From the Departments of Medicine and Biochemistry & Molecular Biology, W. K. Warren Medical Research Institute, University of Oklahoma Health Sciences Center, and Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104
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ABSTRACT |
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P-selectin, an adhesion receptor for
leukocytes, is constitutively expressed in megakaryocytes and
endothelial cells. Tumor necrosis factor- (TNF-
) or
lipopolysaccharide (LPS) increases synthesis of P-selectin in
murine but not in human endothelial cells. To identify potential
species-specific and conserved mechanisms for regulation of expression
of P-selectin, we cloned the 5'-flanking region of the murine
P-selectin gene and compared its features with those previously
reported for the human gene. The murine and human genes shared
conserved Stat-like, Hox, Ets, GATA, and GT-IIC elements. In the murine
gene, a conserved GATA element bound to GATA-2 and functioned as a
positive regulatory element, whereas a conserved Ets element bound to
GA-binding protein and functioned as a negative regulatory element.
Significantly, the murine P-selectin gene had several features not
found in the human gene. These included an insertion from
987 to
649 that contained tandem GATA and tandem AP1-like sequences, which
resembled enhancers in
-globin locus control regions. Both tandem
elements bound specifically to nuclear proteins. The murine gene lacked
the unique
B site specific for p50 or p52 homodimers found in the
human gene. Instead, it contained two tandem
B elements and a
variant activating transcription factor/cAMP response element site,
which closely resembled sites in the E-selectin gene that are required for TNF-
- or LPS-inducible expression. TNF-
or LPS augmented expression of a reporter gene driven by the murine, but not the human,
P-selectin promoter in transfected endothelial cells. Deletional analysis of the murine 5'-flanking region revealed several sequences that were required for either constitutive or inducible expression. These data suggest that both species-specific and conserved mechanisms regulate transcription of the human and murine P-selectin genes.
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INTRODUCTION |
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Leukocyte extravasation is mediated by the sequential
engagement of adhesion molecules, whose expression and/or function are regulated by inflammatory mediators (1-3). For example, tumor necrosis
factor- (TNF-
)1 or LPS
stimulates endothelial cells to transcribe the genes encoding the
adhesion molecules E-selectin, intercellular cell adhesion molecule-1,
vascular cell adhesion molecule-1, and mucosal addressin cell adhesion
molecule-1 (2, 4-9). These newly synthesized proteins are directly
transported to the cell surface, where they mediate adhesion of
leukocytes. In contrast, the expression of P-selectin is regulated by
two distinct mechanisms. First, P-selectin is constitutively
synthesized by megakaryocytes (the precursors of platelets) and
endothelial cells, where it is sorted into the membranes of secretory
granules (10-14). When these cells are stimulated by agonists such as
thrombin, P-selectin is rapidly translocated from granules to the
plasma membrane (15), where it initiates tethering and rolling of
leukocytes (16-19). Second, inflammatory mediators such as TNF-
,
LPS, interleukin-4, or oncostatin M increase levels of P-selectin
mRNA and protein in endothelial cells (20-22). The increased
synthesis of P-selectin may saturate the sorting pathway into secretory
granules, leading to direct delivery of the protein to the cell surface
(22, 23). Notably, TNF-
or LPS increases expression of P-selectin in
murine endothelial cells but not in human endothelial cells (22, 24).
The mechanism for this unusual species-specific response is not
known.
We previously isolated and conducted a preliminary analysis of the
5'-flanking region of the human P-selectin gene (25, 26). The sequence
from 309 to
13 relative to the translational start site conferred
tissue-specific expression of a reporter gene in cultured bovine aortic
endothelial cells. Deletions or mutations of this sequence revealed at
least three positive regulatory regions that included a
B site, a
GATA element, and at least two potential Ets elements. Most
B sites
are recognized by inducible homodimeric or heterodimeric NF-
B/Rel
proteins that participate in cytokine-stimulated gene expression.
However, the
B site in the human P-selectin promoter bound
specifically to constitutively expressed homodimers of p50 or p52,
whose functions were differentially regulated by the proto-oncogene
product, Bcl-3 (26). GATA and Ets elements are often observed in genes
whose expression is restricted to hematopoietic and/or endothelial
cells (25, 27-34). The human P-selectin GATA element bound to the
protein GATA-2, and mutation of this element markedly inhibited gene
expression (25). The function of the potential Ets elements in the
P-selectin gene was not determined.
