From the Immunology Research Center, St Vincent's Hospital, Fitzroy 3065, Victoria, Australia
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ABSTRACT |
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The expression of intercellular adhesion molecule
2 (ICAM-2) in adult tissues is restricted to vascular endothelial cells and megakaryocytes. We have previously shown that the
endothelial-specific in vivo activity of the human ICAM-2
promoter is contained within a small (0.33-kilobase (kbp)) 5'-flanking
region of the gene. Here we describe the in vitro
characterization of this region. The ICAM-2 promoter is TATA-less, and
transcription in endothelial cells initiates at four sites. Reporter
gene expression directed by the promoter was 125-fold greater than
vector alone in bovine aortic endothelial cells but less than 2-fold
vector alone in non-endothelial (COS) cells, confirming that
specificity in vivo was paralleled in vitro.
The addition of 2.7 kbp of 5'-flanking region to the 0.33-kbp fragment
had no effect on promoter activity or specificity. The mutation of an
Sp1 motif centered at base pair 194 or an eight-base pair palindrome
at
268 each reduced promoter activity by 70%. Mutation of GATA
motifs at
145 and
53 reduced promoter activity by 78 and 61%,
respectively. Specific binding of bovine aortic endothelial cells
nuclear proteins to the Sp1 and GATA sites was demonstrated by gel
shift analysis. Promoter activity in COS cells was transactivated
3-4-fold by overexpression of GATA-2. The results presented here
suggest that transcription from the ICAM-2 promoter in endothelial
cells is regulated by the interplay of several positive-acting factors and provide the basis for further analysis of endothelial-specific gene
expression.
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INTRODUCTION |
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The endothelial cells that form the lining of all blood vessels perform a wide range of functions. In addition to providing a selective barrier between the bloodstream and tissues, vascular endothelial cells are critical for processes including thrombosis, angiogenesis, leukocyte trafficking, and the maintenance of vascular tone (1). The vascular endothelium also plays an important role in cancer metastasis and in the pathogenesis of non-neoplastic diseases such as rheumatoid arthritis and atherosclerosis (2, 3). To gain an understanding of the regulation of endothelium-specific gene expression and thus provide insights into these processes and conditions, a number of studies have focused on the characterization of endothelial cell-specific promoters (4-14). Promoters capable of targeting gene expression to endothelial cells in vivo as well as in vitro may be particularly useful, not only in mechanistic studies but also in medical applications ranging from transgenic modification of donor organs in xenotransplantation (15, 16) to gene therapy for diseases affecting the cardiovascular system (17).
The promoters of several genes that are expressed predominantly or exclusively in endothelial cells have been examined both in vitro in transient transfection assays and in vivo in transgenic mouse studies. The human and mouse tie1 promoters demonstrated endothelial cell specificity in vivo, with expression in the vasculature varying from organ to organ but surprisingly were not specific in vitro (7). In contrast, the human KDR/flk-1 and von Willebrand factor promoters were endothelial cell-specific in vitro, but their activity in vivo was undetectable (18) or restricted to a subset of endothelial cells (12), respectively. Unlike these promoters, which presumably lack important regulatory elements, the mouse tie2 (tek) promoter/enhancer was capable of driving endothelial cell-specific transgene expression in vitro and in vivo (4), although the strong expression in all vascular endothelium in adult tie2/lacZ transgenic mice was somewhat unexpected because tie2 expression is downregulated in adult animals (19).
