(Received for publication, June 29, 1995; and in revised form, August 30, 1995)
From the
Transcription of the vascular cell adhesion molecule-1 (VCAM-1)
gene in endothelial cells is induced by the inflammatory cytokines
interleukin-1, tumor necrosis factor-
, and
lipopolysaccharide. Previous studies demonstrated that the
cytokine-response region in the VCAM1 promoter contains
binding sites for the transcription factors nuclear factor-
B
(NF-
B) and interferon regulatory factor-1. Using a saturation
mutagenesis approach, we report that the cytokine-inducible enhancer
consists of these previously characterized elements and a novel region
located 3` of the NF-
B sites. Electrophoretic mobility shift
assays and DNase I footprint studies with endothelial nuclear extracts
and recombinant protein revealed that the transcriptional activator Sp1
interacts with this novel element in a specific manner. Transient
transfection assays using vascular endothelial cells revealed that
site-directed mutations in the Sp1 binding element decreased tumor
necrosis factor-
-induced activity of the VCAM1 promoter.
The cytokine-induced enhancer of the VCAM1 gene requires
constitutively bound Sp1 and induced heterodimeric NF-
B for
maximal promoter activity.
Vascular cell adhesion molecule-1 (VCAM-1) ()is a
110-kDa cell surface glycoprotein (Osborn et al., 1989; Rice
and Bevilacqua, 1989), which interacts with cells bearing the integrin
counter-receptor
(very late
antigen-4 (VLA-4)) and
(Carlos et al., 1990; Elices et al., 1990; Chan et
al., 1992). VCAM-1 is constitutively expressed in
antigen-presenting cells (Freedman et al., 1990; Koopman et al., 1991) and embryonic tissues (Rosen et al.,
1992; Gurtner et al., 1995). Vascular endothelium in vivo exhibits little or no VCAM-1 expression under normal conditions
but can be rapidly induced by lipopolysaccharide (Fries et
al., 1993). The endothelial expression of VCAM-1 has been
implicated in the recruitment of circulating monocytes and lymphocytes
from the bloodstream (reviewed in Carlos and Harlan, 1994; Springer,
1995) and associated with a variety of chronic inflammatory processes
(Rice et al., 1991; Cybulsky and Gimbrone, 1991).
VCAM-1
has a distinct pattern of cytokine induction in cultured endothelial
cells. Naive cells do not express VCAM-1 messenger RNA; however,
exposure of cells to inflammatory mediators or cytokines
(lipopolysaccharide, poly(I:C), interleukin-1, and tumor necrosis
factor-
(TNF-
)) results in rapid up-regulation of mRNA and
surface protein within 3 h (Osborn et al., 1989; Read et
al., 1994). To study the molecular mechanisms controlling the
VCAM-1 response to cytokine, we have characterized the structure of the
human VCAM1 gene (Cybulsky et al., 1991) and begun
analysis of the 5`-regulatory region (Neish et al., 1992; Shu et al., 1993; Neish et al., 1995). In previous work,
we and others demonstrated that TNF-
induction of VCAM1 expression occurs at the transcriptional level and is dependent on
tandem NF-
B motifs located at positions -65 and -75
relative to the single transcriptional start site (Neish et
al., 1992; Iademarco et al., 1992). Heterodimeric p50 and
p65 (RelA) associate with these elements in the nuclei of endothelial
cells exposed to TNF-
. Moreover, NF-
B physically and
functionally cooperates with the transcriptional activator IRF-1 to
achieve full induction of VCAM1 transcription (Neish et
al., 1995).
The regulatory regions conferring cytokine-inducible transcription in the leukocyte adhesion molecule genes are composed of multiple binding elements, which are recognized by a surprisingly finite number of transcription factors (reviewed in Collins et al., 1995). To provide a detailed characterizaton of the cytokine-induced transcriptional enhancer in the VCAM1 promoter, we employed a saturation mutagenesis approach to completely map the boundaries of regulatory elements that occur within the VCAM1 cytokine-response region. Using this approach, we have identified a novel Sp1 binding motif in the proximal promoter and have demonstrated that optimal cytokine activation of the VCAM1 gene requires Sp1.
