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B-Dependent Adhesion Molecule Gene Expression in Microvascular Endothelial Cells Through Intracellular Calcium Mobilization1
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Department of Dermatology and the Emory Skin Diseases Research Core Center, Emory University School of Medicine, Atlanta, GA 30322;
Veterans Administration Medical Center, Atlanta, GA 30322; and
Departments of Physiology and Surgery, University of California, San Francisco, CA 94143-0660
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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), and NF-
B (p65/p50) to tandem NF-B binding
sites at -76/-52 of the VCAM-1 gene. The SP-elicited intracellular
Ca2+ signal was required for activation and subsequent
binding of both NF-AT and NF-B. The transacting factor induction by
SP was specific, since a selective NK-1R antagonist blocked SP
activation and subsequent NF-AT and NF-B activation and binding.
These data demonstrate coincident activation of NF-AT and NF-B via
SP-induced intracellular Ca2+ mobilization and indicate a
crucial role for neuropeptides in modulating localized cutaneous
inflammatory responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The final targets of cell surface signals are often the activated
proteins that associate with DNA regulatory elements and regulate
transcription. Adhesion molecules ICAM-1 and VCAM-1 are highly
regulated at the transcriptional level by a number of mediators
(8, 9, 10, 11, 12, 13, 14, 15, 16, 17). TNF- induces ICAM-1 and VCAM-1 gene expression
via activation of distinct members of the Rel family (14, 18, 19, 20, 21). In most cells, NF-B is retained in the cytoplasm in
an inactive form by the inhibitory protein IB, which, in turn, is
regulated by the IB kinase or IKK (22). Following a
variety of extracellular stimuli, NF-B dissociates from IB,
translocates to the nucleus, and activates target genes. Activation of
NF-B is thus IKK activation dependent. The minimal DNA binding
domain of NF-B (p65/p65) on the ICAM-1 promoter corresponds exactly
to the NF-AT1 high affinity consensus (TGGAAA) site
(23, 24, 25). Within particular NF-B DNA binding domains,
an NF-AT monomer can bind to the 5' half-site of the DNA binding
domain, and a second NF-AT monomer can bind to the symmetrical 3'
half-site, which is why NF-AT transcription factors are often termed
monomeric Rel proteins (reviewed in Refs. 26). NF-AT was
originally described as an activator of IL-2 transcription
(27) and is now known to be expressed in most immune
cells, playing a pivotal role, like NF-B, in the transcription of
genes critical for inflammatory responses (reviewed in Ref.
26).
NF-AT nuclear translocation is initiated by stimulation of receptors
coupled to calcium mobilization (28, 29). The phosphatase
calcineurin, which controls the translocation of NF-AT proteins from
the cytoplasm to the nucleus of activated cells (28, 29),
is the major target of the immunosuppressive drug cyclosporin A (CsA).
Thus, the inhibition of calcineurin activity by CsA prevents activation
and subsequent DNA binding by NF-AT. Recently, CsA has been shown to be
an NK-1 and NK-2 receptor antagonist and was capable of blocking
SP-elicited phosphoinositide turnover in human lymphoblastoid and
astrocytoma cells (30). In addition, SP-mediated
induction of NF-B-dependent IL-8 gene expression in astrocytoma
cells was blocked by CsA (31). Thus, CsA may inhibit
Ca2+-dependent SP-mediated gene expression at the
level of NF-AT-transacting factor activation and/or at the level of
extracellular NK receptor blockade.
Elucidation of the mechanisms by which SP modulates adhesion molecule
gene transcription in microvascular endothelial cells is crucial to
understanding the interplay between the nervous and immune systems,
processes that probably play a significant role during the evolution of
inflammation. In this study we demonstrate the coincident activation by
SP of p65/p50 heterodimers binding to VCAM-1 tandem NF-B sites and
NF-AT binding to the ICAM-1 p65 homodimer site, both occurring as a
consequence of Ca2+ mobilization after NK-1R
engagement by SP.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HDMEC isolated from foreskins were obtained from the cell
culture facility of the Emory Skin Disease Research Core Center
(Atlanta, GA) (32). Experiments were conducted with cells
in passages 3–5. HDMEC were cultured on a gelatinized (0.1%) surface
in MCDB 131 (Life Technologies, Gaithersburg, MD) supplemented with
10% normal human serum (Irvine Scientific, Santa Ana, CA), 5 ng/ml
epidermal growth factor (Clonetics, San Diego, CA), 1 mg/ml
hydrocortisone acetate (Sigma, St. Louis, MO), 100 U/ml penicillin, 250
µg/ml amphotericin B, and 100 µg/ml streptomycin (Life
Technologies). Lyophilized SP (Peninsula Laboratories, Belmont, CA) was
diluted in the appropriate volume of HDMEC assay medium immediately
before use. In selected studies, 1 µM NK-1 receptor antagonist
GR82334 (Peninsula Laboratories, Belmont, CA) was added to cultured EC
20 min before the addition of SP or TNF-. CsA (Sandimmune injection,
Sandoz/Novartis, East Hanover, NJ) in vehicle Cremophor EL
(polyoxyethylated castor oil) was purchased from Sigma and was diluted
in HDMEC medium to 0.1, 1, or 10 µM for dose dependence studies. For
all subsequent studies, CsA was diluted to 1 µM in HDMEC medium. As a
control, vehicle alone (Cremophor EL) from Sandoz/Novartis was diluted
identically to CsA in HDMEC medium. Lyophilized human recombinant
TNF- was obtained from R & D Systems (Minneapolis, MN). TNF- (300
U/ml) served as a positive control for HDMEC ICAM-1 and VCAM-1
induction. mAb 84H10 recognizing human ICAM-1 was provided by Dr.