The first promoter shown to target uniform, high-level transgene expression to the vasculature of transgenic mice was that of the human intercellular adhesion molecule 2 gene (ICAM-2)1 (20). Human ICAM-2 is a counter receptor for lymphocyte function-associated antigen-1 (21) and may provide a costimulatory signal for T cell stimulation by allogeneic class II major histocompatibility complex (22). In human tissues, the expression of ICAM-2 is restricted largely to endothelial cells and megakaryocytes (23). Unlike the mouse tie2 promoter/enhancer, which comprises 2.1 kbp of promoter and 10 kbp of the first intron (4), the human ICAM-2 promoter contains all of the signals necessary for endothelium-specific transgene expression in vivo within a relatively small (<350 bp) region, presenting a novel opportunity for analysis. In this study, we report the first in vitro characterization of the ICAM-2 promoter. We demonstrate that the ICAM-2 promoter is endothelial cell-specific in vitro and identify four elements, an Sp1 site, two GATA sites, and an 8-bp palindromic sequence also present in the tie2 enhancer, that are necessary for full promoter activity. We also show that the ICAM-2 promoter is transactivated in non-endothelial cells by overexpression of two members of the GATA family of transcription factors, GATA-1 and GATA-2.
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EXPERIMENTAL PROCEDURES |
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DNA Preparation, Sequencing, and Analysis-- Plasmid DNA was prepared from bacterial cultures using Qiagen Plasmid Midi or Maxi Kits. DNA sequencing reactions were performed using Thermo Sequenase (Amersham Pharmacia Biotech). T3 and T7 primers were used to sequence inserts cloned into pBluescript II (Stratagene), and the primers GL3-1 and GL3-2 (corresponding to bp 51-68 and to the complement of bp 4785-4803, respectively, in the vector) were used for inserts cloned into pGL3-Basic (Promega). Reactions were electrophoresed at either the Nucleotide Sequencing Facility, Monash University, Victoria, or the Molecular Biology Facility, Griffith University, Queensland, Australia. The DNA sequence was scanned for putative transcription factor binding sites using the MatInspector program (24) and the TRANSFAC data base (25). Comparison of ICAM-2 sequences was carried out using the Clustal W program (26).
Isolation of Human ICAM-2 5'-Flanking Region--
The Human
GenomeWalker kit (CLONTECH) was used to walk
upstream of known ICAM-2 5'-flanking sequence. A primer complementary to the sequence +27/+50 relative to the ICAM-2 translation start site
(21) was used with an outer adaptor primer in primary PCRs using five
adaptor-ligated human genomic libraries as templates. Samples of the
primary PCRs were used in secondary PCRs with a nested ICAM-2-specific
primer (17/+5 relative to the translation start site) and a nested
adaptor primer. Products of the secondary PCRs were resolved by agarose
gel electrophoresis, purified using the QIAquick Gel extraction kit
(Qiagen), and cloned into pBluescript II.
Generation and Testing of Transgenic Mice-- The 5' end of the 0.33-kbp ICAM-2 promoter fragment in an ICAM-2 promoter/human CD59 microinjection construct (20) is delineated by an NcoI site. This site was employed in the precise fusion of 2.7 kbp of upstream flanking sequence (isolated as described above) to the 0.33-kbp region. The resulting construct, designated PK, was used to generate transgenic mice as described (20). Peripheral blood leukocytes from the transgenic mice were examined for the expression of CD59 by flow cytometric analysis as described (20). Fresh frozen tissue sections prepared from mouse organs were stained to detect the pattern of CD59 expression as described (20).
Rapid Amplification of cDNA Ends (RACE)--
The method used
to identify the 5' end of ICAM-2 transcripts was based on the 5' RACE
system (Life Technologies, Inc.). In brief, total RNA (2.5 µg)
prepared from human umbilical vein endothelial cells was
reverse-transcribed from an antisense primer (+27/+50 relative to the
ICAM-2 translation start site) using SuperScript II (Life
Technologies). The cDNA was purified and tailed with dCTP using
terminal deoxynucleotide transferase (Promega). An aliquot of the
tailed cDNA was used as template in a PCR with primers AAP
(5'-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3') and a nested antisense
primer (17/+5 relative to the ICAM-2 translation start site) using
the Advantage Tth polymerase mix
(CLONTECH) to maximize fidelity. PCR was carried
out in a DNA engine thermal cycler (MJ Research) using the following
parameters: 94 °C for 2 min; 5 cycles of 92 °C/10 s, 55 °C/10
s, 72 °C/30 s; and 25 cycles of 92 °C/5 s, 55 °C/5 s,
72 °C/30 s. An aliquot (0.1%) of the first PCR was used directly in
a second PCR with primers AUAP (5'-GGCCACGCGTCGACTAGTAC-3') and the
nested antisense (
17/+5) primer under the same conditions except that
the annealing temperature was increased to 60 °C. Products of the
second PCR were purified using the High Pure PCR product purification
kit (Boehringer Mannheim) and cloned into pBluescript II.
