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Vascular Research Division, Department of Pathology, Brigham and Women’s Hospital, and
Program in Cardiovascular Gene Therapy, Cardiovascular Research Center, Massachusetts General Hospital-East, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to their function in supporting the physical adhesion of leukocytes to the luminal surface of the vascular endothelium, recent studies suggest that selectins may also be playing a role in signal transduction during leukocyte-endothelial interactions. For example, our laboratory has shown that leukocyte adhesion to cytokine-activated HUVEC induces clustering of E-selectin molecules in the vicinity of leukocyte-endothelial cell attachment sites (8). Leukocyte adhesion to cytokine-activated HUVEC, or the Ab induced cross-linking of cell surface E-selectin molecules, results in a transmembrane linkage of E-selectin to the endothelial cytoskeleton via its cytoplasmic domain (8). More recently, Yoshida et al. (9) have demonstrated that phosphorylation on serine residues in the cytoplasmic domain of E-selectin is modulated in HUVEC during engagement of E-selectin by leukocytes, Ab cross-linking or PSGL-coated beads. Taken together, these data suggest that E-selectin can transduce transmembrane signals via its cytoplasmic domain into the endothelial cell. Lorenzon et al. (10) also have shown that the ligation of either P-selectin or E-selectin with mAbs can induce a transient increase of intracellular ionized calcium in endothelial cell, thus further indicating a signaling function for these vascular selectins. Extensive studies of L-selectin-dependent signaling in leukocytes also have been undertaken. For example, Ab ligation of L-selectin on the leukocyte surface generates various transmembrane signals (11, 12), including, increased intracellular ionized calcium and production of superoxide (13), activation of ß2 integrin-dependent adhesion (14), and activation of mitogen-activated protein kinase (MAPK) (15) and c-Jun N-terminal kinase (JNK) (16) signaling pathways.
The MAPK cascade (also known as the extracellular signal-regulated protein kinase, ERK, pathway) was originally described in cells responding to soluble agonists, such as growth factors and cytokines (17, 18, 19). The MAPK cascade consists of a three-kinase module that includes a MAPK, which is activated by a MAPK/ERK kinase (MEK), which in turn is activated by a MEK kinase (MEKK). Among the MEKKs, best characterized are the Raf protein isoforms (20). The MAPK pathway can mediate various cellular responses, including cell motility and shape change, commitment to cell cycle or programmed cell death, as well as the regulation of multiple genes encoding biologically active products (21). Activation of MAPK in endothelial cells also has been demonstrated after stimulation by biomechanical force (22) as well as cytokine and growth factors (23, 24). Recently, ligand binding by integrins or their Ab-mediated cross-linking has been shown to activate MAPK in fibroblasts and endothelial cells (25, 26), and a similar phenomenon has been observed following Ab cross-linking of the Ig-type adhesion molecule, ICAM-1, in cultured HUVEC (27).
In this study, we have examined the ability of E-selectin-dependent leukocyte adhesion or cross-linking of cell surface E-selectin molecules, to initiate outside-in signaling and activation of the MAPK pathway in cultured human endothelial cells. Our data support a role for the intact, transmembrane E-selectin molecule as a signal transducer that potentially can influence multiple events, including gene regulation, in the endothelial cell during inflammatory leukocyte recruitment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Medium 199, RPMI 1640, and Dulbecco’s PBS (DPBS) were obtained from BioWhittaker (Walkersville, MD). FBS was purchased from Life Technologies (Grand Island, NY). Endothelial cell growth factor was obtained from Biomedical Technologies (Stoughton, MA). Paraformaldehyde (laboratory grade) was purchased from Fisher Scientific (Springfield, NJ). Recombinant human IL-1ß was a gift from Biogen (Cambridge, MA). Biscarboxyethyl-carboxyfluorescein acetoxymethyl ester (BCECF) was purchased from Molecular Probes (Eugene, OR). Goat anti-murine (GAM)-IgG immunoglobulin, PD98059, and c-fos oligonucleotide probe were purchased from Calbiochem (La Jolla, CA). The p44/42 MAP kinase assay kit was obtained from New England Biolabs (Beverly, MA). Protein A/G-PLUS-Agarose was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Cultured cells
HUVEC were isolated and established in culture as previously described (28). Primary cultures were serially passaged (<1:3 split ratio) and maintained in Medium 199 buffered with 25 mmol/L HEPES buffer and supplemented with 20% FBS, endothelial cell growth factor (25 µg/ml), and porcine intestinal heparin (50 µg/ml). For experimental use, subcultured (passage 2 or 3) endothelial cells were plated on gelatin-coated 35-mm or 100-mm tissue culture dishes (Difco Laboratories, Detroit, MI). HL60, a human promyelocytic leukocyte cell line, was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and grown in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 20 mM L-glutamine. JY human lymphocytic cells, kindly provided by Dr. T. A. Springer (Center for Blood Research, Boston, MA), were maintained in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 20 mM L-glutamine.
