Limits...
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.

Show MeSH

Related in: MedlinePlus

UXT attenuates the expression of the Notch target genes. (A) UXT impairs the TP-1 luciferase reporter of Notch signaling. The indicated plasmids and/or siRNAs were transfected into Cos-7 cells together with TP-1 reporter plasmids, in the presence or absence of NICD. (B) UXT does not influence BMP signaling. The indicated plasmids and/or siRNAs were transfected into Cos-7 cells together with the BRE reporter plasmids. After transfection, cells were starved for 12 h and then treated with or without BMP4 (10 ng). (C) UXT does not influence WNT signaling. The indicated plasmids and/or siRNAs were transfected into HEK293T cell lines together with the TOPflash reporter plasmids, in the presence or absence of ΔN-β-catenin. Data in A-C were normalized to empty vectors and are presented as the mean±s.e.m. (n=3). (D,E) UXT attenuates the expression of Notch target genes. The control and UXT-deficient zebrafish embryos (from 12 hpf to 72 hpf) were harvested, and the expression of hey1 (D) and her6 (E) were measured by RT-PCR. (F-J) UXT does not influence the expression of notch, rbpja or rbpjb but does affect dll4. The control and UXT-deficient zebrafish embryos (from 12 hpf to 72 hpf) were harvested, and the expression of notch1a (G), notch1b (F), rbpja (H), rbpjb (I) and dll4 (J) were measured by RT-PCR. (K) DLL4 attenuates the expression of UXT. The control and DLL4-deficient zebrafish embryos (from 12 hpf to 36 hpf) were harvested, and the expression of uxt was measured by RT-PCR. (L) The relative mRNA expression level of vegfaa in control and UXT-deficient embryos (from 12 hpf to 36 hpf). Data shown in D-L are the mean±s.e.m. (at least three independent experiments). (M) E-ChIP assays at 24 hpf. Embryos injected with 4 ng of control MO, 4 ng of UXT MO or Notch1b MO were probed with the antibody against UXT and polymerase II, respectively. 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). For all panels, *P<0.05; **P<0.01; ***P<0.001; ns, non-significant versus the corresponding control or as indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4325377&req=5

DEV112532F5: UXT attenuates the expression of the Notch target genes. (A) UXT impairs the TP-1 luciferase reporter of Notch signaling. The indicated plasmids and/or siRNAs were transfected into Cos-7 cells together with TP-1 reporter plasmids, in the presence or absence of NICD. (B) UXT does not influence BMP signaling. The indicated plasmids and/or siRNAs were transfected into Cos-7 cells together with the BRE reporter plasmids. After transfection, cells were starved for 12 h and then treated with or without BMP4 (10 ng). (C) UXT does not influence WNT signaling. The indicated plasmids and/or siRNAs were transfected into HEK293T cell lines together with the TOPflash reporter plasmids, in the presence or absence of ΔN-β-catenin. Data in A-C were normalized to empty vectors and are presented as the mean±s.e.m. (n=3). (D,E) UXT attenuates the expression of Notch target genes. The control and UXT-deficient zebrafish embryos (from 12 hpf to 72 hpf) were harvested, and the expression of hey1 (D) and her6 (E) were measured by RT-PCR. (F-J) UXT does not influence the expression of notch, rbpja or rbpjb but does affect dll4. The control and UXT-deficient zebrafish embryos (from 12 hpf to 72 hpf) were harvested, and the expression of notch1a (G), notch1b (F), rbpja (H), rbpjb (I) and dll4 (J) were measured by RT-PCR. (K) DLL4 attenuates the expression of UXT. The control and DLL4-deficient zebrafish embryos (from 12 hpf to 36 hpf) were harvested, and the expression of uxt was measured by RT-PCR. (L) The relative mRNA expression level of vegfaa in control and UXT-deficient embryos (from 12 hpf to 36 hpf). Data shown in D-L are the mean±s.e.m. (at least three independent experiments). (M) E-ChIP assays at 24 hpf. Embryos injected with 4 ng of control MO, 4 ng of UXT MO or Notch1b MO were probed with the antibody against UXT and polymerase II, respectively. 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). For all panels, *P<0.05; **P<0.01; ***P<0.001; ns, non-significant versus the corresponding control or as indicated.

Mentions: In light of the microarray analysis and the impact of UXT deficiency on Notch signaling activation, we reasoned that UXT might modulate the Notch signaling pathway. To explore this possibility, Notch-TP-1-luciferase reporter plasmids were introduced into UXT-deficient cells, in the presence or absence of NICD. Interestingly, knockdown of UXT potentiated the activation of the Notch-TP-1-luciferase reporter, whereas ectopic expression of UXT attenuated the same reporter expression (Fig. 5A). Notably, ectopic expression of UXT-2M displayed only a marginal effect on the expression of the Notch-TP-1-luciferase reporter (Fig. 5A), which was consistent with its inability to rescue the developmental abnormalities induced by the UXT morpholino (Fig. 1F). BMP signaling and Wnt signaling have been reported to be crucial signaling pathways modulating angiogenesis (Phng et al., 2009; Rothhammer et al., 2007). However, UXT did not affect the activation of BMP signaling or Wnt signaling, according to the results from a BRE reporter assay or a TOPflash reporter assay, respectively (Fig. 5B,C).Fig. 5.