Cell Culture and Transient Transfection-- Bovine aortic endothelial cells (BAEC) were isolated using a modification of a previously described procedure (27). Freshly isolated bovine aortae were ligated, rinsed 3-5 times with phosphate-buffered saline, pH 7.4, and filled with growth medium (see below) lacking fetal calf serum and containing 1 mg/ml collagenase (Boehringer Mannheim). After a 10-15-min incubation in 5% CO2 at 37 °C, the contents of the aortae were removed and centrifuged at 1500 × g for 10 min. The cell pellet was resuspended in growth medium consisting of Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 15% heat-inactivated fetal calf serum (Life Technologies), 15 mM HEPES (Trace Scientific, Australia), 60 µg/ml penicillin (CSL, Australia), 100 µg/ml streptomycin (CSL), and 20 µg/ml cis-4-hydroxy-L-proline (Sigma) and dispensed into gelatin-coated flasks. BAEC were used at passage 3-8. COS-7 (African green monkey fibroblast) cells were obtained from the American Type Culture Collection and cultured in the same medium as used for BAEC except that the concentration of heat-inactivated fetal calf serum was 10%, and no cis-hydroxyproline was added.
Transient transfections were performed at least twice, in triplicate. For transfection of BAEC, approximately 105 cells were plated/well in 6-well trays and incubated in 5% CO2 at 37 °C until 40-50% confluent (16-20 h). For each well, 4 µl of LipofectAMINE (Life Technologies) and 1.5 µg of plasmid DNA (1 µg of luciferase construct plus 0.5 µg of pSVMutagenesis-- A two-step PCR method was used to mutagenize the 0.33-kbp ICAM-2 promoter cloned in pBluescript II such that the 5' end is closest to the M13 forward priming site (20). In the first step, PCRs were carried out using an internal mutagenic primer with an external vector-targeted primer. The internal forward primers (mutated bases underlined) were P8-Fmut, 5'-TTCCCCAGACGCGGGGCTTGT-3'; P12-Fmut, 5'-GTCTCATATTCGCATGCGAACACCCATTG-3'; Sp1-Fmut, 5'-CATTGCCTGCCCTATCCCTTGCACA-3'; GATA U-Fmut, 5'-GCCCTGCTTTAGAGCAGCTTC-3'; and GATA D-Fmut, 5'-CTCTCAGTATATGAGAGGA-3'. The internal reverse primers (P8-Rmut, etc.) were complementary to the forward primers. The forward and reverse primers were used in independent PCRs with the M13/pUC reverse and forward amplification primers (Life Technologies), respectively. The two products from each set of first-step PCRs (e.g., P8-Fmut/M13-R and P8-Rmut/M13-F) were purified, mixed, and used in the second-step PCR with the external primers only. Products from the second-step PCRs were cloned into pGL3-Basic and sequenced to confirm the presence of the desired mutations.
