Published online before print December 18, 2008, doi: 10.1161/CIRCRESAHA.108.185405
© 2009 American Heart Association, Inc.
Cellular Biology |
Wnt5a Is Required for Endothelial Differentiation of Embryonic Stem Cells and Vascularization via Pathways Involving Both Wnt/β-Catenin and Protein Kinase C
From the National Research Laboratory of Molecular Complex Control and Department of Biotechnology (D.-H.Y., J.-Y.Y., S.-H.L., K.-Y.C.) and Department of Biochemistry (Y.-G.K.), College of Life Science and Biotechnology, Yonsei University, Seoul, South Korea; Academy of Sciences of the Czech Republic and Institute of Experimental Biology (V.B.), Faculty of Science, Masaryk University, Brno, Czech Republic; and Laboratory of Molecular Neurobiology (E.R.A., E.A.), Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Correspondence to Kang-Yell Choi, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-752, Korea. E-mail kychoi{at}yonsei.ac.kr
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Abstract |
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In this study, we examined the signaling pathways activated by Wnt5a in endothelial differentiation of embryonic stem (ES) cells and the function of Wnt5a during vascular development. We first found that Wnt5a–/– mouse embryonic stem (mES) cells exhibited a defect in endothelial differentiation, which was rescued by addition of Wnt5a, suggesting that Wnt5a is required for endothelial differentiation of ES cells. Involvement of both β-catenin and protein kinase (PK)C
pathways in endothelial differentiation of mES cells requiring Wnt5a was indicated by activation of both β-catenin and PKC in Wnt5a+/– but not in Wnt5a–/– mES cells. We also found that β-catenin or PKC knockdowns inhibited the Wnt5a-induced endothelial differentiation of ES cells. Moreover, the lack of endothelial differentiation of Wnt5a–/– mES cells was rescued only by transfection of both β-catenin and PKC, indicating that both genes are required for Wnt5a-mediated endothelial differentiation. Wnt5a was also found to be essential for the differentiation of mES cells into immature endothelial progenitor cells, which are known to play a role in repair of damaged endothelium. Furthermore, a defect in the vascularization of the neural tissue was detected at embryonic day 14.5 in Wnt5a–/– mice, implicating Wnt5a in vascular development in vivo. Thus, we conclude that Wnt5a is involved in the endothelial differentiation of ES cells via both Wnt/β-catenin and PKC signaling pathways and regulates embryonic vascular development.
Key Words: Wnt5a • embryonic stem cells • β-catenin • PKC • endothelial differentiation
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Introduction |
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The Wnt family of proteins comprises a large family of cysteine-rich secreted proteins that control multiple processes, including embryonic patterning, growth, migration, and cell differentiation.1 Wnts are known to activate several different pathways. One of them, the canonical pathway, is characterized by in stabilization of β-catenin as a result of the transmission of the signal through cell surface receptors and in subsequent transcriptional activation of target genes. In other pathways, often called noncanonical pathways, Wnt proteins function via cell surface receptors to stimulate the Wnt/Ca2+ pathway through the activation of protein kinase (PK)C2 or the Wnt/PCP pathway through the activation of c-Jun N-terminal kinase (JNK).3,4 Wnt5a has been reported to function through the both noncanonical pathway involving PKC5 and the canonical pathway involving β-catenin.1,6 At a functional level, Wnt5a has been implicated in the regulation of development, proliferation, and cell differentiation.7–12 During development, Wnt5a is involved in the differentiation of chondrocytes,13 as well as dopaminergic neuron differentiation of ventral midbrain.14–16 Wnt5a is also highly expressed in human primary endothelial cells17 and Flk-1+ cells18 and induces proliferation, migration, and survival of endothelial cells.19 However, the role of Wnt5a in endothelial differentiation of ES cells and its signaling mechanism was unclear.
