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Address correspondence to Vikas P. Sukhatme, 330 Brookline Ave., Dana 517, Beth Israel Deaconess Medical Center, Boston, MA 02215. Tel.: (617) 667-2105. Fax: (617) 667-7843. E-mail: vsukhatm{at}caregroup.harvard.edu
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Abstract |
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Key Words: endostatin; ß-catenin; migration; angiogenesis; Xenopus
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Introduction |
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Both collagen XVIII and ES are involved in many aspects of embryonic development. Collagen XVIII is present in the tubular kidney basement membrane and is necessary for ureter morphogenesis (Lin et al., 2001). ES has been purified from the cultured supernatant of a kidney ureteric bud cell line and shown to modulate the mesenchymalepithelial transition (Yung, J., T. Novak, M. Dhanabal, V. Sukhatme, and J. Barasch. 2000. Endostatin stimulates growth of epithelial precursors. American Society of Nephrology Annual Meeting, Toronto, Ontario, Canada. A2187. [Abstr.].). Moreover, we have shown recently that ES blocks renal branching morphogenesis and tubulogenesis (Karihaloo et al., 2001; Karumanchi et al., 2001). A splice mutation in human collagen XVIII that leads to a truncated protein lacking the ES fragment occasionally causes abnormalities in the renal collecting duct (Knobloch syndrome) (Sertie et al., 2000). The major phenotype in this disease is a failure of neural tube closure and vitreal and retinal degradation. At present, the physiological role of collagen type XVIII in control of vasculogenesis remains unclear. Nevertheless, mice lacking collagen type XVIII gene display retinal vessel abnormality (Fukai et al., 2002). In Caenorhabditis elegans, loss of the ES domain in the cle-1 gene, a collagen XVIII homologue, results in multiple cell migration and axon guidance defects (Ackley et al., 2001). Besides ES, the collagen XVIII gene contains an alternatively spliced fragment encoding a domain similar to the extracellular region of the frizzled (Frz) family members (Zatterstrom et al., 2000). Since Frz proteins function as cell surface receptors for secreted Wnt ligands (Bhanot et al., 1996; Wang et al., 1996; Zatterstrom et al., 2000), the structure of collagen XVIII suggests its possible involvement in Wnt signaling.
Wnt signaling pathways play important roles in the regulation of cellular proliferation, differentiation, motility, and morphogenesis (Wodarz and Nusse, 1998; Akiyama, 2000; Bienz and Clevers, 2000; Polakis, 2000). Signaling by the Frz receptors results in activation of the cytoplasmic Dishevelled (Dsh) proteins. Dsh antagonizes the effects of glycogen synthase kinase (GSK)3, thus leading to ß-catenin stabilization (Cadigan and Nusse, 1997; Gumbiner, 1997; Sokol, 1999). Stability of ß-catenin is a critical point in Wnt signaling that is regulated by many cytoplasmic proteins including Axin, Frat/GBP, protein phosphatase 2A, adenomatous polyposis coli (APC), and GSK3 (Yost et al., 1996, 1998; Zeng et al., 1997; Kishida et al., 1999; Smalley et al., 1999; Ikeda et al., 2000; Itoh et al., 2000; Polakis, 2000). Stabilized ß-catenin translocates to the nucleus where it binds to members of the T cellspecific factor (TCF)/lymphoid enhancer binding factor 1 transcription factor family and stimulates transcription of the target genes including c-Myc, cyclin D, and Siamois (Peifer and Polakis, 2000; Polakis, 2000).
Little is known about ES signal transduction. We have reported recently that glypicans are low affinity ES receptors critically important in mediating ES activities, such as the inhibitory effects on both endothelial cell and renal tubular branching morphogenesis (Karihaloo et al., 2001; Karumanchi et al., 2001). Although ES has been reported to activate tyrosine kinase signaling through the Shb adaptor protein (Dixelius et al., 2000), it remains to be defined how ES signals are transduced inside the cell. To gain insight into signal transduction pathways driven by ES, we used Xenopus embryogenesis as a model system that is well characterized with respect to several signaling pathways (Harland and Gerhart, 1997; Kimelman and Griffin, 2000). Here, we show that at high concentrations ES can antagonize the Wnt pathway in Xenopus embryos and in mammalian cells. Furthermore, our results suggest that ES may inhibit endothelial cell migration and arrest the cell cycle by inhibiting TCF-dependent transcription.