To construct the Sp1/GATA U (upstream) double mutant, the upstream GATA site mutant cloned in pGL3-Basic was used as template. The mutagenic primers were Sp1-Fmut and Sp1-Rmut, and the external primers were GL3-1 and GL3-2. The second-step PCR product was cloned and sequenced as described above.Preparation of Nuclear Extracts--
BAEC nuclear extracts were
prepared as follows. A confluent monolayer of cells in a 25 cm2 flask were harvested by scraping, washed in cold
phosphate-buffered saline (pH 8.0), and resuspended in 50 µl of cold
Buffer A (10 mM Tris-HCl, pH 9.0, 2 mM
MgCl2, 5 mM KCl, 10% glycerol, and 1 mM EDTA). 50 µl of cold Buffer B (Buffer A plus 1%
Nonidet P-40) was added, and the cells were allowed to swell on ice for
15 min then vortexed vigorously. The resulting lysate was centrifuged at 700 × g for 10 min at 4 °C. The nuclear pellet
was resuspended in 0.5 ml of Buffer A plus 0.5 ml of Buffer B and
recentrifuged at 700 × g for 5 min at 4 °C. The
pellet was resuspended in 50 µl of cold Buffer C (20 mM
HEPES, 0.4 M NaCl, 1 mM EDTA, and 1 mM EGTA), and nuclear proteins were extracted by vigorous
shaking for 30 min at 4 °C. Cell debris was pelleted by
centrifugation at 18,000 × g for 5 min at 4 °C, and
the nuclear extract was aliquoted, frozen in liquid nitrogen, and
stored at 70 °C. Buffers A, B, and C were adjusted to 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml aprotinin before use.
Electrophoretic Mobility Shift Assay--
The binding mixture
(20 µl) contained ~15 µg of BAEC nuclear extract, 0.5 µg
poly(dI:dC), and 105 cpm of 32P-labeled
double-stranded oligonucleotide probe in 10 mM Tris-HCl, pH
8.0, 5 mM MgCl2, 1 mM
dithiothreitol, 1 mM EDTA, and 1 mM bovine serum albumin. All of the ingredients except the probe were mixed and
incubated for 15 min at room temperature, after which the probe was
added, and incubation was continued for 15-30 min. Band shifts were
resolved by electrophoresis at 4 °C on a non-denaturing 7%
polyacrylamide gel. The probes were as follows (coordinates relative to
the primary transcription start site): Sp1, bp 207 to
183; GATA U
(upstream), bp
155 to
135; and GATA D (downstream), bp
61 to
43. Competition studies were performed by adding a 100-fold molar
excess of unlabeled double-stranded oligonucleotides to the binding
reactions before the addition of labeled probe. DNA-protein-complexes
were judged to be specific if their formation was competitively
inhibited by cold unlabeled probe but not by an unrelated
oligonucleotide. Mutant competitors
(Sp1-Fmut/Sp1-Rmut etc.) are described in the
previous section.
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RESULTS |
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Isolation of 3 kbp of 5'-Flanking Region of the Human ICAM-2
Gene--
Human ICAM-2 expression in the tissues is restricted largely
to vascular endothelial cells and megakaryocytes (23); in the blood,
ICAM-2 is expressed on platelets (31) and at a low level on resting
lymphocytes and monocytes but not on granulocytes (23). We have
previously shown that 0.33 kbp of 5'-flanking region of the human
ICAM-2 gene is sufficient to target high-level transgene expression to
all vascular endothelium in the heart, kidney, liver, lung, and
pancreas of transgenic mice (20), consistent with the tissue
distribution of human ICAM-2. Unexpectedly, however, transgenic mouse
lymphocytes were negative for transgene expression and granulocytes
were very strongly positive. This discrepancy is unlikely to be due to
differences in the pattern of expression of human and mouse ICAM-2,
because flow cytometric analysis revealed that the hierarchy of ICAM-2
expression levels is similar on human and mouse leukocyte populations
i.e. monocytes > lymphocytes granulocytes.2 An alternative
explanation is that elements regulating ICAM-2 expression on leukocytes
are absent from the 0.33-kbp region. To investigate this possibility
and to generate additional material for in vitro analysis of
the ICAM-2 promoter, a larger 5'-flanking region of human ICAM-2 was
isolated. Nested primers targeted to known ICAM-2 promoter sequence
were used with adaptor primers to amplify the 5'-flanking sequence from
five adaptor-ligated human genomic libraries. The PvuII,
SspI, and ScaI libraries yielded products of 1.0, 1.8, and 3.1 kbp, respectively, which were cloned and analyzed by
restriction enzyme digestion to produce the map shown in Fig.
