From the
Cytokine activation of vascular cell adhesion molecule-1
(VCAM-1) gene expression by endothelial cells is an important feature
in a variety of vascular inflammatory responses. Cytokines
transcriptionally activate the VCAM-1 promoter in endothelial cells at
least in part through two closely linked NF-
Vascular cell adhesion molecule
The role of the NF-
Two observations suggest that transcriptional regulatory factors
other than the NF-
To explore the relative roles of NF-
Antibody p50 was a generous gift of Dr. C.
Rosen (Human Genome Sciences Inc., Rockville, MD) and was characterized
to completely shift both the p50 homodimer and the
NF-
The sequences of phosphorothioate
antisense p50, p65
(30) , and the unrelated antisense
(intercellular adhesion molecule-1)
(31) were
5`-TGGATCATCTTCTGCCATTCT-3`, 5`-GGGGAACAGTTCGTCCATGGC-3` and
5`-CCCCCACCACTTCCCCTCTC-3`, respectively. These sequences have been
used previously to inhibit the translation of the respective mRNAs
(30, 31) .
As expected,
TNF-
We first determined the
concentration of p65 that was required for the maximal activation of
p85VCAMCAT. p85VCAMCAT was transfected into HeLa cells along with
different concentrations (0.1-10 µg) of the p65 expression
vector. Increasing amounts of p65 activated p85VCAMCAT in a
dose-dependent manner
(7) (data not shown). The amount of p65
required for the maximal CAT activity was approximately 5 µg.
Further increase in the amount of expression vector did not
significantly increase or decrease CAT activity. Based on these
studies, the concentration of p65 expression vector used for the
activation of p85VCAMCAT was 5 µg or lower. To generate NF-
The promoters
CAT activities obtained as a result of these mixing reconstitution
experiments were determined and plotted as a function of the ratio of
transfected p50 and p65 subunit expression vectors (Fig. 3 A).
As expected, based on the predicted formation of p50+p65
heterodimers, p65-mediated activation of p(HIV
To confirm that the
To determine whether p65
homodimer was specifically present in TNF-
Similar results were obtained when
Ig/
We investigated the transcriptional regulatory mechanisms
underlying the cell type-specific activation of the VCAM-1 promoter by
the cytokine TNF-
The
inability of NF-
In our in vivo reconstitution experiments, p50
negatively controlled the ability of p65 to transactivate the VCAM-1
promoter through the
By co-expression studies, we have established that the p65 subunit
alone is a potent transactivator of the VCAM-1 promoter. That p65
mediates this effect through a p65 homodimer is suggested by two
experimental results: 1) when the DNA binding domain of p65 was
replaced with the DNA binding domain of p50, the p65-mediated
activation of p85VCAMCAT was either reduced to minimal level or lost
and 2) in vivo reconstitution of the p50+p65 heterodimer
inhibited p65-mediated transactivation of the minimal VCAM-1 and
To our knowledge, there is no other example to date of a naturally
occurring
It
is likely that interactions between the transcriptional factors bound
to the
Characterization of the
The
results of the supershift assay suggested that in HUVE and HeLa cells
TNF-
Our results employing
co-transfection of p50 and p65 expression vectors suggested that the
NF-
In conclusion, we have
utilized both overexpression and targeted inhibition strategies to
develop a model of cell type-specific VCAM-1 promoter transactivation
as a function of the relative level of p50 versus either a p65
homodimer or p65 heterodimer with a protein other than p50. This model
is consistent with the recently proposed model that also suggests that
the expression of distinct NF-
We are indebted to Dr. Margaret Offermann (Winship
Cancer Center, Emory University) for critical reading of the manuscript
and extensive discussions and to Dr. Charles Kunsch (Human Genome
Sciences, Rockville, MD) for his many helpful insights and comments. We
are thankful to Lynn Olliff for technical support in preparing plasmids
and to Kate W. Harris for editing the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
B enhancer motifs,
L-
R (positions -77 and -63). However, cytokine
activation of the dimeric NF-
B transcriptional factor
(p50+p65 subunits) occurs in almost all cell types, whereas VCAM-1
gene expression exhibits a cell type-specific pattern of expression.
Tumor necrosis factor-
markedly transactivated a transiently
transfected minimal
L-
R motif-driven VCAM-1 promoter,
p85VCAMCAT, in passaged human vascular endothelial cells but not in the
human epithelial cell line, HeLa suggesting that cell type-specific
factors may function through the
L-
R motif. Both cell types
exhibited similar inductions of NF-
B DNA binding activity and
transcriptional activity. However, co-transfection of HeLa cells with
p65 and p50 expression vectors demonstrated that the minimal VCAM-1
promoter was effectively transactivated by p65 alone but that
additional co-expression of p50 blocked this activity. Furthermore,
cytokine activation of the minimal VCAM-1 promoter in HeLa cells was
recovered by inhibition of p50 expression using antisense
oligonucleotide. These studies suggest that the NF-
B(p50+p65
heterodimer) does not support transactivation of the VCAM-1 promoter
with the p50 subunit potentially playing a significant inhibitory role
in suppressing cytokine activation of VCAM-1. In addition, p65
associated transcriptional factors other than NF-
B may serve as
positive, cytokine-inducible, cell type-specific regulators of VCAM-1
gene expression.
-1 (VCAM-1)
(
)
is an inducible cell surface protein of vascular
endothelial cells that mediates the adhesion of mononuclear leukocytes
to endothelial cells in response to a wide variety of inflammatory
signals
(1, 2) . Endothelial expression of VCAM-1 is
observed in early atherosclerotic lesions of the vessel wall, as well
as other inflammatory processes, suggesting the importance of VCAM-1 in
these disease states
(3) . However, the molecular mechanisms
regulating VCAM-1 gene expression are not well understood. The human
VCAM-1 promoter contains two closely linked
B-like elements,
L -
R, separated by five nucleotides, at positions -77
and -63 relative to the transcription start site, respectively
(4) .
