Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:1775-1781
Published online before print August 28, 2003, doi: 10.1161/01.ATV.0000094432.98445.36
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
23/10/1775    most recent
01.ATV.0000094432.98445.36v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lerner-Marmarosh, N.
Right arrow Articles by Abe, J.-i.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lerner-Marmarosh, N.
Right arrow Articles by Abe, J.-i.
Related Collections
Right arrow Other arteriosclerosis
(Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:1775.)
© 2003 American Heart Association, Inc.

Vascular Biology

Inhibition of Tumor Necrosis Factor-{alpha}–Induced SHP-2 Phosphatase Activity by Shear Stress

A Mechanism to Reduce Endothelial Inflammation

Nicole Lerner-Marmarosh*; Masanori Yoshizumi*; Wenyi Che*; James Surapisitchat; Hisaaki Kawakatsu; Masashi Akaike; Bo Ding; Qunhua Huang; Chen Yan*; Bradford C. Berk; Jun-ichi Abe

From the Center for Cardiovascular Research, University of Rochester, NY, and the Lung Biology Center (H.K.), University of California, San Francisco, Calif.

Correspondence to Jun-ichi Abe, MD, PhD, Cardiology Unit, Box 679, 601 Elmwood Ave, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642. E-mail jun-ichi_abe{at}urmc.rochester.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Objectives— Atherosclerosis preferentially occurs in areas of turbulent flow, whereas laminar flow is atheroprotective. Inflammatory cytokines have been shown to stimulate adhesion molecule expression in endothelial cells that may promote atherosclerosis, in part, by stimulating c-Jun N-terminal kinase (JNK) and nuclear factor (NF)-{kappa}B transcriptional activity.

Methods and Results— Because Src kinase family and Src homology region 2-domain phosphatase-2 (SHP-2) may regulate JNK activation, we studied the effect of shear stress on endothelial inflammation and JNK. Human umbilical vein endothelial cells preexposed to flow showed decreased tumor necrosis factor (TNF)-{alpha}–induced c-Jun and NF-{kappa}B transcriptional activation. TNF-{alpha}–mediated JNK, c-Jun, and NF-{kappa}B activation required Src and SHP-2 activity. Shear stress significantly inhibited SHP-2 phosphatase activity without affecting TNF-{alpha}–induced Src family kinase activation. Because MEKK3 and Gab1 are critical for TNF-{alpha}–induced c-Jun and NF-{kappa}B activation, we determined the role of SHP-2 phosphatase activity in MEKK3 signaling. A catalytically inactive form of SHP-2 increased MEKK3/Gab1 interaction and inhibited MEKK3 (but not MEKK1)-mediated c-Jun and NF-{kappa}B activation.

Conclusions— These results suggest that SHP-2 is a key mediator for the inhibitory effects of shear stress on TNF-{alpha} signaling in part via regulating MEKK3/Gab1 interaction, MEKK3 signaling, and subsequent adhesion molecule expression.


Key Words: pathophysiology • cell signaling • endothelium • mechanism of atherosclerosis


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The development of atherosclerosis occurs in a highly consistent focal manner, preferentially affecting the outer edges of vessel bifurcations. In these predisposed areas, the flow pattern is turbulent, not laminar as found in protected regions. Many findings suggest that steady laminar flow in blood vessels activates signal transduction events that lead to expression of atheroprotective genes. Previously, we have found that shear stress inhibits tumor necrosis factor (TNF)-{alpha}–induced ASK1 and c-Jun N-terminal kinase (JNK) activation.1,2 There are likely many mechanisms by which flow may inhibit TNF-{alpha} signaling. An important regulator of the TNF-{alpha} pathway is the Src homology 2 domain–containing protein-tyrosine phosphatase SHP-2, whose activity is required for activation of JNK and nuclear factor (NF)-{kappa}B.3,4 The SHP-2 is an ubiquitously expressed cytosolic protein that contains 2 amino-terminal tandem SH2 domains and a carboxyl-terminal catalytic domain.5 SHP-2 associates with tyrosine-phosphorylated growth factor receptors such as epidermal growth factor receptor and platelet-derived growth factor and with cytokine receptors such as {alpha}/ß interferon receptors.6–8 Given that growth factors and cytokines can potently activate SHP-2 phosphatase activity and that SHP-2 phosphatase activity may be a key mediator to regulate mitogen-activated protein kinases,9 we hypothesized that flow-mediated inhibition of SHP-2 phosphatase activity may be a protective mechanism of laminar shear stress through inhibition of cytokine-mediated inflammatory responses such as adhesion molecule expression in endothelial cells.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Cell Culture
Human umbilical vein endothelial cells (HUVECs) were isolated and maintained in 20% FBS/RPMI 1640 medium as previously described.1 At least 4 different HUVEC preparations were used for the experiments shown.

Flow Experiments
Short-flow experiments (<200 minutes) were performed as previously described.1 For long-flow experiments (>6 hours), large react angular parallel flow chamber (5x14 cm, Glyco Tech) was used for transfection and stimulation of the cells by a constant shear stress (please see the online supplement at http://atvb.ahajournals.org).

