| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Centre Nationale de la Recherche Scientifique, Unité Propre de Recherche 0415, Institut Cochin de Génétique Moléculaire, Paris, France; and
Institute of Ophthalmology, University College London, London, United Kingdom
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Lß2
and 4ß1, and endothelial transmembranous
proteins of the Ig superfamily, ICAM-1 (CD54) and VCAM-1 (CD106),
respectively (3, 4). Adhesion to the endothelium profoundly regulates a
variety of biochemical processes within leukocytes, including
phosphorylation of proteins, cytoskeletal modifications, and gene
regulation (reviewed in 5 . Indeed, over the last few years, it
has become clear that in addition to their role in cell-cell adhesion,
integrins are able to transduce intracellular signaling within
leukocytes (6, 7, 8).
Much less is known about the consequences of this cell-cell interaction
on endothelial cell physiology, especially at the blood-brain barrier.
ICAM-1 is expressed at the luminal surface of endothelial cells and
seems to be directly involved in the migration of leukocytes to
inflammatory sites in the brain (9) or the retina (10). However, these
molecules do not appear to simply provide points of attachment. In
addition, the ICAM-1 intracellular domain has been shown to interact
with cellular filamentous actin (11) and cytoskeleton-associated
proteins such as -actinin and ezrin (12, 13). Furthermore,
following ICAM-1 cross-linking by specific Abs or by activated T
lymphocytes, actin-based cytoskeleton rearrangements, stimulation of
Src kinase activity, and tyrosine phosphorylation of the actin binding
protein cortactin and of several yet unidentified proteins have been
observed in brain endothelial cells
(14).3 Among candidate
tyrosine-phosphorylated proteins are the cytoskeleton-associated
proteins, Focal adhesion kinase
(FAK),4 paxillin, and
p130Cas (Cas).
FAK, paxillin, and Cas, as well as cortactin, were originally identified as tyrosine-phosphorylated proteins in v-Src-transformed cells (15). They have also been identified as tyrosine-phosphorylated proteins in integrin-activated cells (16, 17). FAK is a widely expressed nonreceptor protein tyrosine kinase (18) that participates in the recruitment of signaling proteins to the focal adhesions of adherent cells and in tyrosine phosphorylation of paxillin (16, 19). Cas is an SH3-containing protein with a cluster of multiple putative SH2 binding motifs, suggesting its role as a docking protein for multiple SH2-containing molecules, including Src and Crk (20).
It is most likely that reorganization of the endothelial cell cytoskeletal architecture is required during leukocyte migration. Such remodeling will involve actin cytoskeleton, which is known to be under the regulation of small GTP binding proteins. Among these proteins, Rho has been shown previously to mediate stress fiber and focal adhesion formation (21). It has been proposed that Rho-regulated signal transduction pathway can lead to tyrosine phosphorylation of FAK paxillin and Cas (22). These observations suggest a possible role for Rho in mediating signaling responses following ICAM-1 activation in brain endothelial cells.
In the present study, using two extensively characterized rat brain endothelial cell lines, RBE4 and GP8 cells, which maintain in culture the differentiated phenotype of cerebral endothelium (23, 24, 25, 26), we show that ICAM-1 cross-linking induces several cellular events associated with Rho activation: FAK, paxillin, and Cas tyrosine phosphorylation; stimulation of the Crk signaling pathway; and JNK activation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Mouse mAbs to rat ICAM-1 1A29 and 3H8 were respectively
purchased from Serotec (Wiesbaden, Germany) and provided by Dr. W. F.
Hickey (Dartmouth Medical School, Hanover, NH). Rabbit
anti-mouse Abs (RAM) were obtained from Dako (Trappes, France).
Mouse mAbs specific for phosphotyrosine, Src, and ERK-2 and rabbit
polyclonal Abs specific for SOS and Shc were purchased from Upstate
Biotechnology (Lake Placid, NY). Mouse mAbs specific to Cas and
paxillin and rabbit polyclonal Abs to FAK, C3G, and ERK-2 were obtained
from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-JNK Ab was a
generous gift of Dr. J. Kyriakis (Massachusetts General Hospital,
Charlestown, MA). The GST fusion protein containing Src-SH2 was
provided by Dr. M. Resh (Memorial Sloan-Kettering Cancer Center, New
York, NY) (27). GST-Grb2 was constructed as previously described (28).
