| Migration of endothelial cells (EC) is a fundamental physiological process that plays central roles in embryonic blood vessel development (vasculogenesis).postnatal angiogenesis, wound healing/or the pathlogical process of many diseases. Promoting endothelial cell (EC) migration is important not only for therapeutic angiogenesis, but also for accelerating re-endothelialization after vessel injury, and engineering of functional artificial blood vessels. Vascular endothelial cell growth factor (VEGF) is the most important factor in regulating the fuctions of endothelial cells, and its effects in the process of endothelial cell migration are critical. PTP1B is an important member of the tyrosine phosphatases (PTP) family, which is highly expressed in endothelial cells. Meanwhile, PTP1B is a critical negative regulator in VEGF signaling. Several recent studies have shown that inhibition of protein tyrosine phosphatase 1B (PTP1B) may promote EC migration and angiogenesis by enhancing the vascular endothelial growth factor receptor-2 (VEGFR2) signalling. In the present study, we tested the hypothesis that PTP1B inhibitor might also be able to promote EC motility in the absence of functional VEGFR2 signalling. Using human ECs in culture, we demonstrated that PTP1B inhibitors promoted EC adhesion, spreading and migration, which were abolished by the inhibitor of Racl but not Rho GTPase. PTP1B inhibitor significantly increased phosphorylation of p130Cas, and the interactions among p130Cas, Crk and DOCK180; whereas the phosphorylation levels of focal adhesion kinase, Src, paxillin, or Vav2 were unchanged. Gene silencing of DOCK180, but not Vav2, abrogated the effects of PTP1B inhibitor on EC motility. The effects of PTP1B inhibitor on EC motility and p130Cas/DOCK180 activation persisted in the presence of the VEGFR2 antagonist. In conclusion, we suggest that stimulation of the DOCK180 pathway represents an alternative mechanism of PTP1B inhibitor-stimulated EC motility, which does not require concomitant VEGFR2 activation as a prerequisite. Therefore, PTP1B inhibitor may be a useful therapeutic strategy for promoting EC migration in cardiovascular patients in which the VEGF/VEGFR functions are compromised.Objectives1. To investigate the regulatory function of PTP1B inhibitor on endothelial cell motility.2. To clarify whether the function of PTP1B inhibitor on endothelial cell motility is absolutely dependent on activation of VEGFR2 signalling.3. To explore the underlying mechanisms of PTP1B inhibitors in regulating endothelial cell motilityMethods1. Reagents3-(3,5-dibromo-4-hydroxybenzoyl)-2-ethyl-N-[4-(1,3-thiazol-2-ylsulfamoyl)phenyl]-1-benzofuran-6-sulfonamide (PTP Inhibitor XXII), Rhosin, and NSC23766, PP2, and Ki8751 were all purchased form Merck Millipore (Darmstadt, Germany). TCS4O1 was purchased from Tocris Bioscience (Bristol, UK).2. Cell cultureHuman umbilical vein ECs (HUVECs) and telomerase-immortalized human microvascular endothelial (TIME) cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were maintained in complete ECM medium (Catalogue #1001, ScienCell, Carlsbad, CA, USA) supplemented with 5% foetal bovine serum (FBS), the Endothelial Cell Growth Supplement, penicillin (100 U/ml) and streptomycin (100μg/ml) as used before and in a humidified incubator with 5% CO2 at 37℃. Subculture was carried out when cell density reached 70-80%. HUVEC cells used for the experiments were between passages 3 and 6.3. Transwell cell migration assayTranswell migration assay was performed using Boyden chambers as described previously. Briefly, cells were trypsinized and resuspended in serum-free medium, and 105 cells were seeded in the upper well. Serum was added to the lower chamber to a final concentration of 1% as a chemotactic factor. After 6 hr of incubation, cells migrating across the membrane were fixed in cold methanol and stained with crystal violet. For each membrane,5-10 random high power (400x) fields were photographed under a light microscope, and the number of cells was counted. All experiments were repeated at least for 3 times. Drugs or vehicle were added to both of the upper and lower chambers.4. Cell adhesion assayCells were pretreated with serum free medium containing vehicle or drugs for 1 hr. Then cells were trypsinized and seeded in 24-well plates in serum-free