Stephen Shaw (National Institutes of Health, Bethesda, MD), and mAb
P3C4 directed against human VCAM-1 was a gift from Dr. Elizabeth Wayner
(University of Minnesota, Minneapolis, MN). The anti-TNF-
blocking study used a combination of 10 µg/ml each of anti-TNF
receptor 1 and anti-TNF receptor 2 Abs added simultaneously. Both
Abs, purchased from Santa Cruz Biotechnology (Santa Cruz, CA), were
diluted in HDMEC medium and incubated with HDMEC for 20 min at 37°C
in 5% CO2 before the addition of TNF- or SP.
Abs against AP-1 (c-Fos and c-Jun) as well as Rel and NF-AT family
members were purchased from Santa Cruz Biotechnology and added to
nuclear lysates at a concentration of 100 ng/µl. The selective
intracellular calcium chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N'-tetraacetic
acid-acetoxymethyl (BAPTA-AM) was purchased from Sigma and diluted to a
concentration of 50 µM in HDMEC medium. HDMEC were incubated in the
presence of BAPTA-AM for 20 min at 37°C in 5%
CO2 before SP or TNF- addition.
Measurement of SP-induced ICAM-1 and VCAM-1 on CsA-treated HDMEC by ELISA
HDMEC were plated onto 96-well plates and upon reaching 80%
confluence were left untreated or were treated with 100 nM SP for
18 h for ICAM-1 induction or 10 nM SP for 16 h for VCAM-1
induction, optimal concentrations and durations of SP for induction of
ICAM-1 and VCAM-1 in HDMECs as previously determined in our
laboratories (6, 7). HDMEC treated with 300 U/ml TNF-
for 16 h were used as a positive control for ICAM-1 and VCAM-1
cell surface induction. HDMEC cell adhesion molecule expression was
assessed using mAbs 84H10 for ICAM-1 or P3C4 for VCAM-1 induction by
ELISA as described previously (10). Results represent the
mean of three values ± SD for each variable tested, and results
are representative of three independent assays.
Reporter constructs generated for CAM deletional analyses
The (-1162/+1) bp ICAM-1 chloramphenicol acetyltransferase (CAT) construct (pBS CATP) has been described previously (9) and was used as a template for the generation of ICAM-1-based constructs. The (-288/+20) bp VCAM-1 CAT construct was provided by Dr. Douglas Dean (Washington University, St. Louis, MO) and has been characterized previously (12). VCAM-1- and ICAM-1-based derivative 5-deletional and heterologous promoter/reporter plasmids as well as site-directed mutagenesis of specific transcription factor DNA binding sites were designed and constructed using the techniques and strategies described previously (9, 11, 33, 34, 35, 36). Fragments of the ICAM-1 or VCAM-1 5'-regulatory regions were isolated by appropriate restriction enzyme digestion or generated by PCR and cloned into pBRAMScat2 (37), which contains the CAT reporter gene under control of the herpes simplex virus minimal thymidine kinase promoter and multiple cloning sites designed for analysis of eukaryotic enhancers. Inserts and ligation sites of all constructs generated were confirmed by sequence analysis via the dideoxynucleotide chain termination method using the Sequenase Quick-Denature Kit (Amersham, Arlington Heights, IL).