Immunoprecipitation for analysis of Ras/Raf-1/phospho-MEK association
After cell surface E-selectin cross-linking, HUVEC were rinsed with ice-cold PBS and scraped off the plate in a lysis buffer (20 mM Tris, 5 mM MgCl2, 1 mM PMSF, 20 µg/ml aproptonin, 10 µg/ml leupeptin, 1 mM Na3VO3, and 20 mM ß-glycerophosphate). The lysates were then sonicated and centrifuged at 14,000 rpm for 15 min; then the supernatants were transferred to new tubes and pre-cleared with protein A/G for 1 h at 4°C. Aliquots (200 µl) of these supernatants were incubated with a Raf-1 polyclonal Ab at 4°C overnight. Twenty microliters of protein A/G was incubated with the cell lysates for another hour at 4°C; then the immune complex was washed twice with the lysis buffer and then resuspended in 50 µl of lysis buffer. Sample buffer (187.5 mM Tris-HCl (pH 6.8), 6% SDS, 30% glycerol, 150 mM DTT, and 0.3% bromphenol blue) was added to the complex and samples were boiled for 5 min. The samples were vortex mixed and centrifuged for 2 min, and the supernatant was analyzed on a SDS-PAGE gel.
Immunoprecipitation and in vitro kinase assay for MAPK activity
MAPK activity was quantified using a kit (p44/42 MAP kinase) from New England Biolabs (Beverly, MA), which measures phospho-Elk-1, the phosphorylated product of activated MAPK in a standardized in vitro kinase assay. After treatment, HUVEC were rinsed with ice-cold PBS and lysed with the kit lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerolphosphate, 1 mM Na3VO4, and 1 µg/ml leupeptin) on ice for 5 min. Total cell lysates were sonicated and centrifuged at 14,000 rpm for 15 min; then supernatants were transferred to new tubes and pre-cleared with protein A/G for 1 h at 4°C. Aliquots (200 µl) of these supernatants were incubated overnight at 4°C with the p44/42 MAPK mAb, which specifically recognizes and extracts the phosphorylated ("activated") species of MAPK. Twenty microliters of protein A/G was incubated with the cell lysates for another hour at 4°C, and the immune complex containing activated MAPK was washed twice with lysis buffer and twice with kinase buffer (25 mM Tris (pH 7.5), 5 mM ß-glycerolphosphate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM MgCl2). The pellets were incubated with 48 µl of kinase buffer, 200 µM ATP, and 2 µg of Elk-1 protein (the substrate for activated MAPK) at 30°C for 30 min. The reaction then was terminated by adding 25 µl of 3x SDS sample buffer. The samples were boiled for 5 min, vortex mixed, and centrifuged for 2 min before Western blotting of the product, phosphorylated Elk-1 (Elk-1-PO4).
Western blotting analysis
Aliquots (25 µl) of immunoprecipitates, prepared as above, were separated on a 12% SDS-PAGE gel and then transferred to a nylon membrane (Millipore, Bedford, MA). Membranes were blocked with 5% nonfat milk in TTBS (20 mM Tris, 138 mM NaCl, 0.5% Tween 20 (pH 7.6)) for 1 h at room temperature and then incubated with various primary Abs (1:1000 diluted in blocking buffer), including Ras Ab, MEK 1/2-phospho-specific Ab (Santa Cruz Biotechnology), or a phospho-Elk-1 polyclonal Ab (1:1000) (New England Biolabs) overnight at 4°C. After three washes with TTBS, membranes were incubated with a HRP-conjugated polyclonal goat anti-mouse (or anti-rabbit or anti-rat) Ab (1:1000) (Santa Cruz Biotechnology) in TTBS for an additional hour at room temperature, and again washed three times in TTBS. The labeled proteins were visualized using an enhanced chemilumenscence kit (Amersham, Arlington Heights, IL).