GST fusion proteins containing Crk or Crk-SH2 domain were provided by
Dr. S. Fischer (Institut Cochin de Génétique
Moléculaire, Paris, France), GST-RBD was supplied by J. L.
Bos (Utrecht University, Utrecht, The Netherlands), and GST-c-Jun was
supplied by Dr. M. Karin (University of California, San Diego, CA). The
pGEX-2T-C3 construct was a gift from L. A. Fieg (Tufts University,
Boston, MA). [
-32P]ATP (3000 Ci/mmol) was obtained
from DuPont de Nemours (Les Ulis, France).
Endothelial cell cultures
RBE4 cells were isolated from rat brain cortex and immortalized
with the plasmid pE1A-neo, containing the adenovirus
E1A-encoding sequence followed by a neomycin resistance gene, and have
been extensively characterized (23, 24, 25). GP8 cells were isolated from
rat brain cortex and immortalized with SV40 large T Ag (26). RBE4 and
GP8 cells were grown in the same conditions, as previously described
(14). RBE4 or GP8 cells were seeded at a density of 104
cells/cm2 and after 3–4 days in culture were incubated
with serum- and basic fibroblast growth factor-free culture medium
containing 100 U/ml IFN-, for 48 h. Cells were washed in PBS
before treatments. Cross-linking of adhesion molecules was performed by
treatment with specific mAbs for 30 min and subsequently with RAM Abs
for indicated periods of time.
Expression and purification of C3 exoenzyme
GST-C3 was expressed in Escherichia coli for 5 h using 1 mM isopropyl-ß-D-thiogalactopyranoside. Cells were harvested by centrifugation at 4,000 x g for 15 min and were sonicated three times for 5 min each time in lysis buffer (50 mM Tris-HCl (pH 8), 50 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 1 mM 4-(2-aminoethyl)benzene-sulfonyl fluoride (AEBSF). Bacterial lysates were then centrifuged at 10,000 x g for 30 min, and the supernatant was chromatographed over glutathione-agarose. The column was washed with 5 vol of AEBSF-free lysis buffer and 3 vol of thrombin cleavage buffer (50 mM Tris-HCl (pH 8), 150 mM NaCl, 5 mM MgCl2, 2.5 mM CaCl2, and 1 mM DTT). Bovine plasma thrombin (10 U/ml gel) was added to the column for 16 h at 40°C. The eluate from the column was collected and subsequently washed with 3 vol of PBS. Thrombin was removed from C3 exoenzyme protein released from the column by chromatography over p-aminobenzamidine-agarose and dialyzed into PBS. C3 exoenzyme was then concentrated in ultrafiltration units (Amicon, Beverley, MA). This procedure produced pure C3 exoenzyme protein as assessed by SDS-PAGE. The protein concentration was quantified using bicinchoninic acid reagent (Pierce, Rockford, IL).
[-32P]ADP-ribosylation of Rho in RBE4 endothelial
lysates with C3 exoenzyme
Cells were lysed in 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 2 mM
MgCl2, and 1 mM AEBSF, and protein concentrations
were determined using bicinchoninic acid reagent. Twenty-five
milligrams of lysate protein was added to reactions containing 50 mM
Tris-HCl (pH 7.4), 1 mM EDTA, 2 mM MgCl2, 0.5 mM ATP, 0.3
mM GTP, 1 mM AEBSF, 5 µCi/ml [-32P]NAD, and
250 ng/ml recombinant C3 transferase and incubated at 37°C for 1
h. Reactions were stopped by the addition of 2 vol of 30% (w/v) TCA on
ice for 30 min. Precipitated proteins were separated by centrifugation
at 14,000 x g for 10 min, and pellets were washed
three times with ethanol at -20°C. Pellets were dried; solubilized
in 10 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.4% DTT, and
0.1% bromophenol blue; and proteins were resolved on 15% SDS-PAGE.