For preparation
of cell extracts, human umbilical vein endothelial cells (HUVE) were
cultured in M199 with 20% fetal bovine serum, 100 mg/ml porcine
intestinal heparin, 50 mg/ml endothelial mitogen, 50 units/ml
penicillin, 50 mg/ml streptomycin, and 25 mM HEPES in
gelatin-coated plates (Gimbrone, 1976). Bovine aortic endothelial cells
(BAEC) were isolated and maintained in culture using previously
described procedures (Neish et al., 1992). For experiments
involving cytokine activation, confluent monolayers of endothelial
cells were exposed to recombinant human TNF- (Genentech, San
Francisco, CA) at a final concentration of 200 units/ml in complete
media for the time periods indicated.
Linker-scanning mutants were generated by PCR as follows. For each construct two complementary primers having individual mutations were designed (see below). The coding primer was used in combination with the 3`-BglII primer, whereas the non-coding one was used in combination with the 5`-SacI primer. Two subfragments were generated, both carrying the mutation at the 5`- and 3`-ends, respectively. The fragments were gel purified, aliquots of both were mixed, combined with the two primers 5`-SacI and 3`-BglII, and subjected to the second round of PCR.
The reactions were carried out as recommended in the manufacturer's specifications (Perkin Elmer Corp.). The resulting subfragments were digested with SacI and BglII and ligated into the SacI and BglII sites of pGL2-Basic (Promega). The integrity of the individual mutants was confirmed by DNA sequence analysis.
Oligonucleotides used for in vitro mutagenesis are as follows: 5` SacI, GCCGCCGAGCTCAGTCTAAATCTGTTAACC; 3` BglII, GCCGCCAGATCTGTATTCAGCTCCTGAAG; LS-100, GTTAATGCGGCCGCGCTGGCTCTGCCCTGGGT and GCCAGCGCGGCCGCATTAACAGACACCCAGCC; LS-90, TTTCCGGCGGCCGCGCCTGGGTTTCCCCTTGA and CCAGGCGCGGCCGCCGGAAAAAAGTTTAACAG; LS-80, TCTGCGGCGGCCGCGCCCTTGAAGGGATTTCC and AAGGGCGCGGCCGCCGCAGAGCCAGGGAAAA; LS-70, GTTTCGGCGGCCGCCGATTTCCCTCCGCCTCT and AAATCGGCGGCCGCCGAAACCCAGGGGCAGAGC; LS-60, GAAGGCGCGGCCGCGCGCCTCGCAACAAGAC and AGGCGCGCGGCCGCGCCTTCAAGGGGAAACCC; LS-50, CCCTCGGCGGCCGCTACAAGACCCTTTATAAA and CTTGTAGCGGCCGCCGAGGGAAATCCCTTCAA; LS-40, CTGCATGCGGCCGCATTATAAAGCACAGACTT and TATAATGCGGCCGCATGCAGAGGCGGAGGGAA (letters in lower case designate introduced nucleotide changes).
A fusion gene carrying the region -258 to +42 of the VCAM1 promoter coupled to a CAT reporter gene (pF2) was described and characterized earlier (Neish et al., 1992). A reporter bearing internal point mutations in the Sp1 site, pF2-mSp1, was made from two separate PCR amplification products as described (Neish et al., 1992). The amplified products were gel purified and subcloned into the SmaI site of the reporter plasmid pCAT3 (Promega).
Mutagenizing oligonucleotides used for construct generation are as follows: VCAM-RP-Sp1, AAGGGGAAACCCAGGGCAGAG; VCAM-FP-mSp1, GAAGGGATTTCCCTCattgTCTGCAA.
BAECs were transfected with a
modified calcium phosphate technique (Sambrook et al., 1989).
Cells were transfected with 10 µg of reporter plasmid. Relative
transfection efficiency was determined by cotransfection with pTK-GH (5
µg) (Nichols Institute Diagnostics, San Juan Capistrano, CA). After
transfection, cultures were washed twice with Hanks' balanced
salt solution and refed with complete Dulbecco's modified
Eagle's media with or without TNF-. After 24 h incubation,
samples of the medium were collected and assayed for growth hormone by
using a commercially available radioimmunoassay kit (Nichols Institute
Diagnostics), and cells were harvested. Confluent monolayers were
collected by trypsin dissociation, centrifuged (650
g for 5 min), washed once in phosphate-buffered saline, and
resuspended in 200 µl of 0.25 M Tris-HCl (pH 7.8) with 10
µg/ml aprotinin (Sigma). Cells were lysed by sonication and one
freeze-thaw cycle. Supernatants containing the cell extracts were
obtained after centrifugation for 10 min at 14,000
g and stored at -80 °C. Conversion of radiolabeled acetyl
coenzyme A to acetylated chloramphenicol was assayed by the two-phase
fluor diffusion technique (Sambrook et al., 1989). For each
sample, 30-50 µl of cell extract was incubated at 65 °C
for 15 min to inactivate endogenous transacetylases. The assay was
performed in a reaction mixture of 1.25 mM chloramphenicol
(Sigma), 125 mM Tris-HCl (pH 7.8), and 0.1 mCi of
H-acetyl coenzyme A (DuPont NEN) in a reaction volume of
200 µl. The reaction mixture was overlaid with 5 ml of water
immiscible scintillation fluid (Econofluor, Dupont NEN) and incubated
at 37 °C for 2 h. The activity of the
H-acetylated
chloramphenicol was measured using an LKB RackBeta scintillation
counter.
