Departments of 1 Molecular and Cellular Physiology and 2 Gastroenterology, Louisianna State University Health Sciences Center, Shreveport, Louisiana, 71130-3932; and 3 Guilford Pharmaceuticals, Baltimore, Maryland 21224
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ABSTRACT |
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Mucosal addressin cell adhesion molecule-1
(MAdCAM-1) is a 60-kDa endothelial cell adhesion glycoprotein that
regulates lymphocyte trafficking to Peyer's patches and lymph nodes.
Although it is widely agreed that MAdCAM-1 induction is involved in
chronic gut inflammation, few studies have investigated regulation of
MAdCAM-1 expression. We used two endothelial lines [bEND.3 (brain) and SVEC (high endothelium)] to study the signal paths that regulate MAdCAM-1 expression in response to tumor necrosis factor (TNF)- using RT-PCR, blotting, adhesion, and immunofluorescence. TNF-
induced both MAdCAM-1 mRNA and protein in a dose- and time-dependent manner. This induction was tyrosine kinase (TK), p42/44, p38
mitogen-activated protein kinase (MAPK), and nuclear factor
(NF)-
B/poly-ADP ribose polymerase (PARP) dependent. Because MAdCAM-1
is regulated via MAPKs, we examined mitogen/extracellular
signal-regulated kinase (MEK)-1/2 activation in SVEC. We found that
MEK-1/2 is activated by TNF-
within minutes and is dependent on TK
and p42/44 MAPKs. Similarly, TNF-
activated NF-
B through TK,
p42/44, p38 MAPKs, and PARP pathways in SVEC cells. MAdCAM-1 was also
shown to be frequently distributed to endothelial junctions both in
vitro and in vivo. Cytokines like TNF-
stimulate MAdCAM-1 in
high endothelium via TK, p38, p42/22 MAPKs, and NF-
B/PARP.
MAdCAM-1 expression requires NF-
B translocation through both direct
p42/44 and indirect p38 MAPK pathways in high endothelial cells.
mucosal addressin cell adhesion molecule-1
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INTRODUCTION |
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LYMPHOCYTE TRAFFICKING is mediated by interactions between homing receptors on the lymphocyte and vascular addressins expressed on specific endothelia that are controlled by a variety of inflammatory mediators (10, 42, 51). In particular, lymphocyte homing to mucosal-associated lymphoid tissues, such as Peyer's patches and the lamina propria of the intestine, is critically dependent on a single-chain 60-kDa glycoprotein adhesion molecule, the mucosal vascular addressin or mucosal addressin cell adhesion molecule (MAdCAM-1; see Refs. 52 and 35). MAdCAM-1 is also expressed on endothelial cells within the mesenteric lymph nodes, the lamina propria of both the small and large intestine, and in the mammary gland during lactation. In addition to its normal role in lymphocyte trafficking to mucosal lymphoid tissue, MAdCAM-1 expression is dramatically increased in chronic inflammatory and disease states (39), e.g., inflammatory bowel disease (IBD; see Refs. 9 and 15) and in diabetes (20, 21, 60), and may play important roles in these conditions.
MAdCAM-1 is believed to play a central role in the etiology of colitis
through its ability to direct circulating lymphocytes to enter
gut-associated lymphoid tissues and the gut interstitium tissues
(17, 56). The adhesion between the
4
7-integrin, expressed on a subset of T
cells, and MAdCAM-1 presented on cytokine-activated endothelial cells,
facilitates the emigration of lymphocytes to inflamed sites in the gut
in several models of colitis (9). Furthermore, although
MAdCAM-1 is constitutively expressed in some regions of the colon and
mesenteric lymph nodes, the expression of MAdCAM-1 in these tissues
increases at least 10- to 11-fold during periods of active colitis
(14, 26, 27). In the interleukin (IL)-10-deficient murine
model of spontaneous colitis, the colon expresses very high basal
levels of MAdCAM-1 and during active colitis shows robust increases in
MAdCAM-1 expression (26). MAdCAM-1 has also been detected
at high levels in colon lamina propria venules from mice with
hapten-induced colitis (57) and in the colon of the
IL-2-deficient mouse (32). In another colitis model, the
severe combined immunodeficient (SCID) mouse, reconstituted with
CD4+ CD45RBhigh T cells, develops a severe form
of colitis (27). This injury can be blocked by antibodies
directed against either MAdCAM-1 or its ligand
4
7 (41). These data
demonstrate that these two adhesive determinants mediate cell
trafficking in the chronically inflamed gut. Interestingly,
MAdCAM-1 may sustain chronic inflammation by stimulating proliferation
of extravasated lymphocytes via MAdCAM/
7-mediated signaling (30).
