Glycogen-Synthase Kinase3/-Catenin Axis Promotes Angiogenesis Through Activation of Vascular Endothelial Growth Factor Signaling in Endothel
http://www.100md.com
循环研究杂志 2005年第2期
the Whitaker Cardiovascular Institute (C.S., H.M., E.R., A.B., K.W.), Boston University Medical Center, Boston, Mass
the Department of Medicine (T.F.), Molecular Cardiology Research Institute, Tufts-New England Medical Center, Boston, Mass.
Abstract
Glycogen-Synthase Kinase 3 (GSK3) has been shown to function as a nodal point of converging signaling pathways in endothelial cells to regulate vessel growth, but the signaling mechanisms downstream from GSK3 have not been identified. Here, we show that -catenin is an important downstream target for GSK3 action in angiogenesis and dissect the signal transduction pathways involved in the angiogenic phenotype. Transduction of human umbilical vein endothelial cells (HUVECs) with a kinase-mutant form of the enzyme (KM-GSK3) increased cytosolic -catenin levels, whereas constitutively active GSK3 (S9A-GSK3) reduced -catenin levels. Lymphoid enhancer factor/T-cell factor promoter activity was upregulated by KM-GSK3 and diminished by S9A-GSK3, whereas manipulation of Akt signaling had no effect on this parameter. -Catenin transduction induced capillary formation in a Matrigel-plug assay in vivo and promoted endothelial cell differentiation into network structures on Matrigel-coated plates in vitro. -Catenin activated the expression of vascular endothelial growth factor (VEGF)-A and VEGF-C in endothelial cells, and these effects were mediated at the levels of protein, mRNA, and promoter activity. Consistent with these data, -catenin increased the phosphorylation of the VEGF receptor 2 (VEGF-R2) and promoted its association with PI3-kinase, leading to a dose-dependent activation of the serineeCthreonine kinase Akt. Inhibition of PI3-kinase or Akt signaling led to a significant reduction in the pro-angiogenic activity of -catenin. Collectively, these data show that the growth factoreCPI3-kinaseeCAkt axis functions downstream of GSK3/-catenin signaling in endothelial cells to promote angiogenesis.
Key Words: -catenin Akt endothelial cells vacular endothelial growth factor VEGF receptor 2 angiogenesis
Introduction
Glycogen-synthase kinase 3 (GSK3) is a serine/threonine protein kinase that regulates differentiation and proliferation in diverse tissues.1 Recently, GSK3 has been shown to play an important role in angiogenesis through its control of vascular cell migration and differentiation,2 but the downstream targets that transmit these pro-angiogenic effects have not been elucidated.
The serine/threonine kinase Akt/PKB is an upstream regulator of GSK3 that controls its activity in response to growth factor stimulation. Akt is an important regulator of angiogenic responses in endothelial cells through its ability to promote migration, differentiation, and nitric oxide production.3 Phosphorylation of GSK3 at an amino-terminal Ser-9 residue by Akt results in the auto-inhibition of GSK3.4 GSK3 activity can also be controlled by Wnts through a mechanism that generally differs from that used by mitogenic factor-mediated phosphorylation.5,6 In the absence of Wnt signaling, -catenin is associated within a cytosolic multi-protein complex consisting of adenomatous polyposis coli protein, GSK3, and axin.1 GSK3 constitutively phosphorylates -catenin at both serine and threonine residues in the NH2-terminal region, resulting in ubiquitination and subsequent proteosomal degradation of the protein.7 In the "canonical pathway," disheveled is activated by Wnt signals and disrupts the destruction complex, leading to cytoplasmic accumulation of -catenin and subsequent nuclear translocation on binding of Wnt to the frizzled receptor.8 In the nucleus, stabilized -catenin forms complexes with members of the lymphoid enhancer factor/T-cell factor (LEF/TCF) family of transcription factors to activate gene expression. Besides its role as transcriptional activator, -catenin also binds to the cytoplasmatic tail of vascular endothelial (VE)-cadherineCanchoring cadherins to the cortical cytoskeleton.9 Although GSK-3 function can be differentially regulated by mitogen and Wnt stimuli, others have shown that insulin signaling can modulate -catenin activity in some cell types through Akt-mediated changes of GSK-3 phosphorylation within the axin complex.10
Wnts and -catenin have been implicated in vascular development and remodeling.11,12 Endothelial cells express Wnt5a, Wnt7a, Wnt10b, and several frizzled receptors.13 A mutant frizzled-4 has been shown to disrupt retinal angiogenesis in familial exudative vitreoretinopathy in humans.14 It also has been suggested that endostatin exerts its antiangiogenic effects, at least in part, via inducing degradation of -catenin, and the death of endothelial cells in dilated cardiomyopathy is correlated with a reduction in -catenin.15,16 Finally, -catenin is stabilized by the angiogenic factor fibroblast growth factor-2 (FGF-2)17 and by the E4 region of adenovirus that promotes an angiogenic response.18
-Catenin can regulate vascular patterning through its role at the membrane at sites of endothelial celleCcell attachment, 19 a function that is distinct from its role as a transcriptional activator. Furthermore, the membrane pool of -catenin is required for mitogenic signaling through vascular endothelial growth factor (VEGF) receptor-2 (VEGF-R2) through activation of PI3-kinase and Akt.20 Recently, it was reported that a -catenineCregulated TCF-responsive transcriptional reporter is activated at sites of neoangiogenesis in the embryo,21 suggesting that -catenin action in the nucleus as a transcriptional regulator may also be important for blood vessel growth. However, the regulatory steps downstream from -catenin/TCF that promote angiogenesis have not been elucidated.
Here, we show for the first time to our knowledge that elevated -catenin signaling in endothelial cells is sufficient to promote angiogenesis and elaborate the participation of the growth factor/PI3-kinase/Akt signaling axis downstream of GSK3/-catenin in conferring the pro-angiogenic phenotype to endothelial cells. Furthermore, it is shown that the crosstalk between these signaling pathways is mediated, at least in part, through the transcriptional activation of VEGF in endothelial cells by -catenin.