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 attenuates the expression of the Notch target genes. (A) UXT impairs the TP-1 luciferase reporter of Notch signaling. The indicated plasmids and/or siRNAs were transfected into Cos-7 cells together with TP-1 reporter plasmids, in the presence or absence of NICD. (B) UXT does not influence BMP signaling. The indicated plasmids and/or siRNAs were transfected into Cos-7 cells together with the BRE reporter plasmids. After transfection, cells were starved for 12 h and then treated with or without BMP4 (10 ng). (C) UXT does not influence WNT signaling. The indicated plasmids and/or siRNAs were transfected into HEK293T cell lines together with the TOPflash reporter plasmids, in the presence or absence of ΔN-β-catenin. Data in A-C were normalized to empty vectors and are presented as the mean±s.e.m. (n=3). (D,E) UXT attenuates the expression of Notch target genes. The control and UXT-deficient zebrafish embryos (from 12 hpf to 72 hpf) were harvested, and the expression of hey1 (D) and her6 (E) were measured by RT-PCR. (F-J) UXT does not influence the expression of notch, rbpja or rbpjb but does affect dll4. The control and UXT-deficient zebrafish embryos (from 12 hpf to 72 hpf) were harvested, and the expression of notch1a (G), notch1b (F), rbpja (H), rbpjb (I) and dll4 (J) were measured by RT-PCR. (K) DLL4 attenuates the expression of UXT. The control and DLL4-deficient zebrafish embryos (from 12 hpf to 36 hpf) were harvested, and the expression of uxt was measured by RT-PCR. (L) The relative mRNA expression level of vegfaa in control and UXT-deficient embryos (from 12 hpf to 36 hpf). Data shown in D-L are the mean±s.e.m. (at least three independent experiments). (M) E-ChIP assays at 24 hpf. Embryos injected with 4 ng of control MO, 4 ng of UXT MO or Notch1b MO were probed with the antibody against UXT and polymerase II, respectively. 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). For all panels, *P<0.05; **P<0.01; ***P<0.001; ns, non-significant versus the corresponding control or as indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4325377&req=5

DEV112532F5: UXT attenuates the expression of the Notch target genes. (A) UXT impairs the TP-1 luciferase reporter of Notch signaling. The indicated plasmids and/or siRNAs were transfected into Cos-7 cells together with TP-1 reporter plasmids, in the presence or absence of NICD. (B) UXT does not influence BMP signaling. The indicated plasmids and/or siRNAs were transfected into Cos-7 cells together with the BRE reporter plasmids. After transfection, cells were starved for 12 h and then treated with or without BMP4 (10 ng). (C) UXT does not influence WNT signaling. The indicated plasmids and/or siRNAs were transfected into HEK293T cell lines together with the TOPflash reporter plasmids, in the presence or absence of ΔN-β-catenin. Data in A-C were normalized to empty vectors and are presented as the mean±s.e.m. (n=3). (D,E) UXT attenuates the expression of Notch target genes. The control and UXT-deficient zebrafish embryos (from 12 hpf to 72 hpf) were harvested, and the expression of hey1 (D) and her6 (E) were measured by RT-PCR. (F-J) UXT does not influence the expression of notch, rbpja or rbpjb but does affect dll4. The control and UXT-deficient zebrafish embryos (from 12 hpf to 72 hpf) were harvested, and the expression of notch1a (G), notch1b (F), rbpja (H), rbpjb (I) and dll4 (J) were measured by RT-PCR. (K) DLL4 attenuates the expression of UXT. The control and DLL4-deficient zebrafish embryos (from 12 hpf to 36 hpf) were harvested, and the expression of uxt was measured by RT-PCR. (L) The relative mRNA expression level of vegfaa in control and UXT-deficient embryos (from 12 hpf to 36 hpf). Data shown in D-L are the mean±s.e.m. (at least three independent experiments). (M) E-ChIP assays at 24 hpf. Embryos injected with 4 ng of control MO, 4 ng of UXT MO or Notch1b MO were probed with the antibody against UXT and polymerase II, respectively. 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). For all panels, *P<0.05; **P<0.01; ***P<0.001; ns, non-significant versus the corresponding control or as indicated.
Mentions: In light of the microarray analysis and the impact of UXT deficiency on Notch signaling activation, we reasoned that UXT might modulate the Notch signaling pathway. To explore this possibility, Notch-TP-1-luciferase reporter plasmids were introduced into UXT-deficient cells, in the presence or absence of NICD. Interestingly, knockdown of UXT potentiated the activation of the Notch-TP-1-luciferase reporter, whereas ectopic expression of UXT attenuated the same reporter expression (Fig. 5A). Notably, ectopic expression of UXT-2M displayed only a marginal effect on the expression of the Notch-TP-1-luciferase reporter (Fig. 5A), which was consistent with its inability to rescue the developmental abnormalities induced by the UXT morpholino (Fig. 1F). BMP signaling and Wnt signaling have been reported to be crucial signaling pathways modulating angiogenesis (Phng et al., 2009; Rothhammer et al., 2007). However, UXT did not affect the activation of BMP signaling or Wnt signaling, according to the results from a BRE reporter assay or a TOPflash reporter assay, respectively (Fig. 5B,C).Fig. 5.

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.

Show MeSH
Related in: MedlinePlus