Transient transfection of reporter gene constructs and analysis of CAT expression
HDMEC were transiently transfected with ICAM-1, VCAM-1, and
control CAT reporter gene constructs as described previously
(36). Briefly, HDMEC were transfected with a concentration
of 20 µg of plasmid DNA in the presence of 500 ng/ml DEAE-dextran
(Sigma) at 85% confluence for 30 min at 37°C in 5%
CO2. After 30 min, HDMEC medium containing 8 µM
chloroquine was added, and the transfection was continued for an
additional 2 h. After exposure to plasmid DNA for 2.5 h, the
medium was replenished. At 24 h post-transfection, cells were left
untreated or were treated for 18 h with 100 nM SP for ICAM-1, for
16 h with 10 nM SP for VCAM-1 induction, or for 16 h with 300
U/ml TNF- as a positive control for both ICAM-1 and VCAM-1
induction. In selected experiments, HDMEC were pretreated for 20 min
with the NK-1R antagonist GR82334 (Peninsula Laboratories), CsA
(Sandoz/Novartis), or the CsA vehicle Cremophor before SP or TNF-
addition. Assays and normalization protocols for transfection
efficiency (using cotransfection of a ß-galactosidase control
vector), transfection lysate protein concentrations, and CAT expression
were performed as described previously (11, 33).
Preparation of nuclear extracts and gel mobility shift analysis
Confluent HDMEC were either left untreated or were treated for
4 h with TNF- or SP for CAM induction. In selected experiments,
HDMEC were incubated in the presence of the NK-1R antagonist GR82334 or
CsA for 20 min before the addition of SP or TNF-. For TNF-
blocking studies, anti-TNF- receptor 1 and 2 Abs (see
Cells and reagent) were added to HDMEC 20 min before the
addition of SP or TNF-. Nuclear extracts were prepared as described
previously (33). Oligonucleotides encompassing the single
NF-B region of ICAM-1 (wild-type NF-B/ICAM) and the tandem
NF-B regions of VCAM-1 (wild-type NF-B/VCAM) were designed,
annealed, and extended as previously described (33). All
oligonucleotides were synthesized by the Emory University Microchemical
Facility. The DNA-protein binding reaction was performed as previously
described (11). Briefly, 3–4 µg of HDMEC nuclear
extracts, binding buffer, and 1 x 105 cpm
of radiolabeled oligonucleotide probe were incubated together for 30
min at room temperature. For the competition assays and supershift
experiments, an excess of the appropriate unlabeled oligonucleotide (50
ng) or Ab (1 µg) was added to the binding reaction prior to the
addition of radiolabeled probe. Unlabeled irrelevant competitor
oligonucleotides incubated in excess (50 ng) with lysate were
(-88/-68) of the ICAM-1 gene or (-42/-22) of the VCAM-1 gene. For
some NF-AT supershift experiments, SP-treated HDMEC nuclear lysates
were incubated with probe DNA for 30 min, followed by the addition of
the anti- NF-AT1 Ab (1 µg). Samples were subjected to
electrophoresis on a native 4% polyacrylamide gel for 4 h at 120
V and 10–12 A. Gels were dried, and autoradiography was performed at
-70°C for 1–3 days. Autoradiographs were scanned on a La Cie
scanner (La Cie, Beaverton, OR) using Adobe Photoshop software (Adobe
Systems, Mountain View, CA). The digitized image was subsequently
labeled in Microsoft Power Point (Microsoft, Redmond, WA) and printed
on a high resolution laser printer. Each figure represents a
computer-generated image of the autoradiograph, and each is typical of
the autoradiograph in the context of relative band and background
densities.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects of pretreatment with CsA and NK-1R antagonists on the
SP-mediated induction of cell surface ICAM-1 and VCAM-1 expression on
HDMEC was examined by ELISA (Fig. 1
).
Optimal concentrations and durations of SP treatment for induction of
ICAM-1 and VCAM-1 on HDMEC were previously established in our
laboratories (6, 7) and were used in these and subsequent
studies. Untreated HDMEC have a low basal expression of cell surface
ICAM-1 (Fig. 1A), which is up-regulated 5-fold when HDMEC
are treated with 100 nM SP for 18 h. This specific SP-mediated
ICAM-1 induction is abrogated when HDMEC are pretreated with an NK-1R
antagonist (Fig. 1A). CsA was found to dose-dependently
block SP induction of ICAM-1 on HDMEC, whereas pretreatment with the
CsA vehicle had no blocking effect on SP-mediated ICAM-1 induction
(Fig. 1A). TNF- strongly induced HDMEC cell surface
ICAM-1 and served as a positive control (Fig. 1A).
