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UXT potentiates angiogenesis by attenuating Notch signaling.

Zhou Y, Ge R, Wang R, Liu F, Huang Y, Liu H, Hao Y, Zhou Q, Wang C - Development (2015)

Bottom Line: This prevents RBP-Jκ/CSL from activation and thus inhibits the consequent gene inductions.Furthermore, blockade of Notch signaling rescues the angiogenesis defect caused by UXT knockdown both in vitro and in vivo.Taken together, the data presented in this study characterize UXT as a novel repressor of Notch signaling, shedding new light on the molecular regulation of angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.

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UXT interacts with NICD endogenously through the Notch TAD domain. (A) UXT interacts with NICD endogenously. HUVEC cell lysates were immunoprecipitated (IP) with an anti-Notch1 antibody or control IgG antibody. The immunoprecipitates were immunoblotted (IB) with the indicated antibodies. (B) UXT directly attenuates Notch-targeted genes in endothelial cells. The relative expression level of hes1 and hey1 were measured by RT-PCR. The data were normalized on the basis of the corresponding input control, and are presented as the mean±s.e.m. (at least three independent experiments). (C) UXT attenuates Notch signaling in vivo. Confocal images of Tg(TP1:mCherry; fli1:EGFP)y1 fish injected with 4 ng of control MO or 4 ng of UXT MO. (D) UXT interacts specifically with NICD. HA-UXT or HA-UXT-2M was co-transfected with Flag-NICD or Flag-RBP-Jκ. Cell lysates were subjected to an immunoprecipitation assay using the anti-Flag antibody, followed by western blot analysis with anti-HA and anti-Flag antibodies. (E) UXT impairs the interaction between NICD and RBP-Jκ. HUVECs were treated with shUXT or phage-UXT-Flag. The endogenous NICD was immunoprecipitated with the anti-Notch1 antibody, and the immunoprecipitates were probed with the anti-RBP-Jκ antibody. (F) Schematic representation of the NICD deletion constructs used in the following experiments. (G) The TAD domain of NICD mediates its interaction with UXT. HA-UXT was co-transfected with ΔRAM-Flag, ΔANK-Flag, ΔTAD-Flag, ΔPEST-Flag or NICD-Flag. Cell lysates were subjected to an immunoprecipitation assay using an anti-Flag antibody. (H) UXT directly impaired Notch signaling. The indicated plasmids were transfected into Cos-7 cells together with TP-1 reporter plasmids, in the presence of NICD or the deletion mutants. Data are presented as the mean±s.e.m. (n=3); **P<0.01; ***P<0.001; ns, non-significant versus the corresponding control or as indicated.
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DEV112532F6: UXT interacts with NICD endogenously through the Notch TAD domain. (A) UXT interacts with NICD endogenously. HUVEC cell lysates were immunoprecipitated (IP) with an anti-Notch1 antibody or control IgG antibody. The immunoprecipitates were immunoblotted (IB) with the indicated antibodies. (B) UXT directly attenuates Notch-targeted genes in endothelial cells. The relative expression level of hes1 and hey1 were measured by RT-PCR. The data were normalized on the basis of the corresponding input control, and are presented as the mean±s.e.m. (at least three independent experiments). (C) UXT attenuates Notch signaling in vivo. Confocal images of Tg(TP1:mCherry; fli1:EGFP)y1 fish injected with 4 ng of control MO or 4 ng of UXT MO. (D) UXT interacts specifically with NICD. HA-UXT or HA-UXT-2M was co-transfected with Flag-NICD or Flag-RBP-Jκ. Cell lysates were subjected to an immunoprecipitation assay using the anti-Flag antibody, followed by western blot analysis with anti-HA and anti-Flag antibodies. (E) UXT impairs the interaction between NICD and RBP-Jκ. HUVECs were treated with shUXT or phage-UXT-Flag. The endogenous NICD was immunoprecipitated with the anti-Notch1 antibody, and the immunoprecipitates were probed with the anti-RBP-Jκ antibody. (F) Schematic representation of the NICD deletion constructs used in the following experiments. (G) The TAD domain of NICD mediates its interaction with UXT. HA-UXT was co-transfected with ΔRAM-Flag, ΔANK-Flag, ΔTAD-Flag, ΔPEST-Flag or NICD-Flag. Cell lysates were subjected to an immunoprecipitation assay using an anti-Flag antibody. (H) UXT directly impaired Notch signaling. The indicated plasmids were transfected into Cos-7 cells together with TP-1 reporter plasmids, in the presence of NICD or the deletion mutants. Data are presented as the mean±s.e.m. (n=3); **P<0.01; ***P<0.001; ns, non-significant versus the corresponding control or as indicated.

Mentions: Next, we explored whether UXT interacted with any components of the transcriptional protein complex. The endogenous UXT was pulled down by NICD in HUVECs (Fig. 6A), indicating that UXT interacted with NICD. In addition, UXT directly regulated the endogenous expression of Notch target genes, such as hes1, hey1 (Fig. 6B), hey2 and heyl (supplementary material Fig. S8). Knockdown of UXT enhanced the expression of Notch-responsive genes significantly, whereas the expression level of these genes was remarkably reduced by ectopic expression of UXT in HUVECs. To prove that UXT directly regulates Notch-responsive genes, we crossed the transgenic Tg(TP1:mCherry) fish with Tg(fli1:EGFP)y1 fish, and observed a robust activation of the TP1 reporter in UXT-deficient embryos (Fig. 6C; supplementary material Fig. S10).Fig. 6.