Tumor necrosis factor (TNF)- appears to be one of the most potent
cytokines stimulating MAdCAM-1 expression both in vivo and in vitro.
Levels of TNF-
and other proinflammatory cytokines are dramatically
elevated in CD45RBhigh/SCID and IL-10-deficient (
/
)
mice (26, 27). Expression of MAdCAM-1 on endothelial cells
has been reported after TNF-
stimulation (50) and in
the colons of mice injected with TNF-
(15). Besides
MAdCAM-1, TNF-
also induces the expression of several other
endothelial cell adhesion molecules (ECAMs), including E-selectin,
intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion
molecule (VCAM)-1 (44), by activating signals leading to
activation of the transcription factor nuclear factor (NF)-
B.
TNF-
-induced expression of ECAMs like E-selectin, VCAM-1, and ICAM-1
(25, 43) is almost completely blocked by peptide-aldehyde inhibitors like MG-132 and lactacystin, which block the 26S proteasome, a central regulator of NF-
B translocation. The genes for these ECAMs
share specific DNA binding motifs in their promoters that allow for
NF-
B-driven transcriptional initiation of ECAM messages (12,
13). However, although the pathways governing E-selectin, VCAM-1, and ICAM-1 have been studied intensively, the specific cell
regulatory mechanisms through which MAdCAM-1 expression is controlled
have not been as closely examined. Although the MAdCAM-1 promoter is
known to contain NF-
B binding sites and may enhance MAdCAM-1
transcripts via NF-
B based on luciferase assays (54), NF-
B-dependent MAdCAM-1 protein expression has not been demonstrated.
Since the original report on MAdCAM-1 (hereafter MAdCAM) regulation by
Sikorski et al. (50), few other studies have examined the
regulation of MAdCAM expression, especially within high endothelial venule cells, which are relevant to chronic gut inflammation and IBD.
Because entry of lymphocytes into specialized venules bearing MAdCAM in
lymphoid tissues appears to regulate chronic gut inflammation, an
improved understanding of the signals that promote the expression of
this molecule may provide an important therapeutic means for limiting
MAdCAM expression, 7-lymphocyte binding, and activation and inflammation in these tissues. To date, most experimental studies
on MAdCAM expression have been obtained using an endothelioma cell line
(bEnd.3) derived from brain microvessels. Although bEnd.3-based models
are clearly useful, cells derived from brain capillaries may not
accurately model high endothelial venules. Here we have compared MAdCAM
expression in murine high endothelial venule cells (SVEC4-10) with
brain-derived cells (bEnd.3). We determined how several second-message
systems, e.g., NF-kB, poly-ADP ribose polymerase (PARP), p38, p42/44
mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and
protein kinase G (PKG), control MAdCAM expression in response to
TNF-
. We measured lymphocyte binding to MAdCAM expressed in our
cells to document the functional significance of these expression
studies. Last, we have also localized the MAdCAM expressed in
stimulated endothelial cells and in the intestine and found that MAdCAM
is enriched at cell-cell junctions. These data show several novel paths
for MAdCAM regulation and suggest potential pathways for limiting
MAdCAM-dependent gut inflammation in IBD.