TNF--mediated ICAM-1 induction was unaffected when HDMEC were
pretreated with either the NK-1R antagonist or 1 µM CsA,
demonstrating specificity and differential receptor signaling pathways
for the two stimuli as well as a nontoxic effect of reagents (Fig. 1A). Untreated HDMEC show little or no constitutive VCAM-1
expression (Fig. 1B). HDMEC treated with 10 nM SP for
16 h resulted in a 7-fold induction of VCAM-1 cell surface
expression, which was abrogated when HDMEC were pretreated with the
NK-1R antagonist (Fig. 1B). CsA in a dose-dependent fashion
was capable of blocking SP-induced VCAM-1 on HDMEC (Fig. 1B). Again, the CsA vehicle had no blocking effect on
SP/VCAM-1 induction on HDMEC (Fig. 1B). As with ICAM-1,
TNF--treated HDMEC displayed high levels of cell surface VCAM-1
expression, which were unaffected by pretreatment with either the NK-1R
antagonist or 1 µM CsA (Fig. 1B). Pretreatment of HDMEC
with CsA vehicle had no effect on the ability of TNF- to induce
ICAM-1 or VCAM-1 expression (data not shown). These data demonstrate
that SP/NK-1R-mediated up-regulation of ICAM-1 and VCAM-1 cell surface
expression is CsA sensitive and occurs via receptors and signaling
pathways distinct from those used by TNF-.
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Our previous studies in HDMEC revealed that upon a 3-h incubation
with SP (100 nM), mRNA basal steady state levels increased by 3-fold
(6). To determine molecular mechanisms responsible for the
SP-mediated ICAM-1 gene transcription, a sequential series of
5'-deletional ICAM-1 promoter-based reporter gene constructs were
transiently transfected into HDMEC (Fig. 2). Analysis of respective CAT expression
in untreated, SP-treated, and TNF--treated cells revealed a basal
activity of the -384/+1 ICAM-1 construct that was increased after
treatment with SP and TNF- by a factor of 3 (Fig. 2). While the
-277/+1 construct demonstrated comparable constitutive, SP-induced,
and TNF--induced expression, the -182/+1 construct showed a loss of
induced transcriptional activity with both SP and TNF- stimulation
(Fig. 2). The -182/+1 construct interrupts a p65/p65 homodimer-binding
modified NF-B site on the ICAM-1 promoter that is critical for
TNF- induction of ICAM-1, as previously described by our laboratory
(36). The promoterless construct, pCAT BASIC, served as a
negative control for transfection and CAT reporter gene expression.
Both SP- and TNF--induced reporter gene expression were restored
when the -191/-170 TK-CAT construct was transfected into HDMEC (Fig. 2, C and D), yet the TK-CAT minimal promoter
alone displayed no inducibility upon SP or TNF- treatment. These
findings indicate that a critical gene regulatory region necessary and
sufficient for SP-inducible expression is located between bp -191 and
-170 of the ICAM-1 promoter.
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To demonstrate the formation of a specific DNA-protein complex,
the newly identified SP-responsive element of the ICAM-1 promoter was
used (-191/-170) in an EMSA. This site had previously been identified
by our laboratory as containing a modified NF-B binding site that
bound a p65 homodimeric complex upon stimulation of cells with TNF-
and PMA (34, 36). Comparison of this minimal sequence to
that of known transcription factor binding sites revealed a strong
homology to the NF-AT family DNA binding domain overlapping the 5'
portion of the p65 homodimeric modified NF-B binding site
(26). We therefore assessed whether NF-B or NF-AT was
part of the SP-induced DNA binding complex (Fig. 3A), and whether complex
formation, if any, displayed CsA sensitivity. Untreated HDMEC displayed
little or no complex formation (lane 1).
TNF--treated HDMEC nuclear lysates served as a positive control for
complex binding to this ICAM-1 promoter region. TNF--treated complex
formation (lane 2) was supershifted with the
anti-RelA Ab (lane 3), but not the anti-p50
Ab (lane 4), as previously described in HDMEC
(36). The TNF--treated HDMEC complexes were unaffected
upon addition of anti-NF-AT1 (lane 5) and
anti-NF-AT family (lane 6) Abs.
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-mediated complex formation (lanes 7 and
8, respectively). The specificity of TNF--induced
complexes was demonstrated by competition with excess cold identical
oligonucleotide, but not with excess cold irrelevant oligonucleotide
(data not shown). Nuclear lysates from SP-treated HDMEC incubated with
the -191/-170 ICAM-1 labeled probe (lane 9)
displayed specific complex formation distinct from that with
TNF--treated HDMEC (lane 2). The specificity of
the SP-induced complex formation was demonstrated by competition with
excess cold identical oligonucleotide (lane 10). SP
complex formation disappeared upon incubation with anti-NF-AT1 Ab
(lane 11) and with the broad spectrum NF-AT Ab
(lane 12), which cross-reacts with all NF-AT family
members.