Plasmids and Transfection
pcDNA3 HA-Gab1 was a gift from Dr Hirano (Osaka University, Osaka, Japan),10 and we subcloned it into pcDNA3.1/His vector (Invitrogen) to add Xpress and His tag. pRK5-MEKK3 (wild-type [WT]), pRK5-CA-MEKK3 (constitutive active form [CA] by deleting the first 11 amino acids) plasmids11 are subcloned into pCMV-tag2 (Strategene) to add Flag tag. pFR-Luc, pFA2-c-Jun, pFC-dbd, pNF-{kappa}B-Luc, and pFC-MEKK1 were from Stratagene. pJ3-hSHP-2 WT and C459S (SHP-2 [C/S]), which abolishes SHP-2 phosphatase activity, were a gift from Dr Neel (Harvard Medical School, Boston, Mass)9 and were also subcloned into pcDNA3.1/His vector. The phosphatase domain deletion mutant of SHP-2 (SHP-2 [1-411]) was created by polymerase chain reaction from SHP-2 WT and subcloning into pcDNA3.1/hisC vector (Invitrogen). Kinase-negative form of Src was cloned into pcDNA3.1 vector.12,13 All plasmid constructs were verified by DNA sequencing. For transient expression experiments, HUVECs were transfected with Lipofectamine Plus (Gibco BRL) as described previously.14

Immunoprecipitation, Western Blot Analysis, Kinases, and SHP-2 Phosphatase Activity
After treatment with reagents, the cells were washed with PBS and harvested in 0.5 mL of lysis buffer as described previously.14 Immunoprecipitation was performed as described previously with mouse anti-Flag (Sigma).15 Western blot analysis was performed as previously described.14 In brief, the blots were incubated for 4 hours at room temperature with the anti-activated Src antibody clone 28,16 anti-SHP-2 (Santa Cruz), Xpress (Invitrogen), or Flag (Sigma) antibody, followed by incubation with horseradish peroxidase–conjugated secondary antibody (Amersham). Antibodies for assaying NF-{kappa}B activation, anti-I{kappa}B{alpha}, and anti-I{kappa}B kinase (IKK) {alpha} antibody were from Santa Cruz, and the phospho-IKK{alpha}/ß and phospho-I{kappa}B{alpha} (ser32) antibodies were from Cell Signaling.

JNK activity was measured with a commercially available kit based on phosphorylation of recombinant c-Jun (Cell Signaling), as described previously.14 SHP-2 phosphatase activity was measured as previously described17,18 (see the online data supplement).

PathDetect in Vivo Signal Transduction Pathway Reporting System
c-Jun and NF-{kappa}B activity was assayed using PathDetect Signal Transduction Pathway trans-Reporting Systems (Stratagene) as described previously19 (please see the online data supplement).

Preparation of Recombinant Proteins and Coimmunoprecipitation (TNT Coupled Reticulocyte Systems [Promega] Assay)
Using pRK5-MEKK3, pcDNA3.1 His-Gab1, and WT and phosphatase-negative form of SHP-2 (SHP-2C/S), recombinant proteins were expressed by the TNT (Coupled Reticulocyte Lysate System) incorporating [35S] methionine, as previously described.20

Cell-Surface Immunoassay
To detect the expression of adhesion molecules, ELISA was performed as previously described21 (please see the online data supplement).

Statistical Analysis
Data are reported as mean±SD. Statistical analysis was performed with the StatView 4.0 package (ABACUS Concepts). Differences were analyzed with 1-way or at 2-way repeated-measures ANOVA, as appropriate, followed by Schéffe’s correction. P<0.05 is indicated by an asterisk and P<0.01 by 2 asterisks.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Flow Inhibits TNF-{alpha} Stimulation of c-Jun and NF-{kappa}B Transcriptional Activity in HUVECs
Previously, we reported that flow inhibited TNF-{alpha}–induced ASK1 and JNK activity but not ERK1/2 and p38 activation.1,2 In the present study, we first analyzed the effect of flow on TNF-{alpha}–mediated c-Jun and NF-{kappa}B transcriptional activation in endothelial cells.1 We transiently transfected HUVECs with c-Jun and NF-{kappa}B reporter genes. After 24 hours of transfection, cells were exposed to flow (shear stress=5 dyne/cm2) for 20 minutes or maintained in static conditions for 20 minutes. We chose 5 dyne/cm2 of flow instead of 12 dyne/cm2 in the long-term flow regimen, because 5 and 12 dyne/cm2 of laminar flow have similar effects on its downstream events, including ERK5 activation,22 and cell viability and attachment in 5 dyne/cm2 of flow is better than in 12 dyne/cm2 after 6 hours of exposure. Cells were then kept in static or flow condition for 6 hours with TNF-{alpha} (20 ng/mL) or vehicle. c-Jun and NF-{kappa}B transcriptional activity were measured using a luciferase reporter assay system.23 As shown in Figures 1A and 1B, flow alone caused no significant increase in c-Jun and NF-{kappa}B transcriptional activity. TNF-{alpha} alone significantly increased c-Jun (Figure 1A) and NF-{kappa}B (Figure 1B) transcriptional activity after 6 hours of stimulation. We found that exposure to flow significantly inhibited TNF-{alpha}–mediated activation of c-Jun and NF-{kappa}B transcriptional activity. We found a similar inhibitory effect of flow when cells were preexposed to shear stress for 60 minutes before TNF-{alpha} stimulation (data not shown). These observations are consistent with our previous work1,2 and suggest that the inhibitory effect of flow is mediated by events that are relatively long lived.



