Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M314015200 on April 21, 2004

J. Biol. Chem., Vol. 279, Issue 26, 27088-27097, June 25, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/26/27088    most recent
M314015200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, J. F.
Right arrow Articles by Groopman, J. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, J. F.
Right arrow Articles by Groopman, J. E.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Activation of Vascular Endothelial Growth Factor Receptor-3 and Its Downstream Signaling Promote Cell Survival under Oxidative Stress*

Jian Feng Wang, Xuefeng Zhang, and Jerome E. Groopman{ddagger}

From the Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215

Received for publication, December 22, 2003 , and in revised form, April 12, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reactive oxygen species (ROS) mediate cell damage and have been implicated in the pathogenesis of diseases that involve endothelial injury. Cells possess antioxidant systems, including intracellular antioxidants and ROS scavenging enzymes, that control the redox state and prevent cell damage. In addition to intracellular antioxidants, certain growth factor receptors can be activated under oxidative stress and trigger downstream cell survival signaling cascades. Vascular endothelial growth factor receptor-3 (VEGFR-3) is a primary modulator of lymphatic endothelial proliferation and survival. Here, we provide evidence that activation of VEGFR-3 signaling in response to hydrogen peroxide (H2O2) promotes endothelial cell survival. Treatment with H2O2 induced the tyrosine phosphorylation of VEGFR-3 and its association with the signaling adaptor proteins Shc, growth factor receptor binding protein 2, Sos, p85, SHP-2, and phospholipase C-{gamma}. Of note, a hereditary lymphoedema-linked mutant of VEGFR-3 was not phosphorylated by H2O2 treatment. Isoforms of protein kinase C (PKC), {alpha} and {delta}, were also tyrosine-phosphorylated after H2O2 stimulation. However, only the {delta} isoform of PKC was required for H2O2-induced phosphorylation of VEGFR-3. The tyrosine phosphorylation of VEGFR-3 or isoforms of PKC was completely inhibited by treatment with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, a specific inhibitor for Src family kinases, indicating that Src family kinases are upstream of PKC and VEGFR-3. Furthermore, expression of the wild-type but not the lymphoedema-linked mutant form of VEGFR-3 in porcine artery endothelial cells significantly enhanced the activation of Akt after H2O2 stimulation. Consistent with these biochemical changes, we observed that expression and activation of the wild-type but not the mutant form of VEGFR-3 inhibited H2O2-induced apoptosis. These studies suggest that VEGFR-3 protects against oxidative damage in endothelial cells, and that patients with hereditary lymphoedema may be susceptible to ROS-induced cell damage.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reactive oxygen species (ROS)1 include a group of molecules such as hydrogen peroxide (H2O2), superoxide anion (), singlet oxygen (O2), and hydroxyl radicals (OH). Low levels of ROS are regularly produced during physiological metabolism. To control the redox state, cells possess antioxidant systems that comprise intracellular antioxidants, such as ascorbic acid, glutathione, {alpha}-tocopherol, and several ROS scavenging enzymes including catalase, glutathione peroxidase, and superoxide dismutase. Cells undergo oxidative stress when levels of ROS exceed the counter-regulatory antioxidant capacity of the cells, either generated from elevated production and accumulation of ROS or from a diminution in cellular antioxidant defenses (1). Cellular responses to oxidative stress can be as different as cell death, growth arrest, proliferation, or transformation, which are dependent on the stress stimuli, dose or time of exposure, cell type, and surrounding cell environment encountered (1, 2). ROS-mediated cell damage is implicated in the pathogenesis of a variety of diseases, including diabetes, cancer, atherosclerosis, and Alzheimer's disease (2).

Exposure to relatively high doses of ROS, such as H2O2, induces death in many cell types. Oxidative stress-induced cell death involves either necrosis attributable to toxic oxidative damage to cellular lipids, nucleic acids, and proteins or apoptosis resulting from the activation of cell death signaling cascades (1, 3). On the other hand, oxidative stress can also trigger the activation of certain signaling pathways that protect against cell death, including mitogen-activated protein kinase (4), PI3-kinase/Akt (5, 6), nuclear factor-{kappa}B (7), and phospholipase C-{gamma} (8). PI3-kinase and Akt activation appear to play an essential role in cell survival by preventing the cell death induced by oxidative stress. However, the mechanism by which oxidative stress activates these signaling pathways is not well elucidated. Recent studies have demonstrated that activation of certain growth factor receptors by ROS and subsequent downstream signaling significantly contribute to cell survival (6, 9, 10). For example, the receptor for epidermal growth factor (6, 9, 11, 12), platelet-derived growth factor (13, 14), or insulin (15) each can be activated by H2O2 stimulation. In some cell types, the activation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase and Akt in response to ROS appears to be mediated by the stimulation of epidermal growth factor receptor (6, 16). The activation of epidermal growth factor receptor thus protects against ROS-mediated cell death induced by oxidative stress (6) or ultraviolet irradiation (17). Hence, the activation of growth factor receptors and downstream signaling may serve as another protective mechanism against oxidant-mediated cell damage in addition to oxidative scavenger enzymes.

Endothelial cells are primary targets of oxidative stress. Upon exposure to ROS, endothelial cells display a variety of adverse biological effects including the production of inflammatory mediators (18, 19), expression of adhesion molecules (20, 21), and increased cell permeability (22, 23). Addition of exogenous ROS also induces cell death in endothelial cells. ROS-induced endothelium damage or dysfunction is considered to be a primary pathogenetic mechanism in various cardiovascular diseases (24). ROS may also play a role in angiogenesis. ROS has been reported to induce the production of various angiogenic factors (25). Interestingly, recent reports have shown that VEGF stimulation elevates the levels of intracellular ROS in endothelial cells, and that ROS is functional as a second messenger and participates in vascular endothelial growth factor receptor (VEGFR)-2 downstream signaling cascades (26, 27).

VEGFR-3 (VEGFR-3/FLT4) is an orphan receptor for the VEGFRs VEGFR-1 and VEGFR-2 (28). VEGF-C and VEGF-D have been identified as the ligands for VEGFR-3 (29, 30). However, VEGF does not bind to VEGFR-3 (29, 30). VEGFR-3 is predominantly expressed in the lymphatic endothelium of adult tissues and serves as a marker that identifies lymphatic endothelial cells (31). VEGFR-3 is perhaps the most important regulator of lymphatic development and lymphangiogenesis (32, 33). VEGFR-3 signaling mediates both angiogenesis and lymphangiogenesis in tumors and appears to play a significant role in tumor metastasis via the lymphatics (34–36). In this report, we present evidence that H2O2 can stimulate the tyrosine phosphorylation of the VEGFR-3 receptor, induce its downstream signaling cascade to activate Akt, and thereby promote endothelial cell survival under redox stress. The VEGFR-3 mutants identified in patients with hereditary lymphoedema lack this signaling function, making the lymphatic endothelium especially prone to oxidative damage.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Antibodies—Recombinant rat VEGF-C, which can activate human VEGFR-3, was purchased from Calbiochem (La Jolla, CA). Rabbit polyclonal antibody for human VEGFR-3 (FLT4), rabbit polyclonal antibody for human c-Src, and mouse anti-phosphotyrosine (PY) monoclonal antibody PY99 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit anti-human antibody against phospholipase C-{gamma}, Shc, or growth factor receptor binding protein 2 was purchased from BD-Transduction Laboratory (San Diego, CA). Rabbit anti-Akt, anti-phospho-Akt, or anti-phospho-Src family (Tyr-416) antibody was obtained from Cell Signaling Technology (Beverly, MA). Normal rabbit serum and purified normal rabbit IgG were purchased from Santa Cruz Biotechnology, Inc. The recombinant human VEGFR-3 (FLT4)/Fc chimera (VEGFR-3/Ig) containing the extracellular domain of human VEGFR-3 and the Fc portion of human immunoglobulin was purchased from R&D Systems (Minneapolis, MN). 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP2), a Src family kinase (SFK) inhibitor; GF109203X, a protein kinase C (PKC) inhibitor; and PD98059, a mitogen-activated kinase kinase inhibitor, were obtained from Calbiochem. Rottlerin (3'-[(8-cinnamoyl-5,7-dihydroxy-2,2-dimethyl-2H-1-benzopyran-6-yl)methyl]-2',4',6'-trihydroxy-5' methylacetophenone) was obtained from Calbiochem. H2O2 was purchased from Sigma. Electrophoresis reagents and nitrocellulose membrane were obtained from Bio-Rad Laboratories. Protein A-Sepharose CL-4B and GammaBind plus Sepharose were obtained from Amersham Biosciences. The protease inhibitors leupeptin, aprotinin, and alpha 1 antitrypsin and all other reagents were obtained from Sigma. Calcium and potassium-free phosphate-buffered saline was obtained from Invitrogen. [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl]tetrazolium bromide (MTT) was purchased from Sigma.