Transfection experiments were repeated at least three times using at least two independent plasmid preparations. A negative control, the promoterless plasmid pCAT3, showed no inducible activity from any experimental manipulation.
VCAM-NF+Sp1, CTGGGTTTCCCCTTGAAGGGATTTCCCTCCGCCTCTGCAA; VCAM-NF+mSp1, CTGGGTTTCCCCTTGAAGGGATTTCCCTCattgTCTGCAA; VCAM-Sp1, ATTTCCCTCCGCCTCTGCAA; consensus-Sp1, ATTTCCCCCCGCCCGTGCAA; mutant-Sp1, ATTTCCCTCattgTCTGCAA; VCAM-IRF, GGAGTGAAATAGAAAGTCTGTG.
Mutated bases are in lower case. Reverse complement strands were designed to leave G overhangs on one end. All oligonucleotides were polyacrylamide gel purified prior to annealing and labeling.
Footprint binding reactions were performed using the P
5`-labeled probe in a total volume of 20 µl containing 2 µl of
10
footprint binding buffer (50% glycerol, 50 mM MgCl
, 500 mM NaCl, 25 mM HEPES, pH
7.9), 0.5 µg poly(dI:dC), 10 µg bovine serum albumin, and
increasing amounts of the recombinant protein as indicated. The
reaction was allowed to proceed for 1 h at 4 °C. 20 µl of DNase
buffer (25 mM NaCl, 10 mM HEPES, pH 7.9, 5 mM MgCl
, 1 mM CaCl
) containing 0.02
units DNase I (Promega) was added to each tube and incubated for 5 min
at 4 °C. The reaction was terminated by the addition of 10 µl
of stop solution (150 mM EDTA, 5% SDS, 250 µg/ml salmon
sperm DNA) and immediate phenol/chloroform extraction prior to DNA
precipitation with absolute ethanol. The pellets were washed with 70%
ethanol, evaporated to dryness, and resuspended in sequencing sample
buffer. The samples were boiled for 2 min and applied to a 6%
polyacrylamide gel and run at 1500 V. The gels were subsequently dried
and autoradiographed overnight at -80 °C.
Figure 1:
Saturation
mutagenesis defines a minimal cytokine-responsive enhancer. A,
transfected HUVE were exposed to TNF- for 6 h and harvested.
Luciferase activity normalized for transfection efficiency was
determined as described under ``Experimental Procedures.''
Results are reported as fold induction and defined as activity of the
reporter divided by the activity of the uninduced -2150
construct. Error bars denote standard errors from three
independent experiments performed in duplicate. B, schematic
representation of linker scan mutants. The NF-
B sites are boxed, and the putative Sp1 site is underlined.
To
identify cis-regulatory sequences required for cytokine-induced
expression, we performed a detailed mutational analysis of the region
between -100 and -50 (Fig. 1B). In
vitro mutagenesis was used to prepare a systematic series of
promoter mutants, each differing by only 10 bp from the wild-type
construct. The plasmids were transfected into HUVE cells, and
luciferase activity was determined in the presence and absence of
TNF-. As shown in Fig. 1A, most of these promoter
mutants responded to TNF-
treatment in a manner similar to the
wild-type construct. The integrity of one large region of the VCAM1 promoter, -80 to -40 (LS-80, LS-70, LS-60, LS-50), was
essential for TNF-
-induced expression in HUVE cells. The -80
to -60 region corresponds to two adjacent consensus NF-
B
elements that have previously been determined to be important for
cytokine induction (Neish et al., 1992). Interestingly, the
-50 to -40 region (LS-50) represents a previously
unidentified regulatory element in the VCAM1 promoter. In
subsequent studies, we focused on this newly defined regulatory region
in the VCAM1 promoter that appeared to be important in
TNF-
-induced expression.