Immunoprecipitation of endothelial rho proteins and guanine nucleotide analysis
Serum-starved RBE4 (5 x 106) cells were incubated in phosphate-free DMEM overnight in the presence of 0.2 mCi/ml [32P]orthophosphate. Cells were washed in HBSS and recultured in serum-free DMEM. After addition of stimuli, cells were lysed in 100 mM HEPES (pH 7.4), 2% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 300 mM NaCl, 10 mM MgCl2, 2 mM EGTA, 2 mg/ml BSA, and protease inhibitors (20 mM benzamidine, 20 µg/ml leupeptin, 20 µg/ml pepstatin, 20 µg/ml aprotinin, 20 µg/ml soybean trypsin inhibitor, and 2 mM AEBSF. Lysates were centrifuged at 14,000 x g for 5 min to sediment nuclei, and supernatants were adjusted to 500 mM NaCl. Lysates were immunoprecipitated by incubation with 4 µg of rabbit polyclonal anti-Rho (Santa Cruz Laboratories, catalogue no. sc-179) for 2 h at 4°C followed by incubation with 50 µl of 50% protein G-Sepharose (Pharmacia, Piscataway, NJ) for 2 h at 4°C. Immunoprecipitates were washed eight times with 1 ml of 50 mM HEPES (pH 7.4), 0.005% SDS, 500 mM NaCl, and 0.1% Triton X-100. Immunoprecipitates were subsequently heated to 68°C for 20 min in the presence of 5 mM EDTA, 2 mM DTT, 0.2% SDS, 0.5 mM GTP, and 0.5 mM GDP to elute [32P]phosphate-labeled nucleotides. Nucleotides were separated by TLC on 0.1-mm poly(ethyleneimine) cellulose plates in 1.2 M ammonium formate/0.8 M HCl (45) and autoradiographed at -70°C using Fuji-RX film (Fuji, Tokyo, Japan). Autoradiographs were subsequently used as templates. Radioactive areas that comigrated with GTP and GDP standards were scraped, and radioactivity was determined by scintillation spectrometry.
SDS-PAGE and immunoblotting
RBE4 or GP8 cells were washed with ice-cold PBS containing 1 mM orthovanadate and lysed with SDS sample buffer (100 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 1 mM orthovanadate, and 100 mM DTT with bromophenol blue). SDS-PAGE and immunoblotting were performed as previously described (29).
Immunoprecipitation
Cells were washed with ice-cold PBS containing 1 mM orthovanadate and were lysed for 30 min at 4°C in Nonidet P-40 buffer (10 mM Tris-HCl (pH 7.5), 140 mM NaCl, 1 mM orthovanadate, and 1% Nonidet P-40 with a mix of protease inhibitors: 2 mM PMSF, 5 mM EDTA, 10 µg/ml leupeptin, 1 µg/ml pepstatin, and 10 µg/ml aprotinin). Nuclei were discarded following centrifugation at 10,000 x g for 10 min. Lysates were incubated overnight at 4°C with specific Abs and subsequently for 2 h with agarose or RAM-coated agarose, respectively. Immunoprecipitates were collected by centrifugation and were extensively washed in Nonidet P-40 buffer. Immunoprecipitated proteins were eluted with SDS-sample buffer and analyzed by 10% SDS-PAGE followed by immunoblotting.
GST fusion protein binding
Expression of GST fusion proteins was induced by the addition of 1 mM isopropyl-ß-D-thiogalactopyranoside, and fusion proteins were isolated from bacterial lysates by affinity chromatography using glutathione-agarose beads. GST-C3 transferase was eluted from glutathione-agarose following cleavage with thrombin. The purity of GST fusion proteins was assessed with SDS-PAGE (not shown).
After treatment, cells (6 x 106) were lysed in Nonidet P-40 buffer. Cell lysates were incubated for 14 h at 4°C with GST fusion proteins immobilized on glutathione-agarose beads. The agarose beads were washed several times in Nonidet P-40 buffer containing 1% sodium deoxycholate and decreasing concentrations of NaCl (300, 150, and 10 mM). Bound proteins were eluted with SDS-sample buffer and analyzed by immunoblotting.