Materials and Methods
Cell Culture and Reagents
Human umbilical vein endothelial cells (HUVECs) (Cambrex, Walkersville, Md) passage 2 to 5 were used in this study and cultured in endothelial growth medium-2 SingleQuots (Clonetics, Walkersville, Md) containing VEGF-A, FGF, epidermal growth factor, insulin-like growth factor-1, hydrocortisone, ascorbic acid, and heparin supplemented with 5% fetal bovine serum and 1% penicillineCstreptomycin. For migration assays and in vitro tube formation, endothelial growth medium was used. Typically, HUVECs were grown to subconfluence in 1.5% gelatin-coated 10-cm dishes, 6-well plates, or slide chambers. Infection with replication defective adenoviral vectors was performed overnight. For Western blot analysis cells were harvested at 16 hours after transduction. VEGF secretion into medium was determined 24 hours after transduction with adenoviral using a Quantakine Mouse VEGF enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, Minn) according to the instructions of the manufacturer. Trypsin was purchased from Gibco (Grand Island, NY), and human VEGF and FGF-2 were acquired from R&D Systems.
In Vitro Network Formation Assay
The formation of vascular-like structures by HUVECs on growth factoreCreduced Matrigel (BD Biosciences, Bedford, Mass) in vitro was performed as previously described.22 In short, 12-well culture plates were coated with Matrigel according to the manufacturer’s instructions. The indicated adenovirus-transduced HUVECs were seeded on coated plates at 3x104 cells/cm2 in endothelial growth medium containing VEGF 50 ng/mL and incubated at 37°C overnight. Network formation was assessed using an inverted phase contrast microscope (Nikon, Tokyo, Japan). Images were captured with a video graphic system (DEI-750 CE Digital Output Camera; Optronics, Goleta, Calif). The degree of network formation was quantified by capturing 5 low-power field images, and the area of networks formed was quantified using Scion Corp (National Institutes of Health Image) area analysis software with background subtraction and averaged. Data are presented as density units. Alternatively, the number of network projections in 10 low-power fields was counted for 4 independent experiments in each group.
In Vivo Mouse Angiogenesis Assay
The formation of new vessels in vivo was evaluated by the Matrigel plug assay (Becton Dickinson, Bedford, Mass) using a modification of the procedures described previously.23 For these experiments, equal amounts of heparin (10 U/mL) and basic FGF (1 e/mL) (R&D Systems) were mixed, and 5 mL of this solution was mixed on ice with 10 mL of Matrigel, such that the final concentration of basic FGF was 250 ng/mL. Solutions of adenoviral vectors encoding -galactosidase, GSK3-KM, or GSK3-S9A, WT--catenin, and delta--catenin were mixed in with Matrigel solution on ice (2x108 plaque-forming units of virus/500 e蘈); 500 e蘈 of Matrigel containing growth factor and adenovirus was injected subcutaneously near the right/left mid-abdomen of C57BL6 mice (Jackson Laboratories, Bar Harbor, Me). Mice were euthanized 14 days after injection. The Matrigel plugs with the adjacent subcutaneous tissues were carefully recovered by en bloc resection, fixed in 4% paraformaldehyde, for 1 hour saturated with 30% sucrose, embedded in optimal cutting temperature compound, and quick-frozen in liquid nitrogen. Immunohistochemistry for hemagglutinin, CD31 (platelet endothelial cell adhesion molecule-1), and histochemistry for alkaline phosphatase were performed on adjacent frozen sections. The primary antibodies used were antieCplatelet endothelial cell adhesion molecule-1 goat polyclonal antibody 1:20 dilution (Santa Cruz Biotechnology, Santa Cruz, Calif), antieCvesicular stomatitis virus glycoprotein rabbit polyclonal antibody (abcam, Cambridge, Mass) 1:100, and antieChemagglutinin mouse monoclonal antibody (Roche, Palo Alto, Calif) 1:100. Bound antibody was detected with an ABC Elite kit (Vector) and visualized with DAB. Sections were counterstained with hematoxylin and observed under a light microscope (Nikon). Matrigel plugs were homogenized to determine hemoglobin content using the Drabkin method.23
Statistical Analysis
All data were compared by ANOVA using Stat View 4.5 (Abacus Software, Burlington, Mass). Data are expressed as mean±SE for the number of independent experiments indicated. P<0.05 was considered to be significant.
For a description of adenoviral constructs, immunofluorescence staining, immunoprecipitation, and Western immunoblot analysis, quantitative reverse-transcription PCR (QRT-PCR) analysis, luciferase reporter assays, and migration assays. See the online data supplement available at http://circres.ahajournals.org.
Results
-Catenin Is a Downstream Target of GSK3 Signaling in Endothelial Cells
Regulation of -catenin by GSK3 in endothelial cells was assessed by fluorescence cytochemistry. As depicted in Figure 1A, -catenin was nearly depleted in HUVECs transduced with a nonphosphorylatable mutant of GSK3 (S9A-GSK3) that is constitutively active. In contrast, transduction with a dominant-negative mutant of GSK3 (KM-GSK3) led to cytosolic and nuclear accumulation of -catenin. The same distribution pattern was observed when either wild-type (WT) or a nondegradable deletion mutation (delta) of -catenin was overexpressed (Figure 1A and data not shown). As shown in a representative Western blot in Figure 1B, cytosolic -catenin levels were diminished in S9A-GSK3eCtransduced endothelial cells. In contrast, transduction of an inactive mutant form of GSK3 (KM-GSK3) leads to an increase in cytosolic -catenin. The VEGF/PI3-kinase signaling axis has been shown to inactivate GSK3 in endothelial cells.2 Therefore, to test whether Akt-mediated signaling can regulate -catenin levels, adenovirus-mediated transduction of constitutively active Akt (myrAkt) was performed on HUVECs. As shown in Figure 1B, Akt gene transfer did not detectably influence cytosolic -catenin levels. These data were collaborated by measurements of TOP/FOPFLASH promoter activity, which measures the status of -catenin/TCF signaling (Figure 1C). The TOPFLASH and FOPFLASH plasmids contain WT and mutant TCF/LEF sequences, respectively, upstream of a minimal thymidine kinase promoter fragment. Transduction of WT -catenin or the nondegradable mutant delta--catenin activated the TCF/LEF promoter (Figure 1C). Similarly, transduction with Ad-KM-GSK3 significantly increased promoter activity, whereas overexpression of active S9A-GSK3 diminished the activity of the reporter. However, neither a constitutively active nor a dominant-negative form of Akt had any influence on TCF/LEF activation (Figure 1C). Taken together, these data indicate that growth factor/Akt/GSK3 and GSK3/-catenin/TCF-LEF pathways comprise separate signal transduction pathways in endothelial cells.