UXT potentiates angiogenesis by attenuating Notch signaling.

Zhou Y, Ge R, Wang R, Liu F, Huang Y, Liu H, Hao Y, Zhou Q, Wang C - Development (2015)

UXT interacts with NICD endogenously through the Notch TAD domain. (A) UXT interacts with NICD endogenously. HUVEC cell lysates were immunoprecipitated (IP) with an anti-Notch1 antibody or control IgG antibody. The immunoprecipitates were immunoblotted (IB) with the indicated antibodies. (B) UXT directly attenuates Notch-targeted genes in endothelial cells. The relative expression level of hes1 and hey1 were measured by RT-PCR. The data were normalized on the basis of the corresponding input control, and are presented as the mean±s.e.m. (at least three independent experiments). (C) UXT attenuates Notch signaling in vivo. Confocal images of Tg(TP1:mCherry; fli1:EGFP)y1 fish injected with 4 ng of control MO or 4 ng of UXT MO. (D) UXT interacts specifically with NICD. HA-UXT or HA-UXT-2M was co-transfected with Flag-NICD or Flag-RBP-Jκ. Cell lysates were subjected to an immunoprecipitation assay using the anti-Flag antibody, followed by western blot analysis with anti-HA and anti-Flag antibodies. (E) UXT impairs the interaction between NICD and RBP-Jκ. HUVECs were treated with shUXT or phage-UXT-Flag. The endogenous NICD was immunoprecipitated with the anti-Notch1 antibody, and the immunoprecipitates were probed with the anti-RBP-Jκ antibody. (F) Schematic representation of the NICD deletion constructs used in the following experiments. (G) The TAD domain of NICD mediates its interaction with UXT. HA-UXT was co-transfected with ΔRAM-Flag, ΔANK-Flag, ΔTAD-Flag, ΔPEST-Flag or NICD-Flag. Cell lysates were subjected to an immunoprecipitation assay using an anti-Flag antibody. (H) UXT directly impaired Notch signaling. The indicated plasmids were transfected into Cos-7 cells together with TP-1 reporter plasmids, in the presence of NICD or the deletion mutants. Data are presented as the mean±s.e.m. (n=3); **P<0.01; ***P<0.001; ns, non-significant versus the corresponding control or as indicated.
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DEV112532F6: UXT interacts with NICD endogenously through the Notch TAD domain. (A) UXT interacts with NICD endogenously. HUVEC cell lysates were immunoprecipitated (IP) with an anti-Notch1 antibody or control IgG antibody. The immunoprecipitates were immunoblotted (IB) with the indicated antibodies. (B) UXT directly attenuates Notch-targeted genes in endothelial cells. The relative expression level of hes1 and hey1 were measured by RT-PCR. The data were normalized on the basis of the corresponding input control, and are presented as the mean±s.e.m. (at least three independent experiments). (C) UXT attenuates Notch signaling in vivo. Confocal images of Tg(TP1:mCherry; fli1:EGFP)y1 fish injected with 4 ng of control MO or 4 ng of UXT MO. (D) UXT interacts specifically with NICD. HA-UXT or HA-UXT-2M was co-transfected with Flag-NICD or Flag-RBP-Jκ. Cell lysates were subjected to an immunoprecipitation assay using the anti-Flag antibody, followed by western blot analysis with anti-HA and anti-Flag antibodies. (E) UXT impairs the interaction between NICD and RBP-Jκ. HUVECs were treated with shUXT or phage-UXT-Flag. The endogenous NICD was immunoprecipitated with the anti-Notch1 antibody, and the immunoprecipitates were probed with the anti-RBP-Jκ antibody. (F) Schematic representation of the NICD deletion constructs used in the following experiments. (G) The TAD domain of NICD mediates its interaction with UXT. HA-UXT was co-transfected with ΔRAM-Flag, ΔANK-Flag, ΔTAD-Flag, ΔPEST-Flag or NICD-Flag. Cell lysates were subjected to an immunoprecipitation assay using an anti-Flag antibody. (H) UXT directly impaired Notch signaling. The indicated plasmids were transfected into Cos-7 cells together with TP-1 reporter plasmids, in the presence of NICD or the deletion mutants. Data are presented as the mean±s.e.m. (n=3); **P<0.01; ***P<0.001; ns, non-significant versus the corresponding control or as indicated.
Mentions: Next, we explored whether UXT interacted with any components of the transcriptional protein complex. The endogenous UXT was pulled down by NICD in HUVECs (Fig. 6A), indicating that UXT interacted with NICD. In addition, UXT directly regulated the endogenous expression of Notch target genes, such as hes1, hey1 (Fig. 6B), hey2 and heyl (supplementary material Fig. S8). Knockdown of UXT enhanced the expression of Notch-responsive genes significantly, whereas the expression level of these genes was remarkably reduced by ectopic expression of UXT in HUVECs. To prove that UXT directly regulates Notch-responsive genes, we crossed the transgenic Tg(TP1:mCherry) fish with Tg(fli1:EGFP)y1 fish, and observed a robust activation of the TP1 reporter in UXT-deficient embryos (Fig. 6C; supplementary material Fig. S10).Fig. 6.

Bottom Line: This prevents RBP-Jκ/CSL from activation and thus inhibits the consequent gene inductions.Furthermore, blockade of Notch signaling rescues the angiogenesis defect caused by UXT knockdown both in vitro and in vivo.Taken together, the data presented in this study characterize UXT as a novel repressor of Notch signaling, shedding new light on the molecular regulation of angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.

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