To better understand potential conserved and nonconserved mechanisms
for expression of the P-selectin gene, we isolated the 5'-flanking
region of the murine P-selectin gene and compared its features with
those previously reported for the human gene (25, 26). Both genes share
functional GATA and Ets sites. However, the murine gene has several
unique putative regulatory elements that may explain why TNF- or LPS
augments transcription of the P-selectin gene in murine but not in
human endothelial cells.
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EXPERIMENTAL PROCEDURES |
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Cells, Reagents, and Antibodies--
Murine bEnd.3 endothelioma
cells, BAEC, and human megakaryocytic HEL and CHRF-288 cells were
cultured as described (22, 25). Recombinant human TNF- and yeast RNA
were obtained from Boehringer Mannheim. LPS from Salmonella
typhosa was purchased from Sigma. Antibodies against human GATA-2,
Ets-1, Ets-2, Fli-1, and p65 (RelA) were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA).
Genomic Cloning, Southern Blot Analysis, and Sequence Comparison-- To clone the murine P-selectin 5'-flanking region, we designed the following two primers: 5'-ACATTTCTGGAAAGCGAATAGG-3' which spanned nucleotides 1-22 of the murine P-selectin cDNA sequence, and 5'-CATGTCCTCTCTGATCCGTCTG-3' which is complementary to nucleotides 105-126 of the cDNA sequence (21). Based on the known human P-selectin gene structure (35), we predicted that the murine P-selectin 5'-untranslated region and the translation initiator ATG would be encoded by one exon. We confirmed this prediction by sequencing the PCR product amplified from mouse genomic DNA using the above two primers. The primers were then used in a PCR screen to identify bacteriophage P1 clones encoding the murine 5'-flanking region (custom service provided by Genome Systems, Inc., St. Louis, MO). The PCR profile involved denaturation at 94 °C for 1.5 min, primer annealing at 50 °C for 2.5 min, and extension at 72 °C for 1 min, with a final concentration of Mg2+ at 1 mM. Two P1 clones named GS5512 and GS5572 were obtained. DNA restriction fragments derived from the GS5512 clone were subcloned into the plasmids pBluescript II SK (Stratagene) or pIBI20 (IBI) for restriction enzyme mapping and DNA sequencing. Southern blot analysis of mouse genomic DNA with a random-labeled probe spanning the entire 5'-untranslated region of the murine P-selectin gene was performed as described (36). Sequence comparison was carried out by using the Bestfit program from Genetics Computer Group (Madison, WI).
RNase Protection Assay--
An RNase protection assay was
performed with a RPAIITM kit (Ambion Inc., Austin, TX)
according to the instructions of the manufacturer. The labeled
165-nucleotide cRNA probe extended from 164 to
13 of the murine
P-selectin 5'-flanking region, plus 14 nucleotides transcribed from the
plasmid. Poly(A)+ RNA from bEnd.3 cells stimulated with LPS
(1 µg/ml) and cycloheximide (10 µg/ml) for 6 h was isolated as
described (25).
Construction of Chimeric Luciferase Expression Vectors,
Transfection, and Luciferase Assay--
The parental vector for
creation of deletion mutants was pMG2, in which a 2032-bp
XbaI fragment that contained the 5'-flanking region, first
exon, and part of intron 1 of the murine P-selectin gene was inserted
between the XbaI site of pBluescript II SK with the
5'-flanking region toward the T3 promoter. Plasmid mp1379LUC was
constructed in the following three steps. 1) A PCR product spanning the
murine P-selectin 5'-flanking region from 164 to
13 was amplified
with a sense primer carrying a native PstI site and an
antisense primer with addition of an KpnI site at the 5' end. The PCR product was digested with PstI and
KpnI and used to replace the shorter
KpnI-PstI fragment in pMG2. 2) The fragment from
1379 to
13 was excised by digestion with KpnI and
XbaI and inserted between the KpnI and
XbaI sites of pIBI20 to create an additional
HindIII site adjacent to the XbaI site. To make other constructs, the PstI site between the XbaI
and HindIII sites was eliminated by ligation of the
compatible XhoI and SalI sites. The resultant
plasmid was designated mp1379IBI. 3) mp1379IBI was digested with
KpnI and HindIII, and the
HindIII-KpnI fragment carrying the 5'-flanking
region from
1379 to
13 was then inserted between the
HindIII and KpnI sites of the plasmid p0LUC (25, 37). Plasmids mp593LUC, mp474LUC, mp349LUC, mp288LUC, and mp229LUC were
constructed by replacement of the shorter
PstI-HindIII fragment in mp1379IBI with the
respective PCR fragment spanning from
593 to
164,
474 to
164,
349 to
164,
288 to
164, or
229 to
164. These fragments were
generated with a sense primer carrying an additional HindIII
site and an antisense primer carrying a native PstI site.