R but not
L completely conforms to the
B
consensus sequence for the binding of the inducible, transcriptional
regulatory factor NF-
B
(5) . Yet, both of these sequences
are necessary for the induction of VCAM-1 promoter by tumor necrosis
factor
(TNF-
) in endothelial cells
(4, 6, 7) .
B
transcriptional factor in transactivating VCAM-1 in response to
cytokine stimulation of endothelial cells is not fully understood.
NF-
B is a member of the Rel family of transcriptional regulatory
proteins and consists of two distinct polypeptides of 50 and 65 kDa,
termed p50 and p65. In most unstimulated cell types, NF-
B is
complexed with an inhibitory protein, I
B, in a non-DNA-binding
form that is localized to the cytosol
(8) . Stimulation of cells
with cytokines
(9) and several other activators
(5, 10) results in the release of NF-
B from I
B and its
translocation into the nucleus where it transcriptionally regulates the
expression of a wide variety of genes through specific
B enhancer
motifs
(5, 11) . An important feature of the Rel family
is its ability to form a wide range of homodimers ( e.g. p65
and p50) as well as heterodimers not only within the Rel family, but
also with other classes of transcriptional factors
(12, 13, 14) . These complexes exhibit
significant differences in their binding affinity for, and
transactivation through, several
B DNA motifs. Recently, it has
been shown that the p65 subunit of NF-
B can form homodimers that
can also function as transactivators
(15, 16, 17, 18, 19, 20) .
Studies with the p50 subunit have shown that it can also act as a
transactivator in vitro (20, 21) as well as in
yeast
(22) , although this has not yet been observed in
mammalian cells
(16, 17, 19, 23) . Thus,
cell type-specific differences in the activation of this family of
NF-
B-like DNA-binding proteins may play an important role in
regulating VCAM-1 gene expression through its
L-
R motif.
B heterodimer itself may mediate transactivation
through the VCAM-1
L-
R elements. First, the
L-
R
elements bind at least two different NF-
B-like binding proteins
(4) . Second, TNF-
transactivates the VCAM-1 promoter
through the
L-
R elements in a cell type-specific manner.
Non-endothelial cell lines such as Jurkat and HeLa activate NF-
B
when stimulated with cytokine TNF-
as well as other activators
(5, 10) . However, although active in
TNF-
-stimulated human vascular endothelial (HUVE) cells,
constructs of the VCAM-1 promoter containing
L-
R are not
transactivated in the TNF-
-stimulated T-cell line Jurkat
(4) . This raises the possibility that instead of NF-
B,
other homo- or heterodimers of members of NF-
B/Rel family may be
involved in activating the VCAM-1 promoter in TNF-
-stimulated HUVE
cells.
B and NF-
B-like
factors in regulating VCAM-1 gene transcription through the
L-
R motif, we have characterized the ability of these factors
to mediate transcription of the VCAM-1 promoter through cytokine
activation of endogenous NF-
B as well as to reconstituted homo-
and heterodimers of p50 and p65 using expression vectors in HeLa cells.
Our studies suggest that while Rel family members, such as the p65
homodimer, are potent transactivators, the NF-
B (p50+p65
heterodimer) does not effectively transactivate the VCAM-1 promoter
through the
L-
R motif. DNA binding studies suggest that
TNF-
induces NF-
B (p50+p65 heterodimer) and p65
homodimer or homodimer-like protein both in HUVE and HeLa cells.
However, functional p65 homodimer was mainly present in HUVE cells.
Thus, VCAM-1 transactivation through
L-
R may be a function of
differential activation of NF-
B (p50+p65 heterodimer)
relative to other members of the Rel family, such as p65 homodimers.
Cell Culture
HeLa cells (CCL2) were obtained
from American Type Culture Collection (Rockville, MD) and maintained in
minimal essential medium supplemented with 10% fetal bovine serum
(Irvine Scientific, Santa Ana, CA). HUVE cells were purchased from
Clonetics (San Diego, CA) and were cultured in M199 medium supplemented
with 20% fetal bovine serum, 16 units/ml heparin (ESI Pharmaceuticals,
Cherry Hill, NJ), 50 µg/ml endothelial cell growth supplement
(Collaborative Research Incorporated, Bedford, MA), and 25 m
M HEPES buffer. The medium for both cells contains 2 m
M
L-glutamine, 100 units/ml penicillin and 100 µg/ml
streptomycin. HUVE cells were grown on tissue culture plates coated
with 0.1% gelatin and were used within the first 6 passages. Human
recombinant TNF- was obtained from Boehringer Mannheim. All other
reagents were of reagent grade.
CAT Assay
One day prior to transfection, HeLa and
HUVE cells were split at the ratio that would give 60-70%
confluence. The transfection was done by the calcium phosphate
co-precipitation technique. For HeLa cells, 5-10 µg of
reporter plasmids were used. For HUVE cells, 30 µg of reporter
plasmids were transfected as described previously
(24) . The
promoterless plasmid, poLUC
(25) , was used for adjusting the
amount of transfected DNA. The cells were harvested and cell extracts
were prepared by three cycles of rapid freeze-thaw in 0.25
M Tris, pH 8.0. Protein content was determined using the Bradford
(26) technique. The same amounts of proteins were assayed for
CAT activity according to standard protocols
(27) . The CAT
activity was expressed as percent of chloramphenicol converted to
acetyl chloramphenicol. Acetylated and unacetylated forms of
chloramphenicol were separated on thin layer chromatography and their
amounts were determined after scraping by counting the radioactivity of
the respective bands in scintillation vials. Each assay was performed
in duplicate or triplicate and the results reported are the average of
at least two separate experiments.
Expression Vectors and CAT Reporter Genes
The
eukaryotic expression vectors CMV-p50, CMV-p65, CMV-p50/65, and
CMV-rel/p65 contain the respective cDNAs cloned between a
cytomegalovirus (CMV) promoter, -globin intron and simian virus 40
poly(A) signal
(17) . CMV-p50/65 encodes a chimeric protein
consisting of the DNA binding domain of p50 (amino acids 1-370)
and the transactivation domain of p65 (amino acids 309-550)
(28) . The reporter plasmid, p(HIV
B)
CAT,
contains four tandem copies of the
B DNA sequences cloned upstream
of the human immunodeficiency virus type-1 (HIV-1) long terminal repeat
and fused to the coding region of the bacterial CAT gene
(28) .