Transduction of HUVEC with wild-type and mutant E-selectin via recombinant adenoviral vectors
To mediate efficient cell surface expression of E-selectin
without activation of HUVEC, two replication-defective recombinant type
5 adenoviruses (AdRSV) were used in this study: adenoviral E-selectin
wild-type [AdRSV(WT-E)] and adenoviral E-selectin cytoplasmic
deletion mutant [AdRSV(
Cyto-E)]. Both constructs use the pJM17
backbone, contain E1/E3 deletions, and were generated as described
previously (9). Large-scale production of recombinant
virus and density gradient purification were performed. High titer
(1.5–2.5 x 1012 particles/ml) stocks of
each vector were used for these studies. Contamination by wild-type
adenovirus was excluded by absence of PCR-detectable E1a sequence in
viral stocks. In preliminary experiments, the optimal dose of
adenoviruses to transduce HUVEC was titrated by a fluorescence
immunobinding assay to obtain a comparable level of cell surface
E-selectin expression to that observed on IL-1ß-activated (10 U/ml,
4 h, 37°C) HUVEC (multiplicity of infection, 64–100 particles
per cell). HUVEC (70% confluent) were transduced in M199 containing
10% FCS and used for experimentation 48–72 h posttransduction, at
which time there were no morphologically detectable differences between
infected and control cultures.
Total RNA isolation and Northern blot analysis
Total cell RNA was isolated from HUVEC according to the
manufacturer’s instructions using RNA STAT-60 (Tel-Test, Friendswood,
TX). Ten milligrams of total RNA was loaded on a 1.2% agarose gel and
transferred to a nylon membrane. The membrane was subjected to
pre-hybridization for at least 4 h at 42oC.
Human c-fos probe was labeled with 5 µCi of
[
-32P]ATP and 30 U/µl of 3'-terminal
deoxynucleotidyl transferase at 37oC for 1.5
h. The probe was purified using a CHROMA SPIN 10 column (Clontech, Palo
Alto, CA) and hybridized with a membrane in hybridization buffer
overnight at 42°C. The membrane was then washed in 2x SSPE and 0.1%
SDS for 15 min twice at 42°C and 0.2x SSPE and 0.1% SDS for 30 min
at 42°C, then exposed to x-ray film.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether MAPK is activated as a consequence of
leukocyte-endothelial adhesion, HUVEC monolayers were activated with
IL-1ß (10 U/ml, 37°C, 4 h) to stimulate maximal E-selectin
cell surface expression, and then incubated with either HL60 cells (a
cultured human leukocyte cell line that expresses ligand for
E-selectin) (28) or JY cells (another cultured human
leukocyte cell line that adheres primarily via LFA-1/ICAM-1)
(29). After adhesion under static conditions for 30 min at
37°C, levels of phospho-Elk-1 were measured as an index of MAPK
activity. When HL60 cells adhered to IL-1ß-activated HUVEC,
MAPK activity increased in proportion to the input concentration
of HL60 cells (Fig. 1
, lanes
3–5), with robust activation occurring with as few as 2 x
104 cells per well. In contrast, JY cells, which
adhered at comparable density for a given input concentration (data not
shown), showed significantly less MAPK activation in these 4-h
IL-1ß-activated HUVEC monolayers (Fig. 1, lanes 6–8).
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A,
live HL60 cells exhibited a robust increase in MAPK activity upon PMA
stimulation, whereas fixed HL60 cells failed to do so. This
demonstrated that paraformaldehyde-fixed HL60 cells were not able to
activate MAPK even with the input of a strong stimulus, PMA. We then
incubated fixed HL60 with IL-1ß-activated HUVEC at 37°C for the
times indicated in Fig. 2B. The adhesion of fixed HL60 to
IL-1ß-activated HUVEC monolayer activated MAPK in a time-dependent
manner (Fig. 2B, lanes 2–4). When an adhesion
blocking mAb to E-selectin, 7A9 (12.5 µg/ml, ATCC), was pre-incubated
with IL-1ß-treated HUVEC, HL60 adhesion was inhibited by 56%
(average of triplicate samples from two separate experiments, 30-min
adhesion), as measured by under static assay conditions, and MAPK
activity was inhibited by 50–60% (Fig. 2B, lanes
2–4 and 6–8, respectively, as measured by
densitometry). Incubation of fixed HL60 cells with unactivated HUVEC
(lacking detectable surface E-selectin expression) resulted in
negligible adhesion and no detectable MAPK activation (data not
shown).