JNK kinase assay
After stimulation, cells (6 x 106) were washed
with ice-cold PBS and lysed at 4°C for 30 min in 200 µl of lysis
buffer (25 mM HEPES (pH 7.7), 300 mM NaCl, 4.5 mM MgCl2, 20
mM ß-glycerophosphate, 0.1 mM orthovanadate, 1% Triton X-100, and
the mixture of protease inhibitors indicated above). After
centrifugation (10 min, 10,000 x g), supernatants were
added to 600 µl of a solution containing 20 mM HEPES (pH 7.7), 2.5 mM
MgCl2, 20 mM ß-glycerophosphate, 0.1 mM orthovanadate,
1% Triton X-100, and protease inhibitors containing 0.3 mM EDTA. After
10-min incubation and centrifugation (10 min, 10 000 x
g) at 4°C, cleared lysates were rocked overnight with
GST-c-Jun fusion protein bound to glutathione-agarose beads. Beads were
washed four times with a buffer containing 2 mM HEPES (pH 7.7), 50 mM
NaCl, 25 mM MgCl2, 0.1 mM EDTA, and 0.05% Triton X-100.
Samples were resuspended in 50 µl of JNK kinase reaction buffer (2 mM
HEPES (pH 7.7), 20 mM MgCl2, 2 mM DTT, 20 mM
ß-glycerophosphate, 20 mM paranitrophenylphosphate, 0.1 mM
orthovanadate, 20 µM ATP, and 4 µCi [-32P]ATP).
After 20-min incubation at 30°C, samples were centrifuged, and
reactions were stopped by addition of 50 µl of 2x SDS sample buffer.
Samples were heated at 95°C for 10 min, analyzed by SDS-PAGE, and
transfered to nitrocellulose. Autoradiography of the blot was performed
with an intensifying screen.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To investigate tyrosine phosphorylation of potential cellular Src
substrates in response to ICAM-1 cross-linking, brain endothelial RBE4
and GP8 cells were treated with anti-ICAM-1 Ab, then with RAM Abs
for 30 min (14). After treatments, cell lysates were incubated with
anti-FAK, anti-paxillin, or anti-Cas Abs, and
immunoprecipitates were subjected to immunoblot analysis with
anti-phosphotyrosine Abs (Fig. 1
A, upper panels).
In both cell lines, tyrosine phosphorylation of FAK, paxillin, and Cas
was clearly enhanced in lysates from cells treated with anti-ICAM-1
Ab cross-linked with RAM, compared with that in lysates from nontreated
cells or cells treated with anti-ICAM-1 or RAM Abs alone.
Immunoblot analysis with Abs against FAK, paxillin, or Cas revealed
similar amounts of proteins loaded in all lanes (Fig. 1A, lower panels). As previously reported,
paxillin exhibited a diffused pattern, and Cas was found to migrate as
two bands, presumably due to differences in post-translational
modifications. The specificity of these phosphorylations was confirmed
by the observation that an additional anti-ICAM-1 mAb, 3H8 induced
similar responses, whereas cross-linking of the transferrin receptor,
another cell surface transmembrane protein, or incubation with an
isotype-matched irrelevant Ab did not (data not shown).
|
B). Tyrosine
phosphorylation of all three proteins was detectable 5 min after ICAM-1
cross-linking, reaching a maximal level between 15 and 30 min, then
decreasing at 60 min. Control immunoblots with anti-FAK, paxillin,
and Cas Abs demonstrated that equal protein amounts of protein were
immunoprecipitated in all cases (data not shown). The kinetics of these
phosphorylations were similar to those of cortactin phosphorylation
(14). Also, they occurred concomitantly with Src activation, and the
tyrosine-phosphorylated proteins were capable of binding the SH2 domain
of Src in vitro (data not shown), as previously described (20, 27, 30). These results demonstrate that ICAM-1 cross-linking induces concomitant tyrosine phosphorylation of FAK, paxillin, and Cas in RBE4 and GP8 cells.