-Catenin Signaling Promotes Angiogenesis In Vivo
Inhibition of GSK3 has been shown to induce an angiogenic phenotype in endothelial cells.2 Thus, a Matrigel plug assay was used in mice to directly test whether -catenin signaling is sufficient to promote angiogenesis in vivo. Adenoviral vectors (2x108 plaque-forming units) were incorporated in the Matrigel plugs along with FGF-2 (250 ng/mL) before subcutaneous implantation in the abdomen of C57BL6 mice for 10 days. In this assay, the Matrigel serves as a reservoir for the viral vector, and endothelial cells that infiltrate the plug become transduced and express the transgene. The expression of the vesicular stomatitis virus-tagged -catenin transgene products within Matrigel plug was shown by immunohistochemistry (Figure 2A). Vesicular stomatitis viruseCpositive immunostaining was detectable in plugs formulated with adenoviral vectors encoding WT--catenin and delta--catenin, but no signal was detected in plugs formulated with the -galactosidaseeCexpressing control adenovirus (Ad-gal). Endothelial cell infiltration of these plugs was assessed by immunohistochemical analysis of CD31-positive and alkaline phosphatase-positive cells. Plugs formulated with WT--catenin and delta--catenin exhibited significantly higher densities of CD31-positive endothelial cells than control plugs (Figure 2B), and these CD31-positive cells surrounded lumens containing erythrocytes (not shown), suggesting the formation of functional vessels. Quantification of hemoglobin in the Matrigel pellets revealed 81.5±18.7, 197.6±23.4 (P<0.05 versus Ad-gal), and 303.7±47.8 (P<0.01 versus Ad-gal) mg hemoglobin/g Matrigel for plugs cast with adenoviral vectors expressing -galactosidase, WT--catenin, and delta--catenin, respectively. These data were corroborated by analyzing the densities of alkaline phosphatase-positive capillaries within these plugs (Figure 2C). With both histological markers, as well as hemoglobin content, the delta--catenin mutant was more effective at promoting angiogenesis than WT--catenin in the in vivo Matrigel assay.
-Catenin Signaling Promotes Endothelial Cell Differentiation and Migration In Vitro
To elucidate the molecular pathways that transmit the angiogenic phenotype downstream of GSK3/-catenin, vascular network formation assays were performed on Matrigel-coated tissue culture plates. In this assay, Matrigel serves as a matrix for endothelial cells to migrate and align such that network structures are formed (Figure 3A). After -catenin transduction, a significant increase in network formation on Matrigel was observed (Figure 3B). The indicated statistically significant differences were observed between experimental conditions whether the data were analyzed on the basis of density units or the number of network projections per low-power field (see Materials and Methods). Morphologically, -catenin transduced cells were able to form larger aggregates of cells and bundles and produced more pronounced network structures (Figure 3A). The effect of -catenin was dependent on the dose of the expression vector and, at lower doses of vector, the delta--catenin was more effective than the WT (data not shown). Consistent with previous data,2 S9AeCGSK3 inhibited network formation, whereas transduction with KM-GSK3 increased their formation. Importantly, the inhibitory effect of S9AeCGSK3 could be partly reversed by cotransduction with -catenin, indicating that -catenin functions as a downstream effector of GSK3 signaling in the angiogenic response. Surprisingly, the formation of -catenineCinduced networks could be inhibited by the PI3-kinase inhibitor LY294002. Although these data establish -catenin as a pro-angiogenic effector that functions downstream of GSK-3, they also suggest that PI3-kinase is involved in this process as well.
Because vascular structure formation requires cellular migration, we investigated the ability of transduced HUVECs to migrate toward angiogenic growth factors using a modified Boyden chamber assay (Figure 4). Transduction with -catenin resulted in a highly significant increase in chemotaxis toward VEGF that was dependent on the dose of the delta--catenin vector. As shown in Figure 4, the increase in migration after -catenin gene transfer could be blocked by cotransduction with a dominant-negative form of Akt (dnAkt).
-Catenin Activates the PI3-kinase/Akt Signaling Axis
As discussed, the regulation of -catenin in HUVECs is independent of the PI3-kinaseeCAkt signaling axis. However, incubation of HUVECs with the PI3-kinase inhibitor LY294002 or dnAkt significantly attenuated -catenineCmediated pro-angiogenic responses in vitro. Thus, the ability of -catenin to modulate PI3-kinase/Akt signaling in endothelial cells was evaluated. A marked increase in the phosphorylation of Akt was observed in HUVECs after -catenin transduction that was dependent on the dose of the -catenin expression vector (Figure 5A). Because KM-GSK3 gene transfer increases cytosolic -catenin levels (Figure 1B), we tested whether KM-GSK3 could also activate Akt signaling. A dose-dependent increase in Akt phosphorylation was also observed after expression of KM-GSK3 (Figure 5B). Collectively, these data suggested that GSK3/-catenin signaling activates the growth factoreCPI3-kinaseeCAkt regulatory axis. To test this hypothesis, immunoprecipitation for VEGF receptor 2 (VEGF-R2) and Western immunoblot analyses for PI3-kinase were performed. As shown in Figure 5C, transduction with -catenin led to increased PI3-kinase recruitment to VEGF-R2.
