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Am J Physiol Cell Physiol 294: C241-C250, 2008. First published November 14, 2007; doi:10.1152/ajpcell.00187.2007
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VASCULAR BIOLOGY

VEGF and thrombin induce MKP-1 through distinct signaling pathways: role for MKP-1 in endothelial cell migration

Corttrell M. Kinney,1,2 Unni M. Chandrasekharan,1 Lori Mavrakis,1 and Paul E. DiCorleto1,2

1Department of Cell Biology, Lerner Research Institute and Cleveland Clinic Lerner College of Medicine, and 2Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio

Submitted 9 May 2007 ; accepted in final form 6 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have previously reported that MAPK phosphatase-1 (MKP-1/CL100) is a thrombin-responsive gene in endothelial cells (ECs). We now show that VEGF is another efficacious activator of MKP-1 expression in human umbilical vein ECs. VEGF-A and VEGF-E maximally induced MKP-1 expression in ECs; however, the other VEGF subtypes had no effect. Using specific neutralizing antibodies, we determined that VEGF induced MKP-1 specifically through VEGF receptor 2 (VEGFR-2), leading to the downstream activation of JNK. The VEGF-A165 isoform stimulated MKP-1 expression, whereas the VEGF-A162 isoform induced the gene to a lesser extent, and the VEGF-A121 isoform had no effect. Furthermore, specific blocking antibodies against neuropilins, VEGFR-2 coreceptors, blocked MKP-1 induction. A Src kinase inhibitor (PP1) completely blocked both VEGF- and thrombin-induced MKP-1 expression. A dominant negative approach revealed that Src kinase was required for VEGF-induced MKP-1 expression, whereas Fyn kinase was critical for thrombin-induced MKP-1 expression. Moreover, VEGF-induced MKP-1 expression required JNK, whereas ERK was critical for thrombin-induced MKP-1 expression. In ECs treated with short interfering (si)RNA targeting MKP-1, JNK, ERK, and p38 phosphorylation were prolonged following VEGF stimulation. An ex vivo aortic angiogenesis assay revealed a reduction in VEGF- and thrombin-induced sprout outgrowth in segments from MKP-1-null mice versus wild-type controls. MKP-1 siRNA also significantly reduced VEGF-induced EC migration using a transwell assay system. Overall, these results demonstrate distinct MAPK signaling pathways for thrombin versus VEGF induction of MKP-1 in ECs and point to the importance of MKP-1 induction in VEGF-stimulated EC migration.

angiogenesis; neuropilin; Src kinase; mitogen-activated protein kinase phosphatase-1

ENDOTHELIAL CELLS (ECs) are organized as a monolayer that lines the vasculature and serves as a regulatory interface between the bloodstream and the underlying tissue. Activation of ECs in response to perturbations from biological, chemical, and mechanical stimuli can alter endothelial function and change vascular homeostasis (13). EC gene transcription is modulated by growth factors, proteases, cytokines, viruses, and shear stress, causing multiple cellular responses, such as increased cellular permeability, cell migration, angiogenesis, proliferation, expression of adhesion molecules, and release of growth factors (9). The growth factor VEGF and the serine protease thrombin are pivotal players in the regulation of EC function. These two agonists trigger many signaling cascades, several of which are in common. The specific pathways required for precise gene induction remain to be elucidated.

VEGF and its signaling receptors are crucial for differentiation of EC progenitors, EC sprouting, and the formation of new blood vessels (i.e., angiogenesis) (3). VEGF plays a central role in the development and maturation of a healthy vascular network as well as vascular pathophysiological conditions. Two tyrosine kinase receptors, VEGF receptor-1 (VEGFR-1; Flt) and VEGFR-2 (Flk/KDR), have been identified to bind VEGF (11). The multiple signaling pathways activated upon VEGF stimulation include phosphoinositide 3-kinase (PI3K), Akt, PLC-{gamma}, Src kinase, ERK, p38, JNK, and focal adhesion kinase (FAK) (11).

Thrombin is a serine protease that is a key regulator in both the intrinsic and extrinsic pathways of blood coagulation. Thrombin also acts as a signaling activator in ECs by cleaving the NH2-terminal domain of the seven-transmembrane domain, G protein-coupled receptors, protease-activated receptors (PARs) (8). The newly cleaved NH2-terminus remains tethered and activates the receptor, triggering numerous intracellular modifications of signaling proteins, culminating in the regulation of gene expression and cellular dynamics (40). The activation of ECs by thrombin leads to the control of multiple genes that are important in the regulation of normal and pathological vascular function. In a previous report, we (5) have shown that MKP-1 is induced in ECs stimulated by thrombin. Here, we show that VEGF also stimulates the induction of MAPK phosphatase-1 (MKP-1) but through a different pathway than thrombin.

MKP-1 is representative of a family of dual-specificity protein phosphatases that are known to dephosphorylate MAPK family members (10). MKP-1 is encoded by an immediate-early gene and is localized in the nucleus (37). MKP-1 has been shown to be stimulated under conditions of inflammation and stress, such as treatment with lipopolysaccharide, dexamethasone, angiotensin, oxidative stress, heat shock, UV light, and growth factors (18, 21, 22, 29, 36). MKP-1 dephosphorylates the tyrosine and threonine residues of MAPK, rendering the enzyme inactive (15). In this report, we show that there is a differential MAPK signaling cascade that leads to the induction of MKP-1 in VEGF-treated versus thrombin-treated ECs and, furthermore, that MKP-1 plays a critical role in EC migration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Recombinant VEGF subtype and isoform neutralizing antibodies (VEGF-A165, VEGF-B, VEGF-C, VEGF-D, VEGF-A121, VEGF-A162, and VEGFR-2) were all purchased from R&D Systems (Minneapolis, MN). VEGF-E was obtained from Research Diagnostics (Concord, MA). VEGFR-1 neutralizing antibody was purchased from Abcam (Cambridge, MA). Thrombin receptor-activating peptide (TRAP) with the peptide sequence SFLLRN-NH2 was custom synthesized by Peptide (Louisville, KY). The inhibitors for the following signaling pathways were purchased from Calbiochem (San Diego, CA): Ro-31-8220 (PKC inhibitor/MKP-1 induction inhibitor), LY-294002 (PI3K inhibitor), PD-98059 (ERK inhibitor), SP-600125 (JNK inhibitor), and SB-203580 (p38 inhibitor). PP1 (Src kinase inhibitor) and GF-109203X (PKC inhibitor) were purchased from BioMol (Plymouth Meeting, PA). JNK-1, MKP-1, neuropilin (NRP)1, and NRP2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Radiolabeled [32P]dCTP was obtained from NEN (Wellesley, MA). Matrigel basement membrane matrix and dispase were purchase from BD Biosciences (Bedford, MA). All other chemicals and reagents were purchased from Sigma (St. Louis, MO). Plasmid constructs containing dominant negative (DN) Src (mutation A430V) and DN Fyn (mutation K to D) were kindly provided by Dr. Andrew Larner (Cleveland Clinic, Lerner Research Institute). Plasmids containing cDNA for wild-type (WT) JNK and DN JNK (K55M) were obtained from Biomyx Technology (San Diego, CA).