medium containing the same treatment agent at a density of 106/mL. After 30 min incubation, wells were washed with PBS and fixed with cold methanol. Cells were stained with 1% crystal violet. For each well,5-10 random high power fields were photographed and the number of attached cells counted.5. Cell spreading assayCells were pretreated with drugs or vehicle in serum-free medium for 1 hr. Then the cells were trypsinized and replated in 8-well Lab-Tek II chamber slides (Thermo Scientific, Waltham, MA, USA) and further incubated with the same treatment agent in serum free medium for 1 hr. Cells were fixed with paraformaldehyde and rinsed in PBS, stained with Rhodamine phalloidin (from Cytoskeleton, Denver, CO, USA), counterstained with DAPI, and observed under a confocal microscope (Model LSM710, Zeiss, Jena, Germany). The spreading response was evaluated by measuring the average cell area using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA). For each experiment,150-200 cells from different random fields were analyzed. To monitor the cell motility behaviour in real-time, TIME cells were pretreated with PTP1B inhibitor or vehicle for 1 hr, harvested and re-suspended in serum free medium and seeded in 6-well plates at a concentration of 105 cells per well, and allowed to adhere for 3 min. Digital videos were recorded for 20 minutes under a phase contrast light microscope (Olympus Lifescience, Tokyo, Japan) equipped with a Cannon digital camera.6. Cell viability assayCells were cultured in 96-well plates to-100% confluent. Cell viability was assessed with the tetrazolium-based CellTiter 96 Aqueous kit (from Promega, Madison, WI, USA) according to the manufacturer’s direction. For cell proliferation assay, cells were plated in 96-well plates at the density of 2000-3000 per well and cultured with serum medium containing vehicle or drugs. Cell viability was assessed with tetrazolium-based CellTiter 96 Aqueous kit every day for 5 days. Cell culture medium was changed every other day.7. Small GTPase pull-down assayCells were cultured to approximately 80% confluence. Before experimentation, cells were washed with serum free medium, and then incubated with vehicle or 10 pM PTPI22 for 20 or 40 min. Pull-down assay for GTP-bound Racl was performed using a Racl Activation Assay Kit from EMD Millipore (Billerica, MA, USA). For western blot detection we used an anti-Racl antibody from Cell Signalling Technology (Cat# 2465) (Beverley, MA, USA) instead of the original antibody provided in the kit.8. Transfection with siRNAThree sequences of siRNA targeting DOCK180 or Vav2 were purchased from GenePharma (Shanghai, China). Transfection was performed using Lipofectamine RNAiMAX Reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. Experiments were carried out 48 hr after transfection. The gene silencing efficiency was determined by western blot.9. Western blot and immunoprecipitationTotal proteins were extracted in cold lysis buffer containing 50 mM Tris, pH 7.5,2 mM EDTA,100 mM NaCl,50 mM NaF,1% Triton X-100,1 mM Na3V04 and 40 mM β-glycerol phosphate, with added protease inhibitor cocktail (Roche, Mannheim, Germany). For immunoprecipitation, equal amount of protein samples were precleared and incubated with 2 μg of antibody and 20 μl of 50% protein A/G agarose bead slurry (Pierce Biotechnology, Rockford, IL, USA) at 4℃ for overnight with gentle agitation. The beads were washed with ice cold lysis buffer for 3 times and boiled in 3×Laemmli buffer. Protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes. Then membranes were blocked with blocking buffer (5% w/v nonfat dry milk,1 X TBS, 0.1% Tween 20) for 1 hour at room temperature. After blocking, membranes were washed with TBST for 3 times and incubated with primary antibody (at the appropriate dilution and diluent as recommended in the product datasheet) in dilution buffer with gentle agitation overnight at 4℃. The next day membranes were developed with HRP-conjugated secondary antibodies and ECL Prime reagents (GE, Piscataway, NJ, USA). Signals were detected with a LAS-4000 luminescent image analyzer (Fujifilm, Stamford, CT, USA). The densitometry analysis was performed using Image-J software (NIH). The following antibodies were used:anti-phospho-tyrosine (#9411), Src (#2110), FAK (#3285), Vav2 (#2848), and p130Cas (#13383) were