To determine specificity of complex formation, HDMEC were preincubated
with a selective NK-1R antagonist that completely prevented complex
formation (Fig. 3
A, lane 13). HDMEC pretreated
with CsA (1 or 10 µM) displayed no SP-dependent complex formation as
well (lanes 14 and 15). The 1 µM dose of
CsA was chosen as optimal for subsequent experiments. These data
demonstrate that the SP-induced ICAM-1-binding complex contains a
protein(s) recognized by anti-NF-AT1 and anti-NF-AT family
member-specific Abs. This specific SP-induced NF-AT-containing complex,
which binds the region -191/-170 of the ICAM-1 promoter, can be
blocked by both CsA and a selective NK-1R antagonist. These data also
demonstrate that two independent transcription factor complexes, p65
homodimers induced by TNF- and an NF-AT-containing complex induced
by SP, can bind to the same critical regulatory region (-191/-170) of
the ICAM-1 gene, and that the activation of these two transcription
factors occurs in a mutually independent fashion.
Typically, NF-AT associates cooperatively with heterologous DNA binding
proteins, particularly AP-1. The SP-inducible NF-AT-containing complex
was unaffected when incubated in the presence of Abs to AP-1 members,
anti-c-Fos, or anti-c-Jun (Fig. 3B). Disappearance
of the SP-induced complex when using anti-NF-AT Abs seen in Fig. 3A presumptively occurred through physical interference of
the bound Ab with the DNA recognition motif. The NF-AT-containing
complex activated by SP was visibly supershifted upon incubation with
an anti-NF-AT1 Ab (Fig. 3B, lane 5) when the
order of addition of reagents was altered, such that the protein/DNA
complex was allowed to form before addition of the anti-NF-AT1
Ab.
Although enhancer trap reporter expression studies had shown that the
-191/-170 region was sufficient to confer both SP and TNF-
inducibility (Fig. 2, C and D), we tested whether
other regions of the wild-type ICAM-1 promoter might be contributing to
native SP transcription induction of ICAM-1. To do so, we compared SP
and TNF- inducibility of CAT reporter expression in HDMEC after
transfection with either wild type -384/+1 ICAM CAT or a derivative
plasmid, mutB-384/+1 ICAM CAT, in which the overlapping regions of
the NF-AT and NF-B consensus binding sites (Fig. 2A) had
been mutated by site-directed mutagenesis. Mutagenesis limited to
nucleotides critical for both NF-AT and p65 binding completely
abrogated both SP and TNF- inducibility (data not shown). Since EMSA
data demonstrate that SP induces only NF-AT and not p65 binding to the
critical -191/-170 region (Fig. 3, A and B),
and since mutation of the NF-AT binding site abrogates all SP reporter
gene inducibility, we conclude that not only are NF-AT activation and
binding to this site necessary for SP induction of ICAM-1 expression,
but also that no other regions of the native ICAM-1 promoter within the
-384/+1 fragment are capable of independently mediating gene
responsiveness to SP.
Rel family members do not comprise the SP-inducible ICAM-1 DNA binding complex
To further elucidate which proteins comprise the SP-induced DNA
binding complex, the SP-responsive region (-191/-170) of the ICAM-1
promoter was used in an EMSA. Since TNF- is known to induce NF-B
(Rel family) member binding to (-191/-170) of the same region of the
ICAM-1 promoter, we therefore investigated whether Rel family members
were part of the SP-induced complex formation in HDMEC (Fig. 4). Nuclear lysates from untreated HDMEC
show no complex formation (lane 1). SP-treated HDMEC
complexes (lane 2) are unaffected by the addition of
Abs against Rel family members anti-p65/RelA (lane
3), anti-p50 (lane 4), anti-c-Rel
(lane 5), and anti-RelB (lane
6). HDMEC pretreated with CsA, followed by SP addition, display no
complex formation (lane 7). Thus, SP-mediated complex
formation, which binds to the ICAM-1 promoter region (-191/-170),
does not contain Rel family member proteins, as defined by
anti-Rel Abs.
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Our previous studies demonstrated a significant induction of
VCAM-1 mRNA expression by HDMEC after SP treatment (7). We
thus investigated SP-mediated VCAM-1 transcription induction via
5'-deletional VCAM-1 promoter-based reporter gene constructs after
transient transfection into HDMEC. Analysis of the respective CAT
expression in untreated cells revealed an expected lack of basal
activity of the -288/+20 VCAM-1 construct, and induction of CAT
expression was seen after treatment with SP and TNF- (Fig. 5). The -88/+20 5' deletion construct
demonstrated comparable SP- and TNF--induced expression equivalent
to the -288/+20 construct, whereas the -32/+20 construct showed a
loss of SP and TNF- inducibility (Fig. 5). We conclude that the
required transcriptional regulatory region necessary for SP-mediated
VCAM-1 induction is located between bp -88 and -32 of the VCAM-1
promoter.