Cell culture, transfection, and treatment. Human ECs were isolated by trypsin digestion of umbilical veins as previously described (33). ECs were maintained in MCDB 105 medium (Sigma) containing 15% FBS, 90 µg/ml heparin, and 150 µg/ml EC growth supplement (ECGS). Cells were used between passages 2 and 5. Cells were transfected with WT JNK, mutant JNK (K55M), DN Src, DN Fyn, and green fluorescent protein vectors using Targefect F-2 plus the peptide enhancer according to the manufacturer's protocol (Targeting Systems, Santee, CA). ECs were split into 35-mm dishes at 90% confluency the day before transfection. Experiments were performed 24–36 h posttransfection. Cells were preincubated with inhibitors 30 min prior to agonist treatment.

Mouse aortic EC isolation. Aortic ECs were isolated from mice by an explant technique as previously described (4). Mouse aortic EC specificity was verified by immunostaining and real-time PCR for vonWilebrand factor.

Construction and validation of MKP-1 siRNA. MKP-1 target-specific siRNA duplexes were designed using the general template sequence AA(N19)UU, (where N can represent any nucleotide) thus giving a 21-nt sense and antisense strand that was hybridized to form a duplex. siRNAs were constructed that targeted the following mRNA sequences: MKP-1 600, AAG AGA CGT TGA TCA AGG CAG and MKP-1 838, AAG CTG GAC GAG GCC TTT GAG. The siRNA was chemically synthesized as described by the manufacturer's instructions in the Ambion siRNA Silencer Kit (Ambion). We also designed the scrambled MKP-1 600 and scrambled MKP-1 838 as controls for each experiment. The siRNA transfection was performed using Targefect F-2 plus the peptide enhancer (Targeting systems) in DMEM for 4 h, after which complete media was added. The final concentration of siRNA in the culture medium was 50 nM. The human recombinant myc-tagged MKP-1 plasmid (kindly provided by Jack Dixon) was transfected into EC using Targefect F-2 according to the manufacturers instructions (Targeting Systems, Santee, CA). Subsequently, human MKP-1 siRNA was used to silence protein levels. MKP-1 protein was analyzed by western blot analysis using myc antibody (Millipore-Upstate). MKP-1 mRNA levels were detected by real-time PCR.

Northern blot analysis. Total RNA from untreated, TRAP-treated, or VEGF-treated ECs was isolated using the RNeasy Mini Kit (QIAGEN, Valencia, CA). Total RNA was separated by gel electrophoresis and transferred to a Nytran SuPerCharge membrane (Schleicher & Schuell, Keene, NH). The membrane was hybridized using a 222-bp fragment of human MKP-1 [3'-untranslated region (UTR)], labeled with [32P]dCTP following the manufacturer's protocol from Amersham Biosciences, and incubated overnight. Hybridized membranes were washed several times using RNA wash buffer (0.2% SDS and 1x SSC) and exposed to a phosphorimaging screen overnight. Data were collected and quantified using a phosphoimaging system with Image-quant software from Amersham Biosciences.

Real-time RT-PCR assay. Total RNA was isolated using the RNeasy Kit (QIAGEN) from two confluent 35-mm wells pooled together, followed by DNase treatment (Ambion). cDNA was prepared using the Superscript first-strand synthesis system for RT-PCR (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. Real-time RT-PCR was carried out on a Perkin-Elmer ABI PRISM 7700 system using SYBR green PCR core reagents (PE Applied Biosystems). MKP-1 primers used for real-time RT-PCR were 5'-CTCCATGCTCCTTGAGAGGAGAAATGC-3' and 5'-GGTAGGTATGTCAAGCATGAAGAG-3', and GAPDH primers used were 5'-TCAACAGCGACACCCACTCC-3' and 5'-TGAGGTCCACCACCCTGTTG-3'.

EC migration. In vitro migration of ECs was studied using a transmigration assay. Human ECs were seeded at 104 cells/membrane on the upper chamber of an 8.0-µm-pore diameter membrane chamber (Becton Dickinson Labware, Franklin Lake, NJ). VEGF (20 ng/ml) was added to the bottom well of the appropriate chambers overnight. Cells were removed from the upper chamber of each well, and membranes were fixed with ice-cold methanol and then stained with hematoxylin (Sigma). Membranes were mounted on cover slides, and cells were counted.

Animal and genotyping analysis. All animals received humane care in accordance with National Institute of Health guidelines. The Institutional Animal Care and Use Committee of the Cleveland Clinic, Lerner Research Institute, approved the experimental protocols. Cryopreserved embryos of Mkp-1 knockout mice were provided by Bristol Myers Squibb Pharmaceutical Institute and regenerated into mice at the Jackson Laboratory as previously described (46). These mice were breed at the Columbus Children's Research Institute to generate Mkp-1+/+ and Mkp-1–/– mice and kindly provided by Dr. Yusen Liu (46). Mice were maintained on a normal rodent chow diet. For genotyping analysis, tissue samples were processed using the DNeasy Tissue Kit (Qiagen). Genotyping was confirmed using PCR analysis with the following primers: forwardprimer 1, 5'-CCAGGTACTGTGTCGGTGGTGC-3'; forward primer 2, 5'-TGCCTGCTCTTTACTGAAGGCTC-3', and reverse primer 1, 5'-CCTGGCACAATCCTCCTAGAC-3' (44).

Aortic segment angiogenesis assay. The descending thoracic aorta was isolated from WT and Mkp-1–/– mice (n = 7 mice/group). Multiple 1-mm-thick aortic segments were prepared under a dissecting microscope. Rings were then placed on a thin layer of Matrigel (BD Bioscience). We measured the area of EC sprouts from the boarder of the aortic tissue to the outer edge of the sprouting area using an automated program (Image-Pro Plus version 6.0).