The sequence of the
B motif of p(HIV
B)
CAT is
5`-GGGGACTTTCC-3`, and is identical to the
B sequence found in the
mouse immunoglobulin gene promoter (Ig
B). These vectors were
generous gifts of Dr. C. Rosen (Human Genome Sciences, Rockville, MD).
The reporter gene, p85VCAMCAT, contains coordinates -85 to
+12 of the human VCAM-1 promoter
(4) . The
L-
R
driven heterologous promoter pTA(-77/-63)CAT has been
described previously
(4) . These reporter genes were generous
gifts of Dr. D. Dean (Washington University, St. Louis, MO).
Nuclear Extracts Preparation
Confluent HUVE and
HeLa cells were exposed to TNF- (100 units/ml) for 1-3 h.
Nuclear extracts were prepared by a modification of the method of
Dignam et al. (29) . Briefly, after washing with
phosphate buffered saline, cells were centrifuged and the cell pellet
suspended in 500 µl of buffer A (10 m
M HEPES, pH 7.9, 1.5
m
M MgCl
, 10 m
M KCl, and 1.0 m
M dithiothreitol). After recentrifugation, the cells were
resuspended in 80 µl buffer A containing 0.1% Triton X-100 by
gentle pipetting up and down. After incubating for 10 min at 4 °C,
the homogenate was centrifuged and the nuclear pellet was washed once
with buffer A and resuspended in 70 µl of buffer C (20 m
M HEPES, pH 7.9, 25% (v/v) glycerol, 0.42
M NaCl, 1.5
m
M MgCl
, 0.2 m
M EDTA, 1 m
M dithiothreitol). This suspension was incubated for 30 min at 4
°C followed by centrifugation at 20,000
g for 10
min. The resulting supernatant (nuclear extract) was stored at
-70 °C. Protein concentrations were determined by the
Bradford
(26) method. To minimize proteolysis, all buffers
contained 1.0 m
M phenylmethylsulfonyl fluoride, aprotinin (10
µg/ml), leupeptin (10 µg/ml), and antipain (10 µg/ml).
Gel Shift Assays
The oligonucleotide containing
L-
R of the VCAM-1 promoter (VCAM-1 wild type oligo) was
synthesized. Its sequence is as follows:
5`CTGCCCTGGGTTTCCCCTTGAAGGGATT-TCCCTCCGCCTCTGCAACAAGCTCGAGATCCTATG-3`.
The sequences of
L and
R are underlined with a single line.
The double underlined sequences represent an unrelated tail sequence
added to serve as a template for synthesis of the double-stranded DNA.
To prepare double-stranded DNA, first an oligonucleotide
5`-CATAGGATCTCGAGC-3` (complementary to the 3`-unrelated tail, double
underlined sequence) was annealed to VCAM-1 wild type oligo. The second
strand was extended with DNA polymerase (Klenow fragment) in a reaction
mixture containing 50 µCi of [
P]dCTP and 0.5
m
M of cold dATP, dGTP, and dTTP. The reaction was followed by
the addition of 0.5 m
M cold dCTP to insure completion of the
second strand. Unincorporated nucleotides were removed by column
chromatography over a Sephadex G-50 column. The DNA binding reaction
was performed at 30 °C for 15 min in a volume of 20 µl, which
contained 225 µg/ml bovine serum albumin, 1.0
10
cpm of
P-labeled probe, 0.1 µg/ml poly(dI-dC),
and 15 µl of binding buffer (12 m
M HEPES pH 7.9, 4 m
M Tris, 60 m
M KCl, 1 m
M EDTA, 12% glycerol, 1
m
M dithiothreitol, and 1 m
M phenylmethylsulfonyl
fluoride). After the binding reaction, the samples were subjected to
electrophoresis in 1
Tris-glycine buffer using 4% native
polyacrylamide gels.
B(p50+p65 heterodimer) in gel shift assays (data not
shown). Antibodies against p65, c-Rel (raised against a peptide in the
carboxyl terminus region), and RelB were purchased from Santa Cruz
Biotechnology, Inc., Santa Cruz, CA. Bacterially expressed proteins,
p50 and truncated p65 (containing amino acids 1-309), were
prepared and purified as described previously
(17) . Due to the
low amounts of available purified proteins, the concentration of the
purified proteins was roughly estimated from the concentration of
proteins before dialysis, the final step of purification. The homo- and
heterodimers of p50 and truncated p65 were prepared by incubating them
alone or in combination with each other for one hour at 37 °C as
described earlier
(20) except that DNA binding buffer was used
for dimerization. These dimers were used for DNA binding studies. The
purified proteins were generous gifts of Dr. C. Rosen (Human Genome
Sciences Inc., Rockville, MD).
TNF-
To explore the mechanisms underlying cell type-specific
expression of VCAM-1, we chose to compare the activation of the VCAM-1
promoter in passaged HUVE cells that markedly induce VCAM-1 expression
in response to cytokines
(4) with the epithelial cell line,
HeLa, that does not express VCAM-1 at either the mRNA or protein levels
(data not shown). This lack of VCAM-1 expression in HeLa cells occurs
despite its well described NF- Activates NF-
B-mediated Transcription in
Both HUVE and HeLa Cells but Induces the VCAM-1 Promoter Only in HUVE
Cells
B activation in response to several
cytokines and to the phorbol ester phorbol 12-myristate 13-acetate
(32, 33, 34, 35) . HeLa and HUVE cells
were transfected with p85VCAMCAT, a deletion construct of the human
VCAM-1 promoter (coordinates -85 to +12) that contains the
L-
R elements and has been previously characterized as the
minimal TNF-
-inducible VCAM-1 promoter
(4) . To assess for
functional NF-
B, both cell types were also transfected in parallel
with p(HIV
B)
CAT, an NF-
B-responsive promoter
construct containing a tetramer of canonical
B elements,
p(HIV
B)
CAT, well characterized to be activated by
NF-
B(p50+p65 heterodimers)
(28) .
markedly induced p(HIV
B)
CAT in both HUVE
(Fig. 1 B, lanes 1 and 2) and HeLa
cells (Fig. 1 A, lanes 1 and 2),
demonstrating functional activation of NF-
B. TNF-
also
markedly induced p85VCAMCAT activity in HUVE cells
(Fig. 1 B, lanes 3 and 4). In contrast,
TNF-
caused little or no activation of p85VCAMCAT (Fig.