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The identified SP-responsive region of the VCAM-1 promoter between
regions bp -88 and -32 contains the previously reported tandem
NF-B regions (14, 38) at position -76/-52. Since our
analysis of the SP-induced ICAM-1 expression had implicated a similar
region, this VCAM-1 region was used as radiolabeled probe along with
HDMEC nuclear lysates in EMSAs (Fig. 6).
Nuclear lysates from untreated HDMEC demonstrated no complex formation
(lane 1). SP-induced complex formation
(lane 2) was abrogated upon preincubation of HDMEC
with an NK-1R antagonist (lane 3) as well as by
competition with excess cold oligonucleotide (lane
8). In contrast to SP-induced ICAM-1 binding complexes,
anti-NF-AT1 (lane 4) and anti-NF-AT
(lane 5) family member-specific Abs did not affect
SP-induced VCAM-1 complex formation. Moreover, both anti-p65
(lane 6) and anti-p50 (lane 7)
supershifted the SP-induced complexes. TNF--treated nuclear extracts
from HDMEC (lane 9) were unaffected by the addition
of anti-NF-AT family Ab (lane 10), yet
TNF--mediated complex formation was supershifted upon incubation
with anti-p65-specific (lane 11) and
anti-p50-specific (lane 12) Abs, as previously
reported (20). TNF--treated complex formation
specificity was demonstrated by preincubation with excess cold
oligonucleotide (lane 13). Therefore, the SP-mediated
VCAM-1 binding complex contains p65 and p50, but not NF-AT family
members, as shown by anti-p65, anti-p50, and anti-NF-AT
family member Abs. In separate experiments, we demonstrated that Abs
against other Rel family members, including c-Rel, RelB, and p52, did
not shift or affect SP-induced complex formation bound to the -76/-52
region of the VCAM-1 promoter (data not shown).
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independent
SP is known to induce cytokine production in various immune cells,
including TNF- production in mast cells (39). TNF-
is the only defined cytokine capable of transcriptionally inducing
VCAM-1 in HDMEC (16), although VCAM-1 can be induced by
other cytokines (e.g., IL-1) in different types of endothelial cells,
such as human umbilical vein endothelial cells (17). Since
SP and TNF--induced identical NF-B complexes binding to the
-76/-52 region of the VCAM-1 promoter in HDMEC, it was possible that
SP was inducing autocrine TNF- expression and stimulation of these
cells. To test this possibility, we used Abs against human
TNF-receptors 1 and -2 added just before SP treatment to block any
subsequent TNF- stimulation of HDMEC. We then assessed the induction
of specific nuclear protein-DNA complex formation using the -76/-52
region of the VCAM-1 promoter (Fig. 7).
Untreated HDMEC nuclear lysates lacked specific complex formation
(lane 1). TNF--treated complex formation
(lane 2) was abrogated when HDMEC were pretreated
with anti-TNF- blocking Abs (lane 3). The
TNF--specific complex (lane 2) was supershifted by
anti-p65 (lane 4) and anti-p50
(lane 5), as shown above. TNF--treated
complex formation supershifts (lanes 4 and
5) by Abs to p65 and p50 were abrogated when HDMEC were
preincubated with anti-TNF- blocking Abs (lanes
6 and 7). Therefore, TNF- blocking Abs are capable
of abrogating TNF--induced p65 and p50 binding to -76/-52 of the
VCAM-1 promoter. SP-treated complex formation (lane
8) was unaffected by HDMEC pretreatment with anti-TNF-
blocking Abs (lane 9). The SP-induced complex was
supershifted by anti-p65 (lane 10) and
anti-p50 (lane 11) Abs, and these supershifts
were unaffected by HDMEC pretreated with anti-TNF- blocking Abs
(lanes 12 and 13). However, SP-treated
complex formation was blocked by HDMEC pretreatment with a selective
NK-1R antagonist (lane 14). These data indicate that
SP-mediated p65 and p50 complex binding to the SP-responsive VCAM-1
element is a direct effect of SP, and that SP-dependent activation of
NF-B does not involve autocrine stimulation by HDMEC-derived
TNF-.