In this study, we investigated the role of Wnt5a in endothelial differentiation of ES cells by generating and analyzing Wnt5a–/– ES cells. We found that the failure of endothelial differentiation in Wnt5a–/– ES cells was rescued by Wnt5a-retroviral infection or extracellular treatment with recombinant Wnt5a. Interestingly, we also observed that both β-catenin and PKC were required for recovery of endothelial differentiation of Wnt5a–/– ES cells. The abilities of Wnt5a–/– ES cells resupplemented with Wnt5a to differentiate into endothelial progenitor cells (EPCs) and to form vascular tubes indicate a role for Wnt5a in endothelial differentiation. Finally, we demonstrate a function of Wnt5a in vascular development in vivo, as evidenced by a defect in the vascularization of embryonic day (E)14.5 Wnt5a–/– mouse embryos. Moreover, here, we address the intricate relationship between noncanonical Wnt/PKC and canonical Wnt/β-catenin pathways during Wnt5a-mediated endothelial differentiation. Thus, we provide the first evidence for a function of Wnt5a in endothelial differentiation of ES cells and in the vascularization of the mouse embryo via pathways involving both Wnt/β-catenin and PKC.
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Materials and Methods |
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Culture of ES Cells
Wnt5a+/– and Wnt5a–/– mouse ES cells were generated as described previously.20
EPC Culture
To collect EPCs, the Sca-1+ cells were sorted from 4-day differentiated ES cells by magnetic labeling cell sorting, as described in detail in the expanded Materials and Methods section, available in the online data supplement at http://circres.ahajournals.org.
Immunohistochemistry
Wnt5a+/– and Wnt5a–/– E14.5 embryos were fixed, embedded, sectioned, and stained as described in detail in the online data supplement.
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Results |
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Generation and Characterization of Wnt5a–/– ES Cells
The role of Wnt5a in endothelial differentiation of ES cells was indicated by a step-wise increase of Wnt5a mRNA during endothelial differentiation of R1 mouse ES cells and a concomitant increase and decrease in mRNA levels of Flk-1, an endothelial marker, and of Oct-4, a stem cell marker, respectively (Figure I, A, in the online data supplement). A Wnt5a–/– ES cell line was generated from a Wnt5a–/– mouse blastocyst.20 The Wnt5a–/– ES cells exhibited similar morphology to the Wnt5a+/– ES cells when the ES cells were grown on mouse embryo fibroblast feeder cells (supplemental Figure I, B). The knockout of Wnt5a in Wnt5a–/– ES cells was validated by the absence of Wnt5a mRNA (supplemental Figure I, C). Moreover, Wnt5a–/– ES cells displayed stem cell characteristics as evidenced by the expression of high Oct-4 mRNA levels, which were similar to those in Wnt5a+/– ES cells (supplemental Figure I, C). To evaluate the pluripotency of the Wnt5a+/– and Wnt5a–/– ES cells in vivo, we induced teratomas by injection of the ES cells. Nude mice transplanted with either Wnt5a+/– or Wnt5a–/– ES cells produced teratomas (d=8
20 mm) after 4 weeks (supplemental Figure II, A and B). The teratomas formed by both cell types displayed a heterogeneous differentiation potential with cells of ectodermal (supplemental Figure II, C and F), mesodermal (supplemental Figure II, D and G), and endodermal (supplemental Figure II, E and H) lineages. The differentiation potentials of the Wnt5a+/– and Wnt5a–/– ES cells were further confirmed in vitro by the expression of increased mRNA levels of Brachyury (T), a mesodermal marker, and Afp and Gata-4, endodermal markers, in embryoid bodies (supplemental Figure III). However, we only observed increased mRNA levels of Flk-1 during embryoid body formation in Wnt5a+/– but not Wnt5a–/– ES cells (supplemental Figure III).21
Wnt5a Is an Important Factor for Endothelial Differentiation of ES Cells and Vascularization
Flk-1, Flt-1, and Tie-2 mRNA levels increased in Wnt5a+/– but not Wnt5a–/– ES cells after 4 days of culture in endothelial differentiation media (Figure 1A). Conversely, Oct-4 mRNA markedly decreased during the differentiation process of Wnt5a+/– ES cells but only modestly in Wnt5a–/– ES cells (Figure 1A). Moreover, endothelin-1 mRNA, a marker of endothelial activation,22 was also induced in differentiated Wnt5a+/– but not Wnt5a–/– ES cells (Figure 1A). The loss of endothelial differentiation of Wnt5a–/– ES cells was more convincingly shown by quantitative RT-PCR analyses (Figure 1B). Fluorescence-activated cell sorting analyses indicated that significantly greater numbers of Flk-1+ cells were present during endothelial differentiation of Wnt5a+/– ES cells compared to Wnt5a–/– ES cells (53.12% versus 