-Catenin Transduction Upregulates VEGF-R2, VEGF-A, and VEGF-C Expression
Analysis of VEGF expression was performed to further investigate the mechanism of the angiogenic response after -catenin gene transfer. As shown in Figure 6A and 6B, transduction of HUVECs with Ad--catenin led to a dose-dependent upregulation of VEGF-A and VEGF-C RNA. Although this did not lead to increased VEGF secretion into medium as assessed by enzyme-linked immunosorbent assay (data not shown), Western blotting revealed an upregulation of intracellular VEGF-A and VEGF-C proteins (Figure 6C), indicative of an autocrine activation mechanism. Consistent with these observations, transient transfection assays revealed a four-fold increase of VEGF-A promoter activity in -catenineCoverexpressing cells (Figure 6D). Taken together, our data indicate that -catenin signaling promotes the expression of the pro-angiogenic factors in endothelial cells.
VEGF transmits angiogenic signals through VEGF-R2, and this leads to an induction in the expression of this receptor.24 In accordance with the VEGF induction data, transduction with -catenin led to a dose-dependent increase in VEGF-R2 phosphorylation, indicative of activation, and an increase in overall VEGF-R2 protein expression (Figure 7A and 7B). QRT-PCR demonstrated a significant upregulation of VEGF-R2 mRNA 16 hours after transduction with -catenin that could be largely inhibited by either coincubation with LY294002 or cotransduction with dnAkt (Figure 7C).
Discussion
This study examined the pro-angiogenic actions of -catenin signaling in endothelial cells. It is shown for the first time to our knowledge that -catenin signaling is sufficient to promote vessel growth in vivo and confer a pro-angiogenic phenotype to endothelial cells in vitro. Here, -catenin regulation in endothelial cells was dependent on GSK3 activity but found to be independent of PI3-kinase/Akt activation, consistent with observations in other cell types.1,25 However, a novel finding of this study is that PI3-kinase and Akt function as downstream effectors of -catenin signaling. -Catenin expression enhanced VEGF-R2 expression and phosphorylation, promoted PI3-kinase recruitment to VEGF-R2, and activated Akt phosphorylation. Therefore, we propose that VEGF/PI3-kinase/Akt signaling is downstream of -catenin, and that it contributes to the pro-angiogenic actions of -catenin on endothelial cells.
GSK3 serves as a nodal point of convergent signaling pathways in endothelial cells to control angiogenic responses.2 The enzyme is a downstream target of PI3-kinase/Akt signaling and is inactivated by phosphorylation.26 Here, we show that -catenin levels are reduced by the transduction of S9A-GSK3, a nonphosphorylatable mutant of GSK3 that is constitutively active. Conversely, transduction of KM-GSK3, a dominant-negative mutant, led to an elevation of -catenin levels. However, neither transduction with constitutively active nor dominant-negative mutants of Akt changed -catenin protein levels, nor influenced TCF/LEF transcriptional activity. These data suggest that endothelial cells contain different cytosolic pools of GSK3 that are regulated by growth factor and Wnt signaling, as has been previously suggested.1,5,8 In contrast, Akt participation in -catenin regulation has been described in other systems.
A Matrigel plug assay was performed to assess the ability of -catenin to form new blood vessels in vivo. Unlike other angiogenesis assays, the mouse Matrigel plug model can be used to study the effects of adenovirus-mediated gene transfer on vessel growth in adult mice because the Matrigel serves as a reservoir for the viral vector, leading to high-efficiency transduction as endothelial cells infiltrate the plug. In these experiments, we confirmed earlier findings of a pro-angiogenic effect of GSK3 kinase mutant and inhibition of angiogenesis by constitutively active GSK3.2 Importantly, WT -catenin was found to promote angiogenesis, and the nonphosphorylatable mutant of -catenin was more effective than WT in promoting capillary formation. This study also found that -catenin controlled endothelial cell migration toward VEGF and endothelial cell differentiation into network structures. Moreover, the anti-angiogenic phenotype conferred by transduction with constitutively active GSK3 was partly reversed by cotransduction with -catenin, suggesting that the GSK3/-catenin signaling axis is functionally significant in endothelial cells. Furthermore, these in vitro effects were not the result of cytoprotective or cytotoxic effects of the different adenoviral vectors because these assays were performed over a short period of time in the presence of a growth factor. Only under conditions of prolonged serum starvation did we observe a pro-survival activity of -catenin (data not shown).
Transduction of endothelial cells with -catenin led to an increase in VEGF-A and VEGF-C expression. The upregulation of VEGF-A and VEGF-C occurred at the levels of protein and transcript, and -catenin activated a fragment of the VEGF-A promoter in cotransfection studies. VEGF-C induction by -catenin has also been reported in transformed epithelial cells.27 VEGF-A is an essential angiogenic factor28 and VEGF-C stimulates angiogenesis and improves ischemic limb revascularization.29,30 VEGF-A and VEGF-C bind to VEGF-R2,31 inducing proliferation, migration, survival, and vascular permeability.31,32 VEGF-R2 is also upregulated by VEGF stimulation, leading to enhanced VEGF signaling and angiogenesis.24 Consistent with increased VEGF signaling, we demonstrate that -catenin transduction produced an upregulation of VEGF-R2 transcript and protein levels in endothelial cells. Moreover, we found that -catenin increased VEGF-R2 tyrosine phosphorylation, which is correlated with endothelial cell migration and tube formation.33 -catenin also recruited PI3-kinase to VEGF-R2, and led to the activation of Akt. Collectively, these data indicate that Wnt signaling and PI3-kinase/Akt signaling converge through -catenineCmediated regulation of VEGF production in endothelial cells.
Previous studies have shown that the growth factor/PI3-kinase/Akt signaling pathway is a key regulator of the angiogenic phenotype.3 Here, it is shown that -catenineCenhanced endothelial cell differentiation and migration are impaired by inhibitors of PI3-kinase and Akt, respectively, indicating the functional significance of mitogenic signaling downstream of -catenin. These data suggest that Wnt/GSK3 signaling may promote blood vessel growth via the induction of angiogenic growth factors and autocrine stimulation of PI3-kinase/Akt signaling in endothelial cells.