Tyrosine phosphorylation of FAK, paxillin, and Cas is associated with Rho activation
Since FAK, paxillin, and Cas are proteins known to localize at
focal adhesions, and Rho proteins have been implicated in the formation
of actin stress fibers, focal adhesions, we investigated whether ICAM-1
cross-linking can induce Rho activation in RBE4 cells. Compared with
nontreated cells, an increase in GTP-loaded Rho proteins was observed
in response to ICAM-1 cross-linking (Fig. 2A). Sample were normalized
for the amount of Rho immunoprecipitated, and quantitative analysis
indicated a twofold increase in Rho-GTP loading following ICAM-1
cross-linking (Fig. 2A: GTP/GDT + GTP = 0.44 in control
cells and 0.9 in ICAM-1-cross-linked cells). This observation was
confirmed in GP8 cells, strongly suggesting that ICAM-1 cross-linking
induces Rho activation in brain endothelial cells.
|
B, left lane) (33). In RBE4 cells after
pretreatment with C3 (50 µg/ml) for 8 h, we observed that Rho
could not be further ADP-ribosylated in the presence of
[32P]NAD (Fig. 2B, right lane).
This result indicates that C3 pretreatment (50 µg/ml, 8 h) of
RBE4 cells was sufficient to provoke the full inactivation of Rho.
To investigate the role of Rho in FAK, paxillin, and Cas
phosphorylation induced by ICAM-1 cross-linking, cells were pretreated
cells with C3 exoenzyme. In these conditions, ICAM-1 cross-linking
failed to induce tyrosine phosphorylation of FAK, paxillin, and Cas
(Fig. 2C). In contrast, C3 pretreatment did not abolish
ICAM-1-induced cortactin phosphorylation (not shown), indicating that
C3 pretreatment differentially effect the various ICAM-1-coupled
events.
Coimmunoprecipitation of FAK, paxillin, and Cas
Western blot analysis of FAK immunoprecipitates from RBE4 cells
activated by ICAM-1 cross-linking revealed coimmunoprecipitated
tyrosine-phosphorylated proteins of 68–70 and 130 kDa (Fig. 3, upper panels). These
phosphorylated proteins comigrated with paxillin and Cas, respectively.
Reciprocally, these three proteins, FAK, paxillin, and Cas, were
detected in paxillin as well as Cas immunoprecipitates from activated
cells. None of these proteins could be detected in immunoprecipitates
using irrelevant Abs (data not shown), excluding the possibility that
these proteins were nonspecifically trapped. These results indicate
that in parallel with their tyrosine phosphorylation, FAK, paxillin,
and Cas can associate in bi- or trimolecular complexes upon ICAM-1
cross-linking.
|
Phosphorylated tyrosine residues are potential binding sites for
SH2 domain-containing proteins such as the adaptor proteins Grb-2 and
Crk. To assess whether these adaptor proteins were involved in
ICAM-1-coupled signaling cascades, we determined the capacity of FAK,
paxillin, and Cas to interact with GST-Grb2 or GST-Crk fusion proteins
(Fig. 4A). Following ICAM-1
cross-linking, Cas and paxillin were retained on GST-Crk, but not on
GST-Grb-2 (Fig. 4A, left
panels). Under these conditions, no association of FAK with either
Grb-2 or Crk was detectable. These results were confirmed by the
observation that another anti-ICAM-1 Ab, 3H8, induced similar
responses (not shown). Treatment of cells with anti-ICAM-1 or RAM
alone did not lead to any association of these proteins with either
GST-Crk or GST-Grb-2 (not shown). Moreover, we observed that paxillin
and Cas bound to GST-Crk-SH2 (Fig. 4A, right
panels) concomitantly with their tyrosine phosphorylation (not
shown). These results indicate that upon ICAM-1 cross-linking,
tyrosine-phosphorylated paxillin and Cas can associate with Crk via its
SH2 domain, while FAK apparently does not couple to any of the
Crk or Grb-2 signaling pathways. Consistent with our observation that
C3 exoenzyme pretreatment inhibits ICAM-1-induced tyrosine
phosphorylation of paxillin and Cas (Fig. 2C), we observed
that this pretreatment also completely abolished the binding of these
proteins to Crk (Fig. 4C).
|
B, left panel). As
expected, no interaction of these proteins was observed with the SH2
domain of Crk.