Cells and Cell Culture—HMEC-1, a human dermal microvascular endothelial cell line, was kindly provided by the Centers for Disease Control and Prevention (Atlanta, GA) and grown in Dulbecco's modified Eagle's medium with 10% FCS. Human dermal microvascular endothelial cells were purchased from Clonetics Inc. (Palo Alto, CA) and expanded in EGM-2 medium. Five to eight passages were used in the described experiments. Porcine aortic endothelial cells (PAECs) and culture medium were obtained from Cell Applications, Inc. (San Diego, CA). All cells were cultured at 37 °C in a humidified atmosphere of 5% CO2.

Stimulation of Cells by H2O2—Subconfluently cultured endothelial cells were starved in serum-free Dulbecco's modified Eagle's medium for 5 h. Various concentrations of H2O2 were prepared from an 8 M stock solution (Sigma) just before each experiment. Cells were then stimulated with various doses of H2O2 or VEGF-C for different time periods as indicated at 37 °C. In some treatments, cells were incubated with various inhibitors as indicated in each figure legend. After stimulation, cells were washed with ice-cold phosphate-buffered saline containing 0.1 mM sodium orthovanadate, and cell lysates were prepared in a lysis buffer containing 50 mM Hepes, pH 7.0, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 10 µg/ml each of aprotinin, leupeptin, and pepstatin. Total cell lysates were clarified by centrifugation at 10,000 x g for 10 min. Protein concentrations were determined by protein assay (Bio-Rad Laboratories).

Immunoprecipitation and Western Blot Analysis—Immunoprecipitation and Western blotting were performed as described previously (37). Briefly, identical amounts of protein from each sample were incubated with different primary antibodies overnight at 4 °C. Immunoprecipitation of the antibody-antigen complexes was performed by incubation for 2 h at 4 °C with 75 µl of protein A-Sepharose or GammaBind plus Sepharose. After washing with lysis buffer, the bound proteins were solubilized in Laemmli sample buffer by boiling for 5 min. Samples were separated on SDS-PAGE, transferred to nitrocellulose membranes, and probed with primary antibody at 4 °C overnight. Immunoreactive bands were visualized using horseradish peroxidase-conjugated secondary antibody and the enhanced chemiluminescent (ECL) system (Amersham Biosciences). Each experiment was repeated at least three times, and the presented blots are representative ones.

DNA Constructs, Site-directed Mutagenesis, and Transfection—The expression construct of the wild-type VEGFR-3 or the mutant form of VEGFR-3, with a Gly to Arg mutation at the 857 site, was generated as described previously (37). For transient expression of wild-type or mutant VEGFR-3 in PAECs, Effectene Transfection Reagent (Qiagen, Valencia, CA) was used according to the manufacturer's instructions. For the cell survival assays, cells were trypsinized and replated in 96-well plates and then cultured overnight before the treatment with H2O2. Dominant-negative Src (K296R/Y528F) cDNA in the pUSEamp or control vector was obtained from Upstate Biotechnology (Lake Placid, NY). For transient expression of dominant-negative Src in HMEC-1 cells, FuGENE 6 Transfection Reagent (Roche Molecular Biochemicals, Indianapolis, IN) was used according to the manufacturer's instructions. 48 h after transfection, cells were serum-starved for 5 h and then stimulated with H2O2. Cell lysates were prepared as described above for the immunoprecipitation and Western blot analysis.

Total Cellular Phosphatase Activity Assay—Serum-starved HMEC-1 cells were stimulated with H2O2. Then, cells were lysed in phosphatase lysis buffer (20 mM HEPES (pH 7.4), 10% glycerol, 0.1% Nonidet P-40, 1 mM EGTA, 30 mM {beta}-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and leupeptin, antipain, and pepstatin (2 µg/ml each)). The protein phosphatase activity was measured by using para-nitrophenyl phosphate as the substrate with the Phosphatase Assay kit (Upstate Biotechnology) according to the manufacturer's instructions. The amount of para-nitrophenyl phosphate produced in the assay reaction was determined by measuring the absorbance at 405 nm.

Cell Survival Assays—HMEC-1 cells or PAECs were plated in collagen-coated 96-well plates and cultured overnight before treatment with various doses of H2O2. H2O2 or control medium was directly added into the culture medium and then incubated for 30 min at 37 °C. Cells were next washed twice with culture medium and cultured for 24 h. Cell survival was determined by using the MTT assay, which determines mitochondrial activity in living cells. 0.1 mg/ml MTT was incubated with the analyzed cells for 4 h at 37 °C. Dark blue formazan crystals that formed in the living cells were dissolved by adding 100 µl of Me2SO. The optical absorbency was measured at 550–650 nm. Results represent the means of three experiments, and cell survival is expressed as the percentage of control.

Statistical Analysis—The results are expressed as the means ± S.D. of data obtained from three or more experiments performed in triplicate. Statistical significance was determined using Student's t test. Differences were considered as significant at p of 0.05 or less.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
H2O2 Induces the Tyrosine Phosphorylation of VEGFR-3 in HMEC-1 Cells and DMVECs—HMEC-1, an immortalized endothelial cell line, was originally derived from dermal microvascular endothelial cells (DMVECs) (38). HMEC-1 cells possess many of the properties of normal primary endothelial cells (38, 39). We found that HMEC-1 expressed robust levels of VEGFR-3. To assess whether H2O2 activated VEGFR-3, we first examined the tyrosine phosphorylation of VEGFR-3 in HMEC-1 cells after stimulation with 1.0 mM H2O2 for various times or, in another experiment, used different doses of H2O2 for a set time of 10 min. The tyrosine phosphorylation of VEGFR-3 was measured by precipitation with an anti-VEGFR-3 antibody, followed by Western blotting with an anti-PY monoclonal antibody (PY99). Western blotting with an anti-VEGFR-3 antibody was used to verify equal protein loading. As shown, H2O2 stimulation increased the tyrosine phosphorylation of VEGFR-3 in both a time- and dose-dependent manner (Fig. 1, A and B). A significant increase in the tyrosine phosphorylation of VEGFR-3 was detected with H2O2 concentrations as low as 0.05 mM (Fig. 1B, upper panel). The H2O2-induced tyrosine phosphorylation of VEGFR-3 was detected as early as 1 min, reached a maximum at 30 min, and declined thereafter but was still detectable after 60 min (Fig. 1A, upper panel). This was in contrast to the VEGF-C-induced tyrosine phosphorylation of VEGFR-3, which peaked at 5 min and declined to basal level 30 min after stimulation (data not shown). To confirm that the H2O2-induced tyrosine phosphorylation of VEGFR-3 was attributable to ROS, the stimulation with H2O2 was performed in the presence of catalase or N-acetylcysteine, a scavenger of ROS and precursor of glutathione. As shown in Fig. 1C, both catalase (1000 units/ml) and N-acetylcysteine (4 mM) inhibited the H2O2-induced tyrosine phosphorylation of VEGFR-3. We verified that equal amounts of VEGFR-3 were present in each lane of these blots (Fig. 1, A–D, lower panels).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 1.
H2O2 induced tyrosine phosphorylation of VEGFR-3 in HMEC-1 cells. The tyrosine phosphorylation of VEGFR-3 was measured by precipitation (IP) with an anti-VEGFR-3 antibody, followed by Western blotting (WB) with an anti-PY monoclonal antibody (PY99) (upper panel). Western blotting with an anti-VEGFR-3 antibody was used to verify equal protein loading (lower panel). A, cells were stimulated with 1.0 mM H2O2 for various times as indicated. B, cells were stimulated with different doses of H2O2 for 10 min. C, stimulation of cells with H2O2 in the absence or presence of catalase (1000 units/ml), N-acetylcysteine (NAC) (4 mM), a scavenger of ROS, or control. D, cells were stimulated with H2O2 (1.0 mM) or VEGF-C (150 ng/ml) in the absence or presence of the recombinant human VEGFR-3/Fc chimera protein (VEGFR-3/Ig).