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To eliminate the possible contribution of leukocyte
cell-associated stimuli (e.g., growth factors absorbed to the surfaces
of the paraformaldehyde-fixed HL60 cells), and to mimic the multivalent
receptor-ligand binding and clustering that occurs at
endothelial-leukocyte surfaces during their adhesive interactions
(8), a saturating amount of a function blocking murine mAb
H18/7 (which recognizes adhesion supporting epitopes in the
extracellular portion of the E-selectin molecule) was incubated with
IL-1ß-activated HUVEC and then cross-linked by a GAM-IgG Ab
essentially as described previously (8, 9). This
cross-linking procedure resulted in marked MAPK activation (Fig. 3, lane 5), while incubation
with either H18/7, or the secondary Ab alone did not do so (Fig. 3, lanes 3 and 4). In contrast, when W6/32, a murine
mAb that recognizes surface HLA class I molecules on HUVEC, was
similarly cross-linked, MAPK was not significantly activated (Fig. 3, lane 7), although IL-1ß-activated HUVEC exhibit comparable
cell surface levels of E-selectin and HLA-class I molecules by
fluorescence immunoassay (data not shown). Pre-incubation with a
specific MEK inhibitor, PD98059 (20 µM), completely inhibited MAPK
activation in response to cross-linking of cell surface E-selectin
(Fig. 4, lane 3 vs lane
1).
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To further investigate the signaling role of the cytoplasmic
domain of E-selectin and the possible influence of other concomitants
of cytokine activation of HUVEC on E-selectin-dependent MAPK
activation, we utilized two adenoviral vectors, AdRSV(WT-E), a full
length (WT-E) E-selectin, and AdRSV(Cyto-E), a cytoplasmic deletion
mutant form of E-selectin, to transduce unactivated cultured HUVEC.
Both WT-E- and Cyto-E-transduced HUVEC expressed comparable levels
of surface E-selectin, as confirmed by a fluorescence immunobinding
assay, with the same murine E-selectin mAb, H18/7, that was used for
cross-linking cell surface E-selectin (Fig. 5A). These cell surface levels
of E-selectin were similar in magnitude to those observed after
standard IL-1ß stimulation of HUVEC and were not accompanied by any
significant change in other activation markers, such as ICAM-1 or
VCAM-1 (data not shown), consistent with our previous published results
(9, 30). Cell surface E-selectin in these unactivated
HUVECs was then cross-linked with a murine E-selectin mAb, H18/7,
followed by GAM-IgG. MAPK activity in HUVEC transduced with
WT-E-selectin was significantly increased (as was observed in
IL-1ß-activated HUVEC), while HUVEC transduced with
Cyto-E-selectin failed to generate any MAPK activation signal (Fig. 5B).
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To investigate the signaling pathway upstream of MAPK activation,
we examined the activation of Ras and Raf-1 by determining the
association of both molecules, since it has been well documented that
only GTP-bound Ras is associated with Raf-1 (31, 32, 33). Cell
surface E-selectin in IL-1ß-activated HUVEC was cross-linked by a
murine Ab, H18/7, as described in Fig. 3
. Raf-1 was then
immunoprecipitated from the total cell lysates using a polyclonal Ab
and this Raf-1-containing immunocomplex was analyzed by SDS-PAGE
followed by immunoblotting using specific Abs against Ras and
phospho-MEK. Cross-linking cell surface E-selectin resulted in
increased amounts of Ras in the Raf-1-containing immunocomplex (Fig. 6A). Similarly, probing with
an anti-phospho-MEK-specific Ab also revealed increased amounts of
this component in the Raf-1-containing complex (Fig. 6A).
Re-probing the same blots with a Raf-1 Ab, after stripping, confirmed
comparable immunoprecipitation of Raf-1 from both control and
cross-linked samples (Fig. 6A). A reciprocal
immunoprecipitation, in which phospho-MEK was immunoprecipitated from
total cell lysate and the resultant immunocomplex analyzed using Abs
against Ras and Raf-1, revealed increased association of Ras and Raf-1
with phospho-MEK following E-selectin cross-linking (data not shown).