Acknowledgments
This work was supported by Public Health Service grants AR40197, AG17241, and AG15052 from the National Institutes of Health (to K.W).
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the Department of Medicine (T.F.), Molecular Cardiology Research Institute, Tufts-New England Medical Center, Boston, Mass.
Abstract
Glycogen-Synthase Kinase 3 (GSK3) has been shown to function as a nodal point of converging signaling pathways in endothelial cells to regulate vessel growth, but the signaling mechanisms downstream from GSK3 have not been identified. Here, we show that -catenin is an important downstream target for GSK3 action in angiogenesis and dissect the signal transduction pathways involved in the angiogenic phenotype. Transduction of human umbilical vein endothelial cells (HUVECs) with a kinase-mutant form of the enzyme (KM-GSK3) increased cytosolic -catenin levels, whereas constitutively active GSK3 (S9A-GSK3) reduced -catenin levels. Lymphoid enhancer factor/T-cell factor promoter activity was upregulated by KM-GSK3 and diminished by S9A-GSK3, whereas manipulation of Akt signaling had no effect on this parameter. -Catenin transduction induced capillary formation in a Matrigel-plug assay in vivo and promoted endothelial cell differentiation into network structures on Matrigel-coated plates in vitro. -Catenin activated the expression of vascular endothelial growth factor (VEGF)-A and VEGF-C in endothelial cells, and these effects were mediated at the levels of protein, mRNA, and promoter activity. Consistent with these data, -catenin increased the phosphorylation of the VEGF receptor 2 (VEGF-R2) and promoted its association with PI3-kinase, leading to a dose-dependent activation of the serineeCthreonine kinase Akt. Inhibition of PI3-kinase or Akt signaling led to a significant reduction in the pro-angiogenic activity of -catenin. Collectively, these data show that the growth factoreCPI3-kinaseeCAkt axis functions downstream of GSK3/-catenin signaling in endothelial cells to promote angiogenesis.
Key Words: -catenin Akt endothelial cells vacular endothelial growth factor VEGF receptor 2 angiogenesis
Introduction
Glycogen-synthase kinase 3 (GSK3) is a serine/threonine protein kinase that regulates differentiation and proliferation in diverse tissues.1 Recently, GSK3 has been shown to play an important role in angiogenesis through its control of vascular cell migration and differentiation,2 but the downstream targets that transmit these pro-angiogenic effects have not been elucidated.
The serine/threonine kinase Akt/PKB is an upstream regulator of GSK3 that controls its activity in response to growth factor stimulation. Akt is an important regulator of angiogenic responses in endothelial cells through its ability to promote migration, differentiation, and nitric oxide production.3 Phosphorylation of GSK3 at an amino-terminal Ser-9 residue by Akt results in the auto-inhibition of GSK3.4 GSK3 activity can also be controlled by Wnts through a mechanism that generally differs from that used by mitogenic factor-mediated phosphorylation.5,6 In the absence of Wnt signaling, -catenin is associated within a cytosolic multi-protein complex consisting of adenomatous polyposis coli protein, GSK3, and axin.1 GSK3 constitutively phosphorylates -catenin at both serine and threonine residues in the NH2-terminal region, resulting in ubiquitination and subsequent proteosomal degradation of the protein.7 In the "canonical pathway," disheveled is activated by Wnt signals and disrupts the destruction complex, leading to cytoplasmic accumulation of -catenin and subsequent nuclear translocation on binding of Wnt to the frizzled receptor.8 In the nucleus, stabilized -catenin forms complexes with members of the lymphoid enhancer factor/T-cell factor (LEF/TCF) family of transcription factors to activate gene expression. Besides its role as transcriptional activator, -catenin also binds to the cytoplasmatic tail of vascular endothelial (VE)-cadherineCanchoring cadherins to the cortical cytoskeleton.9 Although GSK-3 function can be differentially regulated by mitogen and Wnt stimuli, others have shown that insulin signaling can modulate -catenin activity in some cell types through Akt-mediated changes of GSK-3 phosphorylation within the axin complex.10
Wnts and -catenin have been implicated in vascular development and remodeling.11,12 Endothelial cells express Wnt5a, Wnt7a, Wnt10b, and several frizzled receptors.13 A mutant frizzled-4 has been shown to disrupt retinal angiogenesis in familial exudative vitreoretinopathy in humans.14 It also has been suggested that endostatin exerts its antiangiogenic effects, at least in part, via inducing degradation of -catenin, and the death of endothelial cells in dilated cardiomyopathy is correlated with a reduction in -catenin.15,16 Finally, -catenin is stabilized by the angiogenic factor fibroblast growth factor-2 (FGF-2)17 and by the E4 region of adenovirus that promotes an angiogenic response.18
-Catenin can regulate vascular patterning through its role at the membrane at sites of endothelial celleCcell attachment, 19 a function that is distinct from its role as a transcriptional activator. Furthermore, the membrane pool of -catenin is required for mitogenic signaling through vascular endothelial growth factor (VEGF) receptor-2 (VEGF-R2) through activation of PI3-kinase and Akt.20 Recently, it was reported that a -catenineCregulated TCF-responsive transcriptional reporter is activated at sites of neoangiogenesis in the embryo,21 suggesting that -catenin action in the nucleus as a transcriptional regulator may also be important for blood vessel growth. However, the regulatory steps downstream from -catenin/TCF that promote angiogenesis have not been elucidated.
Here, we show for the first time to our knowledge that elevated -catenin signaling in endothelial cells is sufficient to promote angiogenesis and elaborate the participation of the growth factor/PI3-kinase/Akt signaling axis downstream of GSK3/-catenin in conferring the pro-angiogenic phenotype to endothelial cells. Furthermore, it is shown that the crosstalk between these signaling pathways is mediated, at least in part, through the transcriptional activation of VEGF in endothelial cells by -catenin.