0.46%, respectively; supplemental Figure IV). The role of Wnt5a in endothelial differentiation of ES cells was further confirmed by immunocytochemical staining of Wnt5a+/– and Wnt5a–/– ES cells (Figure 1C). The intensity of Flk-1 staining significantly increased in Wnt5a+/– ES cells after 4 days of endothelial differentiation, but this increase was not observed in Wnt5a–/– ES cells (Figure 1C, upper images). Simultaneously, Oct-4 clearly decreased during the differentiation process of Wnt5a+/– ES cells but did not decrease as much inWnt5a–/– ES cells (Figure 1C, middle images). Proliferation of ES cells, as monitored by bromodeoxyuridine staining, was reduced during endothelial differentiation of Wnt5a+/– ES cells but maintained in Wnt5a–/– ES cells (Figure 1C, lower images). We examined the ability of Wnt5a+/– and Wnt5a–/– ES cells to form vascular-like structures in vitro to further characterize the role of Wnt5a in vascularization. Whereas Wnt5a+/– ES cells formed rod-shape endothelial tubes on a Matrigel after 4 days of culture in differentiation condition, Wnt5a–/– ES cells did not form such tubes (Figure 1D).
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To further investigate the role of Wnt5a in endothelial differentiation, we tested whether the defect in endothelial differentiation of Wnt5a–/– ES cells could be rescued by supplementation of Wnt5a. Whereas the mRNA levels of Flk-1, Flt-1, and Tie-2 remained low in Wnt5a–/– ES cells infected with LacZ retroviruses, they significantly increased after infection with Wnt5a-LacZ retroviruses (Figure 2A). The recovery of endothelial differentiation by Wnt5a supplementation was also confirmed by immunocytochemical analyses (Figure 2B). The loss of stem cell characteristics in Wnt5a-treated Wnt5a–/– ES cells was also indicated by reductions of Oct-4 mRNA and protein levels (Figure 2A and 2B). The lack of endothelial differentiation of Wnt5a–/– ES cells could also be rescued by administration of purified Wnt5a protein to the cultures (Figure 2C). Furthermore, we observed induction of Flk-1 protein in Wnt5a-transfected Wnt5a–/– ES cells (Figure 2D). Overall, our data demonstrates that Wnt5a is an essential factor for endothelial differentiation of ES cells.
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Both the β-Catenin– and the PKC-Mediated Pathways Are Required for Endothelial Differentiation of ES Cells by Wnt5a
To identify the signaling pathways involved in the Wnt5a-induced endothelial differentiation of ES cells, we monitored changes in the status of β-catenin and PKC during this process. The levels of β-catenin, as well as its reporter activity,23 were highly upregulated in differentiated Wnt5a+/– ES cells compared to Wnt5a–/– ES cells (Figure 3A and 3B, respectively). To avoid any possibility that the activation of the Wnt/ β-catenin signaling might be caused by secondary effects rather than direct effects of Wnt5a, we measured the immediate activation of β-catenin by recombinant Wnt5a. The level of β-catenin in Wnt5a–/– ES cells started to increase at 30 minutes after treatment with recombinant Wnt5a and then continued to increase until 8 hour after stimulation (supplemental Figure V), indicating that Wnt5a activates the Wnt/β-catenin pathway in these cells. PKC activity, as assessed by phosphorylated PKC (p-PKC) at Thr-500, Thr-641, and Ser-660,24 was significantly increased after 4 days of differentiation of Wnt5a+/– ES cells, but that increase was not observed in Wnt5a–/– ES cells in identical culture conditions (Figure 3A). The phosphorylation of the JNK, a downstream target of PKC signaling,25,26 did not change during endothelial differentiation of ES cells regardless of Wnt5a genotype. To further investigate the involvement of the β-catenin and PKC pathways in ES cell endothelial differentiation through Wnt5a, we measured the mRNA levels of proteins involved in those signaling pathways (Figure 3C). As expected, we did not observe any significant induction of β-catenin mRNA levels. Interestingly, PKC mRNA was induced during endothelial differentiation of Wnt5a+/– ES cells but not Wnt5a–/– ES cells. However, we did not observe any change in the mRNA level of PKC
following endothelial differentiation of Wnt5a+/– ES cells (Figure 3C). The mRNA level of endothelin-1, a known transcriptional target of Wnt/β-catenin signaling,27 also increased during the differentiation process in a Wnt5a-dependent manner (Figure 3C). The role of Wnt5a in the transcriptional regulation of PKC and endothelin-1 was further confirmed by the recovery of PKC and endothelin-1 mRNA levels in Wnt5a–/– ES cells transfected with Wnt5a (Figure 3D, upper gels). The protein levels of β-catenin and PKC were also increased by transfection of Wnt5a in Wnt5a–/– ES cells (Figure 3D, lower gels).