Statistical analysis. Ex vivo aortic segment EC sprouting was analyzed using two-way ANOVA. Student's t-test was performed for all other experiments. All tests were performed using GraphPad Prism 4.0c (GraphPad Software). P values of <0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
VEGF induces MKP-1 expression in ECs. VEGF and thrombin share multiple downstream signaling pathways and activate some common genes in ECs. We tested whether VEGF induces the signaling mediator MKP-1 in human umbilical vein ECs in a similar or distinct way to thrombin. MKP-1 mRNA was rapidly induced in ECs stimulated with VEGF-A165 (Fig. 1A). The time course of VEGF-induced MKP-1 message expression was analyzed by Northern blot. MKP-1 mRNA increased by 30 min, peaked at 1 h (~10-fold), and then returned to basal levels by 2 h. VEGF-A165 stimulated MKP-1 mRNA half maximally at 1 ng/ml with maximal induction at 5 ng/ml (Fig. 1B). These results indicate that VEGF activates MKP-1 mRNA expression in ECs.


Figure 1
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  		Fig. 1. VEGF-induced MAPK phosphatase (MKP)-1 mRNA in human endothelial cells (ECs). A: confluent monolayers of ECs were serum starved for 2 h prior to treatment with VEGF (10 ng/ml) at 37°C over the indicated time course. RNA was isolated from cell lysates and subjected to Northern blot analysis, and the blot was reprobed for GAPDH as a loading control. B: concentration-dependent expression of MKP-1 mRNA in ECs treated with VEGF-A165 for 1 h. Northern blot analysis was performed to monitor MKP-1 mRNA steady-state levels in VEGF-treated ECs at specified dosages.

 
VEGFR-2 and NRPs are critical for MKP-1 induction in ECs. We wanted to determine which VEGF family members could stimulate MKP-1 induction in human umbilical vein ECs. Five VEGF family members were compared: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-E. VEGF-A and VEGF-E stimulated the expression of MKP-1 in ECs (~10-fold), whereas the other VEGF family members were not effective (Fig. 2A). All VEGF subtypes induced the activation of ERK activity in ECs, demonstrating that all VEGF's were active (data not shown).


Figure 2
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  		Fig. 2. VEGF induction of MKP-1 is mediated via VEGF receptor (VEGFR)-2. A: MKP-1 mRNA was detected using Northern blot analysis in cells treated with the following VEGF subtypes: VEGF-A, VEGF-B, VEGF-C, VEGF-D, or VEGF-E (10 ng/ml). NT, no treatment. B: neutralizing antibodies were used to block either VEGFR-1 or VEGFR-2 for 3 h followed by VEGF-A165 treatment for 1 h. ECs were lysed, RNA was isolated, and MKP-1 mRNA was detected by Northern blot analysis. C: ECs were preincubated for 3 h with neutralizing antibodies to VEGFR-1 or VEGFR-2 and then stimulated for 1 h with VEGF-E. Subsequently, MKP-1 mRNA was detected using Northern blot analysis. GAPDH was used as a control for loading.

 
The orf virus VEGF-E has been reported to activate VEGFR-2 and not VEGFR-1 or VEGFR-3 (25). This suggested that VEGF induction of MKP-1 was mediated through VEGFR-2, since both VEGF-A and VEGF-E activate this receptor. To resolve which VEGF receptor was responsible for MKP-1 induction, we pretreated ECs 30 min prior to VEGF stimulation with neutralizing antibodies to VEGFR-1 or VEGFR-2. VEGFR-2 was critical for VEGF-A-induced MKP-1 expression in ECs (Fig. 2B). Consistent data also showed that VEGFR-2 is needed for MKP-1 induction by VEGF-E in ECs (Fig. 2C).

Most VEGF isoforms are identified by heparin-binding domains that are encoded by the splicing of exons 6 and 7 of the VEGF gene. VEGF-A165 contains exon 7, has heparin-binding properties, and binds to NRP receptors. Exons 1–5, 6A, 6B, and 8, but not 7, of the VEGF gene encode VEGF-A162 mRNA. VEGF-A121 lacks both exons 6 and 7, does not bind heparin or NRPs, and is secreted freely. To further address the role of VEGF-A signaling in the induction of MKP-1, we tested the following VEGF-A isoforms: VEGF-A165, VEGF-A162, and VEGF-A121. VEGF-A165 robustly stimulated MKP-1 induction, VEGF-A162 was intermediate in activity, and VEGF-A121 showed no induction of MKP-1 mRNA expression (Fig. 3A). VEGF-A121 binds to both VEGFR-1 and VEGFR-2. Next, we tested if VEGF-A121 could block MKP-1 induction by VEGF-A165. We pretreated ECs with VEGF-A121 (10 ng/ml), subsequently stimulated the cells with VEGF-A165, and then measured MKP-1 induction. VEGF-A121 completely blocked the induction of MKP-1 by VEGF-A165 (Fig. 3B). Interestingly, VEGF-A165-induced MKP-1 mRNA levels were dramatically reduced in ECs pretreated with VEGF-B (10 ng/ml), which only binds to VEGFR-1 and NRP1 (Fig. 3C). These experiments led us to investigate a potential role for NRPs in VEGF-induced MKP-1 expression, since NRPs are important coreceptors for VEGFR-2 signaling. We pretreated ECs with neutralizing antibodies against NRP1 and NRP2 and then stimulated the cells with VEGF-A165. The induction of MKP-1 was blocked using both NRP1 and NRP2 neutralizing antibodies (Fig. 3D). NRP-1 and NRP-2 both bind to VEGF-A165 and serve as coreceptors for VEGFR-2. Thus, our results show that NRPs are required for VEGFR-2 induction of MKP-1 in ECs.


Figure 3
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  		Fig. 3. Neuropilin (NRP)1 and NRP2 are required for VEGF-induced MKP-1 expression. A: MKP-1 mRNA was detected in ECs incubated for 1 h with the following VEGF-A isoforms: VEGF-A121, VEGF-A162, and VEGF-A165 (10 ng/ml). B and C: ECs were preincubated for 30 min with either VEGF-A121 (B) or VEGF-B (C) and then stimulated with VEGF-A165 for 1 h to monitor the expression of MKP-1 mRNA by Northern blot analysis. D: neutralizing antibodies were used to block either NRP1 or NRP2 for 3 h of preincubation, and ECs were stimulated with VEGF-A165. Subsequently, MKP-1 mRNA was detected using Northern blot analysis. All blots were reprobed for GAPDH as a loading control.

