MKK3 and -6-dependent activation of p38{alpha} MAP kinase is required for cytoskeletal changes in pulmonary microvascular endothelial cells induced by ICAM-1 ligation

Qin Wang,1 Michael Yerukhimovich,1 William A. Gaarde,2 Ian J. Popoff,3 and Claire M. Doerschuk1

1Division of Integrative Biology, Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio; 2ISIS Pharmaceuticals, Incorporated, Carlsbad; and 3Pfizer, Incorporated, La Jolla, California

Submitted 4 August 2004 ; accepted in final form 25 October 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previous studies demonstrated that neutrophil adherence induces ICAM-1-dependent cytoskeletal changes in TNF-{alpha}-treated pulmonary microvascular endothelial cells that are prevented by a pharmacological inhibitor of p38 MAP kinase. This study determined whether neutrophil adherence induces activation of p38 MAP kinase in endothelial cells, the subcellular localization of phosphorylated p38, which MAP kinase kinases lead to p38 activation, which p38 isoform is activated, and what the downstream targets may be. Confocal microscopy showed that neutrophil adhesion for 2 or 6 min induced an increase in phosphorylated p38 in endothelial cells that was punctate and concentrated in the central region of the endothelial cells. Studies using small interfering RNA (siRNA) to inhibit the protein expression of MAP kinase kinase 3 and 6, either singly or in combination, showed that both MAP kinase kinases were required for p38 phosphorylation. Studies using an antisense oligonucleotide to p38{alpha} demonstrated that inhibition of the protein expression of p38{alpha} 1) inhibited activation of p38 MAP kinase without affecting the protein expression of p38{beta}; 2) prevented phosphorylation of heat shock protein 27, an actin binding protein that may induce actin polymerization upon phosphorylation; 3) attenuated cytoskeletal changes; and 4) attenuated neutrophil migration to the EC borders. Thus MAP kinase kinase3- and 6-dependent activation of the {alpha}-isoform of p38 MAP kinase is required for the cytoskeletal changes induced by neutrophil adherence and influences subsequent neutrophil migration toward endothelial cell junctions.

tumor necrosis factor-{alpha}; neutrophil; actin; inflammation; mitogen-activated protein kinase kinase 3/6; intercellular adhesion molecule


NEUTROPHIL ADHESION to vascular endothelial cells (ECs) plays important roles in regulating neutrophil emigration from blood into tissues during inflammation. EC adhesion molecules, including ICAM-1, not only can support neutrophil adhesion but also are capable of initiating intracellular signaling events in ECs. In cultured ECs, ligation of ICAM-1 induces activation of tyrosine kinases and mitogen-activated protein kinases (MAPK) (11, 37, 38). These signaling events in turn modulate actin binding proteins and the F-actin cytoskeleton (11, 3538). These responses may play important roles in regulating neutrophil migration along ECs to their borders in preparation for transmigration.

Previous studies demonstrated that neutrophil adherence to TNF-{alpha}-activated pulmonary microvascular ECs induces changes in the actin cytoskeleton of ECs (35, 36). These changes require pretreatment of ECs with TNF-{alpha}, since neutrophil adherence to untreated ECs is minimal and does not induce such changes (35, 36). These cytoskeletal changes are inhibited by an anti-ICAM-1 antibody and are mimicked by cross-linking ICAM-1 with antibodies, suggesting that signaling events induced by ICAM-1 are required. Cross-linking ICAM-1 induces activation of p38 and phosphorylation of heat shock protein 27 (HSP27), and inhibition of p38 in ECs by SB-203580 prevents actin cytoskeletal rearrangements induced by ICAM-1 cross-linking or neutrophil adherence. In addition, pretreatment of ECs with SB-203580 attenuates neutrophil migration toward EC borders (37). These studies suggest a role for p38 in mediating cytoskeletal changes induced by neutrophil adherence. However, these studies largely depended on a pharmacological inhibitor. The only studies evaluating the phosphorylation of p38 in ECs utilized cross-linking of ICAM-1 with antibodies to mimic the effect of neutrophil adherence (37). The ability of neutrophils to activate p38 in ECs has not been directly examined.

The MAPK pathway is composed of three sequential dual-specificity kinases: MAP kinase kinase kinase, MAP kinase kinase (MKK), and MAPK (22, 24, 39). Among the seven MKKs identified, MKK3 and MKK6 are highly selective for p38 activation and do not activate other MAPKs under most conditions (22, 24, 39). Interestingly, MKK-independent activation of p38 MAPK has also been reported (16, 17, 34).

To date, four members of the p38 MAPK family have been identified: p38{alpha}, p38{beta}, p38{gamma}, and p38{delta} (reviewed in Refs. 22, 24, 39). Cultured human ECs express {alpha}-, {beta}-, and {delta}-isoforms at protein levels that can be detected by Western blot analysis (19). Although these isoforms share certain structural and functional similarities, differences exist in their upstream kinase specificity as well as their downstream target specificity, suggesting that these isoforms may have nonoverlapping functions (19, 28, 33). MAPK-activated protein kinase 2, a downstream target of p38 MAPK that is responsible for phosphorylating HSP27, is activated by p38{alpha} and p38{beta}, but much less effectively by p38{gamma} and p38{delta} (8, 15, 30). The widely used p38 inhibitor SB-203580 inhibits the activation of p38{alpha} and p38{beta}, but not the other two isoforms (23).

Our previous studies demonstrating that SB-203580 inhibits cytoskeletal rearrangements in ECs upon neutrophil adhesion led to the hypothesis for the present study that neutrophil adherence leads to activation of p38{alpha} and/or p38{beta} and downstream responses in pulmonary microvascular ECs through upstream MKKs.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Neutrophil adherence or ICAM-1 cross-linking. Blood was drawn from healthy humans by venipuncture after informed consent was obtained. Blood was layered on top of histopaque density gradients (Sigma, St. Louis, MO) and centrifuged for 30 min at 1,800 rpm at room temperature (36). The neutrophil layer was collected and washed with HBSS. Red blood cells were lysed, and neutrophils were washed and counted. The purity of isolated neutrophils was >95%.