 
To exclude the possibility that VEGFR-3 phosphorylation induced by H2O2 was mediated through autocrine stimulation by its natural ligands, HMEC-1 cells were stimulated with H2O2 (1.0 mM) or VEGF-C (50 ng/ml) in the absence or presence of the recombinant human VEGFR-3/Fc chimera protein (VEGFR-3/Ig). VEGFR-3/Ig has been shown to bind to both VEGF-D and VEGF-C and to block ligand/receptor binding (33, 37). As shown in Fig. 1D, the tyrosine phosphorylation of VEGFR-3 induced by VEGF-C was blocked by VEGFR-3/Ig, whereas the H2O2-stimulated phosphorylation of VEGFR-3 was not inhibited in the presence of VEGFR-3/Ig. These results indicated that the H2O2-induced phosphorylation of VEGFR-3 was not mediated by the release of cognate ligands.

We then investigated the effect of H2O2 on the activation of VEGFR-3 in primary endothelial cells (DMVECs) exposed to various doses of H2O2 or to VEGF-C for 10 min. We observed that H2O2 induced the tyrosine phosphorylation of VEGFR-3 in DMVECs in a dose-dependent manner (Fig. 2). In addition, VEGFR-3 also was activated when the receptor was transiently expressed in PAECs, 293 cells, or NIH-3T3 cells (data not shown), indicating that the H2O2-induced phosphorylation was not cell type-dependent.



View larger version (49K):
[in this window]
[in a new window]
  		FIG. 2.
H2O2 induced tyrosine phosphorylation of VEGFR-3 in DMVECs. DMVECs were exposed to various doses of H2O2 for 10 min or to VEGF-C (150 ng/ml) for 5 min. Cell lysates were immunoprecipitated (IP) with an anti-VEGFR-3 antibody. The immunoprecipitates were subjected to Western blotting (WB) with anti-phosphotyrosine antibody (anti-PY) (upper panel) and reprobed with an anti-VEGFR-3 antibody (lower panel).

 
Activation of VEGFR-3 by H2O2 Involves Activation of PKC and SFK and Inhibition of Phosphatase—Previous reports have suggested that ROS can activate PKC in different cell types (40, 41). We thus asked whether the tyrosine phosphorylation of VEGFR-3 induced by H2O2 involved the activation of PKC. Before exposure to H2O2 or VEGF-C stimulation, HMEC-1 cells were pretreated with Go6976, a selective inhibitor for PKC{alpha} or rottlerin, a selective inhibitor of PKC{delta}, and the tyrosine phosphorylation of VEGFR-3 was then examined. As shown in Fig. 3A, pretreatment with rottlerin but not Go6976 decreased the level of VEGFR-3 phosphorylation in response to H2O2 stimulation as compared with the Me2SO control, whereas neither inhibitor altered the VEGF-C-induced phosphorylation of VEGFR-3. Pretreatment with GF109203X, a broader selective inhibitor for various isoforms of PKC, also inhibited the H2O2-induced tyrosine phosphorylation of VEGFR-3 (Fig. 3B). These results suggested that H2O2-mediated receptor activation involved PKC, and most likely the {delta} isoform but not the {alpha} isoform.



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 3.
Activation of VEGFR-3 by H2O2 involved PKC in HMEC-1. A, cells were pretreated with Go6976 (Go) (0.5 µM), a selective inhibitor of PKC{alpha}; rottlerin (Rot) (5.0 µM), a selective inhibitor for PKC{delta}, or with control Me2SO for 45 min. B, cells were pretreated with GF109203X (GF)(3 µM), an inhibitor of all PKCs or with control Me2SO for 45 min. After the pretreatments, cells were then unstimulated or stimulated with H2O2 (1.0 mM) for 10 min or VEGF-C (150 ng/ml) for 5 min as indicated. Cell lysates were immunoprecipitated (IP) with an anti-VEGFR-3 antibody, subjected to Western blotting (WB) with anti-phosphotyrosine antibody (anti-PY) (upper panels) and reprobed with an anti-VEGFR-3 antibody (lower panels).

 
H2O2 induces the tyrosine phosphorylation of PKC {alpha} and {delta} and activates their kinase activity in a phospholipid-independent manner (40, 41). Although the mechanism whereby H2O2 activates PKC isoforms is not clear, it has been demonstrated that PKC, at least the {delta} isoform, can be activated by SFKs both in vitro and in vivo (42, 43). Moreover, SFKs are also activated after stimulation with H2O2 in various types of cells (11, 44, 45). We thus asked whether SFKs are involved in the H2O2-induced activation of VEGFR-3, possibly by modulating PKC. We examined the role of Src family tyrosine kinases in H2O2-induced VEGFR-3 phosphorylation by using PP2, a specific inhibitor for SFKs. As shown in Fig. 4A, the H2O2-stimulated tyrosine phosphorylation of VEGFR-3 was completely blocked in the presence of 10 µM PP2. These results suggested that SFKs are also involved in H2O2-induced VEGFR-3 activation. We also analyzed the tyrosine phosphorylation of PKC {alpha} or {delta} after H2O2 stimulation. We found that the tyrosine phosphorylation of PKC {alpha} or {delta} in response to H2O2 stimulation was inhibited by PP2 (Fig. 4, B and C).



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 4.
The H2O2 stimulation-induced tyrosine phosphorylations of VEGFR-3, PKC{alpha}, and PKC{delta} were inhibited by PP2. Before the exposure to H2O2 (1.0 mM), cells were pretreated with PP2 (10 µM) or control Me2SO for 45 min. The tyrosine phosphorylation of VEGFR-3 (A), PKC{alpha} (B), or PKC{delta} (C) was measured by precipitation (IP) with an anti-VEGFR-3, anti-PKC{alpha}, or anti-PKC{delta} antibody, respectively, followed by Western blotting (WB) with an anti-PY monoclonal antibody (upper panels). Western blotting with an anti-VEGFR-3 (A), anti-PKC{alpha} (B), or anti-PKC{delta} (C) antibody was used to verify equal protein loading (lower panels). Normal rabbit IgG (NR IgG) was used as a control for the immunoprecipitation. TCL, total cell lysates.