 
TRAP and VEGF induction of MKP-1 require distinct Src kinase family members. Our previous findings showed an induction of MKP-1 through PAR-1 using a six-mer peptide (referred to as TRAP) homologous to the new NH2-terminus of PAR-1 that mimics the action of thrombin (5). VEGF stimulation of MKP-1 was greater (~10 fold) than TRAP induction (~4 fold) (Fig. 4C). VEGF and TRAP induction of MKP-1 occur through different ligand receptor-mediated pathways, and we wanted to investigate the mechanism of signal transduction in both of these systems. Many reports have shown that VEGF-A stimulation of VEGFR-2 triggers the activation of Src homology 2 domain-containing protein, growth factor receptor-bound protein 2, c-Src, Nck, and two tyrosine phosphatases, SHP-1 and SHP-2 (11). We first examined the cell signaling pathways critical for VEGF signaling using a pharmacological approach. VEGF- and thrombin-induced MKP-1 mRNA expression were completely blocked in human umbilical vein ECs pretreated for 30 min with the Src kinase inhibitor PP1 (10 µM) (Fig. 4, A and B). The PKC inhibitors Ro-31-8220 (10 µM) and GF-109203X (10 µM) were also tested (2). Ro-31-8220 completely inhibited MKP-1 mRNA induction by VEGF or thrombin; however, GF-109203X had no effect (Fig. 4, A and B). GF-109203X blocked VEGF- and TRAP-induced phosphorylation of cAMP response element-binding protein in the PKC signaling pathway (data not shown). Using the inhibitor LY-294002, we demonstrated that the PI3K pathway had no effect on MKP-1 message levels when challenged with VEGF or thrombin (Fig. 4, A and B). This inhibitor did significantly reduce VEGF- and TRAP-stimulated Akt phosphorylation (data not shown).


Figure 4
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  		Fig. 4. VEGF induction of MKP-1 is Src kinase dependent, whereas thrombin induction of MKP-1 is Fyn kinase dependent. A and B: VEGF- and thrombin receptor-activating peptide (TRAP)-induced MKP-1 expression in ECs were Src kinase dependent but not PKC dependent, as determined by incubating ECs with PP1 (a general Src kinase inhibitor) and GF-109203 (GF; a PKC inhibitor) at 10 µM each for 30 min prior to stimulation with either VEGF (10 ng/ml) or TRAP (100 µM). The phosphoinositide 3-kinase inhibitor LY-294002 (LY) had no effect on the induction of MKP-1. Ro-31-8220 (Ro; 10 µM, an inhibitor of MKP-1 induction) was added 30 min before treatment with VEGF or TRAP (1 h). Ro was used as a positive control for MKP-1 inhibition. C: dominant negative (DN) constructs for Src kinase, Fyn kinase, and green fluorescent protein (GFP; negative control) in pcDNA3 were transfected into ECs using Targefect F-2 (Targeting Systems), and cells were allowed to recover for 24 h. Transfected cells were selected using G418 for 7 days. ECs were then stimulated with either VEGF (10 ng/ml) or TRAP (100 µM) for 1 h. Total RNA was isolated, and MKP-1 mRNA levels were determined by real-time RT-PCR with specific MKP-1 primers and normalized to GAPDH mRNA. The fold induction was quantified relative to the untreated control. Data represent means ± SD of 3 experiments, where each measurement was repeated in duplicate.

 
To address the role of Src kinase family members in the induction of TRAP- and VEGF-induced MKP-1 mRNA expression more specifically, we overexpressed mutant Src family members that act as DN inhibitors of Src and Fyn. DN Src, in which Ala430 was mutated to Val430, blocked VEGF induction of MKP-1 with no alteration in TRAP induction. Conversely, TRAP induction of MKP-1 was blocked by DN Fyn kinase, with no effect on VEGF-induced MKP-1 expression (Fig. 4C).

VEGF induction of MKP-1 is JNK dependent, whereas TRAP induction of MKP-1 is ERK dependent. The JNK inhibitor SP-600125 (10 µM), which blocked stimulated c-Jun phosphorylation in our ECs (not shown), significantly blocked VEGF-induced MKP-1 expression; however, TRAP-induced MKP-1 expression was not affected (Fig. 5, A and B). Conversely, the ERK inhibitor PD-98059 suppressed TRAP-induced MKP-1 expression, but VEGF-induced MKP-1 expression was not altered (Fig. 5, A and B). The p38 inhibitor SB-203580 (10 µM) did not block VEGF- or TRAP-induced MKP-1 expression. Although its effectiveness was confirmed by demonstrating inhibition of VEGF- and TRAP-induced phosphorylation of activating transcription factor-2 (data not shown). Furthermore, DN JNK (K55M) blocked VEGF induction of MKP-1, whereas TRAP induction of MKP-1 was not altered (Fig. 5C). Collectively, our results indicate that the JNK signaling pathway is critical for VEGF-induced MKP-1 expression, whereas TRAP-induced MKP-1 expression requires ERK signaling.


Figure 5
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  		Fig. 5. VEGF induction of MKP-1 is JNK dependent, although TRAP induction of MKP-1 is ERK1/2 dependent. A and B: ECs were preincubated with PD-98059 (PD; 10 µM, an ERK inhibitor), SB-203580 (SB; 10 µM, a p38 inhibitor), or SP-600125 (SP; 10 µM, a JNK inhibitor) for 30 min and subsequently stimulated with VEGF (10 ng/ml) or TRAP (100 µM) for 1 h. C: ECs were transfected with either wild-type (WT) JNK or mutant JNK (K55M) using Targefect F-2 for 24 h. ECs were then stimulated with VEGF or TRAP for 1 h. Total RNA was isolated for all samples, and MKP-1 mRNA was determined by real-time RT-PCR using specific MKP-1 primers and normalized to GAPDH mRNA. The fold induction was quantified relative to the control. Data represent means ± SD of 3 experiments, where each measurement was repeated in duplicate.

 
Effect of VEGF-induced MKP-1 on MAPK activity in ECs. To investigate the role that MKP-1 plays in MAPK activation of VEGF-treated ECs, we used specific siRNA to suppress MKP-1 levels and studied the MAPK phosphorylation profile. We generated specific siRNA for MKP-1 that corresponds to the 21-nt sequence from the start codon at position 600 and 838 within the open reading frame of the MKP-1 mRNA sequence. The VEGF-induced MKP-1 message was completely silenced in ECs 24 h after MKP-1 siRNA transfection (Fig. 6A). However, the scrambled MKP-1 siRNA had no effect. The protein levels of a construct expressing WT MKP-1 were decreased in ECs treated with MKP-1 siRNA (Fig. 6B). VEGF-induced MKP-1 was critical for the dephosphorylation of JNK and, to a lesser extent, ERK and p38 dephosphorylation in ECs, as detected by Western blot analysis (Fig. 6C). We have previously shown that thrombin-induced MKP-1 efficiently dephosphorylates ERK (5).