Human pulmonary microvascular ECs (Cambrex, East Rutherford, NJ) were cultured as described (36, 38). Confluent ECs were stimulated with 20 ng/ml TNF-{alpha} for 24 h. In studies examining the effect of neutrophil adhesion, neutrophils (neutrophil-EC = 1:1) were allowed to adhere for the indicated times in the absence of any exogenous neutrophil stimulators, and nonadherent neutrophils were washed off before they were fixed for staining or to evaluate the localization of neutrophils on ECs as described below (37). In studies where neutrophil adhesion was mimicked by cross-linking ICAM-1, ECs were incubated with murine anti-human ICAM-1 antibody for 30 min, washed, and incubated with goat F(ab)2 fragment of IgG against murine IgG F(c) fragment for the indicated times (36, 38).

Visualization of phosphorylated p38 MAPK, F-actin, and localization of neutrophils on ECs. The cells were fixed with 1% paraformaldehyde and incubated sequentially with 1% BSA and 1% normal goat serum in PBS, 10 µg/ml rabbit antiphosphorylated p38 (New England Biolabs, Beverly, MA), and 10 µg/ml fluorescein-conjugated goat anti-rabbit IgG. F-actin was detected with rhodamine-labeled phalloidin (37). The slides were examined under a Leica DM IRE2 or Zeiss LSM 410 confocal microscope. We identified adherent neutrophils by scanning through the samples in the z-direction. For each experiment, the images of all groups were captured under the same conditions. Phosphorylated p38 in a slice of ECs was quantified using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). The percentage of total adherent neutrophils present at EC borders was determined at each time point. For each slide, neutrophils in 10 randomly generated fields were counted and averaged.

Synthesis of oligonucleotides. 2'-Methoxyethyl-modified, uniform phosphorothioate backbone oligonucleotides were prepared as described previously (38). The p38{alpha} antisense oligonucleotide had the sequence 5'-TTCTCTTATCTGAGTCCAAT-3'. A six-base mismatch control oligonucleotide had the sequence 5'-TTATCCTAGCTTAGACCTAT-3'. Nucleotides containing 2'-methoxyethyl modifications are indicated by underlines.

Treatment of ECs with small interfering RNA or oligonucleotides. Small interfering RNA (siRNA) targeting MKK3 or MKK6 was obtained as a pool of four or more siRNA duplexes from Dharmacon (Lafayette, CO). ECs were plated at ~80% confluence. After 24 h, the cells were washed twice and treated for 4 h with 100 nM siRNA or 25–100 nmol/l oligonucleotides premixed with 10 µg/ml Lipofectin (Life Technologies, Gaithersburg, MD). The cells were then incubated with normal culture medium for 72 h before ELISA or immunoblotting was performed (37, 38).

Measurement of p38 MAPK activity, HSP27 phosphorylation, and actin distribution. The activity of p38 MAPK was evaluated with a p38 assay kit (New England Biolabs, Ref. 37). Phosphorylated p38 was immunoprecipitated from equal amount of protein (ezrin-radixin-moesin in the samples was used as a loading control), or to evaluate the activity of p38{beta}, p38{beta} was immunoprecipitated by incubating 250 µg of preabsorbed cell lysates with 2 µg of anti-p38{beta} antibody for 2 h at 4°C and subsequently with 30 µl of protein A/G plus agarose beads overnight. The amount of immunoprecipitated phosphorylated p38 was examined by SDS-PAGE using 4–12% gradient gels (Novex, San Diego, CA) followed by immunoblotting (37). In addition, the activity of immunoprecipitated p38 was evaluated using exogenous activated transcription factor (ATF)-2 as a substrate, and phosphorylation of ATF-2 was evaluated using an antibody that recognizes phospho-ATF-2 at Thr71.

Phosphorylation of HSP27 was detected as described (37).

We examined the distribution of actin in the Triton X-soluble and -insoluble fractions by lysing ECs with buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM {beta}-glycerolphosphate, 1 mM Na3VO4, and protease inhibitors (37). The cell lysates were microcentrifuged, the supernatants were collected, and the pellets were dissolved in equal volumes of SDS buffer. The proteins were separated by SDS-PAGE using 4–12% gradient gels and stained with colloidal blue (Novex, San Diego, CA). The integrated density of actin in the Triton X-insoluble and -soluble fractions was quantified by densitometry, and the percentage of total actin recovered in the Triton X-soluble and -insoluble fraction was calculated for each sample.

Statistical analysis. Data were analyzed by one-way ANOVA or the Student's t-test. A P value <0.05 was considered significant. The data were expressed as means ± SE.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Neutrophil adherence induced an increase in the phosphorylated p38 MAPK in ECs. To determine whether neutrophil adherence induces activation of p38 MAPK in ECs, we examined phosphorylated p38 using immunocytochemistry. Phosphorylated p38 was present in TNF-{alpha}-treated ECs in the absence of neutrophils. Neutrophil adherence for 2 or 6 min induced an increase in the amount of phosphorylated p38 in ECs, and phosphorylated p38 was present primarily in a punctate distribution located in the central regions of the ECs (Fig. 1A). Little colocalization of phosphorylated p38 and F-actin was observed in ECs.



View larger version (64K):
[in this window]
[in a new window]
 
Fig. 1. Neutrophil adherence induced an increase in the phosphorylated p38 MAPK in TNF-{alpha}-pretreated endothelial cells (ECs). Phosphorylated p38 MAPK and F-actin in a slice of ECs was examined by confocal microscopy. A: distribution of phosphorylated p38 (green) and F-actin (red) in ECs without neutrophils adherent or with neutrophils adherent for 2 or 6 min. A representative image through a slice of ECs is shown, and the arrows indicate the position of an adherent neutrophil. B: we quantified phosphorylated p38 in ECs by measuring the percentage of total pixels that have intensity values of 0–34, 35–70, 71–100, 101–140, or 141–255. C: quantification of F-actin staining intensity in ECs without adherent neutrophils or with neutrophils adherent for 2 or 6 min. Data are means ± SE of 8 confocal images for each condition. Open bars, ECs without neutrophil adherence; light gray bars, ECs with neutrophils adherent for 2 min; filled bars, ECs with neutrophils adherent for 6 min. *P < 0.05 when compared with cells without neutrophil adherence. PMN, neutrophil.