 
To demonstrate further the involvement of SFKs in the H2O2-induced phosphorylation of VEGFR-3 and PKC, we transfected HMEC-1 cells with a dominant-negative mutant of Src or the control empty vector. This dominant-negative Src can block the downstream signaling of various SFK members, because it encodes both the K296R and Y528F mutations that can bind to phosphotyrosine docking sites but are unable to phosphorylate downstream targets. As shown, transfection of cells with this dominant-negative construct resulted in a significant reduction in the tyrosine phosphorylation of VEGFR-3 (Fig. 5A), PKC{alpha} (Fig. 5B), and PKC{delta} (Fig. 5C) after H2O2 stimulation. These results indicated that the H2O2-induced activation of both VEGFR-3 and PKC isoforms required SFK activity.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 5.
Expression of dominant-negative c-Src inhibited the tyrosine phosphorylations of VEGFR-3, PKC{alpha}, and PKC{delta} after H2O2 stimulation. HMEC-1 cells were transfected with dominant-negative mutant (K296R/Y528F) Src cDNA (DN-Src) in a pUSEamp or control vector (Vector). 48 h after transfection, cells were serum-starved for 5 h and then stimulated with H2O2 (1.0 mM). Cell lysates were immunoprecipitated with antibody against VEGFR-3 (A), PKC{alpha} (B), or PKC{delta} (C), subjected to Western blotting with anti-phosphotyrosine antibody (anti-PY) for measuring tyrosine phosphorylation (upper panels) and then reprobed with antibody against each molecule to verify equal protein loading (lower panels).

 
Previous studies have shown that protein tyrosine phosphatases (PTPs) are direct targets for ROS (46–48). ROS inhibit PTP activities by inducing reversible oxidation of the catalytic cysteine residues. In HMEC-1 cells, we observed that treatment with H2O2 inhibited total phosphatase activity in a dose-dependent manner (Fig. 6). These data suggested that inhibition of phosphatases is involved in H2O2-induced VEGFR-3 activation. To determine whether inhibition of PTPs is sufficient to induce the tyrosine phosphorylation of VEGFR-3, we treated serum-starved HMEC-1 cells with sodium orthovanadate (Na3VO4), a potent general inhibitor of phosphatases. As shown in Fig. 7A, this treatment enhanced the tyrosine phosphorylation of VEGFR-3. Similar to H2O2, the sodium orthovanadate-induced phosphorylation of VEGFR-3 was also inhibited by pretreatment with PP2 (Fig. 7A). Furthermore, both sodium orthovanadate and H2O2 stimulated the phosphorylation of Tyr-416, a kinase activation site of the SFKs (Fig. 7B). These results indicated that the inhibition of PTPs may, at least in part, account for the observed phosphorylation of VEGFR-3 through activation of SFKs after H2O2 stimulation.



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 6.
Dose-dependent inhibition of total cellular protein phosphatase activity by H2O2 in HMEC-1 cells. Serum-starved HMEC-1 cells were treated with various doses of H2O2 as indicated for 30 min. Cellular protein phosphatase activity was measured with the Phosphatase Assay kit (Upstate Biotechnology, Lake Placid, NY) using para-nitrophenyl phosphate as the substrate. Phosphatase activity was obtained by measuring para-nitrophenyl phosphate production and expressed as a percentage of the control value.

 



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 7.
Sodium orthovanadate or H2O2 induced the tyrosine phosphorylation of VEGFR-3 and activated c-Src. A, serum-starved HMEC-1 cells were pretreated with PP2 (10 µM) or control Me2SO for 45 min. Cells were then stimulated with sodium orthovanadate (vanadate, 0.1 mM) or H2O2 (1.0 mM) for 10 min. The tyrosine phosphorylation of VEGFR-3 was measured by immunoprecipitation (IP) with an anti-VEGFR-3 antibody, followed by Western blotting (WB) with an anti-PY monoclonal antibody (upper panel). Western blotting with an anti-VEGFR-3 antibody was used to verify equal protein loading (lower panel). B, total cell lysates were subjected to Western blotting with an anti-phospho-Src family (Tyr-416) antibody (upper panel). Western blotting with an anti-c-Src antibody was used to verify equal protein loading (lower panel).

 
H2O2 Stimulation Induces the Association of VEGFR-3 with Its Downstream Signaling Molecules—The tyrosine phosphorylation of tyrosine kinase receptors transmits signals by recruiting SH2 domain-containing signaling proteins. To explore whether the H2O2-induced phosphorylation of VEGFR-3 was functional, we examined the immunocomplexes formed between VEGFR-3 and downstream adaptor proteins. HMEC-1 cells were exposed to H2O2 treatment, and then cell lysates were prepared and immunoprecipitated with anti-VEGFR-3 antibody. The immunoprecipitates were resolved on 8% SDS-PAGE gel and subjected to sequential Western blotting with antibodies against various signaling molecules. As shown in Fig. 8, the association of VEGFR-3 with the p85 subunit of PI3-kinase (Fig. 8A), Shc (Fig. 8B), Grb2 (Fig. 8C), SHP-2 (Fig. 8D), or phospholipase C-{gamma} (Fig. 8E) was enhanced after the treatment with H2O2. These results suggested that the H2O2-induced activation of VEGFR-3 was able to initiate downstream signaling by recruiting adaptor molecules.



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 8.
H2O2 stimulation induces the association of VEGFR-3 with SH2 domain-containing downstream signaling molecules. Serum-starved cells were stimulated with H2O2 (1.0 mM) or VEGF-C for various times as indicated. Cell lysates were subjected to immunoprecipitation (IP) with anti-VEGFR-3 antibody and subsequently to immunoblotting (WB) with antibody against the p85 subunit of PI3-kinase (A), Shc (B), growth factor receptor binding protein 2 (Grb2) (C), SHP-2 (D), or phospholipase C-{gamma} (PLC-{gamma}) (E) (upper panel in each blot). The protein loading in the lanes of each blot was examined by immunoblotting with the same antibody used for the immunoprecipitation in each of the blots (lower panels). Normal rabbit IgG (NR IgG) was used as a control for the immunoprecipitation. TCL, total cell lysates.

 
Activation of VEGFR-3 by H2O2 Stimulation Contributes to the Activation of Akt and Enhances Cell Survival—ROS activates multiple signaling pathways. PI3-kinase/Akt promotes cell survival and functions as a major protector of cells in response to oxidative stress (5, 6). H2O2 stimulation activates Akt in different types of cells (5, 6). VEGFR-3 signaling also induces activation of the PI3-kinase/Akt pathway (4, 6). We thus asked whether the activation of VEGFR-3 might trigger Akt activation and enhance cell survival in response to H2O2 exposure.

We first used MAZ51, a recently identified selective inhibitor for VEGFR-3 (49). HMEC-1 cells were pretreated with 5 µM MAZ51 or control Me2SO for 30 min, followed by stimulation with different doses of H2O2 or VEGF-C (150 ng/ml). The phosphorylation of Akt was examined by Western blotting using specific antibody against phospho-Akt. As shown in Fig. 9, H2O2 or VEGF-C stimulated the phosphorylation of Akt, whereas treatment with MAZ51 inhibited the Akt phosphorylation induced either by H2O2 or by VEGF-C. These results suggested that VEGFR-3 might be involved in the activation of Akt induced by H2O2.



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 9.
Inhibition of VEGFR-3 diminished the phosphorylation of Akt in response to H2O2. HMEC-1 cells were pretreated with Me2SO or MAZ51 (5 µM), a selective inhibitor for VEGFR-3, for 45 min. Cells were then unstimulated (-) or stimulated (+) with H2O2 (0.1–1.0 mM) or with VEGF-C (150 ng/ml) for 10 min. The phosphorylation of Akt was detected by Western blotting with anti-phospho-Akt antibody (upper panel), followed by stripping and reblotting with antibody against Akt (lower panel). P-Akt, phosphorylated Akt.