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MATERIALS AND METHODS |
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Reagents. MG-132 (46) was purchased from Biomol (Plymouth Meeting, PA). GPI-6150, the PARP inhibitor, was obtained from Guilford Pharmaceuticals (Baltimore, MD). Genistein (1) was purchased from Sigma (St. Louis, MO). PD-98059 (2), SB-202190 (29), chelerythrin chloride, and KT-5823 were all purchased from Calbiochem (La Jolla, CA).
Cell culture.
SVEC4-10 (SVEC) is a cell line derived by Simian virus-40
transformation of murine high endothelial cells (37). The
mouse brain endothelial cell line bEnd.3 was obtained from Dr. Eugene Butcher (50). LEII cells are an endothelial cell line
derived from murine lung capillary and were supplied by Dr. Thomas
Maciag (55). These cell types were all maintained in DMEM
with 10% FCS with 1% antibiotic/antimycotic. Mouse CD8+ T
cell lymphoma tyrosine kinase (TK)-1 cells that constitutively express
4
7 (3) were also donated by
Dr. Butcher. These cells were cultured in RPMI medium supplemented with
10% FCS, 2 mM L-glutamine, and 0.05 mM 2-mercaptoethanol.
Quantification of mRNA levels by RT-PCR.
SVEC were pretreated for 1 h with or without inhibitors and then
were incubated for 12 h with either vehicle or 20 ng/ml TNF- (Endogen, Stoughton, MA). MAdCAM message was measured by RT-PCR in
response to treatments. Total RNA was extracted from SVEC cells using
the RNeasy Kit (QIAGEN, Valencia, CA). First-strand cDNAs were prepared
from 4 µg of total RNA using a mixture of
oligo(dT)12-18 and random hexamer primers with
Superscript RT (Promega, Madison, WI). The following oligonucleotides
were synthesized and used as primers: P1,
5'-CCTAGTACCCTACCAGCTCA-3'; P2, 5'-ATCTCCTCTTCTTGCTCTGG-3' (P1-P2; 474 bp); P3,
5'-AGAAGAGGAGATACAAGAGG-3'; and P4,
5'-TAGGTGTGTACATGAGCTAGTGTCTGGGCG-3' (P3-P4; 717 and 286 bp).
Western analysis of cell lysates.
Each protein sample (75 µg each) was separated on 7.5% SDS-PAGE and
transferred to nitrocellulose membranes. Membranes were incubated with
1° anti-mouse MAdCAM monoclonal antibody (mAb; 10 µg/ml MECA-367;
Pharmingen, San Diego, CA; see Ref. 52) or 0.05 µg/ml
anti-activated MAPK polyclonal antibody (Promega, Pittsburgh, PA). Goat
anti-rat or anti-rabbit horseradish peroxidase-conjugated 2° antibody
(Sigma) was added at a 1:2,000 dilution. Last, membranes were
developed by enhanced chemiluminescence (Amersham, La Jolla, CA). MAdCAM staining density was measured by scanning the 58- to
60-kDa band and densitometry using Image Pro Plus (Media Cybernetics, Bethesda, MD). The data are expressed as the percentage of the level of
density induced by TNF- (set at 100%). All experiments were
performed at least in triplicate.
Trypsin protection assay.
Cell surface and cytoplasmic MAdCAM were compared by trypsinizing
TNF--stimulated endothelial monolayers using the technique described
by Takeichi (53). After treatment, cells were trypsinized with 0.1% trypsin (type I; Sigma) in Hanks' balanced salt solution (HBSS) for 5 min. Trypsinized monolayers were then neutralized with 5 mg/ml soybean trypsin inhibitor for 5 min, and samples were solubilized
in electrophoresis sample buffer. These samples were Western blotted as
described and were compared with monolayers of equivalent surface area
that were not exposed to trypsin.
TK-1 lymphocyte adhesion assay.