 
Because the staining of phosphorylated p38 was very localized, we quantified the amount of phosphorylated p38 by measuring the percentage of total pixels that had intensity values of 0–34, 35–70, 71–100, 101–140, or 141–255 (Fig. 1B). Neutrophil adherence induced an increase in the percentage of pixels that had higher staining intensities, indicating activation of p38 MAPK in ECs occurred. Consistent with our previous observations (37), neutrophil adherence also induced an increase in F-actin in ECs, and the mean staining intensity of F-actin in a slice of ECs increased from 35.7 ± 2.1 to 50.4 ± 4.0 or 56.0 ± 4.7 after neutrophil adherence for 2 or 6 min, respectively (P < 0.05, Fig. 1C). These results indicate that neutrophil adhesion induces activation of p38 in ECs, as well as changes in the F-actin cytoskeleton.

Role of MKK3 and MKK6 in mediating activation of p38 MAPK. These data, along with our previous observations, clearly demonstrate that activation of p38 MAPK occurs in TNF-{alpha}-treated ECs upon neutrophil adherence or ICAM-1 cross-linking and is required for the cytoskeletal changes (37). The following experiments were performed to determine the upstream MKKs leading to p38 activation. In particular, the role of MKK3 and MKK6 was examined using siRNA since these two MKKs selectively activate p38 (24). Treatment with MKK3 or MKK6 siRNA completely and selectively inhibited the protein expression of their respective target (Fig. 2A). Combined treatment with both siRNA inhibited both MKKs (Fig. 2A). The expression neither of p38 (Fig. 2A) nor of ICAM-1 (Fig. 2B) was affected by these siRNA.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 2. Inhibition of MAP kinase kinase (MKK) 3 or MKK6 protein expression by small interfering (si) RNA prevented p38 phosphorylation induced by ICAM-1 cross-linking. A: ECs were treated with 100 nM control, MKK3, MKK6, or MKK3 + MKK6 siRNA, and the protein expression of MKK3 and MKK6 was examined by immunoblot. The expression of p38 was also examined as a control. B: ECs were treated with siRNA as described in A, and the expression of ICAM-1 following TNF-{alpha} treatment for 24 h was examined by immunoblotting. The data are expressed as means ± SE and presented as fold changes over the control siRNA-treated samples (n = 4–6 in each sample). C: ECs were treated with siRNA as described in A, and ICAM-1 cross-linking was performed. Phosphorylation of p38 MAPK before (open bars) or after ICAM-1 cross-linking for 6 min (closed bars) was examined by immunoblotting and normalized to total p38 MAPK for each sample. The data are expressed as means ± SE and presented as fold changes over the control siRNA-treated samples without ICAM-1 cross-linking (n = 4 or 5 in each sample). *P < 0.05 when compared with noncross-linked controls in each group by ANOVA; **P < 0.05 only by paired t-test when compared with noncross-linked controls in the group; #P < 0.05 when compared with the corresponding groups in control siRNA-treated samples.

 
The effect of these MKK siRNA on p38 activation following ICAM-1 cross-linking in TNF-{alpha}-treated ECs was then examined. ECs treated with control, MKK3, MKK6, or combined MKK3 and MKK6 siRNA were stimulated with TNF-{alpha}, and ICAM-1 cross-linking was performed. In ECs before ICAM-1 cross-linking, treatment with MKK3 siRNA, but not MKK6 siRNA, significantly reduced p38 phosphorylation from 1 ± 0.04 to 0.52 ± 0.09 (n = 5, P < 0.05, Fig. 2C), and combined treatment of MKK3 and MKK6 siRNA did not induce a further reduction (Fig. 2C). These data indicate that MKK3 contributes to p38 phosphorylation before ICAM-1 cross-linking and that MKK3/MKK6-independent phosphorylation occurs in these ECs.

ICAM-1 cross-linking induced a significant increase in p38 phosphorylation in control siRNA-treated samples. This increase was completely prevented by MKK3 siRNA, and a similar degree of inhibition was observed in cells treated with both MKK3 and MKK6 siRNA (Fig. 2C). In MKK6 siRNA-treated samples, ICAM-1 cross-linking-induced p38 phosphorylation was significantly reduced when compared with the control siRNA-treated samples, although the degree of inhibition was less when compared with MKK3 siRNA- or combined MKK3 and MKK6 siRNA-treated samples (Fig. 2C). These data indicate that both MKK3 and MKK6 are required for p38 activation induced by ICAM-1 cross-linking.

Antisense to p38{alpha} inhibited activation of p38 MAPK upon neutrophil adhesion or ICAM-1 cross-linking. The role of the {alpha}-isoform of p38 MAPK in mediating the cytoskeletal changes in ECs upon neutrophil adherence was examined with an antisense oligonucleotide. Treatment with antisense to p38{alpha} inhibited the protein expression of p38{alpha}, but not p38{beta} (Fig. 3).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3. Antisense to p38{alpha} inhibited the protein expression of p38{alpha} in ECs. EC were either left untreated (C) or treated with 25–100 nM antisense to p38{alpha} or 100 nM control oligonucleotide, and the protein expression of p38{alpha} and p38{beta} was examined.

 
Treatment with p38{alpha} antisense had no effect on ICAM-1 expression. TNF-{alpha} induced a similar increase in ICAM-1 expression on ECs not treated with oligonucleotides [346 + 11 to 2,359 + 107 mean fluorescence units (MFU)] or on ECs treated with 100 nM control oligonucleotide (557 ± 37 to 2,397 ± 243 MFU) or p38{alpha} antisense (443 ± 52 to 2,141 ± 84 MFU).

