(Received for publication, May 5, 1995)
From the
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 B site (
GGGGGTGACCCC
) in the
promoter of the human P-selectin gene. The
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 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-
B/Rel family. Mutations that eliminated binding to
NF-
B/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
B 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
B
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
B site of the P-selectin promoter. This site may be a
prototype for
B elements in other genes that bind specifically to
p50 and/or p52 homodimers.
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), ()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, 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-
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-
do not increase P-selectin transcripts in
cultured HUVEC, (
)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-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 I
B
and related
proteins. Upon cellular stimulation, I
B
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 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 I
B
, 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.
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 B element (overlined) that is aligned with the sequence of the
B
site of the H2-K
promoter. The three central residues in
the H2-K
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
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.
Figure 2:
The B elements in the
H2-K
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
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-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
I
B
and release of p50/p65 complexes into the nucleus (Fig. 3A). The labeled H2-K
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
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 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.
Figure 3:
The P-selectin B element binds
constitutive nuclear proteins but not inducible nuclear proteins. A, the labeled Seq B or H2-K
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
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
or
Seq B oligonucleotide.
To confirm the differential
specificities of the Seq B and H2-K 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
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
B
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 B element binds p50
and p52 homodimers but not p65 homodimers. A, the labeled Seq
B and H2-K
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.
Figure 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.
The three core nucleotides at -213 to -211 in the
P-selectin B element differ from those in the H2-K
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 -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.
Figure 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 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.
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 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).
Figure 7:
Interactions of p52 and Bcl-3 with the
B 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
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 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 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 P-selectin promoter is differentially regulated by interactions of
Bcl-3 with p52 and p50 homodimers.
Figure 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 µg)
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 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.
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.
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 I
B
. Upon cellular
stimulation, I
B
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
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
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
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 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
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
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 5`-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.