Lymphocyte adhesion assays were performed as previously described
(50). Briefly, TK-1 cells were radiolabeled with 30 µCi Na51CrO4/ml (New England Nuclear, Natick, MA)
at 37°C for 60 min. The cells were then washed two times with
ice-cold HBSS and resuspended in HBSS. Labeled TK-1 cells were then
added to the endothelium at a lymphocyte-to-endothelial cell ratio of
5:1 (28) and were allowed to bind for 30 min. After
incubation, monolayers were washed two times with HBSS, and the
adherent TK-1 cells were solubilized with 1 N NaOH. The
51Cr activity of the labeled cells was measured by
-counting. The percentage of adhered TK-1 cells was quantified as
follows: %adhesion = {[counts/min (cpm) in adhered
cells]/(cpm of total added cells)} × 100. All data represent
the averages of four identically treated monolayers.
Electrophoretic mobility shift assay.
SVEC were pretreated for 1 h with or without inhibitors and then
were incubated for 1 h with either vehicle or TNF- (20 ng/ml). Nuclear extracts were prepared as described previously
(48). The NF-
B consensus oligonucleotide
5'-AGTTGAGGGGACTTTCCCAGGC was end labeled with
[
-32P]ATP using T4 polynucleotide kinase
according to the manufacturer's instructions (Promega). Labeled
oligonucleotide (35 fmol) was incubated with 20 µg nuclear extracts
for 10 min on ice in binding buffer [1 mM MgCl2, 0.5 mM
EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris · HCl (pH
7.5), 0.05 µg/µl poly(dI-dC) · poly(dI-dC), and 4%
glycerol] in a total volume of 30 µl. A 50-fold molar excess of
nonlabeled consensus or mutated NF-
B consensus oligonucleotide (Santa Cruz Biotechnology, Santa Cruz, CA) was included in respective reactions. Samples were subsequently incubated for 30 min at 25°C. For supershift studies, antibody specific for either the p50 (Santa Cruz Biotechnologies) or p65 (Rockland, Gilbertsville, PA) subunits was
added after the initial incubation on ice. Protein-DNA complexes were
resolved on 4% nondenaturing polyacrylamide gels by electrophoresis in
0.5× Tris-borate-EDTA. Gels were dried and visualized by a PhosphorImager (Molecular Dynamics, Hercules, CA). Activation of
NF-
B (relative to non-TNF-treated controls) was determined by
performing densitometric analysis (ImageQuant software; Molecular Dynamics, Sunnyvale, CA) on shifted bands from scanned autoradiograms. Experiments shown were repeated three times using independent extracts.
Immunofluorescence staining of MAdCAM.
IL-10(/
) mice (n = 4) were anesthetized, and 10 µg of MAdCAM mAb (Pharmingen) was injected in the jugular vein. After
5 min, the vasculature was completely flushed with bicarbonate-buffered saline, and colons were excised as previously described
(26). Frozen samples were sectioned in a cryostat at 10 µm. The sections were blocked with 10% normal donkey serum and then
incubated with a cytomegalovirus (Cy3) anti-rat secondary antibody
(Jackson Laboratories, Westgrove, PA) for 1 h. Photographs were
taken with a Sen Sys digital camera. SVEC were grown to confluency on
coverslips, stimulated with TNF-
(20 ng/ml, 24 h), and stained
for MAdCAM without fixation. Rat anti-mouse MAdCAM mAb was used at a
concentration of 10 µg/ml. Cy3 goat anti-rat secondary antibody was
used at a 1:100 dilution. The specificity of the reaction was tested by
incubation with HBSS or rat IgG1.
Statistical Analysis. All values are expressed as means ± SE. Data were analyzed using one-way ANOVA with Bonferroni's correction for multiple comparisons. Significance was accepted at P < 0.05.
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RESULTS |
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Analysis of MAdCAM expression on bEnd.3 cells.
