is very similar to a kappaB 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 -222 to -200 immediately surrounding the putative kappaB 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 kappaB element that is functionally related to the element in the H2-K^b promoter.


Figure 2: The kappaB elements in the H2-K^b gene and the P-selectin gene compete for binding to nuclear proteins from unstimulated cells. In the left panel, nuclear extracts were incubated with the labeled Seq I probe in the presence or absence of a 50-, 100-, or 200-fold excess of the unlabeled Seq I or H-2K^b oligonucleotide. In the right panel, nuclear extracts were incubated with the labeled Seq B probe in the presence or absence of a 100-fold excess of the indicated unlabeled oligonucleotides. The positions of the specific DNA-protein complexes are indicated.



To determine whether Seq B bound constitutive p50 or p52 homodimers as well as inducible NF-kappaB 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 IkappaBalpha 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 prevented 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 kappaB site, unlike the H2-K^b kappaB 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-alpha 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.


Figure 3: The P-selectin kappaB element binds constitutive nuclear proteins but not inducible nuclear proteins. A, the labeled Seq B or H2-K^b probes were incubated with nuclear extracts from unstimulated BAEC or from BAEC stimulated with phorbol myristate acetate (PMA) for 1 h. B, the labeled H2-K^b probe was incubated with nuclear extracts from phorbol myristate acetate-stimulated BAEC in the presence or absence of a 50-, 100-, or 200-fold excess of the unlabeled H-2K^b or Seq B oligonucleotide.



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 complexes 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 kappaB element interacts with constitutively expressed nuclear complexes consisting of p50 or p52 homodimers, but not inducible nuclear complexes containing p65.


Figure 4: The P-selectin kappaB 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.



Characterization of the Nucleotides in the P-selectin kappaB Sequence That Contact p50 and p52 Homodimers

We used methylation interference analysis to characterize the sites on the P-selectin kappaB element that bind p50 and p52 homodimers. As shown in Fig. 5, methylation of five guanines at -218 to -214 in the coding strand and four guanines at -210 to -207 in the non-coding strand suppressed binding to p52 and p50. Methylation of the guanine at -212 on the coding strand also prevented binding to both proteins, although the effect was more pronounced for p52.


Figure 5: Methylation interference analysis of the nucleotides in the P-selectin kappaB 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 kappaB 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.



The three core nucleotides at -213 to -211 in the P-selectin kappaB 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 kappaB element of Seq I. Substitution of the G at -212 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 kappaB Element Is Required for Optimal Constitutive Expression of the P-selectin Gene

To determine whether the kappaB element was required for optimal constitutive expression of P-selectin, we changed two guanines at -217 and -216 to cytosines (Fig. 6A). These mutations specifically affected the binding site for NF-kappaB 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 5`-flanking sequence from -309 to -13. When transfected into BAEC, the mutant construct was expressed at levels 40% 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 -309 to -197(11) . These data indicate that a functional kappaB element is required for optimal constitutive expression of the P-selectin gene in BAEC.


Figure 6: A mutation in the P-selectin kappaB 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 5`-flanking sequence from -309 to -13. 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 ± S.D. of three independent experiments. Triplicate transfections were performed in each experiment.



Function of the P-selectin kappaB 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 kappaB-dependent gene expression(14) . The previously studied genes have kappaB elements that bind both p50 and p52 homodimers as well as heterodimers containing p65. In contrast, the kappaB site in the P-selectin promoter binds only p50 and p52 homodimers. Therefore, we asked whether p50, p52, and Bcl-3 regulate kappaB-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 kappaB element.

Co-expression of p52 augmented luciferase expression driven by the wild-type promoter, but not the promoter with the mutated kappaB 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 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 kappaB element (Fig. 7B). The mutated Seq B oligonucleotide also failed to interact with p50 or p52 in gel shift assays (data not shown).