Figure 6
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  		Fig. 6. VEGF-induced MKP-1 regulates JNK and p38; however, thrombin-induced MKP-1 regulates ERK. A: ECs were transfected with MKP-1 short interfering (si)RNA (siR) and MKP-1 scrambled (scr) siRNA and then treated with VEGF. ECs were lysed, and mRNA was isolated and detected using real-time PCR analysis. B: ECs were transfected with recombinant myc-tagged human MKP-1 under the cytomegalovirus promoter followed by transfection with MKP-1 siRNA or scrambled MKP-1 siRNA. After 24 h, cell lysates were collected and subjected to SDS-PAGE. Western blots were performed using a myc antibody with GAPDH antibody as a loading control. C: ECs were transfected with scrambled MKP-1 siRNA or MKP-1-targeted siRNA. Next, ECs were stimulated with either VEGF or TRAP over a 3-h time course. Protein lysates were prepared and subjected to SDS-PAGE and subsequent immunoblotting (IB) techniques using specific phosphorylated ERK (p-ERK), JNK (p-JNK), and p38 (p-p38) antibodies. Total ERK, JNK 1, and p38 were used as loading controls. Quantification was performed for 3 separate immunoblot experiments. Quantity One (Bio-Rad) was used to analyze band intensity.

 
MKP-1 plays a critical role in VEGF- and thrombin-induced angiogenesis ex vivo. First, we wanted to detect the VEGF and thrombin induction of MKP-1 protein in mouse ECs. VEGF and thrombin induced MKP-1 protein levels in mouse aortic ECs (Fig. 7A). We measured sprouting density from cultured aortic segments of WT control or Mkp-1–/– mice treated with VEGF-A (40 ng/ml) or thrombin (6 U/ml). EC sprouts migrated from the aortic segments after 3 days in culture. VEGF and thrombin, to a lesser extent, caused an increase in WT mouse aortic EC sprouting density (Fig. 7B). The sprouting outgrowth in Mkp-1–/– mice treated with VEGF and thrombin was decreased compared with WT control mice (Fig. 7C).


Figure 7
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  		Fig. 7. VEGF- and thrombin-induced MKP-1 expression is critical for ex vivo EC sprouting. A: Western blot analysis was performed to test the induction of MKP-1 protein levels in mouse aortic ECs. B: segments of the descending aorta were isolated from Mkp-1 knockout and WT control mice. Aortic segments were cultured on Matrigel and incubated with VEGF or thrombin for 3 days. C: areas of sprouting were quantified using Image-Pro Plus version 6 analysis software. Data are expressed as mean EC sprout densities ± SE. Two-way ANOVA demonstrated a significant effect on genotype after VEFG or thrombin stimulation (*P < 0.001; n = 7 in each group).

 
Role for MKP-1 in VEGF-induced EC migration. We used the transwell system to study the role of MKP-1 in human EC migration. Scrambled (control) MKP-1 siRNA-treated ECs significantly migrated toward the chemoattractant VEGF. However, EC migration was impaired in VEGF-stimulated MKP-1-null cells (Fig. 8). Thrombin had no effect on the induction of EC migration in our system. These data show that MKP-1 is important in VEGF-induced EC migration.


Figure 8
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  		Fig. 8. MKP-1 is important in EC migration toward VEGF. Human ECs were plated in the upper chamber of a Boyden chamber transwell system. VEGF (10 ng/ml) was added to the lower chamber, and human ECs were allowed to migrate overnight (15 h). Cells were fixed using methanol, and insert membranes were stained and mounted on coverslips. All migrated cells were counted in the membrane field by a blind observer at x100 magnification. Data shown represent means ± SD of 3 independent experiments. *P < 0.005.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Thrombin and VEGF are EC activators, and both are implicated in such pathophysiological processes as embryogenesis, angiogenesis, wound healing, and oncogenesis. Although thrombin is a serine protease that activates a G protein-coupled receptor and VEGF is a growth factor that activates a tyrosine kinase receptor, intracellular signaling pathways induced by these two agonists in ECs have been reported to be similar. For example, Minami et al. (26) showed that thrombin and VEGF stimulate similar signaling pathways to induce the gene and protein expression of Down syndrome critical region-1 (DSCR-1), a negative regulator of the calceneurin-nuclear factor of activated T cells signaling pathway. In our study, MKP-1 was identified as a novel VEGF-inducible gene in human ECs. We have shown that VEGF-A and VEGF-E increase MKP-1 mRNA in ECs via VEGFR-2 activation. Furthermore, we determined that NRPs are critical for VEGF-A- and VEGF-E-induced MKP-1 expression. Previously, we reported that thrombin induced MKP-1 in ECs via the ERK pathway (5). In this study, using pharmacological and DN approaches, we elucidated the differential signaling pathways responsible for MKP-1 induction by VEGF and thrombin in ECs.

MKP-1 is a growth factor and stress-inducible immediate-early gene. In agreement with the characteristics of immediate-early genes, the agonist-induced MKP-1 mRNA and protein levels have short half-lives (30 min and 1 h, respectively) (37). The robustness of MKP-1 induction varies with the specific agonists and cell types studied (10). For example, oxidative stress of cultured fibroblasts resulted in an ~20-fold increase in MKP-1 induction (15, 20), whereas thrombin, PDGF-B, TNF-{alpha}, and mechanical stress induced MKP-1 levels in ECs and smooth muscle cells 2- to 6-fold (5, 17, 29, 41). We now report that VEGF treatment of ECs resulted in a robust increase in MKP-1 mRNA steady-state levels (~10 fold). Elevated MKP-1 is critical for the sustained dephosphorylation of MAPK. Because MKP-1 is a key regulator of MAPK activity (44), we wanted to identify the regulatory mechanisms of MKP-1 induction in ECs.

VEGFRs play a critical role in the growth and maintenance of vascular ECs (3). NRPs are important coreceptors for VEGFR-2 and are critical in the progression of angiogenesis (35). Our results indicated both NRP1 and NRP2 are necessary coreceptors in VEGF-A165 induction of MKP-1. Moreover, only VEGFR-2- and NRP-specific ligands, VEGF-A165 and VEGF-E, were capable of mediating MKP-1 induction in ECs (Fig. 2A). Therefore, NRP1 and NRP2 may enhance the intracellular signaling of MKP-1 induction by augmenting VEGF-A165 binding to VEGFR-2. The short cytoplasmic domains of NRP1 and NRP2 have been shown to have no independent signaling function in ECs (11). Taken together, our results show that VEGFR-2 association with NPRs is the principal pathway for the mediation of VEGF-induced MKP-1 expression and thus may be a critical pathway for the regulation of angiogenesis.