The expression of MAdCAM protein was measured by Western blotting using
the MECA-367 mAb. MAdCAM was not constitutively expressed on
unstimulated bEnd.3 cells but was induced by TNF- (20 ng/ml; Fig.
1A). Expression of MAdCAM in
bEnd.3 cells was dose- and time-dependently increased by TNF-
, with
maximal levels at 24 h with 20 ng/ml TNF-
(data not shown).
These data are similar to previous reports (50).
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Changes in expression of MAdCAM message after TNF- stimulation.
To confirm the effects of these second message inhibitors on
expression, we next examined MAdCAM expression in the mouse high endothelial cell line SVEC4-10. MAdCAM message on SVEC was first measured by RT-PCR. Figure 2A
shows the PCR products obtained with specific primers (primers
3 and 4) that distinguish the longer and shorter
isoforms of the mouse MAdCAM message. MAdCAM message is not transcribed
in unstimulated SVEC but is induced in a time-dependent manner after
treated with mouse TNF-
(20 ng/ml). The kinetics observed for mRNA
accumulation here are consistent with those observed for expression of
MAdCAM protein. With respect to these two MAdCAM isoforms (detected as
286 and 717 bp with RT-PCR), the longer type was dominant after
stimulation with TNF-
.
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Effect of TNF- on MAdCAM expression.
Expression of MAdCAM in SVEC cells was dose- and time-dependently
increased by TNF-
, with maximal levels observed after 24 h of
incubation with 20 ng/ml TNF-
(data not shown). To ensure that the
MAdCAM determined by Western blotting was actually expressed on the
cell surface, the percentage of the total MAdCAM pool on the surface
was determined by 0.1% trypsin treatment (5 min) and immunoblotting
(Fig. 2B). Trypsin-treated cells showed significantly lower
levels of MAdCAM compared with non-trypsin-treated cells (only
15.6 ± 2.5% of controls without trypsin, P < 0.001). According to densitometry, at least 80% of the MAdCAM was
susceptible to trypsin and was therefore present on the cell surface.
Because TK-1 cell adhesion to TNF-
-treated cells was also
significantly reduced by anti-MAdCAM antibody, these data strongly
argue that MAdCAM is indeed present on the surface and is functionally active.
Effect of signal transduction blockers on SVEC.
Although the intracellular signals that mediate MAdCAM induction
have not been completely elucidated in this model, TK, MEK-1, p38 MAPK,
and NF-B have been implicated in the cytokine-induced ECAM
expression in other cell types. To investigate the signal transduction
pathways for MAdCAM, SVEC were pretreated with either the proteasome
inhibitor MG-132, the PARP inhibitor GPI-6150, the TK inhibitor
genistein, the MEK-1 inhibitor PD-98059, the p38 MAPK inhibitor
SB-202190, the nonselective PKC antagonist chelerythrin chloride, or
the PKG inhibitor KT-5823. Figure
3A shows the PCR products
obtained with primers 3 and 4 (see MATERIAL AND METHODS), which amplify the first and second IgG-like domain of MAdCAM (47). A strong MAdCAM transcript band is
detected after stimulation with TNF-
(20 ng/ml) for 12 h.
Pretreatment with 5 µM MG-132, 60 µM GPI-6150, 30 µM genistein,
20 µM PD-98058, or 10 µM SB-202190 blocked the TNF-
(20 ng/ml, 12 h)-induced MAdCAM transcript (to 11.68, 11.9, 23.85, 27.0, and 13.4% of TNF-
-treated levels, respectively).
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Inhibition of NF-kB activation.
We evaluated the effect of these inhibitors on NF-B activation using
the electrophoretic mobility shift assay using an NF-
B-radiolabeled double-stranded oligonucleotide probe. As shown in Fig. 4B,
nuclear protein from untreated (control) cells produced multiple
protein-DNA complexes, of which one complex was dramatically enhanced
in extracts from TNF-
-treated cells (compare lanes 1 and
2). The increased complex was specifically competed with a
50-fold molar excess of cold competitor (lane 3) but did not
compete with the same amount of a mutated probe (lane 4).