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To identify the role of PKC in the Wnt5a-induced endothelial differentiation of ES cells, we examined the effects of both Gö6976, a PKC-specific inhibitor,28 and PKC siRNA. Treatment of differentiated Wnt5a+/– ES cells with either Gö6976 or PKC siRNA decreased the levels of Flk-1, Flt-1, and endothelin-1 mRNA (Figure 4A and 4B). The reduction of Flk-1 level by PKC siRNA was confirmed in immunoblotting analyses (Figure 4C). Moreover, Gö6976 or PKC siRNA treatment abolished the increase of Flk-1, Flt-1, and endothelin-1 mRNA levels in Wnt5a–/– ES cells transfected with Wnt5a (supplemental Figure VI, A and B). The role of β-catenin in endothelial differentiation was also directly investigated by measuring the effects of β-catenin siRNA (Figure 4D). The mRNA levels of Flk-1, Flt-1, and endothelin-1 were concomitantly reduced by β-catenin siRNA in Wnt5a+/– ES cells differentiated for 4 days. Interestingly, the mRNA level of PKC was also reduced by β-catenin siRNA (Figure 4D). Similarly, protein levels of Flk-1 and PKC were reduced by β-catenin siRNA (Figure 4E).
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To determine the importance of PKC and β-catenin signaling in Wnt5a-mediated endothelial differentiation, we tested whether endothelial differentiation of Wnt5a–/– ES cells could be recovered by transfection with PKC and/or β-catenin. Neither Flk-1 nor Flt-1 mRNAs were induced during the differentiation process of Wnt5a–/– ES cells by PKC or β-catenin alone (Figure 5A and 5B). However, cotransfection of Wnt5a–/– ES cells with both PKC and β-catenin during the 4 day of differentiation process induced an increase in Flk-1, Flt-1, and endothelin-1 mRNA levels (Figure 5C). The induction of Flk-1 by PKC and β-catenin was also confirmed by immunocytochemical analyses (Figure 5D), as well as by fluorescence-activated cell sorting analyses showing significant increment of Flk-1+ cells (from 8.73% to 22.79%) by transfection of both PKC and β-catenin into Wnt5a–/– ES cells (supplemental Figure VII). Overall, our results indicate that both Wnt/β-catenin and PKC signaling are required for endothelial differentiation involving Wnt5a.