Figure 2:
A novel element in the VCAM1 promoter interacts with the transcriptional activator Sp1. EMSA
using an oligonucleotide probe spanning the novel element in the VCAM1 promoter (VCAM-Sp1) is shown. A, EMSA with
non-induced endothelial extracts. The shifted complex is indicated by A. Unlabeled competitor oligonucleotides with sequences listed
under ``Experimental Procedures'' were used at 100
molar excess. B, supershift analysis with nuclear extracts
from non-induced (lanes 2-5) and 1-h TNF-
-induced
endothelial cells (lanes 6-9). Binding reactions were
preincubated for 15 min with the antibodies indicated. The supershifted
complex is indicated by B.
The linker scan analysis defined a novel
element with a GC-rich core suggestive of an Sp1 site. Competitive EMSA
was used to provide suggestive evidence for the involvement of Sp1 in
complex A. Complex A was competed by a 100-fold molar excess of an
oligonucleotide bearing a consensus Sp1 site (Fig. 2A, lane 4). When the Sp1-like core sequence in the
oligonucleotide was mutated (CCGCCT to CattgT), it could no longer
compete for binding (Fig. 2A, lane 5). To
further implicate the involvement of Sp1 in complex A, supershift
analysis was used. The probe containing the putative VCAM1 Sp1
site was incubated with HUVE nuclear extract, from control and
TNF--induced cells, in the presence of polyclonal antisera to DNA
binding proteins. Complex A is identical in control and
TNF-
-induced conditions (Fig. 2B, lanes 2 and 6) and largely supershifted by anti-Sp1 antisera
(designated ``B,'' lanes 3 and 7).
The inability of Sp1 antibody to completely supershift the complex may
be due to comigration of a nucleoprotein complex unrelated to Sp1 or
incomplete recognition due to the phosphorylation state of Sp1. Partial
supershifts involving the commercially available antibody used in this
study have been reported elsewhere (Kramer et al., 1994;
Minowa et al., 1994; Jensen et al., 1995). Complex A
is not affected by the presence of antibodies to Egr-1, a related zinc
finger transcription factor that binds GC-rich sequences (Cao et
al., 1993) (lanes 4 and 8), or anti-p50, a
NF-
B subunit abundant in endothelial cells (lanes 5 and 9) (Read et al., 1994).
In vitro DNase I
footprint analysis was used to determine whether recombinant Sp1 could
interact with the human VCAM1 promoter. Sp1 specifically
protected a distinct region in the promoter from partial digestion by
DNase I in a dose-dependent manner (Fig. 3). Comparison of the
electrophoretic mobility of the protected region with the G ladder (Fig. 3) indicates that the site bound by Sp1 corresponds to the
GC-rich element. The Sp1 binding site was just 3` to the two previously
described B-binding elements (Neish et al., 1995)
protected by recombinant p50 homodimers (Fig. 3). These data
indicate that the element bound by Sp1 is in close proximity to the two
B sites in the VCAM1 promoter.
Figure 3:
Sp1 binds in close proximity to the
3`-NF-B recognition element in the proximal VCAM1 promoter. In vitro DNase I footprint analysis of the VCAM1 promoter using recombinant proteins is shown. The single
end-labeled VCAM1 promoter fragment was incubated with
increasing amounts of Sp1 or p50 homodimers for 60 min at 4 °C
prior to partial digestion with DNase I. Binding and electrophoresis
conditions are described under ``Experimental
Procedures.''
Figure 4:
EMSA with composite probes spanning the
cytokine response region. A, mutation of the Sp1 binding site
does not compromise the ability of NF-B to interact with the VCAM1 promoter. EMSA is shown with nuclear extract derived
from endothelial cells, non-induced (lanes 2 and 6),
1-h TNF-
induction (lanes 3 and 7), and 4-h
TNF-
induction (lanes 4 and 8). Probes are
wild-type (VCAM-NF+Sp1) (lanes 1-4) or mutant Sp1
(VCAM-NF+mSp1) (lanes 5-8). Shifted complexes are
designated with arrows. B, Sp1 and NF-
B can
co-occupy the VCAM1 promoter. EMSA is shown with recombinant
Sp1 and NF-
B. Increasing concentrations of full-length
p50
p65 were incubated alone (lanes 2-4) or
combined with a fixed amount of prebound recombinant Sp1 (lanes
5-8). The probe used was wild type (VCAM-NF+Sp1).
Shifted complexes are indicated with arrows.