The plasmids were digested with KpnI and HindIII, and the shorter KpnI-HindIII fragments were
inserted between the HindIII and KpnI sites of
p0LUC. Plasmids mp164LUC, mp148LUC, mp110LUC, and mp91LUC were
constructed by inserting between the HindIII and
KpnI sites of p0LUC the respective PCR product from
164 to
13,
148 to
13,
110 to
13, or
91 to
13 that was generated
with a sense primer carrying an additional HindIII site and
an antisense primer with an additional KpnI site. Plasmids mpMutGATA and mpMutETS1 were constructed in two steps as follows. 1)
The shorter KpnI-PstI fragment in mp1379IBI was
replaced with the respective PCR fragment carrying a mutation that was
generated according to an overlap extension protocol (25). 2) The
shorter HindIII-KpnI fragment from the resultant
plasmid was excised and inserted between the HindIII and
KpnI sites of p0LUC. The fidelity of all constructs was
confirmed by restriction mapping, and the PCR-generated products were
verified by sequencing. Preparation of plasmids, transfections, and
luciferase assays were performed as described (26).
Gel Mobility Shift Assay-- Nuclear extracts from bEnd.3 cells or BAEC were prepared as described (38). Nuclear extracts from HEL and CHRF-288 cells were prepared by a slightly different protocol (39). Gel mobility shift assays were performed as described (25).
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RESULTS |
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Molecular Cloning of the 5'-Flanking Region of the Murine P-selectin Gene-- Two mouse P1 clones encoding the murine P-selectin gene were obtained using PCR screening. Southern blot analysis of these two clones revealed the same restriction patterns as those obtained with mouse genomic DNA, suggesting that there was no DNA rearrangement during cloning (data not shown). Restriction fragments derived from the P1 clone GS5512 were subcloned into plasmids (Fig. 1). The insert in subclone pMG2 was sequenced on both strands; it contained the first 1256 bp of the 5'-flanking region, all of exon 1, and part of intron 1.2
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Determination of the Transcription Start Site--
We employed an
RNase protection assay to determine the transcription start site of the
murine P-selectin gene. Several protected products were seen from 134
to
75 relative to the ATG translation start codon (Fig.
2). These protected products were
observed in mRNA from LPS/cycloheximide-stimulated bEnd.3 cells but
not in control yeast tRNA. The longest product was the most abundant. These data indicate that transcription of the murine P-selectin gene is
initiated primarily at
134 relative to the translation start
site.
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Comparison of Structural Features of the Murine and Human
5'-Flanking Regions--
Fig. 3 aligns
the 5'-flanking sequence of the murine P-selectin gene with the
corresponding sequence of the human gene (25). There was 63% sequence
identity between the two genes in the first 600 bp of the 5'-flanking
region, exon 1, and the first 500 bp of intron 1. This degree of
similarity is typical for the mouse and human homologues of a gene
(40). Both genes ended the first exon with the translation start codon
ATG and shared several putative regulatory regions. The first conserved
region from 419 to
409 in the mouse gene and from
413 to
403 in
the human gene contained a HOX recognition element (41) and a Stat-like
sequence (42). The second region from
337 to
319 in mouse and from
325 to
307 in human contained a HOX element and an Ets motif (43). The third conserved region from
163 to
151 in mouse and
162 to
150 in human contained a GATA element. The fourth conserved region
from
130 to
92 in mouse and from
129 to
91 in human contained a
sequence similar to the GT-IIC element of the SV40 enhancer (44) and to
Stat consensus motifs, plus an adjacent Ets element flanked by 4-bp A/T
sequences that typically bind to HMG I(Y) proteins (45).