View larger version (52K):
[in this window]
[in a new window]
  		Figure 1. Flow inhibits TNF-{alpha}–mediated c-Jun (A) and NF-{kappa}B (B) activation in HUVECs (see the online data supplement, available at http://atvb.ahajournals.org). A and B, HUVECs were transfected with pFR-Luc plasmid and pFA2-c-Jun plasmid (A) or pNF-{kappa}B Luc plasmid (B). After 24 hours of transfection, HUVECs were treated as in the following protocol: maintained in static conditions for 20 minutes followed by vehicle (lane 1) or TNF-{alpha} (20 ng/mL) stimulation (lane 3) in same static conditions or subjected to flow (shear stress=5 dyne/cm2) for 20 minutes followed by vehicle (lane 2) or TNF-{alpha} stimulation (lane 4) with same flow condition for 6 hours. After 6 hours of vehicle or TNF-{alpha} stimulation, luciferase c-Jun transcriptional activity (A) or NF-{kappa}B transcriptional activity (B) was assayed. C, HUVECs were stimulated by TNF-{alpha} (20 ng/mL) for indicated time points. I{kappa}B (C) phosphorylation in the cell lysates were detected by anti-phospho-I{kappa}B (C, top) antibody. I{kappa}B expression was detected by anti-I{kappa}B antibody (C, bottom). D, HUVECs were treated as in the following protocol: maintained in static conditions for 20 minutes followed by vehicle (lane 1) or TNF-{alpha} (20 ng/mL) stimulation (lane 3) in the same static conditions for 5 minutes or subjected to flow (shear stress=12 dyne/cm2) for 20 minutes followed by vehicle (lane 2) or TNF-{alpha} stimulation (lane 4) with same flow condition for 5 minutes (total, 25 minutes of flow). HUVECs were subjected to the same protocol described above, except 120 minutes (lanes 7 and 8) of TNF-{alpha} stimulation and total time of flow treatment, including 20 minutes of flow pretreatment (total, 140 minutes in lane 6 and 8 for flow). I{kappa}B phosphorylation in the cell lysates was detected by anti-phospho-I{kappa}B antibody (top). I{kappa}B expression was detected by anti-I{kappa}B antibody (bottom).

Flow Inhibited the TNF-{alpha}–Induced Late Phase of I{kappa}B Phosphorylation But Not the Early Phase of IKK{alpha}/ß and I{kappa}B Phosphorylation
NF-{kappa}B activation is initiated by the degradation of I{kappa}B inhibitor protein leading to the nuclear import of NF-{kappa}B and culminating with the resynthesis of I{kappa}B{alpha}. The I{kappa}B degradation is mediated through the IKK activation, which functions to phosphorylate 2 serine residues on I{kappa}B proteins.24

To detect the effect of flow on acute-phase events of TNF-{alpha}–induced NF-{kappa}B signaling, we investigated IKK{alpha}/ß and I{kappa}B phosphorylation mediated by TNF-{alpha}. As shown in Figure IA (available online at http://atvb.ahajournals.org), IKK{alpha}/ß is phosphorylated after {approx}5 to10 minutes of TNF-{alpha} stimulation. Analysis of HUVEC extracts using I{kappa}B-specific antibodies showed that stimulation of the cells with TNF-{alpha} caused rapid degradation of I{kappa}B. Protein levels of I{kappa}B decreased by >80% after 30 minutes; thereafter, I{kappa}B levels increased gradually up for to 240 minutes of incubation, attributable to NF-{kappa}B–induced de novo synthesis of I{kappa}B, as previously described.24 I{kappa}B phosphorylation was rapidly increased after 5 minutes of TNF-{alpha} stimulation (Figure 1C, top). Because the phosphorylated I{kappa}B protein level was decreased by degradation, the level of I{kappa}B was also decreased from {approx}10 to 30 minutes after TNF-{alpha} stimulation, but whereas I{kappa}B levels increased, phosphorylation of I{kappa}B could be detected again {approx}30 to 240 minutes after TNF-{alpha} stimulation. To investigate the effect of flow on these rapid events in NF-{kappa}B signaling, cells were exposed to flow (shear stress=12 dyne/cm2) or maintained in static conditions for 20 minutes. Cells were then kept in static or flow conditions for 10 (Figure IB, available online), 5, or 120 minutes (Figure 1D) with TNF-{alpha} (20 ng/mL) or vehicle to detect IKK{alpha}/ß and I{kappa}B phosphorylation. As shown in Figure IB (available online) and Figure 1D, we found that IKK activation after 10 minutes and I{kappa}B phosphorylation after 5 minutes of TNF-{alpha} stimulation was not inhibited by flow. In contrast, I{kappa}B phosphorylation after 120 minutes of TNF-{alpha} stimulation was completely inhibited by flow. These data suggest that shear stress does not inhibit IKK-I{kappa}B phosphorylation induced after {approx}5 to 10 minutes of TNF-{alpha} stimulation. However, shear stress inhibited the late phase (after 120 minutes of TNF-{alpha} stimulation) of I{kappa}B phosphorylation and the subsequent NF-{kappa}B transcriptional activity (Figure 1B).

TNF-{alpha} Stimulates JNK or c-Jun Activation Via a Src Kinase Family–Dependent Mechanism
Because several investigators, including our group,14 have reported the involvement of Src family kinases in JNK signaling, we determined whether TNF-{alpha} induced Src kinase activation in HUVECs. As shown in Figure IIA (available online), Src family kinase was activated by TNF-{alpha}. TNF-{alpha} increased JNK activity at 15 minutes, and PP2, a specific inhibitor for Src family kinase, inhibited TNF-{alpha}–mediated JNK activation in HUVECs in a dose-dependent manner (Figure IIB, available online). Src kinase inactive form (Src-KN) significantly inhibited TNF-{alpha}–induced c-Jun transcriptional activity in a dose-dependent manner (Figure IIC, available online), additionally indicating that TNF-{alpha}–mediated activation of JNK and c-Jun is dependent on Src kinase family activation.