1 A, lanes 3 and 4) in HeLa cells. Similar
results were obtained when the TNF-
concentration was increased up
to 1000 units/ml (data not shown). Similar to studies in Jurkat T cells
(4) , these results suggest that activated NF-
B in
TNF-
-stimulated HeLa cells is not sufficient to effectively
transactivate the minimal inducible VCAM-1 promoter, containing the
L-
R elements.
Figure 1:
TNF- is unable to induce
p85VCAMCAT in HeLa cells. A, HeLa cells were transfected with
10 µg of p85VCAMCAT or p(HIV
B)
CAT as described
under ``Materials and Methods.'' After overnight
transfection, the cells were stimulated with TNF-
(200 units/ml)
for 16-24 h, and CAT activity was determined. B, HUVE
cells were transfected with 30 µg of p85VCAMCAT or
p(HIV
B)
CAT. After transfection the cells were
stimulated with TNF-
(200 units/ml) for 16-24 h, and CAT
activity was determined.
Co-expression of the p65 Subunit of NF-
As an
endogenous activation of NF-B Potently
Transactivates the VCAM-1 Promoter in HeLa Cells
B in HeLa cells appeared not to be
effective, we undertook to determine which members of the NF-
B/Rel
family could transactivate the VCAM-1 promoter through the
L-
R elements. In functional reconstitution experiments
Rel-like subunits were co-transfected into HeLa cells with p85VCAMCAT
or p(HIV
B)
CAT (a p65 homodimer and NF-
B
(p50+p65 heterodimer) driven reporter gene) as a control. These
included expression vectors for the p50 and p65 subunits of NF-
B
as well as for the chimeric protein, p50/65, that contains the DNA
binding domain of p50 and transactivation domain of p65 and is a
functional mimic of NF-
B (p50+p65 heterodimer)
(28) .
As shown in Fig. 2 B, co-expression of the p65 subunit
markedly transactivated p85VCAMCAT
(7) . In contrast,
co-expression of the chimeric protein (p50/65) failed to significantly
induce p85VCAMCAT. Similarly, p50 did not induce p85VCAMCAT. However,
both p65 and the chimeric protein p50/65 potently induced
p(HIV
B)
CAT (Fig. 2 A)
(28) . p50
did not induce p(HIV
B)
CAT. These data suggest that
expression of p65 either as a homodimer or in combination with a
component distinct from p50 already present in HeLa cells is sufficient
to transactivate the minimal inducible VCAM-1 promoter containing the
L-
R elements.
Figure 2:
The p65 subunit of NF-B is a potent
activator of the VCAM-1 promoter. HeLa cells were transfected with 10
µg of p85VCAMCAT ( Panel B) or p(HIV
B)
CAT
( Panel A) along with 5 µg of the expression vectors
encoding p65 and p50, and the chimeric protein p50/65 as described
under ``Materials and Methods.'' The CAT activity was
determined using equal amount of protein. The CAT activity of
p85VCAMCAT ( Panel B) and p(HIV
B)
CAT
( Panel A) under different conditions is expressed as
percentage of acetylated chloramphenicol.
Reconstitution of a Functional NF-
To further elucidate the relative roles of p65 and
the NF-B (p50+p65
Heterodimer) in Vivo Enhances p(HIV
B)
CAT but Blocks
p85VCAMCAT
B (p50+p65 heterodimer) on transactivation of the
VCAM-1 promoter, functional NF-
B (p50+p65 heterodimer) was
reconstituted in HeLa cells by co-transfection of the expression
vectors encoding p50 and p65. Recent studies have shown that the
NF-
B(p50+p65 heterodimer) can be efficiently formed when
expression vectors of both p50 and p65 are co-transfected in different
cell lines. This has been shown by gel mobility shift assays and by
increases in the transcriptional activation of
p(HIV
B)
CAT reporter genes
(16, 17, 18, 19) . Studies in vitro have also suggested that the p50+p65 heterodimer is
preferentially formed when purified proteins of p50 and p65 are
incubated together
(20) .
B
(p50+p65 heterodimer), HeLa cells were transfected with a constant
amount of p65 and increasing amounts of p50 expression vectors. The
effect of the co-expressed p50+p65 heterodimers on promoter
function was assessed in p85VCAMCAT. The functional formation of the
p50+p65 heterodimer and its ability to drive a canonical
B
motif was assessed using p(HIV
B)
CAT.
B)
CAT
was significantly enhanced using a p50:p65 transfection ratio of 0.25
to 0.5. Further increase in the amount of transfected p50 inhibited
activation of p(HIV
B)
CAT, perhaps by the formation of
p50 homodimers. In dramatic contrast, under similar conditions,
p65-mediated activation of p85VCAMCAT was almost completely inhibited
by the presence of any p50, with inhibition occurring in response to a
p50:p65 transfection ratio as low as 0.2. p(HIV
B)
CAT
was activated at the same ratio. This suggests that the formation of
NF-
B(p50+p65 heterodimers) blocked the p85VCAMCAT activation
mediated by p65. These results suggest that under conditions that
transactivate a canonical NF-
B driven promoter, reconstitution of
a functional NF-
B heterodimer inhibits p65-mediated
transactivation of the VCAM-1 promoter.