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The GATA/TATA Element Is Required for Optimal Murine P-selectin Promoter Activity-- To test whether the murine P-selectin GATA/TATA element bound to nuclear proteins, we synthesized double-stranded oligonucleotide probes encompassing the wild-type sequence or a mutant sequence in which the core TATC was converted to TTAG (Fig. 4A). As a positive control, we used a human endothelin-1 GATA probe that is known to bind specific GATA proteins (25, 53, 54). The labeled P-selectin probes did not form detectable sequence-specific DNA-protein complexes. However, a 100-fold molar excess of the unlabeled wild-type probe, but not the mutant probe, weakly competed with the human endothelin-1 GATA element for binding to GATA proteins (Fig. 4B). These data suggest that the GATA/TATA element interacts weakly with GATA proteins.
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Nuclear Protein Binding Activities of Tandem GATA Motifs and
Tandem AP-1-like Elements in the Insertion--
As illustrated in Fig.
5A, the murine P-selectin
sequence from 923 to
849 contained tandem GATA and tandem AP-1-like
elements, which closely resembled the enhancers in hypersensitive
sites 2 and 3 of the murine
-globin locus control region (46).
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Nuclear Protein Binding Activity and Transcriptional Function of the Proximal Ets Motif-- The human and murine genes for P-selectin share conserved proximal and distal Ets elements. To investigate the role of the proximal Ets element, we determined nuclear protein binding activities with oligonucleotides encoding the murine and human proximal Ets sequences (Fig. 6A). A labeled probe encoding the murine proximal Ets element formed two DNA-protein complexes with nuclear extracts from BAEC (Fig. 6B) and from bEnd.3, HEL, and CHRF-288 cells (data not shown). Formation of these two complexes was sequence-specific, as it was significantly diminished by addition of a 100-fold excess of the unlabeled probe, or of the proximal Ets element from the human gene, but not of an unrelated sequence. Complex formation further depended on the Ets core sequence, because a mutant probe in which the core sequence AGGAAG was converted to AGCTAG did not inhibit binding of the labeled wild-type probe and, when labeled, did not form specific DNA-protein complexes (Fig. 6C). Of note, formation of complex I varied with different batches of nuclear extracts, as previously noted for binding of the Ets-related GABP to its cognate DNA sequences (56). We therefore carried out cross-competition gel shift assays with the proximal Ets probes and the HSV1 IE Ets probe that interacts with GABP (56). Addition of a 50-fold excess of the HSV1 IE Ets probe, but not a 200-fold excess of an unrelated sequence, prevented complex formation of the labeled proximal murine P-selectin Ets probe with BAEC nuclear extracts (Fig. 6D). The labeled HSV1 IE Ets probe formed three DNA-protein complexes with nuclear extracts from BAEC (Fig. 6E) or from HEL, CHRF-288, or bEnd.3 cells (data not shown). However, only complex A was consistently observed. Formation of complex A was prevented or significantly diminished by addition of a 50-200-fold excess of the unlabeled HSV1 IE Ets element or the murine or human proximal Ets probe but not of the mutant proximal Ets probes. These data suggest that the proximal Ets elements bind to one or more members of the Ets family, including GABP.
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TNF- or LPS Increases Luciferase Reporter Gene Expression Driven
by the Murine but Not the Human P-selectin 5'-Flanking Region--
To
test whether differences in the promoters of human and murine
P-selectin gene accounted for the differential response of P-selectin
to TNF-
or LPS, we compared the abilities of TNF-
or LPS to
induce expression of a reporter gene driven by the murine or human
P-selectin 5'-flanking region. Following transfection of the constructs
into BAEC, the cells were treated with fresh medium in the presence or
absence of LPS or TNF-
for various intervals, and the cells were
then harvested for luciferase activity assays. As shown in Fig.
7, the reporter gene driven by the
5'-flanking region of the murine P-selectin gene from
1379 to
13
promoted constitutive expression of luciferase that was further
increased 5-15-fold after the transfected cells were stimulated with
TNF-
or LPS for 4.5 h. In sharp contrast, the reporter gene
driven by the 5'-flanking region of the human P-selectin gene from
1339 to
13 promoted constitutive expression that was not further
increased after the same treatment. Similar results were obtained when
the transfected cells were stimulated with TNF-
or LPS for 6 or
8 h. The failure to induce expression of the human P-selectin
reporter gene was not due to excessive basal expression, because the
levels of constitutive expression were similar for both the murine and human P-selectin reporter genes (data not shown). These data
demonstrate that the 5'-flanking regions of both the murine and human
P-selectin genes confer constitutive expression in endothelial cells.