1A. The DNA sequence obtained
from the 3' end of the two larger clones matched that of the 0.33-kbp
region (20) except for an additional G at bp
281 and an additional C
at bp
275 (Fig. 1B). The presence of these bases,
confirmed in the original 0.33-kbp clone by re-sequencing, revealed a
consensus NF-
B motif (GGGGTTCCCC) at bp
281 to bp
272 (Fig.
1B).
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Identification of the Transcription Start Sites of ICAM-2--
To
identify the transcription start site(s) of the human ICAM-2 gene,
5'-RACE was performed on total RNA from human umbilical vein
endothelial cells, which strongly express ICAM-2 (23). First-strand
cDNA prepared using a primer targeted to the first exon was tailed
with dC and used in PCR with a G-rich primer and a gene-specific nested
primer. After a second PCR with nested primers, the resulting products
were cloned into pBluescript II, and five clones were sequenced,
revealing four transcription start sites (Fig. 1B). A
thymine residue (shown in bold type) located 77 bp upstream
of the ICAM-2 start codon was designated the primary transcription
initiation site (+1), because the start sites for two independent
clones mapped to this point and that for a third mapped immediately
upstream (1). The remaining start sites were located at
37 and
66. Thus like the promoters of several other constitutively expressed
cell adhesion molecule genes including PECAM-1 (32) and CD18 (33), the
ICAM-2 promoter lacks consensus TATA and CAAT boxes and contains
multiple transcription start sites.
Endothelial Cell Specificity of the ICAM-2 Promoter in
Vitro--
The in vitro promoter activity and specificity
of various lengths of ICAM-2 5'-flanking region were tested by
transfecting different fragments cloned in the luciferase reporter
vector pGL3-Basic into BAEC or the simian kidney fibroblast cell line
COS. The constructs were cotransfected with pSVGal to correct for
differences in transfection efficiency, and luciferase activity was
normalized to that of pGL3-Basic alone. The 3.0-, 1.7-, and 0.33-kbp
fragments exhibited similar significant promoter activity
(100-130-fold the level of vector alone) in BAEC and only minimal
activity (approximately 2-fold vector alone) in COS cells (Fig.
4), indicating that the signals necessary
for endothelial cell-specific ICAM-2 promoter activity in
vitro are contained within the 0.33-kbp fragment. In
vitro analysis of the promoter was therefore performed on this fragment.
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Identification of Potential Cis-acting Elements in the ICAM-2
Promoter--
The sequence of the 0.33-kbp promoter fragment was
scanned for potential transcription factor binding sites and other
motifs and compared with the corresponding sequence from mouse (34) to
identify conservation of sites. Two sites for the GATA family of
transcription factors, centered at 145 and
53 bp relative to the
primary start point of transcription in the human promoter, were
conserved in position and orientation in the mouse ICAM-2 gene (Fig.
1B). Four conserved sites for the Ets family were located at
135,
127,
70, and
44. An 8-bp palindrome (CAGATCTG, referred to
here as P8) was detected at
268; this sequence is also
present in the first intron of the endothelial-specific tek
(tie2) gene and has been shown to be important in conferring
endothelial specificity in vivo (4). Seven of the
nucleotides in P8 were conserved in the mouse ICAM-2
sequence, although the distance between this motif and a highly
conserved block containing the GATA and Ets sites was 86 bp greater in
mouse than human. Another palindrome (CACGCATGCGTG, referred to as
P12) present at
217 was only partly conserved (9/12 bp)
in the mouse sequence, as was the NF-
B site (8/10 bp) at
276. An
Sp1 motif at
194 in the human promoter was not conserved within the
corresponding region in mouse. The locations of the putative regulatory
elements within the human ICAM-2 promoter are indicated schematically
in Fig. 1C.