L-
R elements were the sequences in the VCAM-1 promoter that
were responsible for the observed responses to transfected p65 and p50,
the
L-
R driven heterologous promoter CAT construct,
pTA(-77/-63)CAT, was used in co-transfection studies.
pTA(-77/-63)CAT contains the
L-
R enhancer
elements driving an SV40 promoter and is transactivated by TNF-
in
HUVE cells
(4) . HeLa cells were transfected with 10 µg of
pTA(-77/-63)CAT and cotransfected with p65 and p50. As
expected, transfection of p65 alone (5 µg) resulted in 30-fold
induction of pTA(-77/-63)CAT above the low background
(Fig. 3 B, left panel). Co-expression of p50 and
p65 at the ratio of 0.5 resulted in almost complete inhibition of
p65-mediated transactivation of pTA(-77/-63)CAT
(Fig. 3 B, left panel), whereas
p(HIV
B)
CAT activity was enhanced
(Fig. 3 B, right panel). This suggests that
reconstituted functional NF-
B (p50+p65 heterodimer) blocks
the p65-mediated transactivation through the
L-
R elements of
both the heterologous and homologous VCAM-1 promoters.
Figure 3:
The concentrations of p50 which stimulate
the p65-mediated activation of p(HIVB)
CAT inhibit the
p65-mediated activation of p85VCAMCAT. A, relative CAT
activity of p(HIV
B)
CAT and p85VCAMCAT at different
ratios of co-transfected p50 and p65. HeLa cells were transfected with
10 µg of p(HIV
B)
CAT or p85VCAMCAT along with a
constant amount of p65 and increasing amounts of p50. To control
transfection efficiencies, the total amount of transfected DNA was kept
constant by using poLUC, a promoterless plasmid. The CAT assays were
performed by using the same amount of protein. The percentage of the
C-labeled chloramphenicol converted to its acetylated form
was determined and plotted against the ratio of co-transfected p50 and
p65 (p65 activation of the reporter genes = 100%). The
transactivation of p(HIV
B)
CAT was determined by using
2 µg of p65 and 0, 0.5, 1.0, 2.0, and 5 µg of p50,
respectively. The transactivation of p85VCAMCAT was determined by using
5 µg of p65 and 0, 1.0, 5.0, and 10 µg of p50, respectively.
B, the concentrations of p50 which stimulate the p65-mediated
activation of p(HIV
B)
CAT inhibit the p65-mediated
activation of pTA77/63CAT. HeLa cells were transfected with 10 µg
of pTA77/63CAT ( left panel) or p(HIV
B)
CAT
( right panel) along with p65 and/or p50. To control
transfection efficiencies, the total amount of transfected DNA was kept
constant by using poLUC, a promoterless plasmid. The CAT assays were
performed by using the same amount of protein. Columns 1 and
4 show the basal levels of the respective CAT reporter gene.
Columns 2 and 5 show the CAT activity mediated by p65
(5 µg). Columns 3 and 6 show the CAT activity
mediated by the combination of p65 (5 µg) and p50 (1.0
µg).
To determine
which part of the p50 protein was responsible for the negative effect
on the transactivation potential of p65 within the NF-B(p50/p65
heterodimer) bound to
L-
R, functional reconstitution
experiments were performed using expression vectors of p65 and chimeric
protein p50/65. HeLa cells were transfected with p85VCAMCAT along with
expression vectors encoding p65 and/or chimeric protein p50/65. As
expected, p65 (5 µg) potently induced the CAT activity of
p85VCAMCAT (data not shown). Co-expression of 1 µg of p50/65 did
not affect the transactivation potential of p65 (5 µg). However,
the co-expression of 5 µg of p50/65 enhanced nearly 2-fold the
p65(5 µg)-stimulated CAT activity of p85VCAMCAT(data not shown).
These results suggest that by replacing the carboxyl terminus of p50
with the activation domain of p65, the inhibitory effect of p50 on the
transactivation potential of p65 bound to
L-
R is lost.
p50 and p65 Homodimers and the NF-
To establish
whether p50 and p65 homo- and heterodimers could directly bind to the
VCAM-1 B Heterodimer
Directly Bind to the
L-
R Motif in Vitro
L-
R motif, homo- and heterodimers were prepared by
incubating purified recombinant p50 and p65 proteins, either alone or
mixed together, for one h at 37 °C
(20) . Gel mobility shift
assays were performed using
P-labeled double stranded DNA
containing
L-
R elements (VCAM wild type probe) (see
``Matrials and Methods''). p65 is a truncated protein that
contains amino acids 1-309, and is capable of both dimerization
(17) and of binding to DNA
(15, 36, 37) .
As shown in Fig. 4( lanes 1-5), p65 homodimers
were able to bind to the VCAM wild type probe in a dose dependent
manner. However, the amount of p65 that bound was substantially less
than the amount of p50+p65 heterodimer or p50 homodimer that
bound. The p50+p65 heterodimer that was generated by incubating a
constant amount of p65 (400 pg) with increasing amounts of p50
(40-800 pg) also showed binding to the VCAM wild type probe. As
the concentration of p50 exceeded that of p65, a second band appeared
in the gel shift assay that corresponded to p50 homodimers ( lane
10). As p50 homodimers appeared, there was no decrease in the
intensity of p50+p65 heterodimer binding. The complex of p50
homodimer with VCAM wild type probe is shown in lane 11. The
mobility of the complex of the p50+p65 heterodimer was
intermediate between the complexes of p50 and p65 homodimers, the p65
homodimer complex migrated fastest. These mobilities are consistent
with the mobilities of the complexes of homo- and heterodimers of p50
and p65 (1-309) with oligonucleotide containing canonical
B
motif
(17, 28) . Interestingly, two bands, which
corresponded to the p50 homodimer and the p50+p65 heterodimer,
co-existed in lane 10. At high concentrations of p50 and p65,
small amounts of complexes with mobility lower than that of the p50
homodimer were also detectable ( lanes 6-10). These
studies demonstrate that the homo- and heterodimers of p50 and p65 all
bind to
L-
R, although the levels of p65 homodimer binding are
lower than the levels of p50+p65 heterodimer or p50 homodimer
binding.