TNF-{alpha} Stimulates JNK, c-Jun, and NF-{kappa}B Transcriptional Activity Via a SHP-2 Phosphatase Activity–Dependent Mechanism
We examined the role of SHP-2 tyrosine phosphatase activity in TNF-{alpha}–mediated signaling events in HUVECs. As shown in Figure III (available online), TNF-{alpha} stimulated SHP-2 phosphatase activity (2.3±0.4-fold, P<0.05). To inhibit SHP-2 function, we cotransfected HUVECs with Flag-tagged JNK and a phosphatase inactive mutant of SHP-2 (SHP-2C/S, cysteine 495 is mutated to serine).18 As shown in Figure 2A, transfection of WT SHP-2 (SHP-2WT) had no significant effect on TNF-{alpha}–induced JNK activation. However, transfection of SHP-2C/S inhibited JNK activation by TNF-{alpha}. In addition, SHP-2C/S, but not SHP-2WT, significantly inhibited TNF-{alpha}–induced c-Jun and NF-{kappa}B transcriptional activity in a dose-dependent manner, suggesting an essential role of SHP-2 phosphatase activity in TNF-{alpha}–mediated JNK, c-Jun, and NF-{kappa}B transcriptional activation in HUVECs (Figures 2B and 2C). To confirm the importance of SHP-2 phosphatase activity in regulating TNF-{alpha}–induced signaling, we used another SHP-2 phosphatase-negative form, a deletion mutant of C-terminal phosphatase domain of SHP-2 (SHP-2 aa1-411), as we have described previously.18 As shown in Figure IV (available online), we found that this phosphatase-negative form of SHP-2 (SHP-2aa1-411) also inhibited TNF-{alpha}–induced c-Jun and NF-{kappa}B transcriptional activation. These data also suggested the critical role of SHP-2 phosphatase activity on TNF-{alpha}–induced c-Jun and NF-{kappa}B transcriptional activation.



View larger version (51K):
[in this window]
[in a new window]
  		Figure 2. SHP-2 phosphatase activity positively regulates JNK, c-Jun, and NF-{kappa}B activation by TNF-{alpha} in HUVECs (see the online data supplement, available at http://atvb.ahajournals.org). A, HUVECs were double-transfected with Flag-JNK and pcDNA3.1 vector alone, SHP-2WT, or SHP-2 C/S, and TNF-{alpha}–induced JNK activation assay was performed (B and C). HUVECs (B and C) were transfected with pFR-Luc plasmid, and pFA2-c-Jun plasmid (B) or pNF-{kappa}B-Luc-plasmid (C) with pcDNA20 m 3.1 SHP-2C/S or SHP-2WT was transfected in the indicated amount; pcDNA3.1 vector was used to provide equal amounts of transfected DNA (B and C); and TNF-{alpha}–induced luciferase c-Jun transcriptional activity (B) or NF-{kappa}B transcriptional activity (C) was assayed.

Shear Stress Did Not Inhibit TNF-{alpha}–Induced Src Kinase Family Activation But Inhibited SHP-2 Tyrosine Phosphatase Activity
To determine the role of Src kinase in the regulation of SHP-2 phosphatase activity and flow-mediated inhibition of TNF-{alpha}–induced JNK activation, we first determined the effect of PP2 on TNF-{alpha}–induced SHP-2 phosphatase activity. As shown in Figure III (available online), TNF-{alpha}–induced SHP-2 phosphatase activity was significantly inhibited by PP2 in a dose-dependent manner. Flow and TNF-{alpha} significantly increased Src family kinase activity in HUVECs (Figures IID and V, available online). In contrast to JNK activation, flow did not change Src family kinase activation by TNF-{alpha} in HUVECs (Figure V, available online). These results suggest that Src family kinase activity is not responsible for flow-mediated inhibition of TNF-{alpha}–induced c-Jun and NF-{kappa}B transcriptional activation.

Next, to determine whether SHP-2 phosphatase activity is a key target of flow-mediated inhibitory effect on TNF-{alpha} signaling, we examined the effect of 20- or 60-minute pretreatment of flow on TNF-{alpha}–induced SHP-2 phosphatase activity. As shown in Figure 3A (left), 20-minute preexposure to flow significantly inhibits TNF-{alpha}–induced SHP-2 phosphatase activity. We also found a similar inhibitory effect after 60 minutes of preexposure to flow on TNF-{alpha}–induced SHP-2 phosphatase activity (Figure 3A, right). These results suggest that SHP-2 is a key target for the inhibitory effect of flow on TNF-{alpha}–induced c-Jun and NF-{kappa}B transcriptional activation in HUVECs.



View larger version (50K):
[in this window]
[in a new window]
  		Figure 3. Flow-inhibited SHP-2 phosphatase activity and the involvement of SHP-2 phosphatase activity in MEKK3-induced c-Jun and NF-{kappa}B activation in HUVECs (see the online data supplement, available at http://atvb.ahajournals.org). A, HUVECs were subjected to flow and TNF-{alpha} treatment with the same protocol described in Figure 1 using 20- or 60-minute flow pretreatment, except for 15 minutes of TNF-{alpha} stimulation. SHP-2 phosphatase activity was measured as in the Methods section. B and C, HUVECs were transfected with pFR-Luc plasmid, and pFA2-c-Jun plasmid (B) or pNF-{kappa}B-Luc-plasmid (C) with pcDNA3.1 SHP-2C/S or SHP-2WT was transfected in the indicated amount. CA-MEKK3 (B and C) was transfected in the indicated amount, pcDNA3.1 vector was used to provide equal amounts of transfected DNA, and luciferase c-Jun transcriptional activity (B) or NF-{kappa}B transcriptional activity (C) was assayed.