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Results |
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ES impinges on Wnt signaling
To evaluate whether ES can modulate the Wnt pathway, we assessed its effect on ß-catenin activity. ß-catenin is an essential mediator of Wnt signaling and is known to induce a secondary axis when overexpressed in Xenopus embryos (Guger and Gumbiner, 1995). Injection of ß-catenin RNA into a ventral blastomere of 4-cell embryos resulted in a significant percentage of embryos with duplicated head-containing body axes (53%, total number = 45) (Fig. 2). In contrast, most embryos coinjected with ES RNA (86%, n = 29) developed only a single axis or a partial secondary axis with trunk and tail structures but without head, thus demonstrating the inhibitory activity of ES. An ES deletion mutant (ES-m) did not have this activity (81% of embryos had secondary axes; n = 25). Similarly, axis-inducing activity of Wnt8 was also suppressed by ES (unpublished data). These findings are consistent with the hypothesis that ES functions as an inhibitor of the Wnt pathway.
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We also examined whether the effects of ES in Xenopus embryos could be reproduced in endothelial cells in which ES activity was initially demonstrated (O'Reilly et al., 1997). TOP-FLASH, a ß-catenin responsive reporter with multimerized TCF sites, but not the control FOP-FLASH construct with mutated TCF sites (van de Wetering et al., 1991, 1996), was activated in human umbilical vein endothelial cells (HUVECs), transiently transfected with a ß-catenin plasmid (Fig. 2 C). At 520 µg/ml doses, ES repressed TOP-FLASH activation in HUVECs by 3060%. Lower ES doses (100 ng/ml) produced similar inhibition in calf pulmonary arterial endothelial (CPAE) cells (unpublished data). On the other hand, ES with double point mutations, which lacks activity in endothelial migration assays (ES3.1) (Karumanchi et al., 2001), had no effect on promoter activation (Fig. 2 C). Together, these experiments suggest that ES can inhibit the Wnt pathway both in Xenopus embryos and endothelial cells.
ß-catenin as a target for ES
The canonical Wnt pathway has been shown to involve several molecular components, which function sequentially (Cadigan and Nusse, 1997; Sokol, 1999). Therefore, we tested the ability of ES to block signal transduction at different levels using both Xenopus embryos and endothelial cells. Injection of TCF-VP16 (TVP) RNA, encoding NH2-terminally deleted Xtcf3 fused to the VP16 transcriptional activator (Vonica et al., 2000), resulted in axis duplication (Fig. 3). TVP lacks the ß-catenin binding site and is a constitutive transcriptional activator that is independent of ß-catenin (Vonica et al., 2000). Importantly, ES failed to inhibit TVP-mediated axis duplication (Fig. 3, AB). In contrast, secondary axes induced by Dsh, an upstream component of the pathway, were inhibited by ES (Fig. 3 B). Similar data were obtained by measuring TOP-FLASH reporter activation in HUVECs (Fig. 3 C). These findings show that ES operates downstream of or parallel to Dsh, possibly at the level of ß-catenin.
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The ßTrCP protein associates with ß-catenin through recognition of specific NH2-terminal sites for GSK3 phosphorylation, thereby targeting it for ubiquitin-proteasomedependent degradation (Jiang and Struhl, 1998; Liu et al., 1999; Maniatis, 1999). In CPAE cells, ß-catenin levels were decreased by ßTrCP, whereas a dominant negative ßTrCP (NßTrCP) interfered with this degradation pathway resulting in upregulation of ß-catenin (Fig. 5 A). ES decreased ß-catenin levels and TOP-FLASH activity in the presence of
NßTrCP, indicating that it can promote ß-catenin degradation even when the ßTrCP function is blocked, lending additional support for GSK3-independent action of ES.
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ES effects on ß-catenin require glypican 1
Previously, we showed that glypicans function as low affinity receptors for ES (Karumanchi et al., 2001). To test whether glypicans are involved in the effect of ES on ß-catenin signaling (Fig. 5 C), we used the antisense approach. In HUVECs stably infected with a glypican 1 antisense virus, surface expression of glypican 1 is reduced by 80% (Karumanchi et al., 2001). ES inhibited TOP-FLASH activation in control HUVECs but not in these HUVECs carrying the antisense retrovirus. These data point to the importance of glypican 1 in ES signaling.
ES inhibits endothelial cell migration and entry into the S phase of the cell cycle by targeting LEF/TCF sites
The antiangiogenic activity of ES has been connected with its ability to modulate endothelial cell migration and cause G1 arrest of the cell cycle (Dhanabal et al., 1999c). Whereas our findings show that ES can decrease ß-catenin stability and inhibit Wnt-dependent transcriptional targets, it is unclear whether the effects of ES on endothelial cell migration or the cell cycle are due to the modulation of the Wnt pathway. Since TVP, a constitutive active transcriptional activator of TCF-dependent transcription, acts in a ß-cateninindependent manner and ES down-regulates ß-catenin, we asked whether this reagent can rescue the inhibitory effects of ES on endothelial cell migration and cell cycle progression.