Analysis of proteins coimmunoprecipitated with paxillin or Cas
following ICAM-1 cross-linking revealed the association of C3G with Cas
(Fig. 4D). This result further demonstrates activation of
the Crk signaling pathway following ICAM-1 cross-linking and points to
C3G as a main downstream element of this pathway.
ICAM-1 cross-linking induces activation of JNK but has no effect on the Erk pathway
Since small G protein are known as upstream regulator of
mitogen-activated protein kinases (Erk and JNK), we investigated the
effect of ICAM-1 cross-linking on these pathways. We first analyzed the
activity of the small G protein Ras, which is an upstream regulator of
mitogen-activated protein kinase cascades. Ras activity was determined
by analyzing its ability to bind the RBD of Raf. Whatever the duration
of the treatment, ICAM-1 cross-linking did not induced any detectable
Ras binding to GST-RBD, whereas epidermal growth factor stimulation did
(Fig. 5). Analysis of Erk-2 kinase activity did not reveal any
significant Erk-2 activation upon ICAM-1 stimulation (data not shown).
|
A). JNK activation was
detectable 15 min after ICAM-1 cross-linking and reached a maximum at
30 min, declining at 60 min (Fig. 6B). The amount of JNK
retained on GST-Jun beads was controlled by immunoblotting
(lower panels). Treatment with anti-ICAM-1 or RAM
Abs alone as well as cross-linking of an irrelevant, isotype-matched Ab
by RAM (not shown) did not induce any detectable elevation in JNK
activity. Investigating the role of Rho in JNK activation, we showed
that C3 pretreatment of cells completely abolished JNK activation
induced by ICAM-1 cross-linking (Fig. 6C). In contrast, C3
pretreatment had no effect on the epidermal growth factor-induced
Ras/Erk pathway (not shown), confirming the specificity of the toxin
toward Rho. Taken together, these results strongly suggest that in
brain endothelial cells, ICAM-1 cross-linking induces JNK activation
via a Ras-independent and Rho-dependent pathway.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
As previously reported for focal adhesion formation, FAK may be
responsible for the recruitment of Src, Cas, and paxillin and their
association (6, 35). We have observed that phosphorylation of FAK,
paxillin, and Cas correlates with their ability to bind the SH2 domain
of Src (data not shown) and occurred concomitantly with Src activation
(14). Our data indicate that FAK, Cas, and paxillin
coimmunoprecipitate, reflecting their stable association within the
cell upon ICAM-1 cross-linking. These results show, for the first time,
that engagement of an adhesion molecule of the CAM family can induce
responses of the same kind as those observed following integrin
stimulation (36). Clustering of ICAM-1 molecules occurs at the luminal
membrane of endothelial cells, not at focal adhesions, suggesting that
a diffusible fraction of FAK, paxillin, and Cas may be recruited by
ICAM-1 cross-linking to the luminal face of brain endothelial cells.
Such a hypothesis has been previously proposed in endothelial cells for
clustering of luminal vß3 integrins (37).
Alternatively, cytoskeletal rearrangements may be involved in a
functional connection between luminal ICAM-1 molecules and tyrosine
phosphorylation at focal adhesions, where clustering of
ß1 integrins is associated with transient tyrosine
phosphorylation of multiple proteins, including FAK, paxillin, and Cas
(16, 17). It has been shown that the ICAM-1 intracellular domain is
directly linked to cytoskeleton-associated proteins -actinin and
ezrin (12, 13). Interestingly, recent observations indicate that
proteins of the ERM family can initiate the activation of Rho through
interaction with Rho GDP dissociation inhibitor (38). We have recently
observed that ICAM-1 cross-linking induced actin-stress fiber formation
in a Rho-dependent manner (see Footnote 3), which suggests that these
cytoskeletal modifications may be responsible for tyrosine
phosphorylation of FAK, paxillin, and Cas at the level of focal
adhesions.