Mutational Analysis of the ICAM-2 Promoter--
To identify
functionally important elements in the ICAM-2 promoter, several of the
motifs described above were mutated, and the effect of each mutation on
promoter activity in endothelial cells was determined. Mutated
derivatives of the 0.33-kbp promoter fragment were constructed by a
two-step PCR method, cloned into pGL3-Basic, and transfected into BAEC.
The mutation introduced into the P8 site (CAGATCTG CAGACGCG), based on that shown to affect tek
promoter activity and endothelial cell specificity in vivo
(4), reduced ICAM-2 promoter activity in BAEC by 70% (Fig.
5). The upstream GATA site mutation
(TTATCA
TTTAGA), which had previously been shown to
reduce the in vitro activity of the P-selectin (14) and eNOS
(11) promoters, caused a 78% reduction in promoter activity (Fig. 5).
Mutation of the core sequence of the Sp1 site (CCCCGCCCC
CCCTATCCC) or that of the downstream GATA site (AGATAA
AGTATA) reduced promoter activity by 70 and 61%,
respectively (Fig. 5). The combination of the Sp1 and upstream GATA
site mutations had a greater effect than either mutation alone,
reducing promoter activity by 87%, although the double mutant retained
measurable promoter activity (more than 10-fold the level of vector
alone).
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Specific Interaction of BAEC Nuclear Proteins with Elements in the ICAM-2 Promoter-- Specific binding of BAEC nuclear proteins to the functional Sp1 and GATA sites was assessed by electrophoretic mobility shift assay. Several retarded bands were observed when BAEC nuclear extract was incubated with a labeled probe containing the Sp1 site, but only the highest molecular weight DNA-protein complex appeared to be specific (Fig. 6A). Excess cold mutant competitor failed to prevent the formation of this complex, indicating that it is likely to contain a protein involved in the regulation of the ICAM-2 promoter in BAEC.
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Transactivation of the ICAM-2 Promoter in COS Cells by
Overexpression of GATA-2--
GATA-2 is abundantly expressed in
endothelial cells (29) and has been implicated in the regulation of
endothelial expression of genes including endothelin-1 (10), eNOS (11),
von Willebrand factor (36), and P-selectin (14). Like GATA-2, GATA-1 is
capable of transactivating an endothelial-specific promoter in
non-endothelial cells in vitro (29). To determine whether
either factor could activate the ICAM-2 promoter in vitro,
expression plasmids for mouse GATA-1 or human GATA-2 were
co-transfected with the ICAM-2 promoter-luciferase reporter into COS
cells. A synthetic GATA-responsive promoter construct (M6-SEAP)
consisting of six GATA sites linked to a minimal promoter driving the
expression of secreted alkaline phosphatase was co-transfected as a
control to confirm expression of the GATA factors. As shown in Fig.
7, expression of GATA-1 or GATA-2
transactivated the ICAM-2 promoter by 7.3- and 3.4-fold, respectively.
The greater activation of both the ICAM-2 and M6
promoters by GATA-1
(Fig. 7) may indicate that GATA-1 is a more potent transactivator
in vitro than GATA-2 as previously suggested (37). However,
since GATA-1 is not expressed in endothelial cells (28), it is likely
that GATA-2 regulates ICAM-2 expression in this cell type in
vivo.