SHP-2 Phosphatase Activity Regulates MEKK3-Mediated But Not MEKK1-Mediated c-Jun and NF-{kappa}B Activation in HUVECs
Previously, we found that MEKK3 was required for c-Jun and NF-{kappa}B transcriptional activity induced by TNF-{alpha} in endothelial cells.23 Because MEKK1 also participates in the JNK and NF-{kappa}B activation by directly stimulating the IKK,25 we investigated the role of SHP-2 phosphatase activity in regulation of MEKK3 and MEKK1 signaling. We transfected HUVECs with SHP-2C/S and a constitutively active form of MEKK3 (CA-MEKK3). As shown in Figures 3B and 3C, CA-MEKK3 stimulated c-Jun and NF-{kappa}B transcriptional activity; SHP-2C/S, but not SHP-2WT, inhibited CA-MEKK3–induced c-Jun and NF-{kappa}B transcriptional activation in a dose-dependent manner. In contrast to CA-MEKK3, SHP-2C/S did not significantly inhibit CA-MEKK1–induced c-Jun and NF-{kappa}B transcriptional activity (Figures VIA and VIB, available online). These data indicate that SHP-2C/S specifically inhibits MEKK3- but not MEKK1-induced c-Jun and NF-{kappa}B transcriptional activity.

SHP-2 Phosphatase Activity Regulates MEKK3 and Gab1 Association and Subsequent c-Jun and NF-{kappa}B Transcriptional Activity in HUVECs
Gab1 associates with SHP-2 and regulates SHP-2–dependent signal transduction.26 Previously, we have shown that Gab1 associates with MEKK3 and inhibits MEKK3 (but not MEKK1)-induced c-Jun and NF-{kappa}B transcriptional activity.23 Therefore, we evaluated whether SHP-2 phosphatase activity regulates the association of Gab1 with MEKK3. As shown in Figures 4A and 4B, the direct interaction between Gab1/MEKK3 in vitro was increased in the presence of SHP-2C/S compared with SHP-2WT. As shown in Figures 4C and 4D, cotransfection of SHP-2WT did not change association of Gab1/MEKK3, but compared with SHP-2WT transfection, the association of Gab1/MEKK3 was increased by cotransfection of SHP-2C/S in vivo. No differences in SHP-2 and Gab1 protein expression were observed in lysates from SHP-2WT–transfected and SHP-2C/S–transfected cells, as determined by immunoprecipitation and Western blot analysis with anti-Flag (Figure 4C, middle) and anti-SHP-2 (Figure 4C, bottom) antibody. These data suggest that SHP-2 phosphatase activity regulates Gab1 and MEKK3 association and consequently TNF-{alpha} signaling in endothelial cells.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 4. Effects of SHP-2C/S on MEKK3/Gab1 interaction in vitro and in vivo. A, TNT-translated (Promega) [35S] methionine–labeled Xpress and His-tagged Gab1 (lanes 1 through 3), SHP-2WT (lane 2), SHP-2C/S (lane 3), and nontagged MEKK3 (lanes 2 and 3) were incubated with anti-His mouse monoclonal IgG2a antibody and protein G-Sepharose beads as described in the Methods section. Input receptor loaded represents one fifth of that used in the immunoprecipitation reaction, and input Gab1 loaded represents one tenth of the final use. B, Densitometric analysis of MEKK3/Gab1 association in vitro. Results were normalized by arbitrarily setting the densitometry of SHP-2WT (lane 2) to 1.0 (shown is the mean±SD, n=5, **P<0.01). C, HUVECs were transfected with the indicated Xpress-tagged Gab1 and Flag-tagged MEKK3 (lanes 1 through 4), SHP-2WT (lanes 1 and 2), or SHP-2C/S (lanes 3 and 4). Growth-arrested cells were incubated with (lanes 2 and 4) or without (lanes 1 and 3) TNF-{alpha} for 20 minutes, harvested, and lysed. Flag-tagged MEKK3 proteins were immunoprecipitated with anti-Flag antibody and analyzed by SDS-PAGE. Immunoblotting with anti-Xpress (C, top) antibody was performed to demonstrate the occurrence of coimmunoprecipitation with Gab1. No difference in the amount of Flag-tagged MEKK3 was observed in immunoprecipitates by Western blot analysis with anti-Flag antibody (C, middle) and the expression of SHP-2WT and SHP-2C/S by Western blot analysis with anti-SHP-2 antibody (C, bottom). D, Densitometric analysis of MEKK3/Gab1 association in vivo as described in panel B.

SHP-2 Phosphatase Activity Regulates CA-MEKK3–Mediated Intracellular Adhesion Molecule-1, Vascular Cellular Adhesion Molecule-1, and E-Selectin Expression in HUVECs
Both c-Jun and NF-{kappa}B are important mediators of adhesion molecule gene expression in HUVECs.27,28 Because we found that Gab1, Src kinase family, and SHP-2 phosphatase activity play critical roles in TNF-{alpha}–induced and MEKK3-induced c-Jun and NF-{kappa}B transcriptional activity, we determined the effect of Src kinase family–specific inhibitor PP2 and overexpression of SHP-2C/S on CA-MEKK3–induced intracellular adhesion molecule-1, vascular cellular adhesion molecule-1, and E-selectin expression in HUVECs. As shown in Figures VIIA through VIIC (available online), SHP-2C/S significantly inhibited CA-MEKK3–induced adhesion molecule expression (measured by ELISA) in a dose-dependent manner. In addition, PP2 also inhibited TNF-{alpha}–induced adhesion molecules expression (Figure VIII, available online). These data suggest that Src kinase family and SHP-2 phosphatase activity regulate TNF-{alpha}–induced and MEKK3-induced adhesion molecule expression.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The major finding of the present study is that SHP-2 functions as a molecular switch for the inhibition of JNK, c-Jun, and NF-{kappa}B activity by shear stress. We propose a scheme (Figure 5) for shear stress–mediated signal transduction leading to inhibition of TNF-{alpha}–induced c-Jun and NF-{kappa}B transcriptional activation based on previous work from our laboratory and other investigators.29–31 A novel aspect of this model is the role of SHP-2 as the target for shear stress effects on TNF-{alpha} signaling pathway.



View larger version (13K):
[in this window]
[in a new window]
  		Figure 5. Model of flow and TNF-{alpha}–mediated signal transduction pathways to regulate adhesion molecule expression in endothelial cells.

Because shear stress did not inhibit TNF-{alpha}–induced Src activity, Src kinase members are not the mediators by which shear stress inhibits the TNF-{alpha} signaling. Instead, we found that flow significantly inhibited TNF-{alpha}–induced SHP-2 phosphatase activity, as shown in Figure 3A. SHP-2 is a non-trans-membrane–type protein-tyrosine phosphatase that contains 2 SH2 domains.32 In the present study, we found that SHP-2 phosphatase activity regulates MEKK3 and Gab1 interaction and regulates TNF-{alpha} and MEKK3 signaling pathway (Figure 5). Previously, Yang et al33 and we23 found that MEKK3 is one of the critical mediators to regulate TNF-{alpha}–induced c-Jun and NF-{kappa}B transcriptional activity and Gab1 has an inhibitory effect on TNF-{alpha}–induced and MEKK3-induced c-Jun and NF-{kappa}B transcriptional activity. The inhibitory effect of SHP-2C/S on MEKK3/Gab1 interaction and MEKK3-induced c-Jun and NF-{kappa}B transcriptional activity suggests the critical role of SHP-2/Gab1/MEKK3 complex in regulating TNF-{alpha} signaling pathway (Figure 5).

The results of the present study differ from those of Liu et al,2 who demonstrated that shear stress did not inhibit NF-{kappa}B DNA binding activity after 15 minutes of TNF-{alpha} stimulation by using EMSA. We found significant shear stress–mediated inhibition of TNF-{alpha}–induced NF-{kappa}B transcriptional activity by using a reporter gene assay. As shown in Figures 1 and I (available online), shear stress did not inhibit the rapid (5- to 10-minute) phosphorylation of IKK{alpha}/ß and I{kappa}B induced by TNF-{alpha}. However, shear stress completely inhibited the slower phase of I{kappa}B phosphorylation after 2 hours of TNF-{alpha} stimulation as well as the total sum of NF-{kappa}B transcriptional activity, which was evaluated by luciferase activity by NF-{kappa}B reporter gene after 6 hours of stimulation. These data suggest that flow-mediated inhibition of NF-{kappa}B transcriptional activation occurred at the late phase (after 120 minutes of stimulation) of NF-{kappa}B signaling activation. Prolonged exposure to certain stimuli, such as TNF-{alpha} and LPS, leads to long-term induction of NF-{kappa}B activation,24 and it has been suggested that the late phase of I{kappa}B phosphorylation is important in controlling persistent activation of NF-{kappa}B.34 IKKs have 3 components, IKK{alpha}, IKKß, and IKK{gamma},24 and IKKß and IKK{gamma} are required for activation of NF-{kappa}B in response to TNF-{alpha} and several cytokines, as determined by studies in IKKß- and IKK{gamma}-deficient cells.35,36 We did not find any inhibitory effect of flow on TNF-{alpha}–induced IKK{alpha}/ß phosphorylation (Figure IB, available online). Therefore, it is possible that another IKK, including IKK{gamma}, is responsible for this late phase of I{kappa}B phosphorylation. Additional studies need to describe this late-phase I{kappa}B phosphorylation induced by flow.


*    Acknowledgments
 
This work was supported by grants NIH/NHLB RO1 HL61319 (to J.A.) and NIH/NHLB RO1 HL64839 (to B.C.B.).