 
To further investigate the role of VEGFR-3 in H2O2-induced Akt activation, we examined the phosphorylation of Akt by H2O2 in the presence of vector, wild-type, or a kinase-inactive mutant form of VEGFR-3 using a transfection cell model. We used PAECs, which do not express endogenous VEGFR-3 (50). In addition, PAECs appear to express very low, if any, levels of VEGFR-1 (51), VEGFR-2 (51), EGFR (52), or platelet-derived growth factor receptor (53). Thus, using PAECs would minimize the possible effects of these growth factor receptors on Akt activation after H2O2 stimulation because receptors such as epidermal growth factor receptor or platelet-derived growth factor receptor are also targets of ROS.

VEGFR-3/G857R is a G-R mutant located in the first kinase domain of VEGFR-3, originally identified in patients with hereditary lymphoedema (54, 55). This mutation results in the loss of receptor kinase activity and unresponsiveness to ligand stimulation (33, 37, 54, 55). As shown in Fig. 10A, when transiently expressed in PAECs, wild-type VEGFR-3 was tyrosine-phosphorylated after H2O2 or VEGF-C stimulation, whereas the mutant form of VEGFR-3 was not phosphorylated by either stimulus. There was no endogenous VEGFR-3 in the control vector-transfected PAECs, as detected by immunoprecipitation and Western blotting using anti-VEGFR-3-specific antibody. These results suggested that receptor kinase activity could be required for the H2O2-induced phosphorylation of VEGFR-3.



View larger version (43K):
[in this window]
[in a new window]
  		FIG. 10.
The hereditary lymphoedema-linked missense mutation in VEGFR-3 abolishes receptor tyrosine phosphorylation and the subsequent activation of Akt in response to H2O2 stimulation. PAECs were transfected with a vector (PRK5) or with the wild-type (WT) or mutant (MT) form of VEGFR-3. 48 h after transfection, the cells were unstimulated (-) or stimulated (+) with H2O2 (0.5 mM) for 10 min or with VEGF-C (150 ng/ml) for 5 min. A, the tyrosine phosphorylation of VEGFR-3 was examined by immunoprecipitation with an anti-VEGFR-3 antibody and by Western blotting with anti-phosphotyrosine antibody (anti-PY) (upper panel). The blot was reprobed with an anti-VEGFR-3 antibody to assure equal protein loading (lower panel). B, PAECs were transfected as in A and then unstimulated (-) or stimulated (+) with H2O2 (0.5 mM) or with VEGF-C (150 ng/ml) for 10 min. The phosphorylation of Akt was detected by Western blotting with anti-phospho-Akt antibody (upper panel), followed by stripping and reblotting with antibody against Akt (lower panel). P-Akt, phosphorylated Akt.

 
To examine activation of Akt, the wild-type or mutant form of VEGFR-3-transfected or control vector-transfected PAECs were stimulated by either H2O2 or VEGF-C. Cell lysates were analyzed by Western blotting using anti-phospho-Akt antibody. As shown in Fig. 10B, the H2O2-induced phosphorylation of Akt was significantly increased in the wild-type but not in the mutant VEGFR-3-transfected cells as compared with the control vector-transfected cells. VEGF-C only stimulated Akt phosphorylation in the wild-type VEGFR-3-transfected cells. These data demonstrated that the stimulation of VEGFR-3 contributed to the activation of Akt in response to H2O2 exposure.

Next, we investigated whether VEGFR-3 signaling could protect cells from ROS-induced cell death in addition to activating Akt. Cell survival assays were performed by applying MAZ51 in HMEC-1 cells or by using the VEGFR-3-transfected PAEC model after exposure to various does of H2O2. As shown, exposure to H2O2 for 30 min induced cell death in both HMEC-1 (Fig. 11A) and PAECs (Fig. 11B) in a dose-dependent manner. In HMEC-1 cells, applying MAZ51 further decreased cell survival after H2O2 treatment (Fig. 11A), although there were no significant effects of MAZ51 on survival in cells untreated with H2O2 (data not shown). These results suggested that blocking VEGFR-3 signaling promoted H2O2-induced cell death. In PAECs, transfection of the wild-type VEGFR-3 enhanced cell survival after H2O2 exposure as compared with cells transfected with either the mutant form of VEGFR-3 or the control expressing vector alone (Fig. 11B). These data indicated that the presence of VEGFR-3 and its activation contributed to cell survival.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 11.
VEGFR-3 protects cells from H2O2-induced death. A, HMEC-1 cells were treated with various doses of H2O2 as indicated in the presence of MAZ51 (5 µM) or Me2SO for 30 min. B, PAECs were transfected with vector (PRK5) or with the wild-type (WT) or mutant (MT) form of VEGFR-3, and 24 h later, were treated with various doses of H2O2 for 30 min as indicated. After H2O2 treatment, cells were washed and then cultured for 24 h in fresh medium containing 2% FCS. Cell viability was measured using colorimetric MTT assays as described in "Experimental Procedures." Results are presented as the percentage of cell survival relative to control (untreated). Data are the means of three independent experiments, and error bars correspond to the standard deviation of the mean. *, significant differences (p < 0.05) in the values between treatment with MAZ51 and control (A) or in the values between wild-type and mutant VEGFR-3 (B), as determined using Student's t test.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Using model HMEC-1 cells, we found that H2O2 treatment induced the tyrosine phosphorylation of VEGFR-3 (Fig. 1). The activation of VEGFR-3 was not attributable to the induction of VEGFR-3 ligands because it was not blocked by the soluble ectodomain of the receptor. Activation was inhibited by the anti-oxidant reagent, N-acetylcysteine, as well as by catalase. The activation of VEGFR-3 by H2O2 was also confirmed in primary endothelial cells, DMVECs (Fig. 2). Thus, VEGFR-3, similar to certain other tyrosine kinase receptors including epidermal growth factor receptor (6, 9, 11, 12), platelet-derived growth factor receptor (13, 14), and the insulin receptor (15), is a target for ROS.

One important issue addressed in our study is whether H2O2-mediated activation of VEGFR-3 is functional in transmitting its downstream signaling. We demonstrated that VEGFR-3 could associate with various SH2-containing molecules, which are believed to mediate the signaling of tyrosine kinase receptors to downstream substrates (Fig. 8), indicating that H2O2-mediated activation of VEGFR-3 was functional. Furthermore, we showed that VEGFR-3 signaling contributed to the activation of Akt (Figs. 9 and 10), a critical pathway in cell survival. The role of Akt in protecting against ROS-induced cell death has been extensively documented (1, 5, 6). Our results clearly demonstrated the involvement of VEGFR-3 in preventing H2O2-induced cell death in endothelial cells (Fig. 11). Thus, similar to other growth factor receptors, VEGFR-3 can serve as a cell survival modulator under oxidative stress.

Considering the critical roles of VEGFR-3 in the development of the lymphatic system and its biological action in maintaining the structural and functional integrity of lymphatic endothelium and lymphangiogenesis (29–33), the findings in this report may have some practical implications in understanding lymphatic-related diseases. For example, chronic lymphoedema is a disease involving lymphatic dysfunction, either developed as a congenital disorder or generated secondary to other diseases such as cancer or filarial parasites. Enhanced formation of ROS and accelerated lipid peroxidation processes have been detected in lymphoedematous tissue, which are considered to contribute to the damage of lymphatic endothelium (56, 57). The expression of VEGFR-3 in lymphatics, along with cellular anti-oxidative defense molecules, likely plays a role in protecting against ROS-induced cell damage. The functional loss of VEGFR-3 may facilitate disease-related pathological processes. Indeed, we found that the mutant form of VEGFR-3 (VEGFR-3/G857R) did not respond to the stimulation by H2O2 and failed both to activate Akt (Fig. 10) and to prevent cell death under oxidative stress (Fig. 11). This mutant and other mutations in the intracellular kinase domains of VEGFR-3 have been identified among families with hereditary lymphoedema (54, 55). These mutations result in the loss of receptor kinase activity and have been proposed as the cause of the disease. Thus, approaches to restore receptor function directly or indirectly through alternative receptor signaling could be beneficial in treating this disease (58, 59).