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DISCUSSION |
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Human ICAM-2 is constitutively expressed on all vascular endothelial cells (23), and the ICAM-2 promoter is unusual among the endothelial cell-specific promoters studied to date in that the signals necessary for specific expression in vivo reside within a very small (0.33-kbp) region of 5'-flanking sequence (20). As the first step toward understanding the mechanism of this specificity, we have characterized the ICAM-2 promoter in vitro. After confirming that the specificity of the 0.33-kbp promoter in vivo was paralleled in vitro, we showed that the addition of up to 2.7 kbp of upstream 5'-flanking region did not increase promoter activity in transfected BAEC (Fig. 4) and did not affect the pattern of transgene expression in transgenic mice (Fig. 3), suggesting that most or all of the positive-acting elements regulating ICAM-2 promoter activity in endothelial cells are indeed located in the 336-bp sequence shown in Fig. 1B.
Based on previous reports, the obvious candidates for these elements included consensus motifs for the Sp1 and GATA families of transcription factors (Fig. 1C). Functional Sp1 binding sites are required for maximum in vitro endothelial activity of the promoters of the KDR/flk-1 (18), platelet-derived growth factor (38), and eNOS (11) genes; likewise, GATA sites have been identified as positive elements in the eNOS (11), von Willebrand factor (36), P-selectin (14), and endothelin-1 (39) promoters. Mutational and gel shift analyses were used to demonstrate that the Sp1 site and two GATA sites in the ICAM-2 promoter function as cis-acting positive regulatory elements in endothelial cells in vitro (Figs. 5 and 6). Mutation of the Sp1 site abolished specific binding of a BAEC nuclear protein and reduced ICAM-2 promoter activity in BAEC by 70%. Mutation of the upstream and downstream GATA sites disrupted BAEC nuclear protein binding and reduced promoter activity in BAEC by 78 and 61%, respectively. Although the identity of the protein(s) binding the GATA sites was not determined, we believe that it is likely to be GATA-2, because overexpression of GATA-2 transactivated the ICAM-2 promoter in non-endothelial (COS) cells in vitro (Fig. 7), and GATA-2 has previously been implicated in the endothelial cell expression of several genes (11, 14, 39). GATA-1 also transactivated the promoter but is not expressed in endothelial cells (28). GATA-1 is believed to play a role in megakaryocyte gene expression (40, 41) and thus may be involved in the regulation of ICAM-2 expression in this cell type.
Another positive element identified by mutagenesis was an 8-bp
palindrome that has been shown to be an important component of an
intronic endothelial cell-specific enhancer in the murine tie2 gene (4). This motif, which we have termed
P8, is also present in the promoter of the
endothelial-specific mouse tie1 gene (7) and (conserved in 7 out of 8 positions) in the promoters of the human tie1 (7)
and mouse ICAM-2 (34) genes. Mutation of P8 reduced ICAM-2
promoter activity in BAEC by 70% (Fig. 5). Mutation of another
palindrome (P12) did not affect promoter activity in BAEC,
and binding of BAEC nuclear proteins to a P12 probe (bp 224 to
209) could not be clearly demonstrated by electrophoretic mobility shift assay (data not shown). However, it is not possible to
conclusively rule out a role for this site, because preliminary DNase I
footprinting experiments indicated that an 18-bp region encompassing
P12 is protected by a protein(s) present in BAEC nuclear
extract.2 Further mutational analysis and identification of
proteins binding to P12 are in progress.
Ets family members are involved in both endothelial (4, 38, 42) and
megakaryocytic (41) gene expression. The function of four Ets motifs in
the human ICAM-2 promoter, which are all largely conserved in the mouse
ICAM-2 gene (Fig. 1B), was not determined in this study, but
it is interesting to note the similarity between the tandem motifs
(GCTTCCCAGCTTCCT) centered at 132 bp in the
ICAM-2 promoter with tandem PEA3 motifs
(GCTTCCTCCCTTTCCT) in the promoters of the
human and mouse tie1 genes (7).