To examine the role of p38{alpha} in mediating ICAM-1-initiated signaling cascades and downstream events, the effect of p38{alpha} antisense on the activation of ECs on neutrophil adherence was first examined. In cells treated with control oligonucleotide, neutrophil adherence induced an increase in the phosphorylated p38 in ECs when examined by confocal microscopy (Table 1). Treatment of ECs with p38{alpha} antisense prevented this increase, indicating that activation of p38 in ECs upon neutrophil adherence requires p38{alpha} (Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1. Phosphorylated p38 and F-actin cytoskeleton in ECs following neutrophil adhesion for 2 min

 
The effect of p38{alpha} antisense on the activation of p38 induced by ICAM-1 cross-linking was also examined. ICAM-1 cross-linking for 6 min induced an increase in phosphorylated p38 in cells treated with control oligonucleotide (Fig. 4A). Treatment with antisense to p38{alpha} completely inhibited this increase, suggesting that p38{alpha} is the isoform activated by ICAM-1 cross-linking (Fig. 4A).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4. Antisense to p38{alpha} inhibited activation of p38 MAPK induced by ICAM-1 cross-linking. ECs treated with 100 nM control or p38{alpha} antisense were treated with TNF-{alpha}, and ICAM-1 cross-linking was performed. Phosphorylated p38 was immunoprecipitated from equal amount of proteins. A: the amount of immunoprecipitated phosphorylated p38 (Pi-p38) was examined by immunoblot. The amount of ezrin-radixin-moesin (ERM) proteins in each sample used for immunoprecipitation was also examined as a loading control. *P < 0.05 compared with the controls without ICAM-1 cross-linking (n = 4). B: activity of immunoprecipitated phosphorylated p38 MAPK was examined by a kinase activity assay using exogenous activating transcription factor (ATF)-2 as a substrate. The activity of p38 MAPK was evaluated by the amount of phosphorylated ATF-2. A representative blot showing ATF-2 phosphorylation [diphosphorylated at Thr71 and Thr69 (Di) and monphosphorylated at Thr71 (Mono)] was included. C: densitometric analysis of ATF-2 phosphorylation as in B. *P < 0.05 compared with the controls without ICAM-1 cross-linking. Open bars, no cross-linking; closed bars, cross-linking ICAM-1 for 6 min. The data are expressed as fold changes over the control oligonucleotide-treated samples without ICAM-1 cross-linking (n = 4).

 
To further evaluate the role of this antisense in p38 activation induced by ICAM-1 cross-linking, we performed an in vitro kinase assay using ATF-2 as a substrate. Phosphorylation of ATF-2 was evaluated with an antibody that recognizes the phospho-ATF-2 at Thr71. By immunoblotting, two forms of phosphorylated ATF-2 were recognized by this antibody: the monophosphorylated form at Thr71 and the diphosphorylated form at Thr71 and Thr69 (Fig. 4B). Without ICAM-1 cross-linking, p38 activity was similar whether the cells were treated with control or p38{alpha} antisense (Fig. 4B). ICAM-1 cross-linking for 6 min induced a 3.6 ± 0.6-fold increase in the monophosphorylated form of ATF-2 in cells treated with control oligonucleotides (Fig. 4, B and C). Treatment with the antisense to p38{alpha} MAPK inhibited this increase (Fig. 4, B and C). In these experiments, the level of the diphosphorylated form of ATF-2 was unchanged by ICAM-1 cross-linking whether the cells were treated with control or the p38{alpha} MAPK antisense (Fig. 4, B and C). These data demonstrate that both phosphorylation and activation of p38 on ICAM-1 cross-linking require the {alpha}-isoform of p38.

Our previous studies using SB-203580 suggest that p38{beta} could also mediate the cytoskeletal changes induced by ICAM-1 ligation. To determine whether p38{beta} is also activated by ICAM-1 cross-linking, we immunoprecipitated p38{beta} and examined its activity by the same in vitro kinase assay using ATF-2 as a substrate. There was a small but significant increase in the baseline activity of p38{beta} before ICAM-1 cross-linking in ECs treated with p38{alpha} antisense (Fig. 5, A and B). ICAM-1 cross-linking, however, did not induce an increase in the activity of p38{beta} whether the cells were treated with control or p38{alpha} antisense (Fig. 5, A and B). These data demonstrated that ICAM-1 cross-linking does not induce activation of p38{beta}.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 5. ICAM-1 cross-linking did not induce activation of p38{beta}, whether the cells were treated with control or p38{alpha} antisense. The activity of p38{beta} was evaluated by a kinase assay using ATF-2 as a substrate. A: representative immunoblot of phospho-ATF-2. The majority of phospho-ATF-2 was in the diphosphorylated form. B: densitometric analysis of ATF-2 phosphorylation (the diphosphorylated form) as in A. Open bars, no cross-linking; closed bars, cross-linking ICAM-1 for 6 min. The data are expressed relative to the values of control oligonucleotide-treated samples without ICAM-1 cross-linking (n = 4). #P < 0.05 when compared with control oligonucleotide-treated samples.

 
Antisense to p38{alpha} MAPK inhibited HSP27 phosphorylation and actin remodeling induced by ICAM-1 ligation. HSP27 is a downstream target of p38 MAPK pathway and may mediate actin polymerization upon phosphorylation. Because antisense to p38{alpha} completely inhibited p38 activation induced by ICAM-1 cross-linking, the effect of this antisense on HSP27 phosphorylation was examined. ICAM-1 cross-linking for 6 min resulted in a 5.3 ± 0.7-fold increase in HSP27 phosphorylation in cells treated with control oligonucleotides (Fig. 6A). Treatment with antisense to p38{alpha} MAPK did not change the protein expression of HSP27 but inhibited the increase in HSP27 phosphorylation upon ICAM-1 cross-linking (Fig. 6A).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 6. p38{alpha} antisense inhibited heat shock protein (HSP) 27 phosphorylation (A) and redistribution of actin into the Triton X-insoluble fraction (B) in response to ICAM-1 cross-linking. A: ECs treated with 100 nM control or p38{alpha} antisense were treated with TNF-{alpha}, and phosphorylation of HSP27 before or after ICAM-1 cross-linking for 6 min was examined. The autorad showing HSP phosphorylation and immunoblot for total HSP27 and p38{alpha} are included. The densitometric data are expressed as fold changes over the control oligonucleotide-treated samples without ICAM-1 cross-linking. B: ECs treated with 100 nM control or p38{alpha} antisense were treated with TNF-{alpha}. The distribution of actin in the Triton X-soluble and -insoluble fractions following ICAM-1 cross-linking for 6 min was determined. A representative image showing the distribution of actin in both fractions is included, and the percentage of total actin in the Triton X-soluble or Triton X-insoluble fraction was measured for each sample. Open bars, no cross-linking; closed bars, cross-linking ICAM-1 for 6 min. *P < 0.05 compared with the controls without ICAM-1 cross-linking (n = 3).