This suggests that the binding activity is dependent on the
NF-
B consensus site. Inclusion of either
-p50 or
-p65
(lanes 5 and 6, respectively) demonstrates that
this complex contains the activating form of NF-
B (4). Interestingly, pretreatment with PD-98059, MG-132, or GPI-6150 reduced
the TNF-
-induced activation of NF-
B by 29.9, 43.6, and 25.0%,
respectively (lanes 7-9). Pretreatment with SB-202190
had no affect on NF-
B activation (lane 10), whereas
pretreatment with genistein had a slight effect (lane 11).
MAdCAM expressed in high endothelial cells is functional.
Having established a role for several signals in the regulation of
MAdCAM expression by endothelial cells, we next examined the effects of
inhibitors on adhesion of the mouse lymphocyte cell line TK-1 to
TNF--treated SVEC. Figure 5 shows that
control adhesion of TK-1 cells was 13.1 ± 1.21%. TNF-
induced
maximal adhesion (37.3 ± 3.25%, P < 0.001 vs.
untreated control) by 24 h. This high level of adhesion was
inhibited by preincubation of SVEC with anti-MAdCAM antibody. This
antibody reduced TK-1 adhesion to 21.5 ± 1.5% (P < 0.001 vs. TNF-
treatment), showing that 65.5% of the
TNF-
-induced TK-1 adhesion was the result of MAdCAM. Treatment of
SVEC with MG-132, GPI-6150, genistein, PD-98058, or SB-202190
significantly reduced adhesion to 15.6 ± 0.96, 13.8 ± 0.66, 16.7 ± 0.60, 18.6 ± 1.01, and 12.6 ± 0.49%,
respectively, of that induced by TNF-
alone (P < 0.001). None of the inhibitors affected adhesion to untreated SVEC
(data not shown).
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Immunofluorescent staining of MAdCAM in vitro.
Immunolocalization of colonic MAdCAM in IL-10(/
) colitic mice shows
vascular staining at large mucosal and submucosal vessels. The staining
appeared to be junctional (Fig. 6,
A and B). MAdCAM expression on SVEC was assessed
by indirect immunofluorescence and was analyzed by a fluorescence
microscope (×200). Control untreated cells exhibited no staining (Fig.
6C). TNF-
(20 ng/ml, 24 h) induced a marked increase
in the immunofluorescent staining of MAdCAM on the surface and junction
to some extent (Fig. 6D). The images shown are
representative of at least 20 different fields observed in each
experiment.
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DISCUSSION |
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MAdCAM, a member of the immunoglobulin-like cell adhesion protein
superfamily, contains several structural motifs that are homologous
with ICAM-1, VCAM-1, and IgA1 and contains a unique mucin-like domain not shared by these molecules (8,
47). MAdCAM is expressed on mucosal venules and directs
lymphocytes to traffic into Peyer's patches and the intestinal
lamina propria (10). MAdCAM expression in vitro has been
previously reported in a mouse brain endothelial cell line
(50). MAdCAM appears to play critical roles in several
chronic immune and inflammatory conditions, especially IBD, through its
ability to direct 7-lymphocytes to the gut, where these
lymphocytes promote several inflammatory processes leading to IBD
(17, 56).
In this study, we have shown that MAdCAM is increased on mouse
"high" endothelial cells in vitro after TNF- stimulation, which
might represent a more relevant model for evaluating potential mechanisms of human IBD than capillary endothelium from the brain (Fig.
2). On the basis of the described role of MAdCAM in forms of chronic
gut inflammation, it is highly likely that TNF-
-induced MAdCAM
expression would be controlled through TKs, PKC, and perhaps PKG,
MAPKs, and NF-
B, as has been reported for other endothelial adhesion
molecules in colitis and IBD. Furthermore, antagonists to these
signaling cascades might provide important therapeutic targets to limit
forms of MAdCAM-dependent inflammation.