However, only the murine sequence has elements that allow inducible
expression in response to TNF-
or LPS.
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Deletional Analysis of the Murine P-selectin 5'-Flanking
Region--
To localize regulatory elements required for the
constitutive or inducible expression of the murine P-selectin gene, we
prepared reporter constructs in which serially truncated fragments of
the 5'-flanking region were linked to luciferase. Following
transfection of the constructs into BAEC, the cells were incubated with
or without TNF- or LPS and then assayed for luciferase activity. As
shown in Fig. 8, deletion to position
593 decreased constitutive expression of the luciferase reporter gene
by
50% but did not affect the degree of induction by LPS or
TNF-
. Deletion to
474 did not further decrease constitutive
expression but did significantly reduce inducible expression. Deletion
to
349 slightly increased constitutive expression but did not further
affect inducible expression. Deletions to
288,
229,
164 or
148
only modestly affected constitutive or inducible expression. Deletions
to
110 or
91 drastically reduced both constitutive and inducible
expression. These data suggest that the sequence from
1379 to
593
contains a positive regulatory element(s) for constitutive expression,
and the sequence from
593 to
474 contains a regulatory element(s)
for TNF-
- or LPS-mediated inducible expression. Sequences proximal
to
229 appear to be required for both constitutive and inducible
expression.
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DISCUSSION |
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We cloned the 5'-flanking region of the murine gene and compared
its features with those previously reported for the human gene. Both
genes shared several conserved regulatory elements, including
functional GATA and Ets elements. However, several unique aspects of
the murine gene were revealed, including several elements that may
explain why TNF- or LPS augments transcription of the P-selectin
gene in murine but not in human endothelial cells.
The human P-selectin promoter initiates transcription at multiple sites
(25). In contrast, the murine promoter initiates transcription
predominantly at a single site. Both genes have functional proximal
GATA sites. However, unlike the human GATA site, the murine site
deviates one nucleotide from the consensus GATA sequence, leading to
weaker binding to GATA proteins. Furthermore, the murine GATA site
overlaps a weak TATA box located 21 bp upstream of the transcription
start site. We refer to the murine GATA site as a GATA/TATA element,
because it resembles the GATA/TATA elements in the rat platelet
factor-4 and chicken -globin genes that can bind to either TFIID or
GATA proteins (28, 57, 58). This dual-binding property may allow the
GATA/TATA element to communicate with a distal tissue-specific
enhancer. In the chicken
-globin gene, the protein GATA-1, when
bound to a proximal GATA/TATA element, may interact with GATA-1 or
other proteins bound to a distal 3' enhancer that contains both GATA
elements and NF-E2 (AP1-like) elements (58). This interaction allows
the two control regions to form a DNA loop. Once the DNA loop is
formed, TFIID and adapter proteins displace GATA-1 and produce an
active initiation complex under control of the erythroid-specific
enhancer. Intriguingly, the murine P-selectin promoter contains an
upstream region composed of tandem GATA elements and tandem AP-1-like
elements. Furthermore, the murine P-selectin tandem GATA elements bound
to at least one GATA protein (GATA-2), and the tandem AP-1-like
elements bound to a still uncharacterized nuclear protein. Deletion of
the sequence from
1379 to
593, which contains these elements,
decreased expression of a luciferase reporter gene in BAEC by 50%.
These data suggest that the murine GATA/TATA element may cooperate with
this putative enhancer, in a manner much like that proposed for the
-globin gene. The murine putative enhancer is located in an
insertion that is not present in the corresponding portion of the human P-selectin gene. It is not known whether the human gene has a similar
enhancer in a different location.
Ets elements, which are often associated with GATA elements, are in the
promoters of many genes whose expression is restricted to hematopoietic
and/or endothelial cells (25, 27-34). The functions of most Ets
elements have not been well characterized, although many Ets proteins
are known transcriptional activators. Many Ets elements are adjacent to
other regulatory elements, which refine their binding specificities for
particular Ets proteins (43). Two conserved Ets elements were
identified in both the mouse and human P-selectin genes. Interestingly,
the distances between the two murine Ets elements (224 bp) and the two
human elements (213 bp) differ by exactly one turn of the DNA helix.