Materials and Methods
Cell Culture and Reagents
Human umbilical vein endothelial cells (HUVECs) (Cambrex, Walkersville, Md) passage 2 to 5 were used in this study and cultured in endothelial growth medium-2 SingleQuots (Clonetics, Walkersville, Md) containing VEGF-A, FGF, epidermal growth factor, insulin-like growth factor-1, hydrocortisone, ascorbic acid, and heparin supplemented with 5% fetal bovine serum and 1% penicillineCstreptomycin. For migration assays and in vitro tube formation, endothelial growth medium was used. Typically, HUVECs were grown to subconfluence in 1.5% gelatin-coated 10-cm dishes, 6-well plates, or slide chambers. Infection with replication defective adenoviral vectors was performed overnight. For Western blot analysis cells were harvested at 16 hours after transduction. VEGF secretion into medium was determined 24 hours after transduction with adenoviral using a Quantakine Mouse VEGF enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, Minn) according to the instructions of the manufacturer. Trypsin was purchased from Gibco (Grand Island, NY), and human VEGF and FGF-2 were acquired from R&D Systems.
In Vitro Network Formation Assay
The formation of vascular-like structures by HUVECs on growth factoreCreduced Matrigel (BD Biosciences, Bedford, Mass) in vitro was performed as previously described.22 In short, 12-well culture plates were coated with Matrigel according to the manufacturer’s instructions. The indicated adenovirus-transduced HUVECs were seeded on coated plates at 3x104 cells/cm2 in endothelial growth medium containing VEGF 50 ng/mL and incubated at 37°C overnight. Network formation was assessed using an inverted phase contrast microscope (Nikon, Tokyo, Japan). Images were captured with a video graphic system (DEI-750 CE Digital Output Camera; Optronics, Goleta, Calif). The degree of network formation was quantified by capturing 5 low-power field images, and the area of networks formed was quantified using Scion Corp (National Institutes of Health Image) area analysis software with background subtraction and averaged. Data are presented as density units. Alternatively, the number of network projections in 10 low-power fields was counted for 4 independent experiments in each group.
In Vivo Mouse Angiogenesis Assay
The formation of new vessels in vivo was evaluated by the Matrigel plug assay (Becton Dickinson, Bedford, Mass) using a modification of the procedures described previously.23 For these experiments, equal amounts of heparin (10 U/mL) and basic FGF (1 e/mL) (R&D Systems) were mixed, and 5 mL of this solution was mixed on ice with 10 mL of Matrigel, such that the final concentration of basic FGF was 250 ng/mL. Solutions of adenoviral vectors encoding -galactosidase, GSK3-KM, or GSK3-S9A, WT--catenin, and delta--catenin were mixed in with Matrigel solution on ice (2x108 plaque-forming units of virus/500 e蘈); 500 e蘈 of Matrigel containing growth factor and adenovirus was injected subcutaneously near the right/left mid-abdomen of C57BL6 mice (Jackson Laboratories, Bar Harbor, Me). Mice were euthanized 14 days after injection. The Matrigel plugs with the adjacent subcutaneous tissues were carefully recovered by en bloc resection, fixed in 4% paraformaldehyde, for 1 hour saturated with 30% sucrose, embedded in optimal cutting temperature compound, and quick-frozen in liquid nitrogen. Immunohistochemistry for hemagglutinin, CD31 (platelet endothelial cell adhesion molecule-1), and histochemistry for alkaline phosphatase were performed on adjacent frozen sections. The primary antibodies used were antieCplatelet endothelial cell adhesion molecule-1 goat polyclonal antibody 1:20 dilution (Santa Cruz Biotechnology, Santa Cruz, Calif), antieCvesicular stomatitis virus glycoprotein rabbit polyclonal antibody (abcam, Cambridge, Mass) 1:100, and antieChemagglutinin mouse monoclonal antibody (Roche, Palo Alto, Calif) 1:100. Bound antibody was detected with an ABC Elite kit (Vector) and visualized with DAB. Sections were counterstained with hematoxylin and observed under a light microscope (Nikon). Matrigel plugs were homogenized to determine hemoglobin content using the Drabkin method.23
Statistical Analysis
All data were compared by ANOVA using Stat View 4.5 (Abacus Software, Burlington, Mass). Data are expressed as mean±SE for the number of independent experiments indicated. P<0.05 was considered to be significant.
For a description of adenoviral constructs, immunofluorescence staining, immunoprecipitation, and Western immunoblot analysis, quantitative reverse-transcription PCR (QRT-PCR) analysis, luciferase reporter assays, and migration assays. See the online data supplement available at http://circres.ahajournals.org.
Results
-Catenin Is a Downstream Target of GSK3 Signaling in Endothelial Cells
Regulation of -catenin by GSK3 in endothelial cells was assessed by fluorescence cytochemistry. As depicted in Figure 1A, -catenin was nearly depleted in HUVECs transduced with a nonphosphorylatable mutant of GSK3 (S9A-GSK3) that is constitutively active. In contrast, transduction with a dominant-negative mutant of GSK3 (KM-GSK3) led to cytosolic and nuclear accumulation of -catenin. The same distribution pattern was observed when either wild-type (WT) or a nondegradable deletion mutation (delta) of -catenin was overexpressed (Figure 1A and data not shown). As shown in a representative Western blot in Figure 1B, cytosolic -catenin levels were diminished in S9A-GSK3eCtransduced endothelial cells. In contrast, transduction of an inactive mutant form of GSK3 (KM-GSK3) leads to an increase in cytosolic -catenin. The VEGF/PI3-kinase signaling axis has been shown to inactivate GSK3 in endothelial cells.2 Therefore, to test whether Akt-mediated signaling can regulate -catenin levels, adenovirus-mediated transduction of constitutively active Akt (myrAkt) was performed on HUVECs. As shown in Figure 1B, Akt gene transfer did not detectably influence cytosolic -catenin levels. These data were collaborated by measurements of TOP/FOPFLASH promoter activity, which measures the status of -catenin/TCF signaling (Figure 1C). The TOPFLASH and FOPFLASH plasmids contain WT and mutant TCF/LEF sequences, respectively, upstream of a minimal thymidine kinase promoter fragment. Transduction of WT -catenin or the nondegradable mutant delta--catenin activated the TCF/LEF promoter (Figure 1C). Similarly, transduction with Ad-KM-GSK3 significantly increased promoter activity, whereas overexpression of active S9A-GSK3 diminished the activity of the reporter. However, neither a constitutively active nor a dominant-negative form of Akt had any influence on TCF/LEF activation (Figure 1C). Taken together, these data indicate that growth factor/Akt/GSK3 and GSK3/-catenin/TCF-LEF pathways comprise separate signal transduction pathways in endothelial cells.