Recent studies on TNF-/NF-
B signals have revealed that
receptor-mediated stimuli that mobilize NF-
B activate several levels of protein kinases (11, 58), which result in the release
and proteasomal degradation of inhibitor protein (I)
B from the
inactive NF-
B-I
B complex. The release of I
B permits the
translocation of the activated p50/p65 NF-
B complex to the nucleus
(18). Read et al. (44) reported that MG-132,
an inhibitor of the proteasome, prevented I
B
degradation induced
by TNF-
and nuclear accumulation of NF-
B, efficiently inhibiting
NF-
B function.
Oliver et al. (38) and Hassa and Hottiger
(22) reported that PARP is also required for the
NF-B-dependent transcription using both PARP gene-deficient mice and
cells. Interestingly, Hassa and Hottiger reported that PARP is actually
a novel binding partner for NF-
B and, apparently, must be activated
to activate NF-kB activation. Taken together, our data with MG-132 and
GPI-6150 strongly suggest that proteasome activity and PARP are
necessary for maximal NF-
B activation in the TNF-
-mediated
induction of MAdCAM in this model.
We also demonstrated that MAdCAM transcription and translation are
regulated through the TK MEK-1, p38, p42/44 MAPK, and NF-B signal
transduction cascades (Figs. 1-4). PKC and PKG inhibitors did not
influence MAdCAM expression. Our data on the regulation of MAdCAM
expression in SVEC cells parallel studies of ICAM-1 and VCAM-1
regulation in human umbilical vein endothelial cells. As shown for
SVEC, ICAM-1, and VCAM-1, expression is upregulated by TNF-
through
TK and NF-
B signal transduction cascades (58) involving
TNF-
-induced ERK1 activation (which is inhibited by genistein; see
Ref. 31). The effects of the second message inhibitors on
MAdCAM mRNA induction are also the same in both bEnd.3 and SVEC cells.
However, the effects of genistein and PD-98059 differ slightly in
bEnd.3 cells and SVEC, suggesting that the signal transduction pathways
in TNF-
-induced MAdCAM expression in these two cell types may differ
somewhat in their sensitivity to TK/MEK-1 inhibition.
Interestingly, in previous reports, ERK1 inhibition did not block the
upregulation of ICAM-1 in IL-1-treated cardiac myocytes and
fibroblasts (24). In that study, ERK1 inhibition prevented both TNF-
-induced NF-
B DNA binding and MAdCAM synthesis.
Therefore, these data suggest that ERK1 activation precedes NF-
B
activation in MAdCAM mobilization.
It has recently been reported that p38 MAPK may be required for
NF-B-dependent gene expression (11), and inhibition of the p38 MAPK pathway prevented IL-1
-induced ICAM and VCAM
translation (24). Here we have shown that, in addition to
NF-
B, p38 MAPK was necessary for MAdCAM expression. Our data
also indicate that p38 MAPK inhibition can prevent MAdCAM
expression induced by TNF-
at the level of transcription but did not
interfere with NF-
B binding to DNA. The effect of the p38 blocker
SB-202190 was restricted to the inhibition of
trans-activating activity, since activation of NF-
B DNA
binding (as measured in gel assays) was unaffected. These data support
the model proposed by Beyaert et al. (7), which indicated
that p38 MAPK was not involved in the release of I
B into the
cytoplasm, and the work by Wesselborg et al. (59) and
Schulze-Osthoff et al. (49), which both showed that p38 MAPK could activate MAPKAPK, which may function as an NF-
B
transactivation factor at the nuclear level. Taken together,
our observations suggest that several forms of MAPK activation are
important in regulating NF-
B activity and MAdCAM expression.