Figure 4:
The
homo- and heterodimer of purified recombinant p50 and p65 proteins bind
to VCAM-1 wild type probe containing L-
R. The homo- and
heterodimers of p50 and p65 were prepared by incubating the purified
p50 or p65 proteins alone or in combination with each other for one
hour at 37 °C and their binding to
L-
R was monitored by
using
P-labeled VCAM-1 wild type probe in gel shift
assays. Lanes 1, 2, 3, 4, and 5 show the binding of 40, 100, 200, 400, and 800 pg of p65 to the
VCAM wild type probe, respectively. Lanes 6, 7,
8, 9, and 10 had a constant amount (400 pg)
of p65 and increasing amounts of p50 (40, 100, 200, 400, and 800 pg),
respectively. Lane 11 had 400 pg of p50. The complexes of
homo- and heterodimers of p50 and p65 with VCAM wild type probe are
marked on the right side of the figure. A shorter exposure of
lanes 9, 10, and 11 is shown in a box on the right side.
TNF-
The
expression of NF- Induces p65-like Homodimers and NF-
B
(p50+p65 Heterodimers) in HUVE and HeLa Cells
B/Rel proteins was determined in both HeLa and
HUVE cells by gel mobility shift assay. As shown in Fig. 5 A ( lanes 1 and 3), the nuclear extract of
uninduced HUVE cells has no basal level of
L-
R-binding
nuclear proteins compared with that of the uninduced HeLa cells.
TNF-
induces
L-
R-binding nuclear proteins (DNA-protein
complex A1) in HUVE and in Hela cells ( lanes 2 and
4). However, the concentration of A1 in TNF-
-activated
HUVE cells was less than half that of HeLa cells ( lanes 2 and
4). The other two complexes, A2 and A3, in TNF-
-activated
HUVE ( lane 2) and HeLa cells ( lane 4) were minor
compared with the complex A1. These results suggest that the major
protein component of the complex A1 may not be the transactivator
protein, since it is induced in large amounts in TNF-
-activated
HeLa cells where the VCAM-1 promoter and gene is not induced.
Figure 5:
TNF- induces homo-and heterodimers of
p50 and p65 in HUVE and HeLa cells and relatively higher concentrations
of NF-
B/rel proteins in HeLa cells. A,
L-
R
binding nuclear protiens of TNF-
-activated HeLa and HUVE cells
were compared using equal amounts of nuclear extract (2 µg of
protein) ( lanes 1-4).
L-
R binding nuclear
proteins of TNF-
-activated HUVE cells ( lane 5). Super
shift with antibodies against p50, p65, c-Rel(c), and RelB is shown in
lanes 6-9, respectively. Specific complexes A1, A2, and
A3, and nonspecific ( NS) and free probes are marked.
L-
R-binding nuclear proteins of TNF-
activated HeLa
cells ( lane 10). Super shift with antibodies p50, p65,
c-rel(c) (raised against a peptide in the carboxyl terminus of c-Rel),
and RelB is shown in lanes 11 to 14, respectively.
Specific complexes A1 and A2, NS, and free probe are marked.
B, Ig/
B binding nuclear proteins of TNF-
-treated
HUVE and HeLa cells. Labeled Ig/
B probe without nuclear proteins
( lane 1). Lanes 2-6 and 7-11 contain HUVE and HeLa cell nuclear extract (2 µg each lane),
respectively. The volume of each antibody used was 1 µl.
TNF-
-activated Ig/
B binding nuclear proteins of HUVE
( lane 2) and HeLa ( lane 7) cells and their super
shift with antibodies against p50, p65, and p49 are
shown.
To
further characterize the proteins in complexes A1-A3, supershift
assays were performed using antibodies of NF-B/Rel proteins. The
antibodies against p50 (Abp50) and p65 (Abp65) recognized their
epitopes in NF-
B (p50+p65 heterodimer) as well as in the
homodimers of their respective subunits (data not shown). The binding
of Abp65 to the NF-
B (p50+p65 heterodimer) resulted in a
decrease in the binding of the NF-
B to
L-
R. As shown in
Fig. 5A ( lane 5), TNF-
-activated HUVE
cells contain the major complex, A1, and the minor complexes, A2 and
A3. The complex A1 is shifted by both antibodies against p50 and p65
suggesting that the major part of the complex A1 is NF-
B
(p50+p65 heterodimer) ( lanes 6 and 7). Because
Abp50 could not completely shift complex A1, this suggested that
complex A1 also contains p65 homodimer or a heterodimer of p65 with a
protein other than p50. The TNF-
-activated HUVE cells did not
contain any
L-
R-binding nuclear c-Rel or RelB proteins
( lanes 8 and 9).
-activated HUVE cells,
the
L-
R binding nuclear proteins of TNF-
-activated HeLa
cells were also characterized using antibodies of NF-
B/Rel
proteins. As shown in Fig. 5 A, lane 10,
TNF-
induces a major complex, A1, and a minor complex, A2. All of
the complex A1 was shifted by antibody Abp65, and the major part of it
by Abp50, suggesting that the complex A1 was mixture of NF-
B (p50
+ p65 heterodimer) and p65 homodimer or p65 heterodimer with a
protein other than p50 ( lanes 11 and 12). The nuclear
extract of HeLa cells did not contain any detectable amount of c-Rel or
RelB ( lanes 13 and 14). Although TNF-
induces
homo- and heterodimers of p50 and p65 in both HUVE and HeLa cells, it
induces lower concentrations of NF-
B (p50+p65 heterodimer) in
HUVE compared with HeLa cells.
B was used as a probe, instead of
L-
R. As shown in
Fig. 5B, Ig/
B made a major complex A1 that had
intensity approximately 2-fold higher in HeLa ( lane 7)
compared with HUVE ( lane 2) cells. The complex A1 supershifted
completely when the antibodies Abp50 and Abp65 were used together
( lanes 5 and 10), and it disappeared instead of
supershifting when Abp65 was used alone, likely due to loss in the DNA
binding ability of the NF-
B proteins ( lanes 4 and
9). The antibody Abp50 also shifted the major part of complex
A1 ( lanes 3 and 8). However, Abp50 was unable to
shift a small part of the complex A1. The antibody Abp49 did not shift
any part of the complex A1 ( lanes 6 and 11). These
results also suggest that the complex A1 consists of p50+p65
heterodimers and p65 homodimers or p65 heterodimers with a protein
other than p50 and, also, that the major part of the complex A1 is the
p50+p65 heterodimer.