Tyrosine phosphorylation of paxillin and Cas allows for the binding of these proteins with the adaptor protein Crk and indirectly with the exchange factors SOS and C3G. Since the only association detected by coimmunoprecipitation was among Cas, Crk, and C3G, it is tempting to consider this complex as the major if not the only one induced by ICAM-1 cross-linking. Indeed, Ras, a known target of SOS, was not activated after ICAM-1 cross-linking. Accordingly, we did not detect any significant activation of Erk. In contrast, in parallel with the C3-sensitive activation of the Crk signaling pathway, we observed a C3-sensitive activation of JNK. These observations suggest that in the ICAM-1 signaling cascade, JNK is downstream of the guanine nucleotide exchange protein C3G, as recently reported in transfected NIH-3T3 cells (39). Moreover, our results are in agreement with the recent report that C3G-mediated JNK activation is Ras independent (40).
Taken together, our results may explain the fundamental role of ICAM-1
in T lymphocyte migration through brain endothelium in vitro (10).
Since JNK participates in the transcriptional regulation of multiple
genes by phosphorylating several transcription factors (41), ICAM-1
cross-linking in brain endothelial cells might transcriptionally
regulate the expression of various agents, such as IL-1ß or IL-6,
which are known to increase blood-brain barrier permeability (42). In
addition, interendothelial junctional complexes can be directly
affected by JNK activation and tyrosine phosphorylation of
cytoskeleton-associated proteins, as shown for adherens junctions in
epithelial cells (43, 44). In line with these observations, our results
suggest that ICAM-1 cross-linking can contribute to cell junction
destabilization and blood-brain barrier opening via a Rho-dependent
mechanism. Our recent observation that this pathway is involved in T
lymphocyte migration through brain endothelium supports this hypothesis
(see footnote 3). Extrapolation of our in vitro data to an in vivo
situation would suggest that in inflammatory situations such as viral
infection or multiple sclerosis, ICAM-1 engagement with
Lß2-expressing activated lymphocytes might
support lymphocyte trafficking to the brain by triggering signal
transduction cascades in brain endothelial cells.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Sandrine Etienne, UPR 0415, Institut Cochin de Génétique Moléculaire, 22 rue Mechain, 75014 Paris, France. E-mail address:
3 P. Adamson, S. Etienne, P.-O. Couraud, V. Calder, and J. Greenwood. T-lymphocyte migration through CNS endothelial cells involves signaling through endothelial ICAM-1 via a Rho-dependent pathway. Submitted for publication.
4 Abbreviations used in this paper: FAK, focal adhesion kinase; Cas, p130Cas; RAM, rabbit anti-mouse; Erk, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; GST, glutathione S-transferase; RBD, Ras-binding domain; NAD, nicotinamide-adenine dinucleotide phosphate; AEBSF, 4-(2-aminoethyl)benzene-sulfonyl fluoride.