It is well known that tumors generate significantly higher levels of ROS than normal tissues (60). ROS likely contribute to the increased angiogenesis observed in malignancy by inducing the production of angiogenic factors such as VEGF, IL-8, and the expression of metalloproteinases (61). More recent evidence implicates VEGFR-3-mediated lymphangiogenesis in tumor metastasis (34–36). The level of H2O2 in some tumors could reach levels beyond those required to activate VEGFR-3 (Fig. 1). On the basis of the observations in this study, it is possible that ROS may also contribute to lymphangiogenesis through activation of VEGFR-3 in tumor tissue.

Although the precise mechanism whereby ROS activate receptor tyrosine kinases is not clear, a plausible explanation is the inactivation of PTPs (26, 27, 62). The catalytic sites of PTPs contain reactive cysteine residues and form thiol-phosphate intermediates during catalysis. The thiol oxidation of these residues leads to inactivation of PTPs. H2O2 has been shown to reversibly inactivate PTPs (46). PTPs are believed to inhibit tyrosine kinase receptors by virtue of dephosphorylation; therefore, inactivation of PTPs would thereby increase receptor phosphorylation (47, 48). Indeed, we demonstrated that H2O2 could inhibit total protein phosphatase activity in HMEC-1 cells. Treatment of cells with sodium orthovanadate, a potent general inhibitor of phosphatases, also induced the tyrosine phosphorylation of VEGFR-3. These results suggested that inhibition of PTPs was involved in the H2O2-induced phosphorylation of VEGFR-3, similar to findings from other receptor tyrosine kinases (47, 48). However, the inactivation of PTPs and the subsequent inhibited dephosphorylation of tyrosine kinase receptors may not be the only mechanism for the increases in receptor tyrosine phosphorylation induced by ROS. Our results demonstrated that the H2O2-induced phosphorylation of VEGFR-3 could be inhibited either by treatment with the SFK-specific inhibitor, PP2, or by overexpression of a dominant-negative Src construct (with K296R and Y528F mutations), thereby indicating that the activation of VEGFR-3 requires SFKs (Figs. 4 and 5). Furthermore, we observed a significant increase in Src kinase activity in HMEC-1 cells after H2O2 stimulation (Fig. 7B). Moreover, the sodium orthovanadate-induced phosphorylation of VEGFR-3 was also inhibited by PP2 (Fig. 7A). Our studies further showed that, similar to H2O2, sodium orthovanadate activated c-Src kinase (Fig. 7B). Taken together, our results suggest that the H2O2-induced phosphorylation of VEGFR-3 requires activation of SFKs and that these effects probably are, at least in part, mediated by the inhibition of PTPs. Previous studies have shown that other tyrosine kinase receptors such as epidermal growth factor receptor also require SFKs for H2O2-induced activation (11). These results suggest that SFK-dependent activation may serve as a common mechanism among tyrosine kinase receptors in their response to ROS stimulation. However, it is not clear how SFKs induce the phosphorylation of tyrosine kinase receptors, although it has been observed that some growth factor receptors such as epidermal growth factor receptor (63) and Ron (64) are directly phosphorylated by Src.

Our results indicated that the phosphorylation of VEGFR-3 induced by H2O2 also required PKC in addition to SFKs (Figs. 3 and 4). H2O2 can activate various isoforms of PKC along with their phosphorylation on tyrosine (40, 41). Consistent with a previous report (13), we observed that the H2O2-induced tyrosine phosphorylation of PKC {alpha} or {delta} was inhibited by PP2 (Fig. 4, B and C) or by overexpression of a dominant-negative mutant of c-Src (Fig. 5). Because Src can directly phosphorylate these PKC isoforms on tyrosine and activate their kinase activities both in vivo and in vitro (42, 65), it is plausible to propose that the contribution of SFKs to the H2O2-mediated phosphorylation of VEGFR-3 may take place upstream of PKC. While determining which isoform of PKC might modulate the phosphorylation of VEGFR-3 after H2O2 stimulation, we observed that H2O2-induced phosphorylation of VEGFR-3 was diminished by inhibition of PKC{delta} but not {alpha}. PKC{delta} was requisite for H2O2-induced but not for VEGF-C-induced tyrosine phosphorylation of VEGFR-3 (Fig. 3A). This further suggested that H2O2 activated this receptor through distinct mechanisms other than through receptor ligands. Thus, our study indicated that the activation of VEGFR-3 in response to H2O2 was probably initiated by intracellular kinases, such as SFKs and PKC{delta}, rather than by extracellular ligand stimulation.

Because ROS and VEGFR-3 are strongly implicated in lymphatic diseases as well as in normal lymph development and lymphangiogenesis, the potent activation of VEGFR-3 and its functional signaling by ROS may provide new mechanistic insights into both physiological and pathological processes in lymphatic endothelium.


   FOOTNOTES
 
* This work was supported in part by NIH Grant R01HL61940 and the Diller-Von Furstenburg Family Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Division of Experimental Medicine, Harvard Institutes of Medicine, Beth Israel Deaconess Medical Center, 4 Blackfan Circle, Boston, MA 02115. E-mail: jgroopma{at}bidmc.harvard.edu.