Receptor-specific signaling pathways activate upstream Src kinase family members that are critical in MKP-1 induction. Our previous results demonstrated the importance of Src kinase in thrombin-induced MKP-1 expression in ECs using pharmacological inhibitors (5). In this report, we identified Fyn kinase as the Src family member that induces MKP-1 expression in thrombin-stimulated ECs (5). Others have shown that Fyn kinase is necessary for MKP-1 induction during melanocyte differentiation and proliferation (43). We further show VEGF induction of MKP-1 is also dependent on a Src kinase family member and not the PKC or PI3K signaling pathways. However, VEGF activation of MKP-1 is dependent on Src kinase and not Fyn kinase. Our results are consistent with the findings of others (7) showing that VEGFR-2 binds to Src kinase but does not associate with Fyn kinase. In previous studies, it was reported that VEGFR-2 is associated with Src kinase, whereas VEGFR-1 binds Fyn kinase. PAR-1 has not been reported to bind directly to either Src or Fyn kinases. Therefore, we propose that β-arrestin-1 coupling with PAR-1 and Fyn kinase may be critical for thrombin-induced MKP-1 expression (27, 38).

ERK, JNK, and p38 signaling pathways have all been shown to modulate MKP-1 induction depending on the cell type and agonist (5, 6, 18). VEGF and thrombin stimulate ERK, JNK, and p38 cascades in ECs, and most reports have shown a similarity in the convergence of the signal and immediate-early gene activation. For example, ERK1/2 but not p38, PI3K, or PKC pathways mediated early growth response factor (Egr)-1 upregulation by thrombin-induced PAR-1 activation in ECs (45). VEGF has been shown to induce Egr-1 in a MEK/ERK- and PKC-dependent, but p38-independent, manner (24). Others have also reported that VEGF and thrombin induction of another immediate-early gene, c-jun, in bovine aortic ECs is also dependent on ERK1/2 activation (28). Here, we demonstrate distinct MAPK pathways for VEGF- and thrombin-induced MKP-1 expression in ECs. The ERK inhibitor demonstrated a complete blockage of thrombin-induced MKP-1 expression but had no consequence on VEGF-induced MKP-1 expression in ECs. Conversely, a JNK inhibitor, as well as expression of a DN construct, suppressed VEGF-induced MKP-1 expression and did not affect thrombin-mediated MKP-1 expression. The p38 inhibitor had no effect on VEGF- or thrombin-induced MKP-1 mRNA levels, indicating that agonist-specific MKP-1 induction is important in the regulation of the MAPK pathway.

A family of upstream MAPK kinases regulates MAPK signaling pathways. In a similar manner, it is plausible to propose that the MKP family has a selective preference when inactivating specific MAPKs. MKP-1 can dephosphorylate all MAPK family members in vitro, but evidence suggests a preference in the dephosphorylation of p38 and JNK over ERK (41). MAPK dephosphorylation by MKP-1 appears to be cell type and agonist specific. In this report, we showed that VEGF-induced MKP-1 is critical for JNK, ERK, and p38 dephosphorylation. This is consistent with reports showing that IL-1β-induced MKP-1 dephosphorylated p38 and JNK in ECs (41). Others have demonstrated in ECs that atrial natriuretic peptide attenuates TNF-{alpha}-induced p38 activation via MKP-1 induction (16, 42). T-kininogen purified from rat serum has been shown to inhibit basal levels and bradykinin- or T-kinin-induced levels of ERK in a MKP-1-dependent manner in ECs (19, 39). Furthermore, thrombin-induced MKP-1 expression has been shown to play a key role in the inactivation of ERK in ECs. Cytochrome P-450-derived epoxyeicosatrienoic acids induced MKP-1 in ECs, thus regulating JNK activation, but p38 and ERK activity remained unaltered (30). Still, the molecular mechanism of agonist-specific MAPK dephosphorylation by MKPs in ECs remains unclear.

Many reports have been published that demonstrate the proangiogenic effect of VEGF and thrombin on ECs (1, 3). Similarly, in this report, we show that both thrombin and VEGF stimulate EC sprouting from aortic segments of WT mice and that this effect is significantly decreased in mice lacking the Mkp-1 gene. Others have reported that MKP-1 has an opposing effect on the regulation of Toll-like receptor-induced inflammation in vivo (32, 46). Mkp-1–/– mice challenged with lipopolysaccharides had decreased survival rates compared with WT controls (32, 46). Our results indicate that VEGF-induced EC migration is dependent on MKP-1 induction. Previous reports have shown that p38 plays an important role in VEGF-induced EC migration (23, 31). MKP-1 most likely acts as a signaling "switch" that attenuates the phosphorylation of p38, JNK, and ERK, thus allowing for the propagation of genes necessary for EC migration. Therefore, we propose that MKP-1 functions as a molecular stress sensor that maintains the balance of MAPK activity and subsequently inflammation in ECs.

Thrombin and VEGF signaling in ECs has been linked to tumor angiogenesis. Recent studies have shown that hypoxic conditions in tumors and the surrounding microenvironment contribute to the increased expression of proangiogeneic factors such as thrombin and VEGF (3, 14). In addition, hypoxic conditions have been shown to induce MKP-1 in different cell types in vitro (1, 12, 34). We propose that pathological hypoxic conditions, such as ischemia and oncogenesis, increase MKP-1, thereby promoting angiogenesis. We have identified that thrombin and VEGF induce MKP-1 in ECs via two distinct signaling pathways. We propose that in thrombin- and VEGF-induced angiogenesis MKP-1 is a critical signaling intermediate for neovascularization and angiogenesis.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health (NIH) Grant HL-29582 (to P. E. DiCorleto). C. M. Kinney was supported in part by NIH Grant T32-HL-07653 and the Bill and Melinda Gates Foundation (Gates Millennium Scholarship).