The presence of an NF-B motif in the ICAM-2 promoter is also
intriguing. NF-
B is a major regulator of the cytokine-inducible expression of various genes including the cell adhesion molecules E-selectin, VCAM-1, ICAM-1, and MadCAM-1 (reviewed in Ref. 43). NF-
B
is also believed to control the constitutive level of expression of
P-selectin in endothelial cells and megakaryocytes by a mechanism involving the interaction of Bcl-3 with promoter-bound p50 or p52
homodimers, and mutation of the P-selectin
B site causes a 40%
reduction in promoter activity in BAEC (44). ICAM-2 is expressed
constitutively at high levels on resting endothelial cells and
megakaryocytes in vivo, and the level of expression in human
umbilical vein endothelial cells, like that of P-selectin (44), was
unchanged by treatment with a variety of inflammatory cytokines (23).
Incubation of nuclear extract from resting BAEC with a probe (bp
285
to
269) containing the ICAM-2
B site resulted in the formation of
a specific DNA/protein
complex.3 Together, these
data suggest that the role (if any) of the ICAM-2
B site in
endothelial expression is similar to that of the P-selectin site,
although this awaits confirmation by mutagenesis and gel supershift
analysis.
The small size and tight specificity of the ICAM-2 promoter make it a
particularly promising tool for gene therapy and other applications
requiring endothelial cell targeting. It is also valuable in the study
of the control of gene expression in endothelial cells, as demonstrated
in this study. On the basis of the results presented here, we propose
that the transcription of ICAM-2 in endothelial cells is regulated
primarily by the binding of Sp1 and GATA-2 transcription factors and
possibly a novel P8 binding factor to their cognate sites
in the ICAM-2 promoter. Interaction of these DNA-bound proteins via
their zinc fingers (45) may induce a conformational change, allowing
them to interact more readily with components of the basal
transcriptional machinery. For example, as suggested for GATA-1 (45),
the interaction of GATA-2 with Sp1 may increase the affinity of the
latter for TAFII110 or some other component of the basal
complex. The involvement of other proteins, such as members of the Ets
and NF-B families, remains to be determined.
Although Sp1 and GATA-2 may be two of the major determinants of ICAM-2 promoter activity in endothelial cells, their presence alone cannot account for the specificity of the promoter, since Sp1 is ubiquitously expressed, and GATA-2 is expressed relatively widely (37). It is possible that a threshold level of GATA-2 is required for activation of the promoter. Alternatively, GATA-2 may interact with a cell-specific factor to activate transcription of ICAM-2 in endothelial cells. Such an interaction between GATA-1 and the protein FOG (Friend of GATA-1) has been proposed to explain the transcriptional regulation of hematopoietic-specific gene expression (46). It is not known whether FOG binds DNA in a sequence-specific manner (46), but if so, it is tempting to speculate that either the P8 or P12 motifs in the ICAM-2 promoter may represent the binding site of an endothelial-restricted FOG-like protein. Finally, it is conceivable that ICAM-2 expression in non-endothelial cells is repressed by a negative-acting factor such as that described for the von Willebrand factor promoter (6). The identification of specific proteins interacting with the ICAM-2 promoter using techniques such as gel supershift and DNA footprinting will be helpful in discriminating between these possibilities.
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ACKNOWLEDGEMENTS |
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We thank Helen Barlow, Nella Fisicaro, and Ewa Witort for assistance in the maintenance and screening of transgenic mice and Dr. Merlin Crossley for providing materials and for helpful advice and comments.
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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.
To whom correspondence should be addressed: Immunology Research
Center, St Vincent's Hospital, 41 Victoria Parade, Fitzroy 3065, Victoria, Australia. Tel.: 613 9288 3140; Fax: 613 9288 3151; E-mail: pearsemj{at}svhm.org.au.
1 The abbreviations used are: ICAM-2, intercellular adhesion molecule 2; BAEC, bovine aortic endothelial cells; PCR, polymerase chain reaction; RACE, reverse amplification of cDNA ends; bp, base pair(s); kbp, kilobase pair(s).
2 P. J. Cowan, unpublished results.
3 C. M. Pedic, unpublished results.
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