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We next determined whether an SP- or TNF--elicited
intracellular Ca2+ mobilization was required for
transcription factor activation and binding to the ICAM-1 -191/-170
region by pretreating HDMEC with the membrane-permeable selective
intracellular calcium chelator BAPTA-AM (Fig. 8). Untreated HDMEC (lane
1) displayed no specific complex formation. SP-induced complex
formation (lane 3) was abrogated when HDMEC were
pretreated with BAPTA-AM (lane 2). Specificity of the
SP-induced complex was demonstrated by incubation with cold excess
identical oligonucleotide (lane 4), while excess cold
irrelevant oligonucleotide (ICAM-1 -88/-68) had no effect on this
complex (data not shown). TNF--stimulated p65 homodimer binding to
the identical region on the ICAM-1 gene (lane 6) was
unaffected when HDMEC were pretreated with BAPTA-AM (lane
5). Thus, SP-induced NF-AT activation and binding to -191/-170
of the ICAM-1 promotor, but not TNF--induced p65 homodimer
activation and binding to the same region, is intracellular
Ca2+ mobilization dependent.
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-induced, p65/p50 heterodimer activation
and binding to the VCAM-1 -76/-52 region is intracellular
Ca2+ mobilization dependent
We have demonstrated that SP and TNF- stimulation of HDMEC
induces identical NF-B family member binding to the -76/-52 region
of the VCAM-1 promoter and that SP induction of such binding was
TNF- independent. We next assessed whether the SP- or
TNF--stimulated p65/p50 heterodimer activation and binding to the
VCAM-1 -76/-52 region was dependent upon the mobilization of
intracellular calcium (Fig. 9). Untreated
HDMEC nuclear lysates displayed no specific complex formation
(lane 1). SP-induced complex formation
(lanes 2 and 4) was inhibited when HDMEC
were pretreated with BAPTA-AM before the addition of SP
(lane 3). The specificity of SP-induced complex
formation is demonstrated when lysates are incubated in the presence of
cold excess -76/-52 oligonucleotide (lane 5), but
not irrelevant -42/-22 oligonucleotide (data not shown). In contrast,
TNF--elicited complex formation (lane 6) was
unaffected when HDMEC were pretreated with BAPTA-AM (lane
7). These data indicate that although both SP and TNF-
stimulation of HDMEC are capable of inducing the activation of
identical transcription factors that bind to the same region
(-76/-52) of the VCAM-1 gene, the signaling mechanisms are distinct.
SP induction of p65/p50 heterodimers requires intracellular calcium
mobilization, whereas TNF- induction of the identical NF-B
complex is not calcium mobilization dependent.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
IL-1, respectively (39, 48). SP activation in the CNS of
the transcription factor NF-B and IL-8 (B-dependent) gene
expression in human astrocytoma cells has been recently reported
(31). SP-mediated transcription factor induction and
subsequent inflammatory gene expression involving neuropeptides
secreted by the peripheral nervous system have not, as of yet, been
described. We have previously demonstrated a difference in concentration dependence induction of mRNA and cell surface HDMEC ICAM-1 and VCAM-1 by SP (6, 7). Optimal induction of VCAM-1 occurred at a 10-fold lower concentration of SP (10 nM) compared with ICAM-1 induction by SP (100 nM). We also demonstrated that SP-mediated induction of VCAM-1 occurred at earlier time points, 12–16 vs 16–18 h for ICAM-1 induction (6, 7). In vivo studies have indicated a preferential infiltration of eosinophils upon injection of SP into human skin (49). Neutrophils represent >70% of the immune cells in blood, yet these cells lack the integrin VLA-4, which is present on eosinophils. One can speculate that SP-mediated eosinophil influx is regulated at the level of VCAM-1/VLA-4 interactions, because SP induces VCAM-1 at lower concentrations and earlier time points, and preferentially activates the integrin VLA-4. It would thus appear from our data that in HDMEC the p65/p50 heterodimers required for VCAM-1 induction display a threshold for activation by SP that is not be seen for the p65 homodimers required for ICAM-1 induction. Indeed, our data demonstrate that at 10-fold higher SP concentrations, NF-AT1, rather than p65 homodimers, is activated and mediates the induction of ICAM-1 expression. Why SP selectively activates p65/p50 heterodimer, but not p65 homodimer, formation and binding in HDMEC is currently under further investigation.