The distal Ets element is adjacent to a conserved AT-rich HOX
recognition sequence (41), and the proximal Ets element is flanked by
conserved 4-bp A/T sequences. Mutation of the proximal Ets element
eliminated binding to an Ets protein, most likely the ubiquitously
expressed GABP, and increased reporter gene expression directed by the
murine P-selectin promoter by 2-10-fold in transfected BAEC. The
-subunit of GABP (GABP
) shares sequence similarity with the Ets
family of transcription factors, and the
-subunit contains four
ankyrin repeats that mediate the interaction with GABP
(56). GABP
dimerizes to form an
2
2 tetramer that binds with high affinity to
two Ets elements separated by variable distances (56, 59). Although the
two conserved Ets elements in the P-selectin genes are separated by
~200 bp in linear sequence, a nucleosome (60, 61) could bring the two
elements sufficiently close to interact simultaneously with a GABP
tetramer to form a DNA loop. By a similar mechanism, the three
DNA-binding domains of the architectural HMG I(Y) protein may interact
with the three A/T-rich sequences adjacent to the Ets elements to
stabilize the loop (62). Formation of this putative loop may be
regulated. For example, oxidation of GABP
destroys its DNA binding
activity (63), a Hox protein may bind to the conserved 4-bp A/T
sequence adjacent to the distal Ets element (41), and
interleukin-4-dependent signaling may modulate the DNA
binding of HMG I(Y) through phosphorylation (64). Mutation of the
murine proximal Ets element increased reporter gene expression. This
suggests that the putative Ets-mediated DNA looping may repress
transcription. Transcriptional repression mediated by DNA looping has
been observed in other genes (65). Although deletion of the distal Ets
element did not alter reporter gene expression, large deletions may
alter other regulatory elements. Furthermore, data from transient
transfection of reporter genes may not fully reflect the architecture
and function of genes in the intact chromosome (66, 67).
Our findings also suggest a molecular explanation for the ability of
TNF- or LPS to augment expression of P-selectin in murine but not in
human endothelial cells. TNF-
or LPS increased expression of a
reporter gene driven by the murine, but not the human, P-selectin promoter in transfected BAEC. Serial deletions of the murine
5'-flanking region identified two segments that were required for the
TNF-
- or LPS-mediated induction of transcription. The first segment from
593 to
474 contained two tandem putative
B sites, whereas the second segment proximal to
229 contained a reverse-oriented
B
site and a variant activating transcription factor/cAMP sequence. In
the accompanying paper (68), we demonstrate that all these elements are
required for TNF-
or LPS to maximally induce transcription of the
murine P-selectin gene. These sites are not present in the 5'-flanking
region of the human P-selectin gene. Instead, the human gene has a
novel
B site that binds homodimers of p50 or p52 but not homodimers
or heterodimers containing p65 (26). Thus, the differential ability of
TNF-
or LPS to induce expression of P-selectin in mice, but not in
humans, may result in part from differences in the promoters of the
respective genes.
The species-specific transcriptional regulation of P-selectin in
response to TNF- or LPS is both unusual and surprising, given the
apparent similarities of many selectin functions across species
(69-71). However, there are other known genes in which species-specific changes in regulatory elements affect responsiveness to particular mediators (72, 73). Although the biological implications
of these species-specific regulatory mechanisms remain unexplained, the
presence of such mechanisms suggests caution in extrapolating the
results of some inducible gene responses in mouse models to humans.
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ACKNOWLEDGEMENTS |
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We thank Ginger Hampton for technical assistance. We are grateful to Dr. James Morrissey 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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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF031662.
To whom correspondence should be addressed: W. K. Warren
Medical Research Institute, University of Oklahoma Health Sciences Center, 825 N.E. 13th St., Oklahoma City, OK 73104. Tel.: 405-271-6480; Fax: 405-271-3137; E-mail: rodger-mcever{at}ouhsc.edu.
1
The abbreviations used are: TNF-, tumor
necrosis factor-
; bp, base pair(s); BAEC, bovine aortic endothelial
cells; GABP, GA-binding protein; LPS, lipopolysaccharide; PCR,
polymerase chain reaction.
2 The sequence of the 2032-bp insert containing the 5'-flanking region, exon 1, and part of intron 1 has been deposited in GenBankTM, with the accession number AF031662. Fig. 3 depicts a 1338-bp portion of the sequence.
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REFERENCES |
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