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Wnt5a Is Involved in Differentiation of ES Cells Into Endothelial Progenitor Cells
Several studies have identified a role of endothelial progenitor cells (EPCs) in repair of damaged endothelium.29,30 Therefore, we examined the involvement of Wnt5a in endothelial differentiation of ES cells into EPCs to identify any potential role of Wnt5a in the recovery of damaged endothelium. The Sca-1–positive EPCs from Wnt5a+/– cells (W-Sca-1+) significantly induced Flk-1 mRNA to levels equivalent to those of 4-day differentiated Wnt5a+/– ES cells and human umbilical vein endothelial cells (HUVECs) (Figure 6A). The Sca-1–positive EPCs from Wnt5a–/– cells transduced with Wnt5a (K-Sca-1+) also induced Flk-1, Flt-1, and Wnt5a mRNAs (Figure 6A). However, no Sca-1–positive cells could be isolated from untransduced Wnt5a–/– ES cells (data not shown). The Sca-1–positive EPCs (W-Sca-1+ and K-Sca-1+) exhibited rapid labeling by acetylated low-density lipoprotein (Dil-Ac-LDL [1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate–labeled acetylated-low density lipoprotein]), in accord with characteristics of endothelial cells (Figure 6B). These endothelial characteristics were confirmed by positive staining of Flk-1 or von Willebrand factor, an alternative endothelial marker, in Sca-1–positive EPCs by immunocytochemistry (Figure 6C and supplemental Figure VIII). The Sca-1–positive EPCs formed capillary-like tube structures, which were similar to those formed by HUVECs on Matrigel (Figure 6D). Overall, these findings indicate that endothelial differentiation of ES cells into EPCs, which are involved in the recovery of damaged endothelium and vascular tube formation, requires Wnt5a.
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Wnt5a Is Required for Vascular Development During Mouse Embryogenesis
To determine the in vivo role of Wnt5a, we examined vascular formation at E14.5 in Wnt5a+/– and Wnt5a–/– mice.11 Wnt5a–/– but not Wnt5a+/– mouse embryo displayed edema, an indicator of a vascular defect,31,32 at the upper dorsal area after basic morphological evaluation (Figure 7A). We first observed the formation of edema in the spinal cord of Wnt5a–/– but not in Wnt5a+/– embryos stained with hematoxylin/eosin (Figure 7B). An antibody against platelet-endothelial cell adhesion molecule (PECAM)-1, an endothelial cell surface marker, labeled the marginal layer of the spinal cord (Figure 7C, upper images) and cells in the dorsal root ganglia (Figure 7C, lower images), a site of vascular sprouting in the nervous system,33,34 in Wnt5a+/– but not in Wnt5a–/– embryos. To identify whether the role of Wnt5a in vascular formation is restricted to the nervous system, we also checked the gut system, which has endothelial cell lineage in its formation.35 However, the staining of PECAM-1 showed no difference between Wnt5a+/– and Wnt5a–/– embryonic intestines (supplemental Figure IX), revealing the specificity of Wnt5a effect on the differentiation of endothelial cells during embryogenesis.
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Discussion |
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Wnt5a controls multiple physiological processes, including chondrocyte13 and neural11 development, by regulating cellular functions such as proliferation and differentiation. The finding of high expression of Wnt2, Wnt5a, and Wnt11 in Flk-1–positive cells has suggested that these Wnt proteins are potentially involved in endothelial commitment from ES cells.18,36 However, the role of Wnt5a in vascular development and the mechanism by which Wnt5a regulates endothelial differentiation of ES cells have remained largely unknown.
The requirement for Wnt5a in endothelial differentiation of ES cells was clearly shown by the defects in differentiation of Wnt5a–/– ES cells. Moreover, the rescue of endothelial differentiation in Wnt5a–/– ES cells by infection with retroviruses encoding Wnt5a, or by treatment with recombinant Wnt5a, confirmed the essential role of Wnt5a in endothelial differentiation of ES cells. We also found that Wnt2 and Wnt7a mRNAs are highly expressed in both Wnt5a+/– and Wnt5a–/– ES cells (supplemental Figure X). The failure of endothelial differentiation of Wnt5a–/– ES cells without the supplementation of Wnt5a excludes the possibility of the compensatory roles of other Wnt proteins in the endothelial differentiation of Wnt5a–/– ES cells.