   ACKNOWLEDGMENTS
 
We thank Sara Bundy for cell culture assistance and Dr. Jianzhong Shen for help with genotyping. We also thank Michael McDermott and Dr. Amit Vasanji for technical help, data analysis, and experimental expertise. We thank Bristol-Myers Squibb Pharmaceutical Research Institute and Dr. Yusen Liu (The Ohio State University) for providing Mkp-1 knockout mice. We thank Dr. Jack Dixon (University of California, San Diego, CA) for kindly providing the human recombinant myc-tagged MKP-1 plasmid. Human umbilical vein ECs were harvested through the Birthing Services Department at the Cleveland Clinic Foundation and the Perinatal Clinical Research Center (National Institutes of Health Research Center Award RR-00080) at the Cleveland Metrohealth Hospital.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. E. DiCorleto, Dept. of Cell Biology, Lerner Research Institute and Cleveland Clinic Lerner College of Medicine, Case Western Reserve Univ., 9500 Euclid Ave., Cleveland, OH 44195 (e-mail: dicorlp{at}ccf.org)

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


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Bassus S, Herkert O, Kronemann N, Gorlach A, Bremerich D, Kirchmaier CM, Busse R, Schini-Kerth VB. Thrombin causes vascular endothelial growth factor expression in vascular smooth muscle cells: role of reactive oxygen species. Arterioscler Thromb Vasc Biol 21: 1550–1555, 2001.[Abstract/Free Full Text]

2. Beltman J, McCormick F, Cook SJ. The selective protein kinase C inhibitor, Ro-31-8220, inhibits mitogen-activated protein kinase phosphatase-1 (MKP-1) expression, induces c-Jun expression, and activates Jun N-terminal kinase. J Biol Chem 271: 27018–27024, 1996.[Abstract/Free Full Text]

3. Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med 6: 389–395, 2000.[CrossRef][Web of Science][Medline]

4. Chandrasekharan UM, Siemionow M, Unsal M, Yang L, Poptic E, Bohn J, Ozer K, Zhou Z, Howe PH, Penn M, DiCorleto PE. Tumor necrosis factor alpha (TNF-alpha) receptor-II is required for TNF-alpha-induced leukocyte-endothelial interaction in vivo. Blood 109: 1938–1944, 2007.[Abstract/Free Full Text]

5. Chandrasekharan UM, Yang L, Walters A, Howe P, DiCorleto PE. Role of CL-100, a dual specificity phosphatase, in thrombin-induced endothelial cell activation. J Biol Chem 279: 46678–46685, 2004.[Abstract/Free Full Text]

6. Chen P, Li J, Barnes J, Kokkonen GC, Lee JC, Liu Y. Restraint of proinflammatory cytokine biosynthesis by mitogen-activated protein kinase phosphatase-1 in lipopolysaccharide-stimulated macrophages. J Immunol 169: 6408–6416, 2002.[Abstract/Free Full Text]

7. Chou MT, Wang J, Fujita DJ. Src kinase becomes preferentially associated with the VEGFR, KDR/Flk-1, following VEGF stimulation of vascular endothelial cells. BMC Biochem 3: 32, 2002.[CrossRef][Medline]

8. Coughlin SR. Thrombin signalling and protease-activated receptors. Nature 407: 258–264, 2000.[CrossRef][Medline]

9. DiCorleto PE, Gimbrone MA Jr. Atherosclerosis and coronary artery disease. In: Atherosclerosis and Coronary Artery Disease, edited by Fuster V, Ross R, Topol EJ. Philadelphia, PA: Lippincott-Raven, 1996, p. 387–399.

10. Farooq A, Zhou MM. Structure and regulation of MAPK phosphatases. Cell Signal 16: 769–779, 2004.[CrossRef][Web of Science][Medline]

11. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 18: 4–25, 1997.[Abstract/Free Full Text]

12. Gorlach A, Diebold I, Schini-Kerth VB, Berchner-Pfannschmidt U, Roth U, Brandes RP, Kietzmann T, Busse R. Thrombin activates the hypoxia-inducible factor-1 signaling pathway in vascular smooth muscle cells: role of the p22phox-containing NADPH oxidase. Circ Res 89: 47–54, 2001.[Abstract/Free Full Text]

13. Gross PL, Aird WC. The endothelium and thrombosis. Semin Thromb Hemost 26: 463–478, 2000.[CrossRef][Web of Science][Medline]

14. Karpatkin S. Role of thrombin in tumor angiogenesis, implantation, and metastasis. Pathophysiol Haemost Thromb 33, Suppl 1: 54–55, 2003.[CrossRef]

15. Keyse SM. Protein phosphatases and the regulation of mitogen-activated protein kinase signalling. Curr Opin Cell Biol 12: 186–192, 2000.[CrossRef][Web of Science][Medline]

16. Kiemer AK, Weber NC, Furst R, Bildner N, Kulhanek-Heinze S, Vollmar AM. Inhibition of p38 MAPK activation via induction of MKP-1: atrial natriuretic peptide reduces TNF-alpha-induced actin polymerization and endothelial permeability. Circ Res 90: 874–881, 2002.[Abstract/Free Full Text]

17. Kim F, Corson MA. Adhesion to fibronectin enhances MKP-1 activation in human endothelial cells. Biochem Biophys Res Commun 273: 539–545, 2000.[CrossRef][Web of Science][Medline]

18. Lasa M, Abraham SM, Boucheron C, Saklatvala J, Clark AR. Dexamethasone causes sustained expression of mitogen-activated protein kinase (MAPK) phosphatase 1 and phosphatase-mediated inhibition of MAPK p38. Mol Cell Biol 22: 7802–7811, 2002.[Abstract/Free Full Text]

19. Leiva-Salcedo E, Perez V, Acuna-Castillo C, Walter R, Sierra F. T-kininogen inhibits kinin-mediated activation of ERK in endothelial cells. Biol Res 35: 287–294, 2002.[Web of Science][Medline]

20. Li J, Gorospe M, Hutter D, Barnes J, Keyse SM, Liu Y. Transcriptional induction of MKP-1 in response to stress is associated with histone H3 phosphorylation-acetylation. Mol Cell Biol 21: 8213–8224, 2001.[Abstract/Free Full Text]

21. Liu PQ, Lu W, Wang TH, Pan JY. [MKP-1 regulates the cardiomyocyte hypertrophic responses induced by angiotensin II]. Sheng Li Xue Bao 52: 365–370, 2000.[Medline]

22. Liu Y, Gorospe M, Yang C, Holbrook NJ. Role of mitogen-activated protein kinase phosphatase during the cellular response to genotoxic stress. Inhibition of c-Jun N-terminal kinase activity and AP-1-dependent gene activation. J Biol Chem 270: 8377–8380, 1995.[Abstract/Free Full Text]

23. McMullen ME, Bryant PW, Glembotski CC, Vincent PA, Pumiglia KM. Activation of p38 has opposing effects on the proliferation and migration of endothelial cells. J Biol Chem 280: 20995–21003, 2005.[Abstract/Free Full Text]

24. Mechtcheriakova D, Schabbauer G, Lucerna M, Clauss M, De Martin R, Binder BR, Hofer E. Specificity, diversity, and convergence in VEGF and TNF-{alpha} signaling events leading to tissue factor upregulation via EGR-1 in endothelial cells. FASEB J 15: 230–242, 2001.[Abstract/Free Full Text]