 
To determine whether p38{alpha} is required for actin remodeling in ECs induced by ICAM-1 cross-linking, we fractionated the cells into Triton X-insoluble and -soluble fractions and examined the effect of the p38{alpha} antisense on actin distribution in these two fractions. Measurement of the percentage of total actin recovered in the Triton X-insoluble fraction indicated that ICAM-1 cross-linking for 6 min significantly increases the percentage from 51.2 ± 2.3 to 68.8 ± 1.3% in cells treated with control oligonucleotides, indicating that changes in actin organization occurred (n = 3 or 4, P < 0.05, Fig. 6B). Treatment with p38{alpha} antisense inhibited this increase (Fig. 6B).

Whether p38{alpha} is required for F-actin redistribution induced by neutrophil adherence was also determined. In the cells treated with control oligonucleotides, neutrophil adherence for 2 or 6 min induced formation of F-actin clusters and thickening of F-actin filaments in ECs (Fig. 7, A-C). Antisense to p38{alpha} attenuated these changes (Fig. 7, D-F). These data demonstrate that p38{alpha} is required for actin reorganization induced by neutrophil adherence. In addition, quantification of the F-actin staining intensity in ECs showed that the increase in the F-actin staining intensity in control oligonucleotide-treated ECs following neutrophil adhesion for 2 min was absent in p38{alpha} antisense-treated ECs (Table 1). Together, these data indicate that changes in the F-actin cytoskeleton including F-actin rearrangement and more F-actin formation in ECs following neutrophil adhesion require p38{alpha} in ECs.



View larger version (131K):
[in this window]
[in a new window]
 
Fig. 7. p38{alpha} antisense inhibited F-actin remodeling in ECs induced by neutrophil adherence. ECs treated with 100 nM control (A-C) or p38{alpha} antisense (D-F) were stimulated with TNF-{alpha}. F-actin distribution in cells left untreated (A, D) or treated with neutrophils adherent for 2 min (B, E) or 6 min (C, F) was examined. Arrows: position of an adherent neutrophil. Images shown represent at least 2 independent experiments for each treatment. Scale bar: 25 µm.

 
Antisense to p38{alpha} MAPK attenuated neutrophil migration toward EC borders. To determine the physiological roles of p38{alpha} in ECs in mediating neutrophil migration on EC monolayer, we determined the effect of p38{alpha} antisense on neutrophil migration toward EC borders. In ECs treated with control oligonucleotides, the percentage of adherent neutrophils located at EC borders increased over time. In ECs treated with antisense to p38{alpha}, this increase was significantly attenuated (Fig. 8). These data suggest that activation of p38{alpha} in ECs upon neutrophil adherence plays important roles in mediating neutrophil migration toward EC borders.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 8. p38{alpha} antisense reduced the percentage of neutrophils observed along the EC edges. ECs treated with 100 nM control or p38{alpha} antisense were stimulated with TNF-{alpha} and washed. Purified neutrophils were added to the wells and allowed to adhere for 1–6 min. The percentage of neutrophils that were present along EC borders was measured. {bullet}, Control-treated ECs; {circ}, p38{alpha} antisense-treated ECs. *P < 0.05 compared between control and p38{alpha} antisense-treated ECs (n = 4).

 
Postulated signaling pathways. The data presented in this study led us to the postulated signaling mechanisms underlying neutrophil-induced changes in the F-actin cytoskeleton in ECs (Fig. 9). During neutrophil adhesion, ligation of ICAM-1 initiates signaling events into ECs, resulting in the activation of both MKK3 and MKK6. This leads to the activation of the {alpha}-isoform of p38 MAPK. Activated p38{alpha} MAPK results in phosphorylation of HSP27, presumably through MAPKAP-2, and is required for changes in the F-actin cytoskeleton in ECs and neutrophil migration along the EC surface to reach the EC borders.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 9. Postulated signaling mechanisms leading to the changes in the F-actin cytoskeleton in TNF-{alpha}-pretreated ECs following neutrophil adhesion. Solid line, based on data presented in this study; dashed line, postulated pathway.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Neutrophil adhesion to TNF-{alpha}-treated ECs induced signaling events into ECs. Using confocal microscopy, this study presents evidence that p38 MAPK is activated in ECs following neutrophil adhesion. Furthermore, MKK3- and MKK6-dependent activation of the {alpha}-isoform of p38 MAPK is required for phosphorylation of HSP27, actin cytoskeletal changes in ECs, and neutrophil migration on the EC surface.

The observations that neutrophil adherence induces activation of p38 in TNF-{alpha}-treated ECs and that activation of p38 is required for cytoskeletal changes in these ECs led to the studies to examine the specific role of upstream MKKs and the {alpha}-isoform of the p38 MAPK. In this study, siRNA and an antisense oligonucleotide were utilized to examine the specific role of MKK3, MKK6, and the {alpha}-isoform of p38 MAPK. The protein expression of MKK3, MKK6, and p38{alpha} was completely and specifically inhibited. These results indicate that siRNA and antisense oligonucleotide can be used to examine the functions of a particular target in ECs.

The studies using MKK3 and MKK6 siRNA and p38{alpha} antisense allowed us to examine the basal p38 phosphorylation before ICAM-1 cross-linking. Inhibition of p38{alpha} protein expression did not result in a decrease in the baseline activity of p38 MAPK in these ECs, suggesting that either these cells have a very low baseline p38{alpha} activity or there are compensatory mechanisms that lead to an increase in the activity of other p38 isoforms (the {beta}-, {gamma}-, and/or {delta}-isoforms). Inhibition of p38{alpha} indeed induced a small but significant increase in the activity of p38{beta}. Recent studies have demonstrated that p38{alpha} negatively regulates the activities of some of its upstream activators including MKK6 and transforming growth factor {beta}-activated protein kinase 1 (TAK1) (2, 6). Thus this negative feedback mechanism may account for the increased activity of other p38 isoforms such as p38{beta} in p38{alpha} antisense-treated samples. In addition, the basal p38 phosphorylation was significantly reduced by MKK3 siRNA and not by MKK6 siRNA. Treatment with both MKK3 and MKK6 siRNA did not result in a further reduction. These data suggest that MKK3, but not MKK6, accounts for a significant portion of the phosphorylated p38 in TNF-{alpha}-pretreated ECs. These data also suggest that MKK3- and MKK6-independent mechanisms contribute to this p38 phosphorylation. These other mechanisms possibly include: 1) other MKKs such as MKK4 and MKK7, since these MKKs can phosphorylate p38 under certain experimental conditions (9, 10, 26), and 2) MKK-independent p38 phosphorylation. Binding of p38 to TAK1-binding protein 1 leads to autophosphorylation and activation of p38 independent of MKKs (16, 17, 34).

Our data suggest that MKK3- and MKK6-dependent mechanisms lead to activation of the {alpha}-isoform of p38 in response to ICAM-1 ligation since 1) treatment with MKK3 or MKK6 siRNA, either singly or in combination, inhibited phosphorylation of p38 induced by ICAM-1 ligation; and 2) activation of p38 induced by neutrophil adherence or ICAM-1 cross-linking was completely inhibited by p38{alpha} antisense. Confocal microscopy allowed us to examine the distribution of phosphorylated p38 in ECs before or after neutrophil adherence. The increase in phosphorylated p38 in ECs following neutrophil adherence occurs in a punctuate manner and was prevented when ECs were treated with p38{alpha} antisense. In addition, p38{alpha} antisense inhibited phosphorylation of p38 and the increase in p38 activity induced by ICAM-1 cross-linking. Thus these data suggest that cross-linking with ICAM-1 or neutrophil adhesion induces activation of the {alpha}-isoform of p38 through MKK3- and MKK6-dependent mechanisms.

Although p38{alpha} and p38{beta} exhibit considerable sequence homology, they clearly have distinct biological functions, and differential activation of p38{alpha} or p38{beta} isoforms can occur in response to a physiological stimulus. For example, in venular ECs, IL-1{beta} preferentially activates the p38{alpha} isoform (19). Estrogen, however, inhibits p38{alpha} activity while stimulating p38{beta} activation in aortic ECs (29). The present study suggests that p38{alpha} is activated upon ICAM-1 ligation. This differential activation of p38 isoforms likely occurs through distinct upstream signaling events including MKKs. When overexpressed in cells, MKK3 can activate p38{alpha}-, {gamma}-, and {delta}-isoforms, whereas MKK6 is capable of activating all isoforms (1, 12, 13). Although both MKK3 and MKK6 are required for p38 phosphorylation in response to ICAM-1 ligation, inhibition of MKK3 resulted in a greater reduction in p38 phosphorylation than inhibition of MKK6. These data suggest that MKK3-dependent mechanisms may be required for most of the p38 phosphorylation and may in part account for the preferential activation of p38{alpha} in response to ICAM-1 ligation.

To determine the role of p38{alpha} in activating downstream events in response to ICAM-1 cross-linking, we examined the effect of p38{alpha} antisense on HSP27 phosphorylation. Both p38{alpha} and p38{beta} are capable of activating MAPKAP-2, which in turn phosphorylates HSP27, and inhibition of HSP27 phosphorylation by SB-203580 has been demonstrated in many studies (see, for example, Refs. 20, 21, 37). In ECs, phosphorylation of HSP27 in response to various stimuli is thought to play important roles in mediating actin rearrangements (21, 25, 31). The study presented here demonstrates that selective activation of p38{alpha} in response to ICAM-1 ligation is required for HSP27 phosphorylation. This specificity may be cell type and stimulus dependent. For example, stimulation of aortic ECs by estrogen activates p38{beta} but inhibits p38{alpha}, and activation of p38{beta} is required for HSP27 phosphorylation (29).

Antisense to p38{alpha} also inhibited actin cytoskeletal rearrangements. This observation is consistent with previous studies demonstrating that activation of p38 is required for cytoskeletal rearrangements induced by neutrophil adherence or ICAM-1 cross-linking. This present study extended previous results to provide quantitative data describing the amount of F-actin and the role of p38{alpha}. ICAM-1 cross-linking increased the amount of Triton X-insoluble actin. Antisense to p38{alpha} inhibited this increase. In addition, confocal imaging of F-actin distribution indicated that while p38{alpha} antisense did not change the TNF-{alpha}-regulated F-actin distribution, it inhibited F-actin remodeling in response to ICAM-1 cross-linking or neutrophil adherence. The exact mechanisms whereby p38{alpha} antisense inhibits actin rearrangements are unclear, although its ability to inhibit HSP27 phosphorylation leads us to hypothesize that HSP27 may be an important modulator. This hypothesis is consistent with studies using cells transfected with a nonphosphorylatable mutant of HSP27 to establish that HSP27 phosphorylation is required for actin rearrangements in response to various stimuli (4, 20, 25, 29).

The data presented in this study have focused on the activation of p38 isoforms and the role of specific upstream MKKs upon ICAM-1 ligation by antibodies or neutrophil adhesion. The signaling mechanisms following ICAM-1 ligation that lead to MKK activation remain to be determined. Activation of Src tyrosine kinases occurs following ICAM-1 ligation and is required for the activation of p38 MAPK (11, 38). Thus Src tyrosine kinases likely lie upstream of MKK 3 and MKK6. Other signaling cascades induced by ICAM-1 ligation in various EC types include activation of Rho GTPases, phospholipase C, and increases in intracellular calcium (7, 14, 18). Whether any of these signaling events may play a role in the activation of p38 MAPK upon ICAM-1 ligation remains to be determined, although our previous studies using pharmacological inhibitors show that intracellular calcium is not required for the cytoskeletal changes in TNF-{alpha}-treated ECs upon neutrophil adherence, suggesting that intracellular calcium may not be required (35).

Treatment of ECs with antisense to p38{alpha} reduced neutrophil migration toward EC borders. These data suggest that signaling events in ECs induced by ICAM-1 ligation upon neutrophil adherence are required for neutrophil migration along the EC surface, and activation of p38{alpha} may be an essential regulatory element. Studies using cells expressing a truncated form of ICAM-1 lacking the cytoplasmic domain show that the cytoplasmic signaling domain of ICAM-1 is required for leukocyte transmigration across ECs, suggesting that signaling through ICAM-1 is essential for leukocyte migration along and/or across ECs (18, 27, 32). Adhesion of leukocytes induces changes in the distribution of ICAM-1 and its association with the F-actin cytoskeleton through actin binding proteins (3, 5, 40). These changes in ICAM-1 likely play important roles in modulating neutrophil migration. Inhibition of p38 MAPK in ECs does not prevent ICAM-1 redistribution on ECs induced by antibodies, suggesting that ICAM-1 redistribution alone is not sufficient to mediate neutrophil migration along ECs (37). Other p38{alpha}-dependent changes that include changes in EC shape, surface characteristics, junctional functions, and/or signaling pathways in ECs all likely to contribute to the migration of neutrophils along ECs to reach the EC junctions in inflammation.

In summary, this study demonstrates that neutrophil adherence or ICAM-1 cross-linking activates the {alpha}-isoform of p38 and not the p38{beta} in ECs. This activation of p38{alpha} requires MKK3 and MKK6 and is essential for phosphorylation of HSP27 and actin rearrangements in ECs upon ICAM-1 cross-linking or neutrophil adherence. Activation of p38{alpha} in ECs induced by neutrophil adherence appears essential for regulating neutrophil migration toward EC borders, likely through cytoskeleton-dependent mechanisms.


    GRANTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was supported by a Parker B. Francis Fellowship from the Francis Families Foundation (Q. Wang), National Heart, Lung, and Blood Institute Grants HL-070009 (Q. Wang) and HL-48160, and a Clinical Scientist Award in Translational Research from the Burroughs Wellcome Fund (C. M. Doerschuk).


    ACKNOWLEDGMENTS
 
The confocal studies were performed at the imaging core facility in the Department of Pediatrics at Case Western Reserve University.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C. M. Doerschuk, Rainbow Babies and Children's Hospital, Rm. 8321, 11100 Euclid Ave., Cleveland, OH 44106 (E-mail: cmd22{at}po.cwru.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alonso G, Ambrosino C, Jones M, and Nebreda AR. Differential activation of p38 mitogen-activated protein kinase isoforms depending on signal strength. J Biol Chem 275: 40641–40648, 2000.[Abstract/Free Full Text]
  2. Ambrosino C, Mace G, Galban S, Fritsch C, Vintersten K, Black E, Gorospe M, and Nebreda AR. Negative feedback regulation of MKK6 mRNA stability by p38alpha mitogen-activated protein kinase. Mol Cell Biol 23: 370–381, 2003.[Abstract/Free Full Text]
  3. Barreiro O, Yanez-Mo M, Serrador JM, Montoya MC, Vicente-Manzanares M, Tejedor R, Furthmayr H, and Sanchez-Madrid F. Dynamic interaction of VCAM-1 and ICAM-1 with moesin and ezrin in a novel endothelial docking structure for adherent leukocytes. J Cell Biol 157: 1233–1245, 2002.[Abstract/Free Full Text]
  4. Benndorf R, Hayess K, Ryazantsev S, Wieske M, Behlke J, and Lutsch G. Phosphorylation and supramolecular organization of murine small heat shock protein HSP25 abolish its actin polymerization-inhibiting activity. J Biol Chem 269: 20780–20784, 1994.[Abstract/Free Full Text]
  5. Carman CV, Jun CD, Salas A, and Springer TA. Endothelial cells proactively form microvilli-like membrane projections upon intercellular adhesion molecule 1 engagement of leukocyte LFA-1. J Immunol 171: 6135–6144, 2003.[Abstract/Free Full Text]
  6. Cheung PC, Campbell DG, Nebreda AR, and Cohen P. Feedback control of the protein kinase TAK1 by SAPK2a/p38alpha. EMBO J 22: 5793–5805, 2003.[Abstract/Free Full Text]
  7. Clayton A, Evans RA, Pettit E, Hallett M, Williams JD, and Steadman R. Cellular activation through the ligation of intercellular adhesion molecule-1. J Cell Sci 111: 443–453, 1998.[Abstract/Free Full Text]
  8. Cuenda A, Cohen P, Buee-Scherrer V, and Goedert M. Activation of stress-activated protein kinase-3 (SAPK3) by cytokines and cellular stresses is mediated via SAPKK3 (MKK6); comparison of the specificities of SAPK3 and SAPK2 (RK/p38). EMBO J 16: 295–305, 1997.[Abstract/Free Full Text]
  9. Dashti SR, Efimova T, and Eckert RL. MEK7-dependent activation of p38 MAP kinase in keratinocytes. J Biol Chem 276: 8059–8063, 2001.[Abstract/Free Full Text]
  10. Derijard B, Raingeaud J, Barrett T, Wu IH, Han J, Ulevitch RJ, and Davis RJ. Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science 267: 682–685, 1995.[ISI][Medline]
  11. Durieu-Trautmann O, Chaverot N, Cazaubon S, Strosberg AD, and Couraud PO. Intercellular adhesion molecule 1 activation induces tyrosine phosphorylation of the cytoskeleton-associated protein cortactin in brain microvessel endothelial cells. J Biol Chem 269: 12536–12540, 1994.[Abstract/Free Full Text]
  12. Enslen H, Brancho DM, and Davis RJ. Molecular determinants that mediate selective activation of p38 MAP kinase isoforms. EMBO J 19: 1301–1311, 2000.[Abstract/Free Full Text]
  13. Enslen H, Raingeaud J, and Davis RJ. Selective activation of p38 mitogen-activated protein (MAP) kinase isoforms by the MAP kinase kinases MKK3 and MKK6. J Biol Chem 273: 1741–1748, 1998.[Abstract/Free Full Text]
  14. Etienne-Manneville S, Manneville JB, Adamson P, Wilbourn B, Greenwood J, and Couraud PO. ICAM-1-coupled cytoskeletal rearrangements and transendothelial lymphocyte migration involve intracellular calcium signaling in brain endothelial cell lines. J Immunol 165: 3375–3383, 2000.[Abstract/Free Full Text]
  15. Freshney NW, Rawlinson L, Guesdon F, Jones E, Cowley S, Hsuan J, and Saklatvala J. Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27. Cell 78: 1039–1049, 1994.[ISI][Medline]
  16. Ge B, Gram H, Di Padova F, Huang B, New L, Ulevitch RJ, Luo Y, and Han J. MAPKK-independent activation of p38alpha mediated by TAB1-dependent autophosphorylation of p38alpha. Science 295: 1291–1294, 2002.[Abstract/Free Full Text]
  17. Ge B, Xiong X, Jing Q, Mosley JL, Filose A, Bian D, Huang S, and Han J. TAB1beta (transforming growth factor-beta-activated protein kinase 1-binding protein 1beta), a novel splicing variant of TAB1 that interacts with p38alpha but not TAK1. J Biol Chem 278: 2286–2293, 2003.[Abstract/Free Full Text]
  18. Greenwood J, Amos CL, Walters CE, Couraud PO, Lyck R, Engelhardt B, and Adamson P. Intracellular domain of brain endothelial intercellular adhesion molecule-1 is essential for T lymphocyte-mediated signaling and migration. J Immunol 171: 2099–2108, 2003.[Abstract/Free Full Text]
  19. Hale KK, Trollinger D, Rihanek M, and Manthey CL. Differential expression and activation of p38 mitogen-activated protein kinase alpha, beta, gamma, and delta in inflammatory cell lineages. J Immunol 162: 4246–4252, 1999.[Abstract/Free Full Text]
  20. Hedges JC, Dechert MA, Yamboliev IA, Martin JL, Hickey E, Weber LA, and Gerthoffer WT. A role for p38(MAPK)/HSP27 pathway in smooth muscle cell migration. J Biol Chem 274: 24211–24219, 1999.[Abstract/Free Full Text]
  21. Huot J, Houle F, Marceau F, and Landry J. Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells. Circ Res 80: 383–392, 1997.[Abstract/Free Full Text]
  22. Johnson GL and Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 298: 1911–1912, 2002.[Abstract/Free Full Text]
  23. Kumar S, McDonnell PC, Gum RJ, Hand AT, Lee JC, and Young PR. Novel homologues of CSBP/p38 MAP kinase: activation, substrate specificity and sensitivity to inhibition by pyridinyl imidazoles. Biochem Biophys Res Commun 235: 533–538, 1997.[CrossRef][ISI][Medline]
  24. Kyriakis JM and Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 81: 807–869, 2001.[Abstract/Free Full Text]
  25. Lavoie JN, Hickey E, Weber LA, and Landry J. Modulation of actin microfilament dynamics and fluid phase pinocytosis by phosphorylation of heat shock protein 27. J Biol Chem 268: 24210–24214, 1993.[Abstract/Free Full Text]
  26. Lin A, Minden A, Martinetto H, Claret FX, Lange-Carter C, Mercurio F, Johnson GL, and Karin M. Identification of a dual specificity kinase that activates the Jun kinases and p38-Mpk2. Science 268: 286–290, 1995.[ISI][Medline]
  27. Lyck R, Reiss Y, Gerwin N, Greenwood J, Adamson P, and Engelhardt B. T-cell interaction with ICAM-1/ICAM-2 double-deficient brain endothelium in vitro: the cytoplasmic tail of endothelial ICAM-1 is necessary for transendothelial migration of T cells. Blood 102: 3675–3683, 2003.[Abstract/Free Full Text]
  28. Mudgett JS, Ding J, Guh-Siesel L, Chartrain NA, Yang L, Gopal S, and Shen MM. Essential role for p38alpha mitogen-activated protein kinase in placental angiogenesis. Proc Natl Acad Sci USA 97: 10454–10459, 2000.[Abstract/Free Full Text]
  29. Razandi M, Pedram A, and Levin ER. Estrogen signals to the preservation of endothelial cell form and function. J Biol Chem 275: 38540–38546, 2000.[Abstract/Free Full Text]
  30. Rouse J, Cohen P, Trigon S, Morange M, Alonso-Llamazares A, Zamanillo D, Hunt T, and Nebreda AR. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 78: 1027–1037, 1994.[ISI][Medline]
  31. Rousseau S, Houle F, Landry J, and Huot J. p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelial cells. Oncogene 15: 2169–2177, 1997.[CrossRef][ISI][Medline]
  32. Sans E, Delachanal E, and Duperray A. Analysis of the roles of ICAM-1 in neutrophil transmigration using a reconstituted mammalian cell expression model: implication of ICAM-1 cytoplasmic domain and Rho-dependent signaling pathway. J Immunol 166: 544–551, 2001.[Abstract/Free Full Text]
  33. Tamura K, Sudo T, Senftleben U, Dadak AM, Johnson R, and Karin M. Requirement for p38alpha in erythropoietin expression: a role for stress kinases in erythropoiesis. Cell 102: 221–231, 2000.[ISI][Medline]
  34. Tanno M, Bassi R, Gorog DA, Saurin AT, Jiang J, Heads RJ, Martin JL, Davis RJ, Flavell RA, and Marber MS. Diverse mechanisms of myocardial p38 mitogen-activated protein kinase activation: evidence for MKK-independent activation by a TAB1-associated mechanism contributing to injury during myocardial ischemia. Circ Res 93: 254–261, 2003.[Abstract/Free Full Text]
  35. Wang Q, Chiang ET, Lim M, Lai J, Rogers R, Janmey PA, Shepro D, and Doerschuk CM. Changes in the biomechanical properties of neutrophils and endothelial cells during adhesion. Blood 97: 660–668, 2001.[Abstract/Free Full Text]
  36. Wang Q and Doerschuk CM. Neutrophil-induced changes in the biomechanical properties of endothelial cells: roles of ICAM-1 and reactive oxygen species. J Immunol 164: 6487–6494, 2000.[Abstract/Free Full Text]
  37. Wang Q and Doerschuk CM. The p38 mitogen-activated protein kinase mediates cytoskeletal remodeling in pulmonary microvascular endothelial cells upon intracellular adhesion molecule-1 ligation. J Immunol 166: 6877–6884, 2001.[Abstract/Free Full Text]
  38. Wang Q, Pfeiffer GR II, and Gaarde WA. Activation of SRC tyrosine kinases in response to ICAM-1 ligation in pulmonary microvascular endothelial cells. J Biol Chem 278: 47731–47743, 2003.[Abstract/Free Full Text]
  39. Widmann C, Gibson S, Jarpe MB, and Johnson GL. Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol Rev 79: 143–180, 1999.[Abstract/Free Full Text]
  40. Wojciak-Stothard B, Williams L, and Ridley AJ. Monocyte adhesion and spreading on human endothelial cells is dependent on Rho-regulated receptor clustering. J Cell Biol 145: 1293–1307, 1999.[Abstract/Free Full Text]




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
288/2/L359    most recent
00292.2004v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Wang, Q.
Articles by Doerschuk, C. M.
Articles citing this Article
PubMed
PubMed Citation
Articles by Wang, Q.
Articles by Doerschuk, C. M.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.