To investigate the role of the cytoplasmic domain of E-selectin in this
macromolecular complex formation, HUVEC were transduced with either
WT-E-selectin or Cyto-E-selectin adenoviral constructs, as described
in Materials and Methods. Cell surface E-selectin molecules
were cross-linked by a murine mAb, H18/7, followed by a GAM-IgG. As
shown in Fig. 6B, cross-linking WT-E-selectin
(left lanes in each panel), but not
Cyto-E-selectin (right lanes in each panel), resulted in
increased association of Raf-1 with Ras and phospho-MEK.
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To determine whether E-selectin-mediated MAPK signaling can result
in gene regulation, total RNA was isolated from HUVEC that were treated
with IL-1ß (10 U/ml, 37°C, 4 h) and then surface cross-linked
with either the anti-E-selectin murine mAb, H18/7, or the
anti-HLA class I murine mAb, W6/32. Northern blotting was conducted
to measure the steady-state mRNA levels of c-fos, an
immediate early response gene that can be regulated via the MAPK
pathway (34, 35). As seen in Fig. 7A, the c-fos mRNA
level was markedly increased only when cell surface E-selectin was
cross-linked (Fig. 7A, lane 4). The MEK
inhibitor, PD98059, completely inhibited this up-regulation (Fig. 7A, lane 7). Cross-linking of cell surface HLA
class I molecules using the mAb, W6/32 did not result in
c-fos up-regulation (Fig. 7A, lane 6).
Cross-linking WT-E-selectin (Fig. 7B, lanes 1 and
2), but not Cyto-E-selectin (Fig. 7B,
lanes 3 and 4), up-regulated c-fos at
mRNA level.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 2). MAPK activation was proportionally inhibited with a specific
adhesion-blocking mAb, 7A9, that binds to the extracellular domain of
E-selectin, thus indicating its direct involvement in this signaling
event. Interestingly, adhesion of JY cells, another human leukocyte
cell line, that adheres to a comparable extent to 4-h IL-1ß-activated
HUVEC but largely via an ICAM-1/LFA-1 interaction (29),
does not result in robust MAPK activation (Fig. 1). However, this does
not rule out a possible signaling role for ICAM-1 during
leukocyte-endothelial adhesion, as suggested in previous studies
(27, 36). Nonetheless, our data do suggest that E-selectin
may play a more dominant role in MAPK activation during the early
phases of anti-inflammatory response, as mimicked by short-term
cytokine stimulation in this in vitro model system.
In our previous studies, the use of Ab-mediated cross-linking to mimic
the clustering of cell surface E-selectin that occurs during
leukocyte-endothelial interaction resulted in cytoskeletal association
and dephosphorylation events that were dependent on the cytoplasmic
domain of this transmembrane protein (8, 9). As seen in
Fig. 3, when surface E-selectin was cross-linked by the same Ab
treatment, MAPK activity increased dramatically. In contrast,
cross-linking another endothelial surface molecule, the HLA class I
heterodimer, present at comparable density on the surface of
IL-1ß-treated HUVEC, did not generate a comparable level of MAPK
activation. Thus, nonspecific perturbation of the cell membrane by Ab
cross-linking does not appear to be responsible for the observed MAPK
activation. Further, since cross-linking surface E-selectin via a
non-adhesion-blocking mAb, H4/18 (which also interacts with the
extracellular domain of E-selectin), comparably activated MAPK, it
appears that receptor-clustering per se is a sufficient stimulus. In
the preliminary experiments, binding of beads coated with an E-selectin
ligand, PSGL-1, also induced MAPK activation (Y. Hu, unpublished
observation). Taken together, we interpret these data to indicate that
clustering of E-selectin molecules at the cell surface, induced by
leukocyte adhesion, or Ab- or ligand-induced cross-linking, can act as
a sufficient stimulus for activation of the MAPK pathway. However, our
current studies do not establish that E-selectin clustering, as occurs
in the context of leukocyte adhesion to the surface of an activated
endothelial cell, is the sole mechanism for MAPK pathway activation.
Further, it is also possible that E-selectin clustering may result in
the activation, in parallel, of signaling pathways (e.g., c-Jun
NH2-terminal kinase/stress-activated protein
kinase) in addition to the MAPK cascade.