Although different Wnts are known to preferentially activate canonical or noncanonical pathways, this distinction is not absolute, and it is likely to be dependent on the presence of specific signaling components, putative receptors, and other cofactors at the cell surface.1 Wnt5a functions in the noncanonical pathways through signaling components such as PKC and JNK,37,38 as well as in the canonical pathway involving β-catenin.1,6 Our results showing specific activation of PKC and β-catenin during endothelial differentiation of Wnt5a+/– ES cells indicate that both the PKC and Wnt/β-catenin pathways are involved in the endothelial differentiation of ES cells by Wnt5a. A role for PKC in vascular tube formation of endothelial cells was previously identified.39–41 However, our results are the first to indicate that PKC is specifically involved in the Wnt5a-induced endothelial differentiation of ES cells. We found that both the enzymatic activation and the transcriptional/translational upregulation of PKC contribute to differentiation of ES cells. Similarly, the activation of the Wnt/β-catenin pathway by Wnt5a and the increase of endothelin-1 mRNA, a transcriptional target of β-catenin,27 indicated that the Wnt/β-catenin pathway plays a prominent role in endothelial differentiation of ES cells. It has been previously reported that β-catenin signaling promotes angiogenesis of primary endothelial HUVECs.42 We also demonstrated here the role of the Wnt/β-catenin pathway in endothelial differentiation of ES cells, as seen in reductions of Flk-1, Flt-1, and endothelin-1 mRNA levels and Flk-1 protein level in β-catenin siRNA-transfected Wnt5a+/– ES cells. We suggest that the endothelial differentiation of ES cells by Wnt5a via the Wnt/β-catenin signaling may be acquired by highly selective expression of target genes such as endothelin-1, because other transcriptional targets of Wnt/β-catenin including c-Myc and cyclinD1 were not upregulated during endothelial differentiation.43,44 However, we observed a weak induction of endothelin-1 mRNA during the endothelial differentiation of Wnt5a–/– ES cells, indicating that small portion of endothelin-1 mRNA is inducible independently of Wnt5a during the differentiation process. The expression of PKC was reduced by β-catenin knockdown during the endothelial differentiation of Wnt5a+/– ES cells. Moreover, the lack of endothelial differentiation in Wnt5a–/– ES cells was rescued only when both PKC and β-catenin were cotransfected. These results indicate that both the PKC and β-catenin pathways are required for endothelial differentiation by Wnt5a. Furthermore, our data suggest that the canonical and noncanonical Wnt pathways function reciprocally in the process of endothelial differentiation by Wnt5a.
Involvement of Wnt5a in vascular development was shown by a defect of the vascular system at E14.5 in Wnt5a–/– mice. Similar phenotypes have been observed in the mutant embryos deficient in vascular endothelial growth factor-C.45 The requirement for Wnt5a in normal vascular development of nervous system was further shown by a strong expression of PECAM-1 in the marginal layer and dorsal root ganglia of the spinal cord of Wnt5a+/– but not the Wnt5a–/– embryos. The marginal layers of the spinal cord are involved in the formation of the neural tube and express endothelial-specific proteins, such as neuropilin-1, vascular endothelial growth factor, hypoxia-inducible factor-1, and PECAM-1.33,46 The endothelial cells participate in formation of tubes that extend from the gut plexus to the surface of the gut.35 We observed no significant difference in the PECAM-1 staining of Wnt5a+/– and Wnt5a–/– embryonic intestines. These results suggest that vascular defect by Wnt5a knock out is not a general phenotype but a phenotype limited to specific systems such as nervous systems. Finally, our data provide first-time evidence that Wnt5a plays an important role in endothelial differentiation of ES cells and embryonic vascular development.
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Acknowledgments |
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We thank Terry Yamaguchi and Andrew McMahon for providing Wnt5a-deficient mice.
Sources of Funding
This work was supported by grants from the Korea Science and Engineering Foundation, funded by the Ministry of Education, Science and Technology of Korea (no. 2005-01564; 2006-02681; R112000078010020) and by the European Union (STROKEMAP project). V.B. is supported by the Academy of Sciences of the Czech Republic (grant KJB501630801), the Ministry of Education, Youth and Sports of the Czech Republic (grant MSM0021622430), and by an EMBO Installation grant. D.-H.Y., J.-Y.Y., and S.-H.L. were supported by a BK21 scholarship from the Ministry of Education and Human Resources Development.
Disclosures
None.
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Footnotes |
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Original received August 14, 2008; revision received November 19, 2008; accepted December 9, 2008.
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References |
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