1 The abbreviations used are: ROS, reactive oxygen species; H2O2, hydrogen peroxide; PI3-kinase, phosphatidylinositol 3-kinase; VEGFR, vascular endothelial growth factor receptor; PY, phosphotyrosine; SFK, Src family kinase; RTK, receptor tyrosine kinase; PKC, protein kinase C; MTT, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl]tetrazolium bromide; HMEC, human microvascular endothelial cell; PAEC, porcine aortic endothelial cell; DMVEC, dermal microvascular endothelial cell; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PTP, protein tyrosine phosphatase. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Martindale, J. L., and Holbrook, N. J. (2002) J. Cell. Physiol. 192, 1-15[CrossRef][Medline] [Order article via Infotrieve]
  2. Finkel, T., and Holbrook, N. J. (2000) Nature 408, 239-247[CrossRef][Medline] [Order article via Infotrieve]
  3. Irani, K. (2000) Circ. Res. 87, 179-183[Abstract/Free Full Text]
  4. Wang, X., Martindale, J. L., Liu, Y., and Holbrook, N. J. (1998) Biochem. J. 333, 291-300[Medline] [Order article via Infotrieve]
  5. Qin, S., and Chock, P. B. (2003) Biochemistry 42, 2995-3003[CrossRef][Medline] [Order article via Infotrieve]
  6. Wang, X., McCullough, K. D., Franke, T. F., and Holbrook, N. J. (2000) J. Biol. Chem. 275, 14624-14631[Abstract/Free Full Text]
  7. Wang, X., McCullough, K. D., Wang, X. J., Carpenter, G., and Holbrook, N. J. (2001) J. Biol. Chem. 246, 28364-28371
  8. Mattson, M. P., Culmsee, C., Yu, Z., and Camandola, S. (2000) J. Neurochem. 74, 443-456[CrossRef][Medline] [Order article via Infotrieve]
  9. Huang, R. P., Peng, A., Golard, A., Hossain, M. Z., Huang, R., Liu, Y. G., and Boynton, A. L. (2001) Mol. Carcinog. 30, 209-217[CrossRef][Medline] [Order article via Infotrieve]
  10. Nair, V. D., Olanow, C. W., and Sealfon, S. C. (2003) Biochem. J. 373, 25-32[CrossRef][Medline] [Order article via Infotrieve]
  11. Chen, K., Vita, J. A., Berk, B. C., and Keaney, J. F., Jr. (2001) J. Biol. Chem. 276, 16045-16050[Abstract/Free Full Text]
  12. Goldkorn, T., Balaban, N., Matsukuma, K., Chea, V., Gould, R., Last, J., Chan, C., and Chavez, C. (1998) Am. J. Respir. Cell Mol. Biol. 19, 786-798[Abstract/Free Full Text]
  13. Saito, S., Frank, G. D., Mifune, M., Ohba, M., Utsunomiya, H., Motley, E. D., Inagami, T., and Eguchi, S. (2002) J. Biol. Chem. 277, 44695-44700[Abstract/Free Full Text]
  14. Gonzalez-Rubio. M., Voit, S., Rodriguez-Puyol, D., Weber, M., and Marx, M. (1996) Kidney Int. 50, 164-173[Medline] [Order article via Infotrieve]
  15. Koshio, O., Akanuma, Y., and Kasuga, M. (1988) Biochem. J. 250, 95-101[Medline] [Order article via Infotrieve]
  16. Frank, G. D., Eguchi, S., Inagami, T., and Motley, E. D. (2001) Biochem. Biophys. Res. Commun. 280, 1116-1119[CrossRef][Medline] [Order article via Infotrieve]
  17. Wan, Y. S., Wang, Z. Q., Shao, Y., Voorhees, J. J., and Fisher, G. J. (2001) Int. J. Oncol. 18, 461-466[Medline] [Order article via Infotrieve]
  18. Shono, T., Ono, M., Izumi, H., Jimi, S. I., Matsushima, K., Okamoto, T., Kohno, K., and Kuwano, M. (1996) Mol. Cell. Biol. 16, 4231-4239[Abstract]
  19. Lu, D., Maulik, N., Moraru, I. I., Kreutzer, D. L., and Das, D. K. (1993) Am. J. Physiol. 264, C715-C722[Medline] [Order article via Infotrieve]
  20. Roebuck, K. A., Rahman, A., Lakshminarayanan, V., Janakidevi, K., and Malik, A. B. (1995) J. Biol. Chem. 270, 18966-18974[Abstract/Free Full Text]
  21. Ng, C. K., Deshpande, S. S., Irani, K., and Alevriadou, B. R. (2002) Am. J. Physiol. Cell. Physiol. 283, C93-C102[Abstract/Free Full Text]
  22. Niwa, K., Inanami, O., Ohta, T., Ito, S., Karino, T., and Kuwabara, M. (2001) Free Radic. Res. 35, 519-527[CrossRef][Medline] [Order article via Infotrieve]
  23. Kevil, C. G., Okayama, N., and Alexander, J. S. (2001) Am. J. Physiol. Cell. Physiol. 281, C1940-C19407[Abstract/Free Full Text]
  24. Lum, H., and Roebuck, K. A. (2001) Am. J. Physiol. Cell. Physiol. 280, C719-C741[Abstract/Free Full Text]
  25. Sorescu, D., and Griendling, K. K. (2002) Congest. Heart Fail. 8, 132-140[Medline] [Order article via Infotrieve]
  26. Ushio-Fukai, M., Tang, Y., Fukai, T., Dikalov, S. I., Ma, Y., Fujimoto, M., Quinn, M. T., Pagano, P. J., Johnson, C., and Alexander, R. W. (2002) Circ. Res. 91, 1160-1167[Abstract/Free Full Text]
  27. Colavitti, R., Pani, G., Bedogni, B., Anzevino, R., Borrello, S., Waltenberger, J., and Galeotti, T. (2002) J. Biol. Chem. 277, 3101-3108[Abstract/Free Full Text]
  28. Galland, F., Karamysheva, A., Pebusque, M. J., Borg, J. P., Rottapel, R., Dubreuil, P., Rosnet, O., and Birnbaum, D. (1993) Oncogene 8, 1233-1240[Medline] [Order article via Infotrieve]
  29. Joukov, V., Pajusola, K., Kaipainen, A., Chilov, D., Lahtinen, I., Kukk, E., Saksela, O., Kalkkinen, N., and Alitalo, K. (1996) EMBO J. 15, 290-298[Medline] [Order article via Infotrieve]
  30. Achen, M. G., Jeltsch, M., Kukk, E., Makinen, T., Vitali, A., Wilks, A. F., Alitalo, K., and Stacker, S. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 548-553[Abstract/Free Full Text]
  31. Kaipainen, A., Korhonen, J., Mustonen, T., van Hinsbergh, V. W., Fang, G., Dumont, D., Breitman, M., and Alitalo, K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3566-3570[Abstract/Free Full Text]
  32. Dumont, D. J., Jussila, L., Taipale, J., Lymboussaki, A., Mustonen, T., Pajusola, K., Breitman, M., and Alitalo, K. (1998) Science 282, 946-949[Abstract/Free Full Text]
  33. Makinen, T., Jussila, L., Veikkola, T., Karpanen, T., Kettunen, M. I., Pulkkanen, K. J., Kauppinen, R., Jackson, D. G., Kubo, H., Nishikawa, S., Yla-Herttuala, S., and Alitalo, K. (2001) Nat. Med. 7, 199-205[CrossRef][Medline] [Order article via Infotrieve]
  34. Stacker, S. A., Caesar, C., Baldwin, M. E., Thornton, G. E., Williams, R. A., Prevo, R., Jackson, D. G., Nishikawa, S., Kubo, H., and Achen, M. G. (2001) Nat. Med. 7, 186-191[CrossRef][Medline] [Order article via Infotrieve]
  35. Skobe, M., Hawighorst, T., Jackson, D. G., Prevo, R., Janes, L., Velasco, P., Riccardi, L., Alitalo, K., Claffey, K., and Detmar, M. (2001) Nat. Med. 7, 192-198[CrossRef][Medline] [Order article via Infotrieve]
  36. Padera, T. P., Kadambi, A., di Tomaso, E., Carreira, C. M., Brown, E. B., Boucher, Y., Choi, N. C., Mathisen, D., Wain, J., Mark, E. J., Munn, L. L., and Jain, R. K. (2002) Science 296, 1883-1886[Abstract/Free Full Text]
  37. Wang, J. F., Zhang, X. F., and Groopman, J. E. (2001) J. Biol. Chem. 276, 41950-41957[Abstract/Free Full Text]
  38. Ades, E. W., Candal, F. J., Swerlick, R. A., George, V. G., Summers, S., Bosse, D. C., and Lawley, T. J. (1992) J. Invest. Dermatol. 99, 683-690[CrossRef][Medline] [Order article via Infotrieve]
  39. Bouis, D., Hospers, G. A., Meijer, C., Molema, G., and Mulder, N. H. (2001) Angiogenesis 4, 91-102[CrossRef][Medline] [Order article via Infotrieve]
  40. Konishi, H., Tanaka, M., Takemura, Y., Matsuzaki, H., Ono, Y., Kikkawa, U., and Nishizuka, Y. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11233-11237[Abstract/Free Full Text]
  41. Min, D. S., Kim, E. G., and Exton, J. H. (1998) J. Biol. Chem. 273, 29986-29994[Abstract/Free Full Text]
  42. Gschwendt, M., Kielbassa, K., Kittstein, W., and Marks, F. (1994) FEBS Lett. 347, 85-89[CrossRef][Medline] [Order article via Infotrieve]
  43. Tapia, J. A., Garcia-Marin, L. J., and Jensen, R. T. (2003) J. Biol. Chem. 278, 35220-35230[Abstract/Free Full Text]
  44. Abe, J., Takahashi, M., Ishida, M., Lee, J. D., and Berk, B. C. (1997) J. Biol. Chem. 272, 20389-20394[Abstract/Free Full Text]
  45. Aikawa, R., Komuro, I., Yamazaki, T., Zou, Y., Kudoh, S., Tanaka, M., Shiojima, I., Hiroi, Y., and Yazaki, Y. (1997) J. Clin. Invest. 100, 1813-1821[Medline] [Order article via Infotrieve]
  46. Denu, J. M., and Tanner, K. G. (1998) Biochemistry 37, 5633-5642[CrossRef][Medline] [Order article via Infotrieve]
  47. Knebel, A., Rahmsdorf, H. J., Ullrich, A., and Herrlich, P. (1996) EMBO J. 15, 5314-5325[Medline] [Order article via Infotrieve]
  48. Meng, T. C., Fukada, T., and Tonks, N. K (2002) Mol. Cell. 9, 387-399[CrossRef][Medline] [Order article via Infotrieve]
  49. Kirkin, V., Mazitschek, R., Krishnan, J., Steffen, A., Waltenberger, J., Pepper, M. S., Giannis, A., and Sleeman, J. P. (2001) Eur. J. Biochem. 268, 5530-5540[Medline] [Order article via Infotrieve]
  50. Makinen, T., Veikkola, T., Mustjoki, S., Karpanen, T., Catimel, B., Nice, E. C., Wise, L., Mercer, A., Kowalski, H., Kerjaschki, D., Stacker, S. A., Achen, M. G., and Alitalo, K. (2001) EMBO J. 20, 4762-4773[CrossRef][Medline] [Order article via Infotrieve]
  51. Kim, K. J., Li, B., Winer, J., Armanini, M., Gillett, N., Phillips, H. S., and Ferrara, N. (1993) Nature 362, 841-844[CrossRef][Medline] [Order article via Infotrieve]
  52. Jiang, X., Huang, F., Marusyk, A., and Sorkin, A. (2003) Mol. Biol. Cell 14, 858-870[Abstract/Free Full Text]
  53. Hooshmand-Rad, R., Yokote, K., Heldin, C. H., and Claesson-Welsh, L. (1998) J. Cell Sci. 111, 607-614[Abstract]
  54. Irrthum, A., Karkkainen, M. J., Devriendt, K., Alitalo, K., and Vikkula, M. (2000) Am. J. Hum. Genet. 67, 295-301[CrossRef][Medline] [Order article via Infotrieve]
  55. Karkkainen, M. J., Ferrell, R. E., Lawrence, E. C., Kimak, M. A., Levinson, K. L., McTigue, M. A., Alitalo, K., and Finegold, D. N. (2000) Nat. Genet. 25, 153-159[CrossRef][Medline] [Order article via Infotrieve]
  56. Siems, W. G., Brenke, R., Beier, A., and Grune, T. (2002) Q. J. M. 95, 803-809
  57. Ohkuma, M. (1993) Lymphology 26, 38-41[Medline] [Order article via Infotrieve]
  58. Saaristo, A., Karkkainen, M. J., and Alitalo, K. (2002) Ann. N. Y. Acad. Sci. 979, 94-110[Medline] [Order article via Infotrieve]
  59. Karkkainen, M. J., Saaristo, A., Jussila, L., Karila, K. A., Lawrence, E. C., Pajusola, K., Bueler, H., Eichmann, A., Kauppinen, R., Kettunen, M. I., Yla-Herttuala, S., Finegold, D. N., Ferrell, R. E., and Alitalo, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 12677-12682[Abstract/Free Full Text]
  60. Szatrowski, T. P., and Nathan, C. F. (1991) Cancer Res. 51, 794-798[Abstract/Free Full Text]
  61. Brown, N. S., and Bicknell, R. (2001) Breast Cancer Res. 3, 323-327[CrossRef][Medline] [Order article via Infotrieve]
  62. Kamata, H., Shibukawa, Y., Oka, S. I., and Hirata, H. (2000) Eur. J. Biochem. 267, 1933-1944[Medline] [Order article via Infotrieve]
  63. Tice, D. A., Biscardi, J. S., Nickles, A. L., and Parsons, S. J. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1415-1420[Abstract/Free Full Text]
  64. Danilkovitch-Miagkova, A., Angeloni, D., Skeel, A., Donley, S., Lerman, M., and Leonard, E. J. (2000) J. Biol. Chem. 275, 14783-14786[Abstract/Free Full Text]
  65. Zang, Q., Frankel, P., and Foster, D. A. (1995) Cell Growth & Differ. 6, 1367-1373[Abstract]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
H. S. Kim and I. K. Lim
Phosphorylated Extracellular Signal-regulated Protein Kinases 1 and 2 Phosphorylate Sp1 on Serine 59 and Regulate Cellular Senescence via Transcription of p21Sdi1/Cip1/Waf1
J. Biol. Chem., June 5, 2009; 284(23): 15475 - 15486.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. Lahdenranta, J. Hagendoorn, T. P. Padera, T. Hoshida, G. Nelson, S. Kashiwagi, R. K. Jain, and D. Fukumura
Endothelial Nitric Oxide Synthase Mediates Lymphangiogenesis and Lymphatic Metastasis
Cancer Res., April 1, 2009; 69(7): 2801 - 2808.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Jakob, P. Schroeder, M. Lukosz, N. Buchner, I. Spyridopoulos, J. Altschmied, and J. Haendeler
Nuclear Protein Tyrosine Phosphatase Shp-2 Is One Important Negative Regulator of Nuclear Export of Telomerase Reverse Transcriptase
J. Biol. Chem., November 28, 2008; 283(48): 33155 - 33161.
[Abstract] [Full Text] [PDF]