*    Footnotes
 
*These authors contributed equally to this study. Back

Received July 16, 2003; accepted August 18, 2003.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Surapisitchat J, Hoefen RJ, Pi X, Yoshizumi M, Yan C, Berk BC. Fluid shear stress inhibits TNF-alpha activation of JNK but not ERK1/2 or p38 in human umbilical vein endothelial cells: inhibitory crosstalk among MAPK family members. Proc Natl Acad Sci U S A. 2001; 98: 6476–6481.[Abstract/Free Full Text]
  2. Liu Y, Yin G, Surapisitchat J, Berk BC, Min W. Laminar flow inhibits TNF-induced ASK1 activation by preventing dissociation of ASK1 from its inhibitor 14-3-3. J Clin Invest. 2001; 107: 917–923.[CrossRef][Medline] [Order article via Infotrieve]
  3. Fukunaga K, Noguchi T, Takeda H, Matozaki T, Hayashi Y, Itoh H, Kasuga M. Requirement for protein-tyrosine phosphatase SHP-2 in insulin-induced activation of c-Jun NH(2)-terminal kinase. J Biol Chem. 2000; 275: 5208–5213.[Abstract/Free Full Text]
  4. You M, Yu DH, Feng GS. Shp-2 tyrosine phosphatase functions as a negative regulator of the interferon-stimulated Jak/STAT pathway. Mol Cell Biol. 1999; 19: 2416–2424.[Abstract/Free Full Text]
  5. Neel BG. Structure and function of SH2-domain containing tyrosine phosphatases. Semin Cell Biol. 1993; 4: 419–432.[CrossRef][Medline] [Order article via Infotrieve]
  6. Case RD, Piccione E, Wolf G, Benett AM, Lechleider RJ, Neel BG, Shoelson SE. SH-PTP2/Syp SH2 domain binding specificity is defined by direct interactions with platelet-derived growth factor beta-receptor, epidermal growth factor receptor, and insulin receptor substrate-1-derived phosphopeptides. J Biol Chem. 1994; 269: 10467–10474.[Abstract/Free Full Text]
  7. Gadina M, Stancato LM, Bacon CM, Larner AC, O’Shea JJ. Involvement of SHP-2 in multiple aspects of IL-2 signaling: evidence for a positive regulatory role. J Immunol. 1998; 160: 4657–4661.[Abstract/Free Full Text]
  8. David M, Zhou G, Pine R, Dixon JE, Larner AC. The SH2 domain-containing tyrosine phosphatase PTP1D is required for interferon alpha/beta-induced gene expression. J Biol Chem. 1996; 271: 15862–15865.[Abstract/Free Full Text]
  9. Oh ES, Gu H, Saxton TM, Timms JF, Hausdorff S, Frevert EU, Kahn BB, Pawson T, Neel BG, Thomas SM. Regulation of early events in integrin signaling by protein tyrosine phosphatase SHP-2. Mol Cell Biol. 1999; 19: 3205–3215.[Abstract/Free Full Text]
  10. Takahashi-Tezuka M, Yoshida Y, Fukada T, Ohtani T, Yamanaka Y, Nishida K, Nakajima K, Hibi M, Hirano T. Gab1 acts as an adapter molecule linking the cytokine receptor gp130 to ERK mitogen-activated protein kinase. Mol Cell Biol. 1998; 18: 4109–4117.[Abstract/Free Full Text]
  11. Chao TH, Hayashi M, Tapping RI, Kato Y, Lee JD. MEKK3 directly regulates MEK5 activity as part of the big mitogen-activated protein kinase 1 (BMK1) signaling pathway. J Biol Chem. 1999; 274: 36035–36038.[Abstract/Free Full Text]
  12. Tai LK, Okuda M, Abe J, Yan C, Berk BC. Fluid shear stress activates proline-rich tyrosine kinase via reactive oxygen species-dependent pathway. Arterioscler Thromb Vasc Biol. 2002; 22: 1790–1796.[Abstract/Free Full Text]
  13. Okuda M, Takahashi M, Suero J, Murry CE, Traub O, Kawakatsu H, Berk BC. Shear stress stimulation of p130(cas) tyrosine phosphorylation requires calcium-dependent c-Src activation. J Biol Chem. 1999; 274: 26803–26809.[Abstract/Free Full Text]
  14. Yoshizumi M, Abe J, Haendeler J, Huang Q, Berk BC. Src and cas mediate JNK activation but not ERK1/2 and p38 kinases by reactive oxygen species. J Biol Chem. 2000; 275: 11706–11712.[Abstract/Free Full Text]
  15. Abe J, Kusuhara M, Ulevitch RJ, Berk BC, Lee JD. Big mitogen-activated protein kinase 1 (BMK1) is a redox-sensitive kinase. J Biol Chem. 1996; 271: 16586–16590.[Abstract/Free Full Text]
  16. Kawakatsu H, Sakai T, Takagaki Y, Shinoda Y, Saito M, Owada MK, Yano J. A new monoclonal antibody which selectively recognizes the active form of Src tyrosine kinase. J Biol Chem. 1996; 271: 5680–5685.[Abstract/Free Full Text]
  17. Tang H, Zhao ZJ, Huang XY, Landon EJ, Inagami T. Fyn kinase-directed activation of SH2 domain-containing protein-tyrosine phosphatase SHP-2 by Gi protein-coupled receptors in Madin-Darby canine kidney cells. J Biol Chem. 1999; 274: 12401–12407.[Abstract/Free Full Text]
  18. Huang Q, Lerner-Marmarosh N, Che W, Ohta S, Osawa M, Yoshizumi M, Glassman M, Yan C, Berk BC, Abe JI. The novel role of C-terminal region of SHP-2: involvement of Gab1 and SHP-2 phosphatase activity in Elk-1 activation. J Biol Chem. 2002; 277: 29330–29341.[Abstract/Free Full Text]
  19. Yan C, Luo H, Lee JD, Abe J, Berk BC. Molecular cloning of mouse ERK5/BMK1 splice variants and characterization of ERK5 functional domains. J Biol Chem. 2001; 276: 10870–10878.[Abstract/Free Full Text]
  20. Heinlein CA, Ting HJ, Yeh S, Chang C. Identification of ARA70 as a ligand-enhanced coactivator for the peroxisome proliferator-activated receptor gamma. J Biol Chem. 1999; 274: 16147–16152.[Abstract/Free Full Text]
  21. Zund G, Uezono S, Stahl GL, Dzus AL, McGowan FX, Hickey PR, Colgan SP. Hypoxia enhances induction of endothelial ICAM-1: role for metabolic acidosis and proteasomes. Am J Physiol. 1997; 273: C1571–C1580.
  22. Yan C, Takahashi M, Okuda M, Lee JD, Berk BC. Fluid shear stress stimulates big mitogen-activated protein kinase 1 (BMK1) activity in endothelial cells: dependence on tyrosine kinases and intracellular calcium. J Biol Chem. 1999; 274: 143–150.[Abstract/Free Full Text]