Apparently, p38 and NF-
B involve separate pathways, and p38 may
govern other coactivating transcription factors, e.g., p300
(40) or A20 (23), which govern
NF-
B-mediated transcription but not DNA binding.
With respect to PKC, although previous studies show that direct PKC
activation (with phorbol esters) increases E-selectin, VCAM-1, and
ICAM-1 expression in endothelial cells (31), TNF-- and
IL-1
-induced expression of E-selectin and ICAM-1 are, however, independent of PKC activation (34, 45). Our data agree
with those findings and fail to show an important role for PKC in
MAdCAM regulation, as previously reported in brain endothelial cells (50).
Although our Western blotting data indicate an increase in absolute
MAdCAM expression, it is possible that this MAdCAM might not be present
on the cell surface or be functional. To address this concern, both
total and cell surface MAdCAM pools were determined by trypsin
treatment. Trypsin treatment removed at least 80% of the total MAdCAM
present on the cell surface induced by TNF- (Fig. 2B).
These observations are further supported by our observations that TK-1
cell adhesion (an
4
7-expressing
lymphocyte line) was significantly reduced by addition of an
anti-MAdCAM antibody (Fig. 5). Similarly, immunofluorescence staining
of MAdCAM was detected without prior cell permeabilization (Fig.
6B). We also found that MAdCAM antibody reduced maximal TK-1
cell adhesion to SVEC induced by TNF-
but that basal adhesion was
unaffected by blocking antibody. With the use of MAdCAM antibody to
block adhesion, >60% of the TNF-
-stimulated adhesion of TK-1 cells
was prevented by anti-MAdCAM, as reported by Sikorski et al.
(50) in bEnd.3 cells.
Another interesting finding in this study was the distribution of
MAdCAM on the cell surface. We showed that TNF--induced MAdCAM
expression often appeared to be concentrated at cell-cell junctions
(Fig. 6). This result is supported by the study of Kawachi et al.
(26), which first showed MAdCAM expressed on dilated mucosal and submucosal vessels in IL-10(
/
) mouse colon in what appears to be cell-cell junctions. This is the first report to confirm
this junctional localization of MAdCAM in vitro.
It has been demonstrated that MAdCAM serves as the main binding partner
for 4
7-integrin on lymphocytes and plays
a role in L-selectin-mediated lymphocyte rolling (5, 6, 16, 19, 57). However, our results show that this junctional MAdCAM
appearance is not uniform on all cells. Explanations for the lack of
homogeneous junctional staining could be cell alterations induced by
transformation or the degree of culture confluency. The possible roles
of MAdCAM in endothelial junctions still remain to be elucidated.
In conclusion, the results of our studies show that MAdCAM expression
is increased on high endothelial cells by TNF- and that this
TNF-
-induced MAdCAM expression is mediated through TK, p38, p42/44
MAP, and NF-
B signal transduction cascades. The increased MAdCAM
expression on these endothelial cells may model the chronic
inflammation caused by lymphocyte immune response seen in IBD. Recent
studies show that NF-
B antagonist NF-
B antisense phosphorothioate
oligonucleotide and 26S proteosome inhibitor can successfully attenuate
inflammation in experimental colitis in vivo (14, 33, 36).
In our studies, although we have not considered the expression of
VCAM-1 in our cells, these data suggest that MAdCAM might be yet
another target of NF-
B inhibition. Although we may speculate that
additional as-yet-undefined signals may be required for the maximal
induction of these genes during exposure to cytokines, it is likely
that inhibitors of TKs, MAPKs, and NF-
B could be effective in
limiting the inflammation associated with human IBD, which is mediated
by upregulation of MAdCAM.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grants HL-47615, DK-43785, and DK-47663.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. S. Alexander, Molecular and Cellular Physiology, Louisiana State Univ. Health Sciences Center, 1501 Kings Hwy., Shreveport, LA, 71130-3932 (E-mail: jalexa{at}lsuhsc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 March 2001; accepted in final form 14 May 2001.
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