-Catenin Signaling Promotes Angiogenesis In Vivo
Inhibition of GSK3 has been shown to induce an angiogenic phenotype in endothelial cells.2 Thus, a Matrigel plug assay was used in mice to directly test whether -catenin signaling is sufficient to promote angiogenesis in vivo. Adenoviral vectors (2x108 plaque-forming units) were incorporated in the Matrigel plugs along with FGF-2 (250 ng/mL) before subcutaneous implantation in the abdomen of C57BL6 mice for 10 days. In this assay, the Matrigel serves as a reservoir for the viral vector, and endothelial cells that infiltrate the plug become transduced and express the transgene. The expression of the vesicular stomatitis virus-tagged -catenin transgene products within Matrigel plug was shown by immunohistochemistry (Figure 2A). Vesicular stomatitis viruseCpositive immunostaining was detectable in plugs formulated with adenoviral vectors encoding WT--catenin and delta--catenin, but no signal was detected in plugs formulated with the -galactosidaseeCexpressing control adenovirus (Ad-gal). Endothelial cell infiltration of these plugs was assessed by immunohistochemical analysis of CD31-positive and alkaline phosphatase-positive cells. Plugs formulated with WT--catenin and delta--catenin exhibited significantly higher densities of CD31-positive endothelial cells than control plugs (Figure 2B), and these CD31-positive cells surrounded lumens containing erythrocytes (not shown), suggesting the formation of functional vessels. Quantification of hemoglobin in the Matrigel pellets revealed 81.5±18.7, 197.6±23.4 (P<0.05 versus Ad-gal), and 303.7±47.8 (P<0.01 versus Ad-gal) mg hemoglobin/g Matrigel for plugs cast with adenoviral vectors expressing -galactosidase, WT--catenin, and delta--catenin, respectively. These data were corroborated by analyzing the densities of alkaline phosphatase-positive capillaries within these plugs (Figure 2C). With both histological markers, as well as hemoglobin content, the delta--catenin mutant was more effective at promoting angiogenesis than WT--catenin in the in vivo Matrigel assay.
-Catenin Signaling Promotes Endothelial Cell Differentiation and Migration In Vitro
To elucidate the molecular pathways that transmit the angiogenic phenotype downstream of GSK3/-catenin, vascular network formation assays were performed on Matrigel-coated tissue culture plates. In this assay, Matrigel serves as a matrix for endothelial cells to migrate and align such that network structures are formed (Figure 3A). After -catenin transduction, a significant increase in network formation on Matrigel was observed (Figure 3B). The indicated statistically significant differences were observed between experimental conditions whether the data were analyzed on the basis of density units or the number of network projections per low-power field (see Materials and Methods). Morphologically, -catenin transduced cells were able to form larger aggregates of cells and bundles and produced more pronounced network structures (Figure 3A). The effect of -catenin was dependent on the dose of the expression vector and, at lower doses of vector, the delta--catenin was more effective than the WT (data not shown). Consistent with previous data,2 S9AeCGSK3 inhibited network formation, whereas transduction with KM-GSK3 increased their formation. Importantly, the inhibitory effect of S9AeCGSK3 could be partly reversed by cotransduction with -catenin, indicating that -catenin functions as a downstream effector of GSK3 signaling in the angiogenic response. Surprisingly, the formation of -catenineCinduced networks could be inhibited by the PI3-kinase inhibitor LY294002. Although these data establish -catenin as a pro-angiogenic effector that functions downstream of GSK-3, they also suggest that PI3-kinase is involved in this process as well.
Because vascular structure formation requires cellular migration, we investigated the ability of transduced HUVECs to migrate toward angiogenic growth factors using a modified Boyden chamber assay (Figure 4). Transduction with -catenin resulted in a highly significant increase in chemotaxis toward VEGF that was dependent on the dose of the delta--catenin vector. As shown in Figure 4, the increase in migration after -catenin gene transfer could be blocked by cotransduction with a dominant-negative form of Akt (dnAkt).
-Catenin Activates the PI3-kinase/Akt Signaling Axis
As discussed, the regulation of -catenin in HUVECs is independent of the PI3-kinaseeCAkt signaling axis. However, incubation of HUVECs with the PI3-kinase inhibitor LY294002 or dnAkt significantly attenuated -catenineCmediated pro-angiogenic responses in vitro. Thus, the ability of -catenin to modulate PI3-kinase/Akt signaling in endothelial cells was evaluated. A marked increase in the phosphorylation of Akt was observed in HUVECs after -catenin transduction that was dependent on the dose of the -catenin expression vector (Figure 5A). Because KM-GSK3 gene transfer increases cytosolic -catenin levels (Figure 1B), we tested whether KM-GSK3 could also activate Akt signaling. A dose-dependent increase in Akt phosphorylation was also observed after expression of KM-GSK3 (Figure 5B). Collectively, these data suggested that GSK3/-catenin signaling activates the growth factoreCPI3-kinaseeCAkt regulatory axis. To test this hypothesis, immunoprecipitation for VEGF receptor 2 (VEGF-R2) and Western immunoblot analyses for PI3-kinase were performed. As shown in Figure 5C, transduction with -catenin led to increased PI3-kinase recruitment to VEGF-R2.