Although selectins share highly homologous mosaic domains in their
extracellular portions, their respective cytoplasmic domains are
distinct (1, 3) and these divergent structures appear to
support different functions. For example, the cytoplasmic domain of
L-selectin plays a critical role in neutrophil rolling in vivo at sites
of inflammation, and in the binding of lymphocytes to high endothelial
venules of peripheral lymph node tissue (37). In contrast,
the deletion of the cytoplasmic domain of P-selectin does not affect
leukocyte adhesion (38). However, the cytoplasmic domain
of P-selectin does appear to play a critical role in the intracellular
sorting of P-selectin to storage granules in endothelial cells and
platelets (38). HUVEC transduced with a cytoplasmic domain
deletion mutant E-selectin show a comparable level of cell surface
expression of E-selectin protein as those transduced with
WT-E-selectin, and also support comparable HL60 cell adhesion under
nonstatic adhesion assay conditions (9). However, deletion
of the cytoplasmic domain of E-selectin does disrupt adhesion-induced
cytoskeletal association and dephosphorylation (8, 9),
thus suggesting that this portion of the molecule is playing an
important role in signaling. In the current study, we now demonstrate
that the deletion of the cytoplasmic domain of E-selectin results in
the loss of the robust MAPK activation that is induced by cross-linking
of cell surface E-selectin (Fig. 5B).
We observed that Ras and Raf-1 form an immunoprecitable complex when
cell surface E-selectin is cross-linked (Fig. 6A). This
association appeared within 10 min and was sustained for at least 30
min after cross-linking, which is consistent with typical time course
of stimulation of Ras (33). It has been shown that, upon
receptor activation, Raf-1 is recruited to the plasma membrane and
becomes associated selectively with GTP-bound Ras (33, 39). Numerous reports also have shown that Ras and Raf-1 can
form a signaling complex with MEK, in which Raf-1 mediates
phosphorylation of MEK on serine residues (40, 41, 42, 43).
Consistent with this, we found that surface E-selectin cross-linking
resulted in increased amounts of phosphorylated MEK in the Raf-1/Ras
complex. Thus, a functional macromolecular complex
(Ras/Raf-1/phosphorylated MEK) forms as a consequence of cell surface
E-selectin cross-linking. Unactivated HUVEC transduced with a
cytoplasmic deletion mutant (Cyto-E) failed to generate this
Ras/Raf-1/phospho-MEK macromolecular complex upon cell surface
E-selectin cross-linking (Fig. 6B), thus indicating that the
cytoplasmic domain may play an important role in the activation of Ras.
It has been previously shown that Ras can become indirectly associated
with L-selectin via adapter proteins during Ab-mediated cross-linking
(15). We are currently exploring the exact mechanism by
which the cytoplasmic domain of E-selectin interacts with Ras in
endothelial cells.
In addition to its involvement in various basic aspects of cell biology
(e.g., cell motility, cell shape, cell cycle, apoptosis), signaling via
the MAPK pathway has been shown to influence the regulation of genes
encoding a broad spectrum of biologically active products, including
chemokines (34, 44) and adhesion molecules (25, 26, 35, 45, 46). c-fos is an example of an immediate
early gene that encodes a transcription factor that is involved in the
transcriptional regulation of multiple genes. MAPK activation can
result in c-fos up-regulation (34, 35, 47, 48).
In our experiments, c-fos mRNA was up-regulated in a
MAPK-dependent manner when cell surface E-selectin molecules, but not
surface HLA class I molecules, were cross-linked (Fig. 7A).
In contrast, the cytoplasmic domain deletion mutant of E-selectin
failed to up-regulate c-fos expression after cross-linking
(Fig. 7B), which again suggests the direct involvement of
this portion of the E-selectin molecule in MAPK signaling. We are
currently characterizing the temporal pattern of expression of multiple
endothelial genes, associated with E-selectin-dependent MAPK activation
via a transcriptional profiling strategy in an effort to define this
aspect of leukocyte adhesion-induced phenotypic modulation.