from Cell Signalling; anti-Src (ab109381) and Crk (ab133581) were from Abcam (Cambridge, UK); anti-paxillin (#05-417) was from EMD Millipore; anti-Tiaml (AF5038) was from R&D Systems (Minneapolis, MN, USA); anti-DOCK180 (sc-6167) was from Santa Cruz (Dallas, Texas USA).10. ImmunofluorescenceCells were cultured on Lab-Tek chamber slides. After treatment with drugs or vehicle, cells were fixed with 4% paraformaldehyde and rinsed in PBS for 3 times. Cells were blocked with 5% w/v BSA and incubated with anti-Crk or anti-DOCK180 antibodies (dilution 1:100) at 4℃ overnight and then with Alexa Fluor 488- or 594-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at room temperature for 2 hr. Cells were counterstained with DAPI for 15 min. Fluorescent images were taken with the confocal microscope.11. Statistical analysisAll experiments were independently repeated at least three times. Data are presented as mean±standard error of the mean (S. E. M.). Data analysis was performed with unpaired t-test or one-way ANOVA followed by post hoc Newman-Keuls test as appropriate. P <0.05 was considered as statistically significant. All tests were two-tailed.Results1. PTP1B inhibitors enhanced EC adhesion and spreadingPTP1B Inhibitor XXII (referred to as PTPI22 thereafter) is a cell-permeable selective inhibitor of PTP1B. We first determined potential cytotoxic effects of PTPI22 with increasing concentrations in TIME cells. We found that PTPI22 under 20 μM had no significant cytotoxic effects at 24 or 48 hr. In the following experiments, therefore, we used PTPI22 at 10μM. We demonstrated that PTPI22 treatment significantly enhanced TIME cell adhesion and spreading on the collagen substratum. To continuously monitor the dynamic changes of cell motility following PTP1B inhibitor treatment, we recorded digital videos of TIME cells with and without PTPI22 treatment. PTPI22 treatment increased the rate of cell spreading process. To further confirm PTP1B inhibitor effects, we also tested PTPI22 in primary HUVECs. PTPI22 exhibited similar enhancing effects on cell adhesion and spreading in HUVECs. To clarify whether the effects of the PTP1B inhibitor were dependent on specific adhesion substratum, we seeded TIME cells in uncoated culture plates. We found that PTPI22 produced similar increasing effects on EC adhesion and spreading on the uncoated surface. In addition, we studied the effects of another small molecule PTP1B inhibitor TCS401. We showed that similar to PTPI22, TCS401 also significantly increased adhesion and spreading responses in TIME cells.2. PTP1B inhibitors enhanced EC migrationNext we examined the effects of PTPI22 and TCS401 on EC migration using the transwell assay. We demonstrated that both PTPI22 and TCS401 significantly enhanced TIME cell migration stimulated by serum.3. The effects of PTP1B inhibitor persisted after VEGFR2 inhibitionThe above results indicate that PTP1B inhibitors exert stimulatory effects on EC motility. To delineate the associated signalling mechanisms, we pretreated TIME cells with the VEGFR2 inhibitor Ki8751 (2 nM). We showed that Ki8751 at this concentration effectively blocked VEGF-induced ERK1/2 phosphorylation. Under the basal condition, Ki8751 significantly reduced the cell spreading response. However, we found that in the presence of Ki8751, PTPI22 still exhibited an enhancing effect on EC spreading. This result indicates that PTPI22 can promote EC motility in the absence of VEGFR2 signalling. To confirm this finding, we repeated the experiments using transwell migration assay. We showed that Ki8751 reduced the basal level of cell migration, while PTPI22 still significantly increased EC migration in the presence of Ki8751.4. The effects of PTP1B inhibitor on EC motility were Rac1-dependentGiven the pivotal role of Rac small GTPase in mediating cell motility, we examined whether Racl activation was involved in the effects of PTP1B inhibitor. TIME cells were pretreated with the Racl inhibitor NSC23766 (100 μM). We found that NSC23766 reduced the basal level of cell spreading, and the stimulatory effect of PTPI22 on cell spreading was abolished by NSC23766. Using Racl GTPase pull-down assay, we demonstrated that PTPI22 significantly increased Racl activation in EC. We also demonstrated that PTPI22 could stimulate Racl activation in the presence