-Catenin Transduction Upregulates VEGF-R2, VEGF-A, and VEGF-C Expression
Analysis of VEGF expression was performed to further investigate the mechanism of the angiogenic response after -catenin gene transfer. As shown in Figure 6A and 6B, transduction of HUVECs with Ad--catenin led to a dose-dependent upregulation of VEGF-A and VEGF-C RNA. Although this did not lead to increased VEGF secretion into medium as assessed by enzyme-linked immunosorbent assay (data not shown), Western blotting revealed an upregulation of intracellular VEGF-A and VEGF-C proteins (Figure 6C), indicative of an autocrine activation mechanism. Consistent with these observations, transient transfection assays revealed a four-fold increase of VEGF-A promoter activity in -catenineCoverexpressing cells (Figure 6D). Taken together, our data indicate that -catenin signaling promotes the expression of the pro-angiogenic factors in endothelial cells.
VEGF transmits angiogenic signals through VEGF-R2, and this leads to an induction in the expression of this receptor.24 In accordance with the VEGF induction data, transduction with -catenin led to a dose-dependent increase in VEGF-R2 phosphorylation, indicative of activation, and an increase in overall VEGF-R2 protein expression (Figure 7A and 7B). QRT-PCR demonstrated a significant upregulation of VEGF-R2 mRNA 16 hours after transduction with -catenin that could be largely inhibited by either coincubation with LY294002 or cotransduction with dnAkt (Figure 7C).
Discussion
This study examined the pro-angiogenic actions of -catenin signaling in endothelial cells. It is shown for the first time to our knowledge that -catenin signaling is sufficient to promote vessel growth in vivo and confer a pro-angiogenic phenotype to endothelial cells in vitro. Here, -catenin regulation in endothelial cells was dependent on GSK3 activity but found to be independent of PI3-kinase/Akt activation, consistent with observations in other cell types.1,25 However, a novel finding of this study is that PI3-kinase and Akt function as downstream effectors of -catenin signaling. -Catenin expression enhanced VEGF-R2 expression and phosphorylation, promoted PI3-kinase recruitment to VEGF-R2, and activated Akt phosphorylation. Therefore, we propose that VEGF/PI3-kinase/Akt signaling is downstream of -catenin, and that it contributes to the pro-angiogenic actions of -catenin on endothelial cells.
GSK3 serves as a nodal point of convergent signaling pathways in endothelial cells to control angiogenic responses.2 The enzyme is a downstream target of PI3-kinase/Akt signaling and is inactivated by phosphorylation.26 Here, we show that -catenin levels are reduced by the transduction of S9A-GSK3, a nonphosphorylatable mutant of GSK3 that is constitutively active. Conversely, transduction of KM-GSK3, a dominant-negative mutant, led to an elevation of -catenin levels. However, neither transduction with constitutively active nor dominant-negative mutants of Akt changed -catenin protein levels, nor influenced TCF/LEF transcriptional activity. These data suggest that endothelial cells contain different cytosolic pools of GSK3 that are regulated by growth factor and Wnt signaling, as has been previously suggested.1,5,8 In contrast, Akt participation in -catenin regulation has been described in other systems.
A Matrigel plug assay was performed to assess the ability of -catenin to form new blood vessels in vivo. Unlike other angiogenesis assays, the mouse Matrigel plug model can be used to study the effects of adenovirus-mediated gene transfer on vessel growth in adult mice because the Matrigel serves as a reservoir for the viral vector, leading to high-efficiency transduction as endothelial cells infiltrate the plug. In these experiments, we confirmed earlier findings of a pro-angiogenic effect of GSK3 kinase mutant and inhibition of angiogenesis by constitutively active GSK3.2 Importantly, WT -catenin was found to promote angiogenesis, and the nonphosphorylatable mutant of -catenin was more effective than WT in promoting capillary formation. This study also found that -catenin controlled endothelial cell migration toward VEGF and endothelial cell differentiation into network structures. Moreover, the anti-angiogenic phenotype conferred by transduction with constitutively active GSK3 was partly reversed by cotransduction with -catenin, suggesting that the GSK3/-catenin signaling axis is functionally significant in endothelial cells. Furthermore, these in vitro effects were not the result of cytoprotective or cytotoxic effects of the different adenoviral vectors because these assays were performed over a short period of time in the presence of a growth factor. Only under conditions of prolonged serum starvation did we observe a pro-survival activity of -catenin (data not shown).
Transduction of endothelial cells with -catenin led to an increase in VEGF-A and VEGF-C expression. The upregulation of VEGF-A and VEGF-C occurred at the levels of protein and transcript, and -catenin activated a fragment of the VEGF-A promoter in cotransfection studies. VEGF-C induction by -catenin has also been reported in transformed epithelial cells.27 VEGF-A is an essential angiogenic factor28 and VEGF-C stimulates angiogenesis and improves ischemic limb revascularization.29,30 VEGF-A and VEGF-C bind to VEGF-R2,31 inducing proliferation, migration, survival, and vascular permeability.31,32 VEGF-R2 is also upregulated by VEGF stimulation, leading to enhanced VEGF signaling and angiogenesis.24 Consistent with increased VEGF signaling, we demonstrate that -catenin transduction produced an upregulation of VEGF-R2 transcript and protein levels in endothelial cells. Moreover, we found that -catenin increased VEGF-R2 tyrosine phosphorylation, which is correlated with endothelial cell migration and tube formation.33 -catenin also recruited PI3-kinase to VEGF-R2, and led to the activation of Akt. Collectively, these data indicate that Wnt signaling and PI3-kinase/Akt signaling converge through -catenineCmediated regulation of VEGF production in endothelial cells.
Previous studies have shown that the growth factor/PI3-kinase/Akt signaling pathway is a key regulator of the angiogenic phenotype.3 Here, it is shown that -catenineCenhanced endothelial cell differentiation and migration are impaired by inhibitors of PI3-kinase and Akt, respectively, indicating the functional significance of mitogenic signaling downstream of -catenin. These data suggest that Wnt/GSK3 signaling may promote blood vessel growth via the induction of angiogenic growth factors and autocrine stimulation of PI3-kinase/Akt signaling in endothelial cells.
Acknowledgments
This work was supported by Public Health Service grants AR40197, AG17241, and AG15052 from the National Institutes of Health (to K.W).
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