TNF-
To assess the role of
endogenous p50 in the TNF- Can Activate p85VCAMCAT in HeLa Cells when
Co-transfected with Antisense p50
-mediated regulation of p85VCAMCAT, HeLa
cells were co-transfected with p85VCAMCAT and phosphorothioate
antisense oligonucleotides of p50 or p65 or an unrelated antisense
(intercellular adhesion molecule-1) and treated with TNF-
. After
16-24 h the cells were assayed for CAT activity. As expected,
p85VCAMCAT had a low level of activity and TNF-
was unable to
effectively induce the CAT activity (Fig. 6). However, in HeLa
cells co-transfected with antisense p50, TNF-
was able to induce
p85VCAMCAT severalfold above the low level of background. The
co-transfection of the p65 or the unrelated antisense did not improve
the ability of TNF-
to induce p85VCAMCAT. These results suggest
that in TNF-
-treated HeLa cells, endogenous p50 inhibited the
ability of TNF-
to induce p85VCAMCAT.
Figure 6:
Co-transfected antisense p50 renders
p85VCAMCAT inducible by TNF- in HeLa cells. HeLa cells
(30-40% confluent) were transfected with 10 µg of p85VCAMCAT
and 100 n
M of phosphorothioate antisense p50 or unrelated
antisense (intercellular adhesion molecule-1) or p65 using calcium
phosphate precipitation method. The cells were also transfected only
with p85VCAMCAT. After overnight transfection, the cells were
maintained in fresh medium for another 24 h. The cells were then
treated with TNF-
and harvested for CAT assays 16-24 h
later. Equal amounts of protein (50 µg) were used in each CAT
assay. The CAT activity is presented as percentage of chloramphenicol
converted to acetyl chloramphenicol. These results are average of three
independent experiments each performed in
duplicate.
. Prior studies established that TNF-
induces the VCAM-1 promoter in HUVE cells but not Jurkat T cells
through two closely linked
B-like elements,
L -
R,
located at positions -77 and -63 relative to the
transcriptional start site, respectively
(4) . We confirmed
these findings in HUVE cells and also established that the VCAM-1
promoter is not transactivated by TNF-
in another non-endothelial
cell type, HeLa. Our data strongly suggest that, while TNF-
markedly activates NF-
B in both cell types
(4, 5, 6, 10, 38, 39) ,
NF-
B itself does not effectively transactivate the VCAM-1 promoter
through these
L-
R elements. Importantly, the p50 subunit
itself may function as a negative transcriptional regulator of this
promoter. Thus, cytokine inducible transactivation of the VCAM-1
promoter, and by extrapolation the VCAM-1 gene, may be a function of
negative (p50 associated) and positive (p65 associated) transcriptional
factors that are activated in a cell type-specific manner.
B to effectively transactivate the VCAM-1 promoter
through the
L-
R elements was established by three functional
experimental observations: 1) activation of endogenous NF-
B in
HeLa cells by TNF-
markedly induced a transiently transfected,
NF-
B driven reporter gene, p(HIV
B)
CAT, but failed
to activate p85VCAMCAT, a minimal,
L -
R containing the VCAM-1
promoter construct, that was induced by TNF-
in HUVE cells; 2)
co-expression of a p50/65 chimeric protein, that has the p50 DNA
binding and p65 transactivation properties of NF-
B, failed to
effectively transactivate p85VCAMCAT in HeLa cells; and 3)
transactivation of p85VCAMCAT in HeLa cells was dramatically inhibited
under conditions of co-expression of p50 and p65, and hence NF-
B
(p50+p65 heterodimer) formation, whereas this coexpression
markedly transactivated an NF-
B driven p(HIV
B)
CAT
reporter gene. In this functional context, we also established that the
VCAM-1
L-
R elements could bind the homodimers of p50 and p65
as well as NF-
B prepared by heterodimerization of purified,
recombinant p50 and p65 proteins. Taken together, these findings
suggest that the binding of NF-
B in vivo to
L-
R
is not sufficient for the activation of p85VCAMCAT. Although the
mechanism mediating this effect is not fully understood, a specific
conformation of NF-
B after binding to the motif may be necessary.
Such a hypothesis was proposed for the in vitro transcriptional activation by p50 of the CAT reporter gene
containing the
B motifs of H-2K gene
(20) . In this study,
binding of p50 to a
B motif in a chymotrypsin-resistant
conformation correlated with its ability to transactivate. Thus, the
different conformations of NF-
B bound to different
B motifs
determine NF-
B's functional role as a transcription
activator.
L-
R elements. This is suggested by the
activation of p85VCAMCAT and
L-
R-driven heterologous
promoter, pTA(-77/-63)CAT, by p65 but not NF-
B. In
contrast, p50 does not inhibit the transactivation potential of p65
within the canonical Ig
B element until excess p50 is expressed
(16, 17, 18, 19) . Indeed, it has been
suggested that within NF-
B, the function of p50 is to improve the
DNA binding of p65, which is otherwise a weak binder
(40) .
Consequently, p65 can act as a more effective transactivator within
NF-
B. Furthermore, the transactivating activity of NF-
B
through various
B motifs led to a model in which the
transactivation domain of p65 is always exposed and available for
transactivation, either as a homodimer or as the p50+p65
heterodimer and regardless of the
B motif to which it is bound
(20) . Our results suggest that within
L-
R-bound
NF-
B, p50 does not function as a helper subunit but may actively
inhibit the transactivation potential of p65. The enhancement by
co-expressed chimeric p50/65 of p65-mediated transactivation of
85VCAMCAT (data not shown) suggests that a carboxyl terminus domain of
p50 was responsible for inhibiting the transactivation potential of p65
within NF-
B bound to
L-
R. This would suggest that the
transactivation domain of p65 in NF-
B can either be sequestered or
exposed, depending on the
B motif to which NF-
B is bound.