HUVECs infected with a TVP-containing retrovirus (TVP-HUVEC) express TVP in cell lysates (Fig. 6 A) and reveal elevated TOP-FLASH reporter activity (Fig. 6 B). We subsequently studied the effects of ES and TVP on endothelial cell migration in the Boyden chamber assay in response to the angiogenic factors VEGF and basic FGF (bFGF) (Yamaguchi et al., 1999; Boilly et al., 2000). Interestingly, in TVP-HUVECs, bFGF but not VEGF augmented the migratory response compared with control vector-infected HUVECs, suggesting that the effects of the two angiogenic factors involve different signaling pathways. ES inhibited both bFGF- and VEGF-induced migration of control HUVECs but had no effect on TVP-HUVECs (Fig. 6 C). This observation shows that TVP overcomes the block in cell migration imposed by ES.
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Discussion |
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The major limitation of our gain of function experiments is that they employ fairly high concentrations of ES and that only modest effects on ß-catenin are observed. However, our experiments are controlled by ES mutants that have no effect at comparable doses. Therefore, the findings may be most relevant to antiangiogenic effects seen in vivo, when pharmacological doses of ES are administered. Moreover, it is possible that ES may interfere with extracellular matrix assembly and thereby elicit antiangiogenic effects through this mechanism. The recent description of collagen XVIII expression in the early frog embryo (Elamaa et al., 2002) is also consistent with a biological role for ES in normal development.
ß-catenin as a target of ES
To investigate the signaling events triggered by ES, we took advantage of Xenopus embryogenesis, which involves a cascade of inductive events with only a small number of signal transduction pathways (Harland and Gerhart, 1997). Microinjection of ES RNA into animal blastomeres of early embryos resulted in specific developmental abnormalities: ectopic cement gland formation and suppression of eye development. These phenotypes are characteristic of embryos in which Wnt signaling has been inhibited by overexpression of GSK3 or partial depletion of ß-catenin (Itoh et al., 1995; Heasman et al., 2000). The hypothesis that ES functions by impinging on Wnt signaling has been supported using several experimental approaches in both frog embryos and human cells. The doses of injected ES RNA (2 ng) are relatively high compared with those typically used for other signaling proteins in the Xenopus embryos. It is possible that the requirement for high levels of ES arises from the fact that mammalian ES is labile in frog cells or that binding of human ES to its frog receptor is suboptimal.
Several inhibitors of ß-catenin signaling are known to inhibit dorsal development; however, animal ventral injections of RNA (Fig. 1) do not allow one to assess dorsal development. Therefore, we tested the effect of ES on embryos injected into dorsal vegetal blastomeres that are known to contribute to dorsal development. ES had very little effect on endogenous axis formation even when injected into dorsovegetal blastomeres (data not shown). Thus, ES appears to affect only the secondary but not the primary body axis. We note that overexpression of GSK3, a well-characterized inhibitor of ß-catenin, also has only mild effects on endogenous axis development (He et al., 1995; Pierce and Kimelman, 1995; unpublished data). Although the reasons for this observation are currently unclear, the lack of the effect may be due to targeting a subcellular pool of ß-catenin that is not directly involved in axis specification (Guger and Gumbiner, 2000). ES would also fail to cause an effect in dorsovegetal cells if they lack the ES receptor or contain an endogenous inhibitor. Alternatively, overexpressed ES may function at a later stage than needed for severe axis disruption or may have a partial effect on ß-catenin.
ES activates a novel molecular pathway leading to ß-catenin degradation
To define at which level ES impinges on Wnt signal transduction, we have shown that ES suppressed axis-inducing activities of Dsh and ß-catenin, yet failed to block the activity of TVP, a downstream component of the pathway. These results, obtained in Xenopus embryos and extended to mammalian endothelial cells, suggest that ES modulates the Wnt pathway at the level of ß-catenin. Consistent with this hypothesis, ß-catenin levels were diminished in the presence of ES. Interestingly, stabilized ß-catenin, which is insensitive to GSK3-mediated phosphorylation and degradation, was also affected by ES, indicating that the underlying pathway is GSK3 independent. Also, ES suppressed ß-catenin signaling in the DLD-1 carcinoma cell line with constitutively high ß-catenin signaling (Morin et al., 1997). This observation is consistent with our conclusion that ES acts via a novel pathway and raises an intriguing possibility that ES may have direct antitumor effects.
Among mechanisms regulating ß-catenin are stabilization by the upstream components of the Wnt pathway Dsh, axin, and GSK3 (Gumbiner, 1997; Salic et al., 2000), or association of ß-catenin and TCF (Lee et al., 2001), and p53-dependent destruction by Siah1, a mammalian homologue of Sina (Matsuzawa and Reed, 2001). Our data suggest that ES targets ß-catenin for proteasome-mediated degradation using pathways independent of ßTrCP and Siah1. We also show that the ES ability to inhibit Wnt signaling depends on glypican 1, since cells infected with a glypican 1 antisense virus fail to down-regulate TCF reporter activity in response to ES (Fig. 5 C). This result is supported by our recent study showing that glypicans function as low affinity ES receptors (Karumanchi et al., 2001). At present, it remains unclear whether the requirement of glypicans for ES binding and function is related to the involvement of glypicans in Wnt signaling (Lin and Perrimon, 1999; Tsuda et al., 1999; Baeg et al., 2001).
Our data argue that a function of ES is to inhibit Wnt signaling. Interestingly, a list of genes rapidly down-regulated by ES (Shichiri and Hirata, 2001) includes c-myc, a known direct target of Wnt signaling (He et al., 1998). Also, we have shown recently that cyclin D1, another direct target of Wnt signaling (Shtutman et al., 1999; Tetsu and McCormick, 1999), is repressed by ES, leading to G1 arrest (Hanai et al., 2002). Moreover, critical cyclin D1 promoter sequences responsible for ES effects have been mapped to the LEF/TCF recognition site (Hanai et al., 2002). Together, these findings support our hypothesis that ES might act by blocking Wnt/ß-catenin signaling.
A role for Wnt signaling in endothelial cells
The Wnt signaling pathway is involved in the control of multiple cellular processes (Cadigan and Nusse, 1997; Sokol, 1999; Peifer and Polakis, 2000). In endothelial cells, Wnt signaling has been reported to increase cell proliferation (Wright et al., 1999), whereas the secreted Frz-related protein FrzA has the opposite effect (Dennis et al., 1999; Duplaa et al., 1999). Moreover, defects of yolk sac and placental angiogenesis in mice lacking the Frz5 gene directly demonstrate the involvement of Wnt signaling in vascular development (Ishikawa et al., 2001). Our results provide further insight into a possible function of the Wnt/ß-catenin pathway in angiogenesis. In particular, our data reveal that endothelial cell migration and cell cycle progression depend on activation of TCF-responsive target genes. Identification and characterization of these targets will be an important focus of future studies.
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Materials and methods |
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Cell culture and DNA transfection
Human umbilical vein endothelial cells (HUVECs) were obtained from Clonetics. CPAE cells and DLD-1 cells were obtained from American Type Culture Collection. HUVECs and CPAE cells were used between passages 2 and 3. HUVECs were maintained in EGM2-MV medium (Clonetics) containing endothelial basal medium (EBM-2), supplemented with 5% FBS, gentamicin, amphothericin B, hydrocortisone, ascorbic acid, and the following growth factors: VEGF, bFGF, hEGF, and IGF-1. CPAE cells were maintained in DME supplemented with 10% FCS and penicillin/streptomycin (P/S). DLD-1 cells were maintained in RPMI 1640 supplemented with 10% FCS and P/S. All cell lines were grown at 37°C in a 100% humidified incubator with 5% CO2. Cells were grown to 8090% confluency, harvested with trypsin, and resuspended to the cell density required for each assay. For transient transfections, 6070% confluent cells in 6-well plates were transfected using Lipofectamine 2000 (Life Technologies) for HUVECs and CPAE cells and Lipofectamine Plus (Life Technologies) for DLD-1 cells. Total amount of transfected DNA in each experimental group was adjusted to the same value by adding vector DNA. MG132 was purchased from Sigma-Aldrich.
Retrovirus production
Retrovirus production was performed as described before (Ory et al., 1996). Briefly, 20 µg of retroviral plasmids were transfected into the 293 GPG packing cell line using the Calphos transfection kit (CLONTECH Laboratories, Inc.). 48 h after transfection, the packing cell line supernatant was collected and used to infect target cells (HUVECs) in complete medium (EGM-2MV).
Xenopus eggs and embryos
Eggs were obtained from Xenopus females injected with 600 U of human chorionic gonadotropin, fertilized in vitro, and cultured in 0.1x Marc's modified Ringer's medium as described previously (Newport and Kirschner, 1982). Embryonic stages were determined according to Nieuwkoop and Faber (1967).
RNA microinjections
Capped synthetic RNAs were generated with SP6 or T7 RNA (Krieg and Melton, 1984) by in vitro transcription of plasmids using mMessage mMachine kits (Ambion). RNA microinjections were performed as described (Itoh et al., 1995).
Luciferase assay
After transient transfection of the plasmids, cells were incubated for 20 h in 10% FCS (for the TOP-FLASH and FOP-FLASH promoters), and luciferase activity in the cell lysates was determined using a luminometer normalized using sea-pansy luciferase activity under the control of the thymidine kinase promoter. The Dual-Luciferase Reporter Assay System was purchased from Promega. For luciferase assays of embryo cell lysate, embryos were injected in the animal pole with 30 pg of the Siamois-luciferase reporter (pSia-Luc) DNA (Fan et al., 1998) and mRNAs for ES and ES mutant, and the luciferase activity was measured in light units as described (Fan and Sokol, 1997).
Immunoprecipitation and immunoblotting
Collected cell lysates or cell lysates immunoprecipitated as described (Kawabata et al., 1998) were separated by PAGE (precast gels; Bio-Rad Laboratories). This was followed by electroblotting onto a polyvinylidenedifluoride membrane. After blocking with 2% BSA in Tris buffered saline/Tween-20 (TBS-T) for 1 h, the polyvinylidenedifluoride membrane was incubated overnight with each primary antibody. After washing with TBS-T, the membrane was incubated with the secondary antimouse Ig at a 1:5,000 dilution for 0.5 h. Protein bands were detected using SuperSignal® West Pico Chemiluminescent Substrate (Pierce Chemical Co.). Primary antiserum against ES was used as described (Dhanabal et al., 1999a). Injected embryos were lysed in 500 µl of lysis buffer (1% Triton X-100, 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.1 mM PMSF, 10 mM NaF, 1 mM Na3VO4) when sibling embryos had developed to late blastula stages (stage 8+). Western blot analysis was performed as described (Itoh et al., 1998).
Migration assays
Migration assays were performed using 12-well Boyden chemotaxis chambers (Neuro Probe, Inc.) with a polycarbonate membrane (25 x 80-mm, PVD free, 8 µm pore size; Osmonics) as described previously (Dhanabal et al., 1999a). Briefly, polycarbonate membrane coated with Vitrogen 100 (Collagen Biomaterials, Inc.) collagen solution (0.3 mg/ml) on both sides was placed over a 0.1% gelatin-coated bottom chamber. The lower chamber was filled with DME containing 0.1% BSA and 20 ng/ml bFGF or 20 ng/ml VEGF (R & D Systems) or nothing. The upper chamber was seeded with 60,000 cells/well with different concentrations of recombinant ES in triplicate. Endothelial cells were labeled with DiI and allowed to migrate for 5 h at 37°C as described (Dhanabal et al., 1999a).
Cell cycle analysis
HUVECs were growth arrested by contact inhibition for 48 h. The 0 h value refers to the percentage of cells in S phase at this time point. Cells (0.1 x 106 cells/well) were harvested and plated into a 6-well plate in 1% FCS/EGM2-MV with recombinant VEGF or bFGF with or without ES. The cells were harvested at various time points and then fixed in ice-cold ethanol. Fixed cells were dehydrated at 4°C for 0.5 h in PBS containing 2% FCS and 0.1% Tween-20 and then centrifuged and resuspended in 0.5 ml of the same buffer. RNase digestion (5 µg/ml) was performed at 37°C for 1 h followed by staining with propidium iodide (5 µg/ml). The cells were analyzed using a FACScan Becton Dickinson flow cytometer.
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Footnotes |
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* Abbreviations used in this paper: APC, adenomatous polyposis coli; bFGF, basic FGF; CPAE, calf pulmonary artery endothelial; Dsh, Dishevelled; ES, endostatin; Frz, frizzled; GSK, glycogen synthase kinase; HUVECs, human umbilical vein endothelial cells; TCF, T cellspecific factor; TVP, TCF-VP16.
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Acknowledgments |
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V.P. Sukhatme has equity in and is a consultant to Ilex Oncology, a company developing angiogenesis inhibitors for cancer. This work was supported by National Institutes of Health grants to V.P. Sukhatme and S. Sokol.
Submitted: 13 March 2002
Revised: 12 June 2002
Accepted: 12 June 2002
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References |
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