of Ki8751. To clarify whether Rho GTPase was also implicated in the effects of PTP1B inhibitor, we pretreated cells with the Rho inhibitor Rhosin (1μM). In both control and PTP122-treated cells, Rhosin decreased the abundance of intracellular stress fibres. However, Rhosin did not change the stimulatory effects of PTPI22 on EC spreading. Moreover, we performed transwell migration assays and confirmed that NSC23766 also abolished the stimulatory effect of PTPI22 on EC migration, which was not changed by Rhosin.5. PTP1B inhibitor did not change the phosphorylation levels of FAK, Src or paxillin in ECBoth of the VEGFR2 and FAK/Src pathways can regulate Racl activation and cell motility. In the following experiments, therefore, we investigated whether PTP1B inhibitor affected the FAK/Src signalling pathway. Using immunoprecipitation and western blot, we found that treatment with PTPI22 had no significant effects on the phosphorylation levels of FAK, Src or paxillin. On the other hand, we demonstrated that Ki8751 significantly reduced the phosphorylation levels of FAK and paxillin in normal ECs.6. The effects of PTPIB inhibitor were dependent on DOCK180In order to define the Racl guanine nucleotide exchange factors (GEFs) involved in PTPI22-induced effect ts, we preformed western blot to detect the expression of endogenous Vav2, Tiaml and DOCK180. We found that DOCK180 was readily detectable in EC; Vav2 could be detected only after immunoprecipitation. However, Tiaml was not detectable even with immunoprecipitation, indicating an extremely low expression level in EC. Next we performed gene silencing experiments for Vav2 and DOCK180 with siRNA. We found that knocking down of DOCK180 significantly blunted the stimulating effects of PTPI22 on EC spreading and migration. In contrast, knocking down of Vav2 did not change the effects of PTPI22. To further corroborate that Vav2 was not involved in the PTPI22 effects, we measured the tyrosine phosphorylation level of Vav2. We found that the endogenous level of Vav2 phosphorylation was undetectable in TIME cells, while treating cells with PTPI22 did not increase the level of Vav2 phosphorylation (repeated 2 times).7. PTPIB inhibitor increased p130Cas phosphorylation and p130Cas-Crk-DOCK180 interactionsThe activity of DOCK180 is under the control by the p130Cas/Crk complex, while p130Cas is a PTP1B substrate. Based on this evidence, we reasoned that PTP1B inhibitor might act to promote the activation of DOCK180. To test this possibility, we first measured the phosphorylation level of p130Cas. PTPI22 treatment significantly increased tyrosine phosphorylation of p130Cas. This effect of PTPI22 was also observed in the presence of Ki8751. Then we immunoprecipitated Crk, and demonstrated that PTPI22 treatment significantly increased the interaction between Crk and p130Cas. PTPI22 also increased binding of Crk with paxillin. To detect the interaction between Crk and DOCK180, we labelled them with immunofluorescence. PTPI22 treatment induced intracellular translocation of Crk toward the cell periphery and the plasma membrane, supporting that the Crk function was activated. Moreover, we demonstrated that treatment with PTPI22 increased co-localisation of Crk and DOCK180. To further verify the interaction between p130Cas and DOCK180, we performed co-immunoprecipitation assays and showed that PTPI22 increased the binding of p130Cas with DOCK180. We confirmed that the effect of PTPI22 was also observable in Ki8751-pretreated cells. Although we found that the phosphorylation level of Src was not affected by the PTP1B inhibitor, this could not exclude that Src was functionally important in the effects of PTP1B inhibitor. To address this question, we pretreated cells with the Src inhibitor PP2. Interestingly, we found that PP2 blocked the stimulatory effect of PTPI22 on EC migration.Conclusion1. PTP1B inhibitor promotes motility of endothelial cells by increasing its adhesion, spreading and migration.2. While PTP1B inhibition may promote EC migration by enhancing VEGFR2 signalling. The major finding of the present study is that PTP1B inhibitor may also stimulate EC motility in the absence of functional VEGFR2 signalling.3. The effect of PTP1B inhibition on ECs motility is DOCK180/Rac1 depended. |
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