L-
R driven heterologous promoters. These results suggest that
for transactivation through
L-
R, both the p65 transactivation
domain and the p65 DNA binding domain were necessary. Although our
studies suggest that differential transactivation of
L-
R is
mediated through direct interactions with the p65 homodimer, they do
not rule out the possibility that p65 mediates transactivation through
an indirect mechanism. This might include the complexing of p65 with an
endogenous rel-like or other transactivating factor expressed in HeLa
cells distinct from p50 or c-Rel
(41) . Finally, co-expression
of transcription factors could modulate the de novo expression
of other transcriptional factors during the course of the transient DNA
transfection. Only through defined, reconstituted in vitro transcriptional assays will these issues be definitively resolved.
B motif that binds to both the homo- and heterodimers of
p50 and p65 and is specifically activated by p65 homodimer and not by
NF-
B. Synthetic and natural
B-like motifs conferring
differential transactivation have been identified
(28, 42) . In vitro synthesized
B motifs
were isolated, using p65 as a binding protein, from a randomly prepared
pool of
B-like DNA sequences using polymerase chain reaction
(28) . Several of these sequences, such as 65-2, specifically
bind p65 homodimer but not NF-
B (p50+p65 heterodimer) in
vitro. Consistent with this pattern of DNA binding, this enhancer
element confers p65, but not NF-
B, transactivation to a
heterologous promoter. However, this differs significantly from
transactivation mediated through
L-
R. Although a detailed
kinetic and affinity study was not performed, our in vitro binding assays demonstrated that the
L-
R motif bound
both p65 homodimers as well as the NF-
B (p50+p65
heterodimer). This suggests that the selective activation mechanism
found in
L-
R is different than that found in the selective
p65-specific
B motifs that have been described
(28) .
L and
R elements define the selective transactivation
response to p65 and NF-
B. The nature of these interactions is
suggested by a comparison of these
B-like motifs with other
natural and synthetic
B elements. Some of the synthetic p65
selected sequences
(28) are either identical or similar to
L or
R. Structurally,
R is identical to 65-4
(28) and the
B motif of the promoter of the plasminogen
activator gene
(43) . This suggests that
R should function
as a canonical
B enhancer element. In contrast,
L is not
identical to any known canonical
B sequence and thus may represent
a new class of
B binding motifs. Indeed, the
R and
L
elements each exhibited distinct protein binding activities, suggesting
that the
L-
R bound protein complex consists of multiple, and
likely interacting, transactivation factors
(4) . Definition of
the mechanism by which
L-
R confers specificity on
L-
R-driven promoters requires further understanding of
binding and functional properties of the individual
B elements and
is under investigation. Nevertheless, the activation of
L-
R
by p65 but not NF-
B suggests that the induction of the VCAM-1
promoter by TNF-
in endothelial cells is not mediated through
NF-
B.
L-
R binding nuclear
proteins of TNF-
-activated HUVE and Hela cells also suggests that
NF-
B is not a transactivator of the VCAM-1 promoter. Instead, high
concentrations of NF-
B can be correlated with suppression of the
VCAM-1 promoter. In our studies, although TNF-
induced NF-
B
in HeLa cells approximately 2-fold more than in HUVE cells, it was
unable to induce the VCAM-1 gene in HeLa cells. Along with NF-
B,
TNF-
also induced p65 both in HeLa and HUVE cells. From these
studies we conclude that both p65 and NF-
B play an important role
in the regulation of the VCAM-1 gene. Although p65 can specifically
transactivate the VCAM-1 promoter, changes in the concentration of
NF-
B can modify the transactivation mediated by p65.
activated both homo- and heterodimers of p50 and p65,
although the concentration of
L-
R binding NF-
B/Rel
proteins was higher in HeLa cells. The major part of the NF-
B/Rel
proteins was the p50+p65 heterodimer. These results also suggest
that the lower expression of p50 compared with p65 gene in HUVE cells
is one of the mechanism by which TNF-
can induce VCAM-1 in this
cell type. Whether this is a general characteristic of other
endothelial cells needs to be determined.
B heterodimer (p50+p65) does not transactivate the minimal
VCAM-1 promoter p85VCAMCAT. By extension, these results suggest that
p50 may also function as a negative regulator of VCAM-1 promoter
activity in HeLa cells. A similar role of p49, the processed product of
p100, similar to p50, has been suggested in the regulation of IL-6
promoter
(44) . To assess this proposed functional role of the
endogenous p50 subunit, we used an antisense oligonucleotide directed
against p50 (ASp50). In HeLa cells, we demonstrated that despite marked
activation of NF-
B-mediated transcriptional activity, the minimal
VCAM-1 promoter p85VCAMCAT was not significantly induced by the
cytokine TNF-
. Strikingly, co-transfection of ASp50, but not an
unrelated antisense or p65 oligonucleotides, conferred cytokine
inducibility to the VCAM-1 promoter. This suggests that the cell
type-specific pattern of cytokine activation of the VCAM-1 promoter,
and by extrapolation the VCAM-1 gene, may be regulated through a p50
subunit dependent mechanism. While it is tempting to speculate that the
ASp50 functions by altering the relative proportion of p50 in the
L-
R complex, we have not directly assessed this in these
studies and thus cannot rule out an indirect effect of p50 on the
expression of other transcriptional factors.
B/Rel transcription factors are
responsible for cell type-specific and inducible gene activation
(45) . Recent demonstration of the physical interaction between
p65 and non-Rel factors suggest that non-Rel factors may also be
involved in the tissue specific regulation of VCAM-1 gene. A detailed
analysis of the NF-
B/Rel proteins and their interacting non-Rel
factors in endothelial cells will be required to identify the exact
nature of the endogenous transactivator of VCAM-1 gene expression and
its relationship to the p50 mediated inhibitory factors.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.