Our previous transcriptional work focused on the identification and
characterization of the ICAM-1 5' regulatory region (9, 10, 35, 36) as well as additional characterization of the VCAM-1
5'-regulatory region (11, 17, 33, 50). In this study we
have identified the minimal DNA binding domains within the regulatory
regions of ICAM-1 and VCAM-1 that confer SP-mediated transcriptional
activity in HDMEC. In addition, we have identified which SP-activated
transcription factors associate with the DNA binding domains of the
ICAM-1 and VCAM-1 regulatory regions in HDMEC. We demonstrate that
5'-regulatory regions between -191/-170 of the ICAM-1 gene and
-76/-52 of the VCAM-1 gene are necessary for activation-dependent
transcription by SP in HDMEC. Both regulatory regions, -191/-170 of
ICAM-1 and -76/-52 of VCAM-1, contain previously defined NF-B
binding domains (14, 18, 21, 37). Multiple differences
exist between the B regions of the ICAM-1 and VCAM-1 promoters. The
VCAM-1 promoter contains two tandem consensus B regions at positions
-73 and -58, and TNF- induces preferential p65/p50 heterodimer
binding to these sites (38). The ICAM-1 promoter contains
a single nonconsensus B region to which TNF- induces preferential
binding of a p65 homodimer (19, 36). The ICAM-1 B
nonconsensus region is flanked by sequences that are required for
TNF--induced p65 homodimer binding (36), whereas
flanking sequences have not been shown to be necessary for
TNF--mediated p65/p50 heterodimer binding to the tandem consensus
region on the VCAM-1 promoter. We demonstrate SP-mediated activation
and binding of NF-AT to the -191/-170 of ICAM-1 and NF-B (p65/p50)
to the -76/-52 of VCAM-1 genes. These events can be specifically
blocked by both the NK-1R antagonist and CsA. Typically, NF-AT is
associated with heterologous DNA binding proteins, especially AP-1
(reviewed in Refs. 48). SP-driven NF-AT/ICAM-1 appears in
vitro to lack cooperative binding with transcription factors of the
AP-1 family. It is possible that in vivo, cooperation with other
transcription factors may occur. However, site-directed mutagenesis of
the NF-AT site, which overlaps with the 5' portion of the p65 homodimer
NF-B binding site, abrogates both SP- and TNF--induced
transcription, even within the context of the remaining native ICAM-1
promoter. Our data also indicate that the SP-elicited intracellular
Ca2+ mobilization is required for NF-AT and
NF-B activation and binding to their respective consensus sites on
the ICAM-1 and VCAM-1 genes. In contrast, the TNF--activated NF-B
pathway was Ca2+ independent, again demonstrating
that SP and TNF- transduce via distinct signaling pathways. Recent
reports have demonstrated an intracellular Ca2+
mobilization requirement for NF-B activation and B-dependent gene
expression in cell types other than EC and by signals other than
neuropeptides (51, 52).
Our model of activation and differential regulation of microvascular
endothelial cell adhesion molecule expression by the neuropeptide SP
differs from the traditional TNF--induced NF-B-mediated
transcriptional induction of adhesion molecules ICAM-1 and VCAM-1 in
microvascular endothelial cells (Fig. 10). The neuropeptide SP upon binding
to its G protein-coupled NK-1R elicits an intracellular calcium signal.
We have demonstrated that this Ca2+ signal
results in differential transcription factor activation and binding to
previously defined regions of ICAM-1 and VCAM-1. As a consequence of
the SP-induced calcium signal, the phosphatase calcineurin becomes
activated and dephosphorylates NF-AT (reviewed in Ref.
26). We propose that NF-AT then translocates to the
nucleus and binds its high affinity TGGAAA consensus site on the ICAM-1
promoter, a site that is colocalized with the 5' portion of the
modified NF-B site that is used with TNF- activation of p65
homodimers. By an unknown mechanism, the SP-elicited calcium signal
also leads to the activation of the NF-B pathway, resulting in
nuclear translocation of the p65/p50 heterodimer, which binds to its
high affinity tandem B sites on the VCAM-1 promoter. This SP-induced
activation of NF-B heterodimers does not extend to the activation of
sufficient quantities of p65 homodimers for the binding and activation
of transcription through the ICAM-1-modified B site. Instead, SP
treatment induces NF-AT activation and ICAM-1 DNA binding. These
transcriptional activation events lead to ICAM-1 and VCAM-1 HDMEC cell
surface expression, and these pathways of gene expression induction can
be specifically blocked by both NK-1R and CsA.
|
B, resulting in induction of ICAM-1 and VCAM-1 gene expression in
microvascular endothelial cells. These findings indicate that
neuropeptides released in skin can activate transcription factors and
modulate adhesion molecule expression, contributing to inflammation and
wound healing.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. S. Wright Caughman, Department of Dermatology, Emory University School of Medicine, 5001 Woodruff Memorial Research Building, Atlanta, GA 30322. E-mail address:
3 Abbreviations used in this paper: SP, substance P; NK-1R, neurokinin receptor-1; HDMEC, human dermal microvascular endothelial cells; IB, inhibitor of NF-B; IKK, IB kinase; CsA, cyclosporin A; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N'-tetraacetic acid-acetoxymethyl; CAT, chloramphenicol acetyltransferase; TK, thymidine kinase; VLA-4, very late Ag-4.
Received for publication April 2, 1999. Accepted for publication September 7, 1999.
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References |
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