  23. Che W, Lerner-Marmarosh N, Huang Q, Osawa M, Ohta S, Yoshizumi M, Glassman M, Lee JD, Yan C, Berk BC, Abe J. Insulin-like growth factor-1 enhances inflammatory responses in endothelial cells: role of Gab1 and MEKK3 in TNF-alpha-induced c-Jun and NF-kappaB activation and adhesion molecule expression. Circ Res. 2002; 90: 1222–1230.[Abstract/Free Full Text]
  24. Rothwarf DM, Karin M. The NF-kappa B activation pathway: a paradigm in information transfer from membrane to nucleus [review]. Sci STKE. 1999; 1999: RE1.
  25. Lee FS, Peters RT, Dang LC, Maniatis T. MEKK1 activates both IkappaB kinase alpha and IkappaB kinase beta. Proc Natl Acad Sci U S A. 1998; 95: 9319–9324.[Abstract/Free Full Text]
  26. Shi ZQ, Yu DH, Park M, Marshall M, Feng GS. Molecular mechanism for the shp-2 tyrosine phosphatase function in promoting growth factor stimulation of ERK activity. Mol Cell Biol. 2000; 20: 1526–1536.[Abstract/Free Full Text]
  27. Min W, Pober JS. TNF initiates E-selectin transcription in human endothelial cells through parallel TRAF-NF-kappa B and TRAF-RAC/CDC42-JNK-c-Jun/ATF2 pathways. J Immunol. 1997; 159: 3508–3518.[Abstract]
  28. Kol A, Bourcier T, Lichtman AH, Libby P. Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth muscle cells, and macrophages. J Clin Invest. 1999; 103: 571–577.[Medline] [Order article via Infotrieve]
  29. Lander HM, Ogiste JS, Teng KK, Novogrodsky A. p21ras as a common signaling target of reactive free radicals and cellular redox stress. J Biol Chem. 1995; 270: 21195–21198.[Abstract/Free Full Text]
  30. Rao GN. Hydrogen peroxide induces complex formation of SHC-Grb2-SOS with receptor tyrosine kinase and activates Ras and extracellular signal-regulated protein kinases group of mitogen-activated protein kinases. Oncogene. 1996; 13: 713–719.[Medline] [Order article via Infotrieve]
  31. Tateno M, Nishida Y, Adachi-Yamada T. Regulation of JNK by src during drosophila development. Science. 2000; 287: 324–327.[Abstract/Free Full Text]
  32. Feng GS. Shp-2 tyrosine phosphatase: signaling one cell or many. Exp Cell Res. 1999; 253: 47–54.[CrossRef][Medline] [Order article via Infotrieve]
  33. Yang J, Lin Y, Guo Z, Cheng J, Huang J, Deng L, Liao W, Chen Z, Liu Z, Su B. The essential role of MEKK3 in TNF-induced NF-kappaB activation. Nat Immunol. 2001; 2: 620–624.[CrossRef][Medline] [Order article via Infotrieve]
  34. Suyang H, Phillips R, Douglas I, Ghosh S. Role of unphosphorylated, newly synthesized I kappa B beta in persistent activation of NF-kappa B. Mol Cell Biol. 1996; 16: 5444–5449.[Abstract]
  35. Woronicz JD, Gao X, Cao Z, Rothe M, Goeddel DV. IkappaB kinase-beta: NF-kappaB activation and complex formation with IkappaB kinase-alpha and NIK. Science. 1997; 278: 866–869.[Abstract/Free Full Text]
  36. Yamaoka S, Courtois G, Bessia C, Whiteside ST, Weil R, Agou F, Kirk HE, Kay RJ, Israel A. Complementation cloning of NEMO, a component of the IkappaB kinase complex essential for NF-kappaB activation. Cell. 1998; 93: 1231–1240.[CrossRef][Medline] [Order article via Infotrieve]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
C.-H. Woo, M. P. Massett, T. Shishido, S. Itoh, B. Ding, C. McClain, W. Che, S. R. Vulapalli, C. Yan, and J.-i. Abe
ERK5 Activation Inhibits Inflammatory Responses via Peroxisome Proliferator-activated Receptor {delta} (PPAR{delta}) Stimulation
J. Biol. Chem., October 27, 2006; 281(43): 32164 - 32174.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
M. Dixit, A. E. Loot, A. Mohamed, B. Fisslthaler, C. M. Boulanger, B. Ceacareanu, A. Hassid, R. Busse, and I. Fleming
Gab1, SHP2, and Protein Kinase A Are Crucial for the Activation of the Endothelial NO Synthase by Fluid Shear Stress
Circ. Res., December 9, 2005; 97(12): 1236 - 1244.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Itoh, S. Lemay, M. Osawa, W. Che, Y. Duan, A. Tompkins, P. S. Brookes, S.-S. Sheu, and J.-i. Abe
Mitochondrial Dok-4 Recruits Src Kinase and Regulates NF-{kappa}B Activation in Endothelial Cells
J. Biol. Chem., July 15, 2005; 280(28): 26383 - 26396.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
D. T. Bolick, S. Srinivasan, K. W. Kim, M. E. Hatley, J. J. Clemens, A. Whetzel, N. Ferger, T. L. Macdonald, M. D. Davis, P. S. Tsao, et al.
Sphingosine-1-Phosphate Prevents Tumor Necrosis Factor-{alpha}-Mediated Monocyte Adhesion to Aortic Endothelium in Mice
Arterioscler. Thromb. Vasc. Biol., May 1, 2005; 25(5): 976 - 981.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
K. Kappert, K. G. Peters, F. D. Bohmer, and A. Ostman
Tyrosine phosphatases in vessel wall signaling
Cardiovasc Res, February 15, 2005; 65(3): 587 - 598.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
M. Akaike, W. Che, N.-L. Marmarosh, S. Ohta, M. Osawa, B. Ding, B. C. Berk, C. Yan, and J.-i. Abe
The Hinge-Helix 1 Region of Peroxisome Proliferator-Activated Receptor {gamma}1 (PPAR{gamma}1) Mediates Interaction with Extracellular Signal-Regulated Kinase 5 and PPAR{gamma}1 Transcriptional Activation: Involvement in Flow-Induced PPAR{gamma} Activation in Endothelial Cells
Mol. Cell. Biol., October 1, 2004; 24(19): 8691 - 8704.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
23/10/1775    most recent
01.ATV.0000094432.98445.36v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lerner-Marmarosh, N.
Right arrow Articles by Abe, J.-i.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lerner-Marmarosh, N.
Right arrow Articles by Abe, J.-i.
Related Collections
Right arrow Other arteriosclerosis

ATVB Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 2003 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.