25. Meyer M, Clauss M, Lepple-Wienhues A, Waltenberger J, Augustin HG, Ziche M, Lanz C, Buttner M, Rziha HJ, Dehio C. A novel vascular endothelial growth factor encoded by Orf virus, VEGF-E, mediates angiogenesis via signalling through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor tyrosine kinases. EMBO J 18: 363–374, 1999.[CrossRef][Web of Science][Medline]

26. Minami T, Horiuchi K, Miura M, Abid MR, Takabe W, Noguchi N, Kohro T, Ge X, Aburatani H, Hamakubo T, Kodama T, Aird WC. Vascular endothelial growth factor- and thrombin-induced termination factor, Down syndrome critical region-1, attenuates endothelial cell proliferation and angiogenesis. J Biol Chem 279: 50537–50554, 2004.[Abstract/Free Full Text]

27. Paing MM, Stutts AB, Kohout TA, Lefkowitz RJ, Trejo J. β-Arrestins regulate protease-activated receptor-1 desensitization but not internalization or down-regulation. J Biol Chem 277: 1292–1300, 2002.[Abstract/Free Full Text]

28. Pedram A, Razandi M, Levin ER. Extracellular signal-regulated protein kinase/Jun kinase cross-talk underlies vascular endothelial cell growth factor-induced endothelial cell proliferation. J Biol Chem 273: 26722–26728, 1998.[Abstract/Free Full Text]

29. Plevin R, Malarkey K, Aidulis D, McLees A, Gould GW. Cyclic AMP inhibitors inhibits PDGF-stimulated mitogen-activated protein kinase activity in rat aortic smooth muscle cells via inactivation of c-Raf-1 kinase and induction of MAP kinase phosphatase-1. Cell Signal 9: 323–328, 1997.[CrossRef][Web of Science][Medline]

30. Potente M, Michaelis UR, Fisslthaler B, Busse R, Fleming I. Cytochrome P450 2C9-induced endothelial cell proliferation involves induction of mitogen-activated protein (MAP) kinase phosphatase-1, inhibition of the c-Jun N-terminal kinase, and up-regulation of cyclin D1. J Biol Chem 277: 15671–15676, 2002.[Abstract/Free Full Text]

31. Rousseau S, Houle F, Landry J, Huot J. p38 MAP kinase activation by vascular endothelial growth factor mediates actin reorganization and cell migration in human endothelial cells. Oncogene 15: 2169–2177, 1997.[CrossRef][Web of Science][Medline]

32. Salojin KV, Owusu IB, Millerchip KA, Potter M, Platt KA, Oravecz T. Essential role of MAPK phosphatase-1 in the negative control of innate immune responses. J Immunol 176: 1899–1907, 2006.[Abstract/Free Full Text]

33. Shankar R, de la Motte CA, Poptic EJ, DiCorleto PE. Thrombin receptor-activating peptides differentially stimulate platelet-derived growth factor production, monocytic cell adhesion, and E-selectin expression in human umbilical vein endothelial cells. J Biol Chem 269: 13936–13941, 1994.[Abstract/Free Full Text]

34. Short MD, Fox SM, Lam CF, Stenmark KR, Das M. Protein kinase Czeta attenuates hypoxia-induced proliferation of fibroblasts by regulating MAP kinase phosphatase-1 expression. Mol Biol Cell 17: 1995–2008, 2006.[Abstract/Free Full Text]

35. Soker S, Takashima S, Miao HQ, Neufeld G, Klagsbrun M. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 92: 735–745, 1998.[CrossRef][Web of Science][Medline]

36. Stawowy P, Goetze S, Margeta C, Fleck E, Graf K. LPS regulate ERK1/2-dependent signaling in cardiac fibroblasts via PKC-mediated MKP-1 induction. Biochem Biophys Res Commun 303: 74–80, 2003.[CrossRef][Web of Science][Medline]

37. Sun H, Charles CH, Lau LF, Tonks NK. MKP-1 (3CH134), an immediate early gene product, is a dual specificity phosphatase that dephosphorylates MAP kinase in vivo. Cell 75: 487–493, 1993.[CrossRef][Web of Science][Medline]

38. Tohgo A, Choy EW, Gesty-Palmer D, Pierce KL, Laporte S, Oakley RH, Caron MG, Lefkowitz RJ, Luttrell LM. The stability of the G protein-coupled receptor-beta-arrestin interaction determines the mechanism and functional consequence of ERK activation. J Biol Chem 278: 6258–6267, 2003.[Abstract/Free Full Text]

39. Torres C, Li M, Walter R, Sierra F. Modulation of the ERK pathway of signal transduction by cysteine proteinase inhibitors. J Cell Biochem 80: 11–23, 2000.[CrossRef][Web of Science][Medline]

40. Vu TK, Wheaton VI, Hung DT, Charo I, Coughlin SR. Domains specifying thrombin-receptor interaction. Nature 353: 674–677, 1991.[CrossRef][Medline]

41. Wadgaonkar R, Pierce JW, Somnay K, Damico RL, Crow MT, Collins T, Garcia JG. Regulation of c-Jun N-terminal kinase and p38 kinase pathways in endothelial cells. Am J Respir Cell Mol Biol 31: 423–431, 2004.[Abstract/Free Full Text]

42. Weber NC, Blumenthal SB, Hartung T, Vollmar AM, Kiemer AK. ANP inhibits TNF-alpha-induced endothelial MCP-1 expression–involvement of p38 MAPK and MKP-1. J Leukoc Biol 74: 932–941, 2003.[Abstract/Free Full Text]

43. Wellbrock C, Weisser C, Geissinger E, Troppmair J, Schartl M. Activation of p59(Fyn) leads to melanocyte dedifferentiation by influencing MKP-1-regulated mitogen-activated protein kinase signaling. J Biol Chem 277: 6443–6454, 2002.[Abstract/Free Full Text]

44. Wu JJ, Bennett AM. Essential role for mitogen-activated protein (MAP) kinase phosphatase-1 in stress-responsive MAP kinase and cell survival signaling. J Biol Chem 280: 16461–16466, 2005.[Abstract/Free Full Text]

45. Wu SQ, Minami T, Donovan DJ, Aird WC. The proximal serum response element in the Egr-1 promoter mediates response to thrombin in primary human endothelial cells. Blood 100: 4454–4461, 2002.[Abstract/Free Full Text]

46. Zhao Q, Wang X, Nelin LD, Yao Y, Matta R, Manson ME, Baliga RS, Meng X, Smith CV, Bauer JA, Chang CH, Liu Y. MAP kinase phosphatase 1 controls innate immune responses and suppresses endotoxic shock. J Exp Med 203: 131–140, 2006.[Abstract/Free Full Text]




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