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The Journal of Immunology, 2000, 165: 2142-2148.
Copyright © 00 by The American Association of Immunologists

E-Selectin-Dependent Signaling Via the Mitogen-Activated Protein Kinase Pathway in Vascular Endothelial Cells1

Yenya Hu*, Jeanne-Marie Kiely*, Brian E. Szente*, Anthony Rosenzweig{dagger} and Michael A. Gimbrone, Jr.2,*

* Vascular Research Division, Department of Pathology, Brigham and Women’s Hospital, and {dagger} Program in Cardiovascular Gene Therapy, Cardiovascular Research Center, Massachusetts General Hospital-East, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
E-selectin, a cytokine-inducible adhesion molecule, supports rolling and stable arrest of leukocytes on activated vascular endothelium. Previous studies have suggested that this transmembrane protein can also transduce signals into the endothelial cell. We now demonstrate activation of the mitogen-activated protein kinase (MAPK) signaling cascade in cultured HUVEC in response to E-selectin-dependent leukocyte adhesion and Ab-mediated cross-linking of cell surface E-selectin. Adhesion of increasing numbers of HL60 cells to IL-1ß-activated HUVEC stimulated robust increases in MAPK activity that were abrogated by an E-selectin blocking Ab. Cross-linking of cell surface E-selectin with Abs, as a mimic of multivalent ligand engagement, strongly stimulated MAPK/extracellular signal-related kinase (ERK) kinase (MEK)-dependent MAPK activation and concomitant up-regulation of mRNA for c-fos, an immediate early response gene, whereas Ab cross-linking of HLA class I molecules (present at comparable density) failed to do so. Coimmunoprecipitation documented Ras, Raf-1 and, phospho-MEK complex formation. Unactivated HUVEC transduced with a full-length adenoviral E-selectin construct also exhibited cross-link-induced MAPK activation, macromolecular complex formation, and c-fos up-regulation, whereas HUVEC transduced with a cytoplasmic domain deletion mutant failed to respond. These observations indicate that E-selectin can transduce an activating stimulus via the MAPK cascade into the endothelial cell during leukocyte adhesion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The selectin family of adhesion molecules, which includes E-selectin, L-selectin, and P-selectin (1), mediates interaction of circulating leukocytes with vascular endothelium in various physiological and pathological settings (1). Unique among the selectin family molecules, E-selectin typically is not detected in unactivated endothelial cells, but is rapidly synthesized in response to certain cytokines and other pro-inflammatory stimuli, thus making it a marker of the "activated" endothelial phenotype (2). Along with other selectin family members, E-selectin exhibits a complex mosaic structure consisting of a large extracellular portion comprised of an amino-terminal lectin domain, an epidermal growth factor domain, and multiple complement regulatory repeats, followed by a transmembrane portion and a relatively short (32 aa) cytoplasmic domain (3). P-selectin glycoprotein ligand-1 (PSGL-1)3 can function as a ligand for both E- and P-selectins (4), but other ligands appear to exist for E-selectin as well (5). E-selectin can support the initial rolling of leukocytes on activated endothelium as demonstrated in various in vitro and in vivo models (3, 6). Milstone et al. (7) have shown that E-selectin may also play a role in mediating the stable arrest of leukocytes on the luminal surface of inflamed microvascular endothelium in the mouse.

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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

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({Delta}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 [{alpha}-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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
E-selectin-dependent leukocyte adhesion activates endothelial MAPK

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. 1Go, 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. 1Go, lanes 6–8).



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  		FIGURE 1. Adhesion of leukocytes to IL-1ß-activated HUVEC results in MAPK activation. All HUVEC were serum-starved overnight before the experiments, and phospho-Elk-1 was measured as an index of MAPK activity (see Materials and Methods). Lane 1, LPS-stimulation of HUVEC (100 ng/ml, 15 min, 37°C), as a positive control for MAPK activation. Lane 2, IL-1ß-activated HUVEC (10 U/ml, 4 h, 37°C). Lanes 3–5 and 6–8, Increasing concentrations of either HL60 or JY cells, respectively (1x = 2 x 104, 10x = 2 x 105, and 100x = 2 x 106 cells per well) were incubated with IL-1ß-activated HUVEC for 30 min at 37°C in a standard (static) adhesion assay.

 
However, the MAPK activity measured in this experimental setting could be contributed by either the adherent leukocytes or HUVEC, or both. To investigate whether adhesive interactions between HL60 cells and activated HUVEC result in MAPK activation in HUVEC, we fixed HL60 cells with 2% paraformaldehyde (4°C, 20 min), and then subjected both live and paraformaldehyde-fixed HL60 cells to PMA (a strong stimulus for MAPK activity; 1 µM, 15 min) and assayed their lysates for MAPK activity. As seen in Fig. 2GoA, 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. 2GoB. The adhesion of fixed HL60 to IL-1ß-activated HUVEC monolayer activated MAPK in a time-dependent manner (Fig. 2GoB, 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. 2GoB, 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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  		FIGURE 2. A, Fixed HL60 cells fail to show agonist-induced MAPK activity. Live or fixed (2% paraformaldehyde, 4°C, 20 min) HL60 cells were either treated with PMA or vehicle for 15 min, and phospho-Elk-1 (Elk-1-PO4) was measured as an index of MAPK activity. B, Adhesion of fixed HL60 cells to IL-1ß-activated HUVEC results in MAPK activation via E-selectin. HUVEC were activated with IL-1ß (10 U/ml, 37°C, 4 h). Lane 1, IL-1ß-activated HUVEC alone. Lanes 2–4, Fixed HL60 cells (2 x 106 per well) were incubated with activated HUVEC for the times indicated. Lane 5, An E-selectin adhesion-blocking mAb, 7A9, was preincubated with activated HUVEC (12.5 µg/ml, 37°C, 30 min) without the addition of leukocytes. Lanes 6–8, After preincubation of HUVEC monolayers with a mAb, 7A9, fixed HL60 cells (2 x 106 cells per well) were added for the times indicated. Phospho-Elk-1 was assayed as an index of MAPK activity and quantified by densitometry; inhibition of leukocyte adhesion was also measured in parallel (see Results).

 
Ab-mediated cross-linking surface E-selectin activates MAPK and this activation is MEK-dependent

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. 3Go, lane 5), while incubation with either H18/7, or the secondary Ab alone did not do so (Fig. 3Go, 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. 3Go, 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. 4Go, lane 3 vs lane 1).



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  		FIGURE 3. Cross-linking of cell surface E-selectin, but not HLA class I molecules, activates MAPK in HUVEC. Lane 1, LPS-stimulated HUVEC (positive control for MAPK activity). Lane 2, IL-1ß-activated HUVEC (10 U/ml, 37°C, 4 h; followed by sham incubations at 4°C, 30 min, and 37°C, 30 min) Lane 3, Murine mAb H18/7, which binds to the extracellular domain of E-selectin, was incubated with activated HUVEC (10 µg/ml, 4°C, 30 min). Lane 4, GAM-IgG was incubated with activated HUVEC (sham incubation at 4°C, 30 min; following 1:200, 37°C, 30 min). Lane 5, Activated HUVEC incubated with murine mAb H18/7 (4°C, 30 min), followed by GAM-IgG (37°C, 30 min). Lane 6, W6/32, murine mAb to HLA class I molecules, was incubated with activated HUVEC (10 µg/ml, 4°C, 30 min; followed by sham incubation at 37°C, 30 min). Lane 7, Cell surface HLA class I molecules were cross-linked (W6/32, 4°C, 30 min, followed by GAM-IgG, 37°C, 30 min). Phospho-Elk-1 (Elk-1-PO4) was assayed as an index of MAPK activity.

 


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  		FIGURE 4. MAPK activation induced by cross-linking cell surface E-selectin is MEK-dependent. Lane 1, Cross-linking of cell surface E-selectin in IL-1ß-activated HUVEC (10 U/ml, 4 h, 37°C) via sequential incubation with a murine anti-E-selectin mAb, H18/7, and a GAM-IgG (4°C, 30 min and 37°C, 30 min, respectively). Lane 2, A specific MEK inhibitor, PD98059 (20 µM) incubated with IL-1ß-activated HUVEC (37°C, 45 min). Lane 3, PD98059 (20 µM) was pre-incubated (37°C, 45 min) with activated HUVEC before the cross-linking procedure. Phospho-Elk-1 (Elk-1-PO4) was assayed as an index of MAPK activity.

 
MAPK activation is induced by cross-linking cell surface E-selectin requires its cytoplasmic domain, but does not require concomitant cytokine activation of HUVEC

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({Delta}Cyto-E), a cytoplasmic deletion mutant form of E-selectin, to transduce unactivated cultured HUVEC. Both WT-E- and {Delta}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. 5GoA). 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 {Delta}Cyto-E-selectin failed to generate any MAPK activation signal (Fig. 5GoB).



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  		FIGURE 5. A, Unactivated, adeno-transfected HUVEC (WT-E and {Delta}Cyto-E) show comparable levels of E-selectin surface expression. HUVEC were transfected with adenoviral constructs encoding either full-length (WT-E) or cytoplasmic domain deletion mutant ({Delta}Cyto-E) E-selectin as described in Materials and Methods. Cell surface E-selectin expression as measured by a fluorescence immunobinding assay as described in Materials and Methods. B, The cytoplasmic domain of E-selectin is necessary for MAPK activation by Ab cross-linking. Lanes 1–3, HUVEC transduced with WT-E-selectin, Lanes 4–6, HUVEC transduced with {Delta}Cyto-E-selectin. Lanes 1 and 4, Cells incubated with a murine mAb, H18/7, alone (4°C, 30 min). Lanes 2 and 5, Cells incubated with GAM-IgG alone (37°C, 30 min). Lanes 3 and 6, Cells incubated sequentially with H18/7 and GAM-IgG. Phospho-Elk-1 (Elk-1-PO4) was assayed as an index of MAPK activity.

 
Cross-linking cell surface E-selectin results in the formation of a Ras/Raf-1/phospho-MEK macromolecular complex and that is cytoplasmic domain-dependent

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. 3Go. 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. 6GoA). Similarly, probing with an anti-phospho-MEK-specific Ab also revealed increased amounts of this component in the Raf-1-containing complex (Fig. 6GoA). 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. 6GoA). 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 {Delta}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. 6GoB, cross-linking WT-E-selectin (left lanes in each panel), but not {Delta}Cyto-E-selectin (right lanes in each panel), resulted in increased association of Raf-1 with Ras and phospho-MEK.



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  		FIGURE 6. A, Cross-linking cell surface E-selectin results in the formation of a macromolecular complex containing Ras, Raf-1, and phospho-MEK. Total cell lysates prepared from HUVEC with and without cross-linking of cell surface E-selectin were subjected to immunoprecipitation (IP) with a Raf-1 polyclonal Ab, and the resulting immunocomplex was analyzed by SDS-PAGE gel and immunoblotting (IB) with appropriate Abs to detect the presence of Ras, Raf-1, and phospho-MEK. Left lane in each panel, IL-1ß-activated HUVEC (10 U/ml, 37°C, 4 h) alone. Right lane in each panel, IL-1ß-treated HUVEC following sequential incubation with murine mAb H18/7 (4°C, 30 min) and GAM-IgG (37°C, 30 min) to cross-link cell surface E-selectin. Note that the association of Ras and phospho-MEK with Raf-1 is strongly enhanced by E-selectin cross-linking, and that comparable amounts of Raf-1 are present in HUVEC lysates before and after E-selectin cross-linking. B, The cytoplasmic domain of E-selectin is required for formation of the Ras, Raf-1, and phospho-MEK macromolecular complex. Cell surface E-selectin molecules were cross-linked by sequential incubation with murine mAb, H18/7 (4°C, 30 min) and GAM-IgG (37°C, 30 min) on HUVEC transduced with either WT-E-selectin or {Delta}Cyto-E-selectin adenoviral constructs. Total cell lysates were subjected to immunoprecipitation (IP) using a Raf-1 polyclonal Ab, and the Raf-1-containing immunocomplex was analyzed by SDS-PAGE and immunoblotted (IB) with either an anti-Ras Ab or an anti-phospho-MEK Ab. Left lanes on each panel, WT-E-selectin transduced HUVEC; Right lanes on each panel, {Delta}Cyto-E-selectin-transduced HUVEC.

 
Cross-linking surface E-selectin up-regulates c-fos at mRNA level and the cytoplasmic domain of E-selectin is required for this up-regulation

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. 7GoA, the c-fos mRNA level was markedly increased only when cell surface E-selectin was cross-linked (Fig. 7GoA, lane 4). The MEK inhibitor, PD98059, completely inhibited this up-regulation (Fig. 7GoA, 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. 7GoA, lane 6). Cross-linking WT-E-selectin (Fig. 7GoB, lanes 1 and 2), but not {Delta}Cyto-E-selectin (Fig. 7GoB, lanes 3 and 4), up-regulated c-fos at mRNA level.



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  		FIGURE 7. A, Cross-linking cell surface E-selectin up-regulates c-fos mRNA in an MAPK-dependent manner. Lanes 1–7, HUVEC was activated with IL-1ß (10 U/ml, 37°C, 4 h). Lane 1, IL-1ß-activated HUVEC alone. Lane 2, H18/7 (10 µg/ml) was incubated with IL-1ß-activated HUVEC (4°C, 30 min). Lane 3, GAM-IgG (37°C, 30 min). Lane 4, Cell surface E-selectin molecules were cross-linked by H18/7 followed by GAM-IgG. Lane 5, PD98059 was incubated with HUVEC (20 µM, 37°C, 45 min). Lane 6, HLA class I molecules were cross-linked using W6/32 followed by GAM-IgG. Lane 7, PD98059 was incubated with HUVEC before the cell surface E-selectin cross-linking. Hybridization of a GAPDH probe to the same blot reveals comparable loading of total RNA in each lane. B, The cytoplasmic domain of E-selectin is required for the E-selectin-dependent up-regulation of c-fos at mRNA level. Total RNA was isolated and analyzed. Lanes 1 and 3, HUVEC transduced with either WT-E-selectin or {Delta}Cyto-E-selectin, respectively. Lanes 2 and 4, Cell surface E-selectin molecules were Ab cross-linked (as in A) on HUVEC transduced either with WT-E-selectin or with {Delta}Cyto-E-selectin, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report we show that E-selectin, an inducible adhesion molecule expressed on the surface of activated endothelial cells, can transduce signals across the cell membrane to activate the MEK-dependent MAPK cascade. Adhesion of both live and fixed (metabolically inert) HL60 cells to IL-1ß-activated HUVEC resulted in the activation of MAPK in a dose- and time-dependent fashion (Figs. 1Go and 2Go). 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. 1Go). 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. 3Go, 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. 5GoB).

We observed that Ras and Raf-1 form an immunoprecitable complex when cell surface E-selectin is cross-linked (Fig. 6GoA). 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 ({Delta}Cyto-E) failed to generate this Ras/Raf-1/phospho-MEK macromolecular complex upon cell surface E-selectin cross-linking (Fig. 6GoB), 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. 7GoA). In contrast, the cytoplasmic domain deletion mutant of E-selectin failed to up-regulate c-fos expression after cross-linking (Fig. 7GoB), 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.


   Acknowledgments
 
We gratefully acknowledge the expert assistance of Kay Case and William Atkinson in cell culture. We also thank Drs. Andrew Connolly and Francis W. Luscinskas for helpful discussions.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health (P01-HL36028 to M.A.G. and HL54202 and AI40970 to A.R.). A.R. is an Established Investigator of the American Heart Association. Back

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. Back

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; {Delta}Cyto-E, E-selectin cytoplasmic deletion. Back

Received for publication January 18, 2000. Accepted for publication May 30, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kansas, G. S.. 1996. Selectins and their ligands: current concepts and controversies. Blood 88:3259.[Free Full Text]
  2. Bevilacqua, M. P., J. S. Pober, D. L. Mendrick, R. S. Cotran, Jr M. A. Gimbrone. 1987. Identification of an inducible endothelial-leukocyte adhesion molecule. Proc. Natl. Acad. Sci. USA 84:9238.[Abstract/Free Full Text]
  3. Bevilacqua, M. P., R. M. Nelson. 1993. Selectins. J. Clin. Invest. 91:379.
  4. McEver, R. P., R. D. Cummings. 1997. Role of PSGL-1 binding to selectins in leukocyte recruitment. J. Clin. Invest. 100:S97.
  5. Yang, J., B. C. Furie, B. Furie. 1999. The biology of P-selectin glycoprotein ligand-1: its role as a selectin counterreceptor in leukocyte-endothelial and leukocyte-platelet interaction. Thromb. Haemostasis 81:1.[Medline]
  6. Jung, U., K. E. Norman, K. Scharffetter-Kochanek, A. L. Beaudet, K. Ley. 1998. Transit time of leukocytes rolling through venules controls cytokine-induced inflammatory cell recruitment in vivo. J. Clin. Invest. 102:1526.[Medline]
  7. Milstone, D. S., D. Fukumura, R. C. Padgett, P. E. O’Donnell, V. M. Davis, O. J. Benavidez, W. L. Monsky, R. J. Melder, R. K. Jain, Jr M. A. Gimbrone. 1998. Mice lacking E-selectin show normal numbers of rolling leukocyte but reduced leukocyte stable adhesion to cytokine-activated microvascular endothelium. Microcirculation 5:153.[Medline]
  8. Yoshida, M., W. F. Westlin, N. Wang, D. E. Ingber, A. Rosenzweig, N. Resnick, Jr M. A. Gimbrone. 1996. Leukocyte adhesion to vascular endothelium induces E-selectin linkage to the actin cytoskeleton. J. Cell Biol. 133:445.[Abstract/Free Full Text]
  9. Yoshida, M., B. E. Szente, J. M. Kiely, A. Rosenzweig, Jr M. A. Gimbrone. 1998. Phosphorylation of the cytoplasmic domain of E-selectin is regulated during leukocyte-endothelial adhesion. J. Immunol. 161:933.[Abstract/Free Full Text]
  10. Lorenzon, P., E. Vecile, E. Nardon, E. Ferrero, J. M. Harlan, F. Tedesco, A. Dobrina. 1998. Endothelial cell E- and P-selectin and vascular cell adhesion molecule-1 function as signaling receptors. J. Cell Biol. 142:1381.[Abstract/Free Full Text]
  11. Laudanna, C., G. Constantin, P. Baron, E. Scarpini, G. Scarlato, G. Cabrini, C. Dechecchi, F. Rossi, M. A. Cassatella, G. Berton. 1994. Sulfatides trigger increase of cytosolic free calcium and enhanced expression of tumor necrosis factor-{alpha} and interleukin-8 mRNA in human neutrophils: evidence for the role of L-selectin as a signaling molecule. J. Biol. Chem. 269:4021.[Abstract/Free Full Text]
  12. Steeber, D. A., P. Engel, A. S. Miller, M. P. Sheetz, T. F. Tedder. 1997. Ligation of L-selectin through conserved regions within the lectin domain activates signal transduction pathways and integrin function in human, mouse, and rat leukocytes. J. Immunol. 159:952.[Abstract]
  13. Crockett-Torabi, E., J. C. Fantone. 1997. L-selectin stimulation of canine neutrophil initiates calcium signal secondary to tyrosine kinase activation. Am. J. Physiol. 272:H1302.[Abstract/Free Full Text]
  14. Simon, S. I., A. R. Burns, A. D. Taylor, P. K. Gopalan, E. B. Lynam, L. A. Sklar, C. W. Smith. 1995. L-selectin (CD62L) cross-linking signals neutrophil adhesive functions via the Mac-1 (CD11b/CD18) ß2 integrin. J. Immunol. 155:1502.[Abstract]
  15. Brenner, B., E. Gulbins, K. Schlottmann, U. Koppenhoefer, G. L. Busch, B. Walzog, M. Steinhausen, K. M. Coggeshall, O. Linderkamp, F. Lang. 1996. L-selectin activates the Ras pathway via the tyrosine kinase p56lck. Proc. Natl. Acad. Sci. USA 93:15376.[Abstract/Free Full Text]
  16. Brenner, B., S. Weinmann, H. Grassme, F. Lang, O. Linderkamp, E. Gulbins. 1997. L-selectin activates JNK via src-like tyrosine kinases and the small G-protein Rac. Immunology 92:214.[Medline]
  17. Xing, J., J. M. Kornhauser, Z. Xia, E. A. Thiele, M. E. Greenberg. 1998. Nerve growth factor activates extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways to stimulate CREB serine 133 phosphorylation. Mol. Cell. Biol. 18:1946.[Abstract/Free Full Text]
  18. Stancato, L. F., M. Sakatsume, M. David, P. Dent, F. Dong, E. F. Petricoin, J. J. Krolewski, O. Silvennoinen, P. Saharinen, J. Pierce, et al 1997. ß Interferon and oncostatin M activate Raf-1 and mitogen-activated protein kinase through a JAK1-dependent pathway. Mol. Cell. Biol. 17:3833.[Abstract]
  19. May, M. J., C. P. Wheeler-Jones, R. A. Houliston, J. D. Pearson. 1998. Activation of p42mapk in human umbilical vein endothelial cells by interleukin-1{alpha} and tumor necrosis factor-{alpha}. Am. J. Physiol. 274:C789.[Abstract/Free Full Text]
  20. Robinson, M. J., M. H. Cobb. 1997. Mitogen-activated protein kinase pathways. Curr. Opin. Cell Biol. 9:180.[Medline]
  21. Yamada, K. M., B. Geiger. 1997. Molecular interactions in cell adhesion complexes. Curr. Opin. Cell Biol. 8:657.
  22. Traub, O., B. C. Berk. 1998. Laminar shear stress: mechanisms by which endothelial cells transduce an atheroprotective force. Arterioscler. Thromb. Vasc. Biol. 18:677.[Abstract/Free Full Text]
  23. Milanini, J., F. Vinals, J. Pouyssegur, G. Pages. 1998. p42/p44 MAP kinase module plays a key role in the transcriptional regulation of the vascular endothelial growth factor gene in fibroblasts. J. Biol. Chem. 273:18165.[Abstract/Free Full Text]
  24. Doanes, A. M., D. D. Hegland, R. Sethi, I. Kovesdi, J. T. Bruder, T. Finkel. 1999. VEGF stimulates MAPK through a pathway that is unique for receptor tyrosine kinases. Biochem. Biophys. Res. Commun. 255:545.[Medline]
  25. Schlaepfer, D. D., S. K. Hanks, T. Hunter, P. van der Geer. 1994. Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase. Nature 372:786.[Medline]
  26. Short, S. M., G. A. Talbott, R. L. Juliano. 1998. Integrin-mediated signaling events in human endothelial cells. Mol. Biol. Cell 9:1969.[Abstract/Free Full Text]
  27. Lawson, C., M. Ainsworth, M. Yacoub, M. Rose. 1999. Ligation of ICAM-1 on endothelial cells leads to expression of VCAM-1 via a nuclear factor-{kappa}B-independent mechanism. J. Immunol. 162:2990.[Abstract/Free Full Text]
  28. Bevilacqua, M. P., J. S. Pober, M. E. Wheeler, R. S. Cotran, Jr M. A. Gimbrone. 1985. Interleukin 1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte cell lines. J. Clin. Invest. 76:2003.
  29. Rothlein, R., T. A. Springer. 1986. The requirement for lymphycyte function associated-antigen 1 in homotypic leukocyte adhesion stimulated by phorbol ester. J. Exp. Med. 163:1132.[Abstract/Free Full Text]
  30. Gerszten, R. E., F. W. Luscinskas, H. T. Ding, D. A. Dichek, L. M. Stoolman, Jr M. A. Gimbrone, A. Rosenzweig. 1996. Adhesion of memory lymphocytes to vascular cell adhesion molecule-1-transduced human vascular endothelial cells under simulated physiological flow conditions in vitro. Circ. Res. 79:1205.[Abstract/Free Full Text]
  31. Koide, H., T. Satoh, M. Nakafuku, Y. Kaziro. 1993. GTP-dependent association of Raf-1 with Ha-Ras: identification of Raf as a target downstream of Ras in mammalian cells. Proc. Natl. Acad. Sci. USA 90:8683.[Abstract/Free Full Text]
  32. Marshall, M.. 1995. Interactions between Ras and Raf: key regulatory proteins in cellular transformation. Mol. Reprod. Dev. 42:493.[Medline]
  33. Denhardt, D. T.. 1996. Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling. Biochem. J. 318:729.
  34. Jalali, S., Y. S. Li, M. Sotoudeh, S. Yuan, S. Li, S. Chien, J. Y. Shyy. 1998. Shear stress activates p60src-Ras-MAPK signaling pathways in vascular endothelial cells. Arterioscler. Thromb. Vasc. Biol. 18:227.[Abstract/Free Full Text]
  35. Huttunen, P., T. Hyypia, P. Vihinen, L. Nissinen, J. Heino. 1998. Echovirus 1 infection induces both stress- and growth-activated mitogen-activated protein kinase pathways and regulates the transcription of cellular immediate-early genes. Virology 250:85.[Medline]
  36. Durieu-Trautmann, O., N. Chaverot, S. Cazaubon, A. D. Strosberg, P. O. Couraud. 1994. Intercellular adhesion molecule 1 activation induces tyrosine phosphorylation of the cytoskeleton-associated protein cortactin in brain microvessel endothelial cells. J. Biol. Chem. 269:12536.[Abstract/Free Full Text]
  37. Kansas, G. S., K. Ley, J. M. Munro, T. F. Tedder. 1993. Regulation of leukocyte rolling and adhesion to high endothelial venules through the cytoplasmic domain of L-selectin. J. Exp. Med. 177:833.[Abstract/Free Full Text]
  38. Hartwell, D. W., T. N. Mayadas, G. Berger, P. S. Frenette, H. Rayburn, R. O. Hynes, D. D. Wagner. 1998. Role of P-selectin cytoplasmic domain in granular targeting in vivo and in early inflammatory responses. J. Cell Biol. 143:1129.[Abstract/Free Full Text]
  39. Overbeck, A. F., T. R. Brtva, A. D. Cox, S. M. Graham, S. H. Huff, R. Khosravi-Far, L. A. Quilliam, P. A. Solski, C. J. Der. 1995. Guanine nucleotide exchange factors: activators of Ras superfamily proteins. Mol. Reprod. Dev. 42:468.[Medline]
  40. Van Aelst, L., M. Barr, S. Marcus, A. Polverino, M. Wigler. 1993. Complex formation between RAS and RAF and other protein kinases. Proc. Natl. Acad. Sci. USA 90:6213.[Abstract/Free Full Text]
  41. Wartmann, M., R. J. Davis. 1994. The native structure of the activated Raf protein kinase is a membrane-bound multi-subunit complex. J. Biol. Chem. 269:6695.[Abstract/Free Full Text]
  42. Moodie, S. A. W., Berthe M. Weber, Michael J. Wolfman Alan. 1993. Complexes of Ras:GTP with Raf-1 and mitogen-activated protein kinase kinase. Science 260:1658.[Abstract/Free Full Text]
  43. Huang, W. A., Alessandro Crews, Craig M. Erikson, R.L. 1993. Raf-1 forms a stable complex with Mek1 and activates Mek1 by serine phosphorylation. Proc. Natl. Acad. Sci. USA 90:10947.
  44. Fischer, T. A., K. Singh, D. S. O’Hara, D. M. Kaye, R. A. Kelly. 1998. Role of AT1 and AT2 receptors in regulation of MAPKs and MKP-1 by ANG II in adult cardiac myocytes. Am. J. Physiol. 275:H906.
  45. Herrera, R., S. Hubbell, S. Decker, L. Petruzzelli. 1998. A role for the MEK/MAPK pathway in PMA-induced cell cycle arrest: modulation of megakaryocytic differentiation of K562 cells. Exp. Cell Res. 238:407.[Medline]
  46. Ahmad, M., P. Theofanidis, R. M. Medford. 1998. Role of activating protein-1 in the regulation of the vascular cell adhesion molecule-1 gene expression by tumor necrosis factor-{alpha}. J. Biol. Chem. 273:4616.[Abstract/Free Full Text]
  47. Zutter, M. M., A. D. Painter, X. Yang. 1999. The megakaryocyte/platelet-specific enhancer of the {alpha}2ß1 integrin gene: two tandem AP1 sites and the mitogen-activated protein kinase signaling cascade. Blood 93:1600.[Abstract/Free Full Text]
  48. Cavigelli, M., F. Dolfi, F. X. Claret, M. Karin. 1995. Induction of c-fos expression through JNK-mediated TCF/Elk-1 phosphorylation. EMBO J. 14:5957.[Medline]



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