Received for publication February 27, 1998. Accepted for publication July 13, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-actinin. J. Cell Biol. 118:1223.vß3 integrin mediate enhances tyrosine phosphorylation of multiple proteins in adherent bovine pulmonary artery endothelial cells. J. Biol. Chem. 270:16781.This article has been cited by other articles:
|
|
 |
|
  C. Rampon, N. Weiss, C. Deboux, N. Chaverot, F. Miller, D. Buchet, H. Tricoire-Leignel, S. Cazaubon, A. Baron-Van Evercooren, and P.-O. Couraud Molecular Mechanism of Systemic Delivery of Neural Precursor Cells to the Brain: Assembly of Brain Endothelial Apical Cups and Control of Transmigration by CD44 Stem Cells, July 1, 2008; 26(7): 1673 - 1682. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. J. Kelly, P. Wu, C. E. Patterson, C. Temm, and J. H. Dominguez LOX-1 and inflammation: a new mechanism for renal injury in obesity and diabetes Am J Physiol Renal Physiol, May 1, 2008; 294(5): F1136 - F1145. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Yamamoto, S. H. Ramirez, S. Sato, T. Kiyota, R. L. Cerny, K. Kaibuchi, Y. Persidsky, and T. Ikezu Phosphorylation of Claudin-5 and Occludin by Rho Kinase in Brain Endothelial Cells Am. J. Pathol., February 1, 2008; 172(2): 521 - 533. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Allingham, J. D. van Buul, and K. Burridge ICAM-1-Mediated, Src- and Pyk2-Dependent Vascular Endothelial Cadherin Tyrosine Phosphorylation Is Required for Leukocyte Transendothelial Migration J. Immunol., September 15, 2007; 179(6): 4053 - 4064. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. van Buul, E. Kanters, and P. L. Hordijk Endothelial Signaling by Ig-Like Cell Adhesion Molecules Arterioscler. Thromb. Vasc. Biol., September 1, 2007; 27(9): 1870 - 1876. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Rao, L. Yang, G. Garcia-Cardena, and F. W. Luscinskas Endothelial-Dependent Mechanisms of Leukocyte Recruitment to the Vascular Wall Circ. Res., August 3, 2007; 101(3): 234 - 247. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-M. Oh, S. Lee, B.-R. Na, H. Wee, S.-H. Kim, S.-C. Choi, K.-M. Lee, and C.-D. Jun RKIKK Motif in the Intracellular Domain Is Critical for Spatial and Dynamic Organization of ICAM-1: Functional Implication for the Leukocyte Adhesion and Transmigration Mol. Biol. Cell, June 1, 2007; 18(6): 2322 - 2335. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Celli, J.-J. Ryckewaert, E. Delachanal, and A. Duperray Evidence of a Functional Role for Interaction between ICAM-1 and Nonmuscle {alpha}-Actinins in Leukocyte Diapedesis J. Immunol., September 15, 2006; 177(6): 4113 - 4121. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Schreibelt, R. J. P. Musters, A. Reijerkerk, L. R. de Groot, S. M. A. van der Pol, E. M. L. Hendrikx, E. D. Dopp, C. D. Dijkstra, B. Drukarch, and H. E. de Vries Lipoic Acid Affects Cellular Migration into the Central Nervous System and Stabilizes Blood-Brain Barrier Integrity J. Immunol., August 15, 2006; 177(4): 2630 - 2637. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Persidsky, D. Heilman, J. Haorah, M. Zelivyanskaya, R. Persidsky, G. A. Weber, H. Shimokawa, K. Kaibuchi, and T. Ikezu Rho-mediated regulation of tight junctions during monocyte migration across the blood-brain barrier in HIV-1 encephalitis (HIVE) Blood, June 15, 2006; 107(12): 4770 - 4780. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Cernuda-Morollon and A. J. Ridley Rho GTPases and Leukocyte Adhesion Receptor Expression and Function in Endothelial Cells Circ. Res., March 31, 2006; 98(6): 757 - 767. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. C. Newcomb, U. Sajjan, S. Nanua, Y. Jia, A. M. Goldsmith, J. K. Bentley, and M. B. Hershenson Phosphatidylinositol 3-Kinase Is Required for Rhinovirus-induced Airway Epithelial Cell Interleukin-8 Expression J. Biol. Chem., November 4, 2005; 280(44): 36952 - 36961. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Yang, R. M. Froio, T. E. Sciuto, A. M. Dvorak, R. Alon, and F. W. Luscinskas ICAM-1 regulates neutrophil adhesion and transcellular migration of TNF-{alpha}-activated vascular endothelium under flow Blood, July 15, 2005; 106(2): 584 - 592. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Cook-Mills and T. L. Deem Active participation of endothelial cells in inflammation J. Leukoc. Biol., April 1, 2005; 77(4): 487 - 495. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. E. Gegg, R. Harry, D. Hankey, H. Zambarakji, G. Pryce, D. Baker, P. Adamson, V. Calder, and J. Greenwood Suppression of Autoimmune Retinal Disease by Lovastatin Does Not Require Th2 Cytokine Induction J. Immunol., February 15, 2005; 174(4): 2327 - 2335. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
|