Taken together, these experiments
demonstrate that endothelial Sp1 binds to a novel site in the VCAM1 promoter immediately adjacent to the 3`-NF-B site. Sp1 binds
equally well to its recognition element under quiescent and
TNF-
-stimulated conditions and is able to occupy the promoter
simultaneously with NF-
B. The findings have not, however,
established the functional significance of the Sp1 element in the
transcriptional regulation of the VCAM1 gene.
Figure 5:
Sp1 binding site is necessary for full
cytokine-induced activity of the VCAM1 promoter. BAEC were
transfected with 10 µg of VCAM-CAT reporter plasmids containing
either wild-type sequences or bearing a mutant Sp1 site. Accumulated
CAT was assayed 24 h post-transfection. Transfected cells were
incubated with either media (lanes 1 and 3, open
bars) or 200 units/ml TNF- (lanes 2 and 4, closed bars) as indicated. Results are normalized to human
growth hormone and represent the mean of three experiments utilizing
independent plasmid preparations. Standard errors are
indicated.
Transcriptional enhancer elements are comprised of multiple
discrete DNA sequence motifs, each representing a cognate site for a
DNA binding protein. The precise number, orientation, and relative
positions of these protein binding modules may be critical for proper
regulatory function (reviewed by Tjian and Maniatis(1994)). To
characterize the cytokine-inducible transcriptional enhancer in the VCAM1 promoter, we have utilized saturation mutagenesis to
define the boundaries of relevant positive regulatory regions prior to
the characterization of interacting nuclear protein species. Here, we
report that both Sp1 and NF-B are required for maximal activity of
the cytokine response region of the VCAM1 promoter. Deletion
or mutation in the Sp1 binding motif results in significant attenuation
in the ability of the promoter to drive induced transcription in
endothelial cells.
A key component of cytokine-inducible enhancers
is the ubiquitous transcription factor NF-B (reviewed in Collins et al., 1995). The heterodimeric form (p65
p50) is
critical for the transcriptional activation of the human VCAM1 promoter (Neish et al., 1995). It is likely that the
specific patterns of transcriptional regulation seen among
NF-
B-dependent genes is mediated by the combinatorial interactions
of NF-
B with other transcriptional activators and chromatin
components. Physical and functional interactions have been shown
between NF-
B and ATF-2 (Du et al., 1993), C/EBP
(Stein et al., 1993a), c-Jun (Stein et al., 1993b),
and HMGI(Y) (Thanos and Maniatis, 1992). Previous work with the VCAM1 promoter in our laboratory demonstrated similar
interactions between NF-
B and IRF-1 (Neish et al., 1995).
Interactions between NF-
B and Sp1 have also been reported.
Cooperative functional and physical interactions between NF-
B and
Sp1 are required for efficient transcription of the HIV-LTR (Perkins et al., 1993, 1994). This effect is mediated by specific
physical interactions between the amino terminus of the p65 Rel domain
(RelA) and the zinc finger region of Sp1. Interestingly, the HIV-LTR
shares structural similarities with the VCAM1 cytokine
response region (Fig. 6); both genes have tandem NF-
B sites
located immediately 5` of an Sp1 motif. Our findings are consistent
with the proposal that the juxtaposition of Sp1 and NF-
B elements
promotes an interaction between these transcription factors (Perkins et al., 1994). The exposed surfaces of the DNA binding domains
of p65 and Sp1 may contact each other in an interaction facilitated by
the close approximation of the
B and Sp1 elements. This close
physical association in both the HIV-LTR and the VCAM1 enhancer would leave the transactivation domains of both p65 and
Sp1 to interact with TATA box binding protein-associated factors, or
TAFs. These proteins are coactivators postulated to interact with
different types of activation domains and thus integrate signals from
distinct bound transcription factors and assemble the basal
transcriptional complex. The Sp1 activation domain has been
demonstrated to directly and specifically interact with
dTAF
110 (Chen et al., 1994). Similarly, p65 has
been reported to interact with the TATA box binding protein and enhance
transcription (Kerr et al., 1993). These results add to our
understanding of the transcription factors necessary for the cytokine
responsiveness of this pathophysiologically important gene and provide
insights into a general mechanism for the activation of some
B-dependent genes.
Figure 6:
Sequence comparison of the basal VCAM1 promoter and the HIV-LTR. Regions immediately 5` of the TATAA box
of both promoters are aligned. NF-B sites are in bold,
and Sp1 sites are underlined.