Figure 7: Interactions of p52 and Bcl-3 with the kappaB element increase P-selectin promoter activity. BAEC were transfected with a reporter gene (3.5 µg) containing the P-selectin promoter with the wild-type or mutated kappaB element (panel A) or with two copies of Seq B containing the wild-type or mutated kappaB element linked to the SV40 minimal promoter (panel B). The cells were co-transfected with the indicated amounts of expression plasmids encoding p52 and/or Bcl-3. Luciferase activity is expressed as light units/25 µg of protein. The data in panel A represent the mean ± S.D. of one experiment. Similar results were obtained in two other experiments. The data in panel B represent the mean ± S.D. of one experiment. Similar results were obtained in three other experiments.



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 kappaB-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 kappaB element in the P-selectin promoter is differentially regulated by interactions of Bcl-3 with p52 and p50 homodimers.


Figure 8: Interactions of p50 with the kappaB element inhibit P-selectin promoter activity, but Bcl-3 prevents this inhibition. BAEC were transfected with a reporter gene (7 µg) containing the P-selectin promoter with the wild-type or mutated kappaB element (panel A) or with two copies of Seq B containing the wild-type or mutated kappaB element linked to the SV40 minimal promoter (panel B). The cells were co-transfected with the indicated amounts of expression plasmids encoding p50 and/or Bcl-3. Luciferase activity is expressed as light units/25 µg of protein. The data in panel A represent the mean ± S.D. of one experiment. Similar results were obtained in two other experiments. The data in panel B represent the mean ± S.D. of one experiment. Similar results were obtained in another experiment.



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 constitutively express P-selectin(41) , identified transcripts for NF-kappaB1 (p105), NF-kappaB2 (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.


Figure 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 P-labeled cDNAs encoding p50, p52, and Bcl-3.




DISCUSSION

We defined a unique kappaB 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 kappaB site. The gene for P-selectin may be a prototype for other genes whose expression may be regulated by kappaB sites that do not bind inducible heterodimeric NF-kappaB complexes.

Inducible NF-kappaB complexes containing p65 activate gene transcription in response to a variety of inflammatory signals(14) . These complexes are normally sequestered in the cytoplasm by IkappaBalpha. Upon cellular stimulation, IkappaBalpha is phosphorylated 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 kappaB elements(30, 31) . For example, p50 homodimers in nuclear extracts of unstimulated T cells bind the kappaB 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 kappaB 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 kappaB element(42, 44) .

A difficulty in interpreting previous studies of p50 and p52 homodimers is that the kappaB 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 kappaB elements. Because the kappaB 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 kappaB 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 kappaB 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 kappaB 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 kappaB site with proteins binding to other elements in the promoter/enhancer. Mutation of the kappaB element reduced but did not eliminate constitutive expression in BAEC. A GATA element downstream of the kappaB 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 kappaB site, formed several other complexes with nuclear proteins. These and other proteins may positively or negatively regulate binding of p50 or p52 homodimers to the kappaB 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 kappaB element, consistent with a preference for these homodimers to bind symmetrical half-sites(42, 50) . The kappaB elements in the P-selectin and H2-K^b genes are similar, except that the three residues separating the two half-sites 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 kappaB 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 kappaB elements that bind only p50 and/or p52 homodimers. One candidate is the gene encoding Bcl-3, which has two putative kappaB elements in its 5`-flanking region(48) . The sequence of one of these elements, GGGGACACCCC, is similar to that of the P-selectin kappaB 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 kappaB element and/or form activating complexes with p52 homodimers bound to the kappaB 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-alpha requires binding of p65-containing NF-kappaB dimers to kappaB 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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-alpha, tumor necrosis factor alpha; VCAM-1, vascular cell adhesion molecule-1.

(^2)
L. Yao, J. Pan, H. Setiadi, K. D. Patel, and R. P. McEver, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Drs. Craig Rosen, Riccardo Dalla-Favera, and Timothy McKeithan for valuable reagents and Ginger Hampton for technical assistance. We are grateful to Drs. James Morrissey and Joan Conaway for critical reading of the manuscript. We also thank Dr. Kenneth Jackson (Molecular Biology Resource Facility, University of Oklahoma Health Sciences Center) for synthesis of oligonucleotides.


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