Home page
 Hum Exp ToxicolHome page
R. Demiralay, N. Gursan, and H. Erdem
Regulation of nicotine-induced apoptosis of pulmonary artery endothelial cells by treatment of N-acetylcysteine and vitamin E
Human and Experimental Toxicology, July 1, 2007; 26(7): 595 - 602.
[Abstract] [PDF]

Home page
 Clin. Cancer Res.Home page
C. Wissmann and M. Detmar
Pathways Targeting Tumor Lymphangiogenesis
Clin. Cancer Res., December 1, 2006; 12(23): 6865 - 6868.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J. Song, J. Li, A. Lulla, B. M. Evers, and D. H. Chung
Protein kinase D protects against oxidative stress-induced intestinal epithelial cell injury via Rho/ROK/PKC-{delta} pathway activation
Am J Physiol Cell Physiol, June 1, 2006; 290(6): C1469 - C1476.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
C. A. Garces, E. V. Kurenova, V. M. Golubovskaya, and W. G. Cance
Vascular Endothelial Growth Factor Receptor-3 and Focal Adhesion Kinase Bind and Suppress Apoptosis in Breast Cancer Cells
Cancer Res., February 1, 2006; 66(3): 1446 - 1454.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
L. DeMaio, M. Rouhanizadeh, S. Reddy, A. Sevanian, J. Hwang, and T. K. Hsiai
Oxidized phospholipids mediate occludin expression and phosphorylation in vascular endothelial cells
Am J Physiol Heart Circ Physiol, February 1, 2006; 290(2): H674 - H683.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/26/27088    most recent
M314015200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, J. F.
Right arrow Articles by Groopman, J. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, J. F.
Right arrow Articles by Groopman, J. E.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement