Hanai J et al. (2002), Endostatin is a potential inhibitor of Wnt sign...

XB-ART-6770

J Cell Biol 2002 Aug 05;1583:529-39. doi: 10.1083/jcb.200203064.

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Endostatin is a potential inhibitor of Wnt signaling.

Hanai J , Gloy J , Karumanchi SA , Kale S , Tang J , Hu G , Chan B , Ramchandran R , Jha V , Sukhatme VP , Sokol S .

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Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. To gain insight into ES-mediated signaling, we studied the effects of ES RNA on Xenopus embryogenesis and observed developmental abnormalities consistent with impaired Wnt signaling. ES RNA blocked the axis duplication induced by beta-catenin, partially suppressed Wnt-dependent transcription, and stimulated degradation of both wild-type and "stabilized" forms of beta-catenin, the latter suggesting that ES signaling does not involve glycogen synthase kinase 3. Moreover, ES uses a pathway independent of the Siah1 protein in targeting beta-catenin for proteasome-mediated degradation. ES failed to suppress the effects of T cell-specific factor (TCF)-VP16 (TVP), a constitutive downstream transcriptional activator that acts independently of beta-catenin. Importantly, these data were replicated in endothelial cells and also in the DLD-1 colon carcinoma cells with the mutated adenomatous polyposis coli protein. Finally, suppression of endothelial cell migration and inhibition of cell cycle by ES were reversed by TVP. Though high levels of ES were used in both the Xenopus and endothelial cell studies and the effects on beta-catenin signaling were modest, these data argue that at pharmacological concentrations ES may impinge on Wnt signaling and promote beta-catenin degradation.

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Species referenced: Xenopus laevis
Genes referenced: acvr1 cat.2 ctnnb1 dld dvl1 dvl2 fgf2 gpc1 myc sia1 siah1 vegfa

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	Figure 1. Developmental effects of ES in Xenopus embryos. Four to eight cell embryos were injected into the animal side of one ventral (V) blastomere (A) with wild-type or mutated ES RNAs (2 ng each) and cultured for 48 h at room temperature to assess cement gland development. (B) Summary of the data. (C) Embryos injected with ES RNA develop ectopic cement glands (arrowheads). (D) Embryos injected with RNA encoding an NH2-terminal deletion construct (ES-m) are indistinguishable from uninjected embryos. (E) Protein expression levels in embryos injected with ES and ES-m RNAs assessed by anti-ES antibody.
	Figure 2. ES inhibits Î²-cateninâ€“dependent signaling. (A) Embryos were injected into one ventral vegetal blastomere with 50 pg of Î²-catenin (Î²-cat) RNA with or without 2 ng of ES or ES-m RNA. Secondary axis induction was assessed after 48 h culture at room temperature. (B) Luciferase activity was measured in embryos microinjected with the Siamois promoterâ€“reporter construct (pSiaLuc). pSia-Luc activation by Î²-catenin was reduced by ES but not by ES-m. (C) The effect of ES on Î²-catenin signaling in endothelial cells. Transcriptional activation of the TOP-FLASH and FOP-FLASH reporters was measured in HUVECs transfected with indicated plasmids in the presence or absence ES or ES mutant (ES 3.1). Numbers show the amount of ES in Î¼g/ml and the amount of transfected Î²-catenin plasmid per well of a 6-well plate. Cells were transfected with 0.8 Î¼g of each reporter plasmid.
	Figure 3. Effects of ES in response to increasing Dsh and TVP activity. (A and B) Embryos were injected into one ventral vegetal blastomere as indicated with the following RNAs: TVP (0.7 pg), Dsh (0.7 ng); ES and ES-m (2 ng). (A) ES failed to inhibit TVP induced secondary axes. (B) The effect of ES constructs on secondary axes induced by Dsh and TVP. Total number of injected embryos varied from 14 to 70 per group. (C) The effect of ES on transcriptional activation of TOP-FLASH in response to Dsh and TVP. HUVECs were transfected with 0.4 Î¼g of TVP or empty vector in the presence (5 or 20 Î¼g/ml) or absence of ES. ES inhibited Dsh-mediated but not TVP-mediated activation of the reporter.
	Figure 4. ES down-regulates Î²-catenin levels. (A) ES decreases steady state Î²-catenin levels in embryo lysates. Embryos were coinjected with Myc-tagged Î²-catenin (Î²-cat) or Î”Nâ€“Î²-catenin (Î”N-Î²-cat) RNAs (300 and 100 pg, respectively) and either wild-type or mutated ES RNA. At embryonic stages 8â€“8.5, Î²-catenin levels were analyzed by Western blotting with anti-Myc antibodies. ES but not ES-m reduced both Î²-catenin and Î”Nâ€“Î²-catenin protein levels. (B) Effect of ES on Î²-catenin levels in HUVECs. ES (5 or 20 Î¼g/ml) but not mutant ES 3.1 (20 Î¼g/ml) reduced Flagâ€“Î²-catenin levels in transfected HUVECs. (C) ES down-regulates endogenous Î²-catenin in CPAE cells. Cells were incubated with indicated amounts of ES (Î¼g/ml) for 24 h, and cell lysate were collected for western analysis. (D and E) ES blocks the signaling activity of stabilized Î²-catenin in Xenopus. Embryos were injected with 150â€“300 pg of Î”Nâ€“Î²-catenin RNA and 2 ng of ES or ES-m RNA as described in the Fig. 2 legend. (D) Suppression of secondary axes induced by Î”Nâ€“Î²-catenin. (E) Summary of the data. (F) ES blocks the signaling activity of stabilized Î²-catenin in HUVECs. Transcriptional activation of TOP-FLASH in HUVECs transfected with 0.4 Î¼g of Î²-catenin S>A mutant (Î²-SA) or empty vector in the presence or absence of 5â€“20 Î¼g/ml of ES. (G) ES down-regulates TOP-FLASH in the colon cancer DLD-1 cells with constitutively high levels of Î²-catenin signaling. DLD-1 cells were transfected with 0.9 Î¼g of TOP-FLASH reporter plasmid and incubated in the presence (10 or 20 Î¼g/ml) or absence of ES. Cell lysates were collected 24 h after transfection for luciferase activity measurement.
	Figure 5. ES effects on Î²-catenin are not mediated through Î²TrCP or Siah1. (A) CPAE cells were transfected with 0.4 Î¼g of mycâ€“Î²-catenin and 0.8 Î¼g of each Flag-Î²TrCP, Flag-Î”NÎ²TrCP, Flag-Siah1, or Flagâ€“Siah1-dR, as indicated, in the presence (20 Î¼g/ml) or absence of ES. ES reduced transcriptional activation of TOP-FLASH by Î²-catenin in the presence of dominant negative forms of Î²TrCP or Siah1. Î²-catenin levels were measured in the same experiment in the presence (20 Î¼g/ml) or absence of ES. (B) Proteasome inhibitor treatment reverses the effect of ES on Î²-catenin. CPAE cells were transfected with the Flagâ€“Î²-catenin and GFP plasmids. After 24 h, cells were incubated with ES (10 Î¼g/ml) and/or the proteasome inhibitor MG132 (MG) for 3 h as indicated. Cell lysates were collected for Western blotting with anti-Flag and anti-GFP antibodies. (C) ES effect on TOP-FLASH requires glypican 1. Transcriptional activity of TOP-FLASH was measured in glypican 1 antisense virus- or control vector-infected HUVEC.
	Figure 6. Activated TCF (TVP) reverses the inhibitory effects of ES on endothelial cell migration and cell cycle. For migration assays and cell cycle analysis, TVP-infected HUVECs (TVP-HUVEC) or empty virus-infected HUVECs were used. TVP-HUVECs expressed Myc-TVP as detected by anti-Myc antibodies (A) and had elevated TOP-FLASH reporter activity (B). (C) Migration assays were performed in Boyden chambers with VEGF (20 ng/ml) or bFGF (20 ng/ml) as stimulants. ES (1 Î¼g/ml) had no effect on TVP-HUVECs. (D) FACSÂ® analysis of the cell cycle stage was performed with VEGF (20 ng/ml) or bFGF (20 ng/ml) as stimulants in the presence or absence of ES (20 Î¼g/ml). ES reduced the number of cells entering the S phase in control HUVECs but not in TVP-HUVECs.

References [+] :

Ackley, The NC1/endostatin domain of Caenorhabditis elegans type XVIII collagen affects cell migration and axon guidance. 2001, Pubmed