In summary, we have demonstrated that E-selectin can act as a transmembrane signal transducer, activating the MAPK cascade and resulting in the up-regulation of the immediate early response gene, c-fos, which is itself further implicated in the transcriptional control of various pro-inflammatory genes. E-selectin-dependent leukocyte adhesion-induced modulation of endothelial phenotype may have important implications for the evolution of the inflammatory process. Further studies of the molecular mechanisms linking leukocyte adhesion-dependent rearrangement of E-selectin at the cell surface to intracellular cascade signaling cascades, such as the MAPK pathway, may provide valuable insights into the orchestration of the inflammatory response at the level of the vascular endothelial lining.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael A. Gimbrone, Jr., Vascular Research Division, Department of Pathology, Brigham and Women’s Hospital, 221 Longwood Avenue, LMRC-401, Boston, MA 02115.
3 Abbreviations used in this paper: PSGL-1, P-selectin glycoprotein ligand-1; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated protein kinase; MEK, MAPK/ERK kinase; GAM, goat anti-murine; WT-E, E-selectin wild type; Cyto-E, E-selectin cytoplasmic deletion.
Received for publication January 18, 2000. Accepted for publication May 30, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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J.-M. Kiely, Y. Hu, G. Garcia-Cardena, and M. A. Gimbrone Jr. Lipid Raft Localization of Cell Surface E-Selectin Is Required for Ligation-Induced Activation of Phospholipase C{gamma} J. Immunol., September 15, 2003; 171(6): 3216 - 3224. [Abstract] [Full Text] [PDF]
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P. Kumar, M. A. Amin, L. A. Harlow, P. J. Polverini, and A. E. Koch Src and phosphatidylinositol 3-kinase mediate soluble E-selectin-induced angiogenesis Blood, May 15, 2003; 101(10): 3960 - 3968. [Abstract] [Full Text] [PDF]
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 M. Yoshida, Y. Takano, T. Sasaoka, T. Izumi, and A. Kimura E-Selectin Polymorphism Associated With Myocardial Infarction Causes Enhanced Leukocyte-Endothelial Interactions Under Flow Conditions Arterioscler Thromb Vasc Biol, May 1, 2003; 23(5): 783 - 788. [Abstract] [Full Text] [PDF]
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B. G. Yipp, S. M. Robbins, M. E. Resek, D. I. Baruch, S. Looareesuwan, and M. Ho Src-family kinase signaling modulates the adhesion of Plasmodium falciparum on human microvascular endothelium under flow Blood, April 1, 2003; 101(7): 2850 - 2857. [Abstract] [Full Text] [PDF]
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 K. V. Vijayan, Y. Liu, J.-F. Dong, and P. F. Bray Enhanced Activation of Mitogen-activated Protein Kinase and Myosin Light Chain Kinase by the Pro33 Polymorphism of Integrin beta 3 J. Biol. Chem., January 31, 2003; 278(6): 3860 - 3867. [Abstract] [Full Text] [PDF]
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Y. Hu, B. Szente, J.-M. Kiely, and M. A. Gimbrone Jr. Molecular Events in Transmembrane Signaling via E-selectin. SHP2 ASSOCIATION, ADAPTOR PROTEIN COMPLEX FORMATION AND ERK1/2 ACTIVATION J. Biol. Chem., December 14, 2001; 276(51): 48549 - 48553. [Abstract] [Full Text] [PDF]
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S. L. Cuvelier and K. D. Patel Shear-dependent Eosinophil Transmigration on Interleukin 4-stimulated Endothelial Cells: A Role for Endothelium-associated Eotaxin-3 J. Exp. Med., December 10, 2001; 194(12): 1699 - 1709. [Abstract] [Full Text] [PDF]
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 B. C. Biedermann Vascular Endothelium: Checkpoint for Inflammation and Immunity Physiology, April 1, 2001; 16(2): 84 - 88. [Abstract] [Full Text] [PDF]
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P. Kumar, S. Hosaka, and A. E. Koch Soluble E-selectin Induces Monocyte Chemotaxis through Src Family Tyrosine Kinases J. Biol. Chem., June 8, 2001; 276(24): 21039 - 21045. [Abstract] [Full Text] [PDF]
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M. Medhora, M. Bousamra II, D. Zhu, L. Somberg, and E. R. Jacobs Upregulation of collagens detected by gene array in a model of flow-induced pulmonary vascular remodeling Am J Physiol Heart Circ Physiol, February 1, 2002; 282(2): H414 - H422. [Abstract] [Full Text] [PDF]
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