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Notch alters VEGF responsiveness in human and murine endothelial cells by direct regulation of VEGFR-3 expression
Carrie J. Shawber, … , Mihaela Skobe, Jan Kitajewski
Carrie J. Shawber, … , Mihaela Skobe, Jan Kitajewski
Published October 18, 2007
Citation Information: J Clin Invest. 2007;117(11):3369-3382. https://doi.org/10.1172/JCI24311.
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Research Article Vascular biology

Notch alters VEGF responsiveness in human and murine endothelial cells by direct regulation of VEGFR-3 expression

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Abstract

The Notch family of cell surface receptors and its ligands are highly conserved proteins that regulate cell fate determination, including those involved in mammalian vascular development. We report that Notch induces VEGFR-3 expression in vitro in human endothelial cells and in vivo in mice. In vitro, Notch in complex with the DNA-binding protein CBF-1/suppressor of hairless/Lag1 (CSL) bound the VEGFR-3 promoter and transactivated VEGFR-3 specifically in endothelial cells. Through induction of VEGFR-3, Notch increased endothelial cell responsiveness to VEGF-C, promoting endothelial cell survival and morphological changes. In vivo, VEGFR-3 was upregulated in endothelial cells with active Notch signaling. Mice heterozygous for null alleles of both Notch1 and VEGFR-3 had significantly reduced viability and displayed midgestational vascular patterning defects analogous to Notch1 nullizygous embryos. We found that Notch1 and Notch4 were expressed in normal and tumor lymphatic endothelial cells and that Notch1 was activated in lymphatic endothelium of invasive mammary micropapillary carcinomas. These results demonstrate that Notch1 and VEGFR-3 interact genetically, that Notch directly induces VEGFR-3 in blood endothelial cells to regulate vascular development, and that Notch may function in tumor lymphangiogenesis.

Authors

Carrie J. Shawber, Yasuhiro Funahashi, Esther Francisco, Marina Vorontchikhina, Yukari Kitamura, Stephanie A. Stowell, Valeriya Borisenko, Nikki Feirt, Simona Podgrabinska, Kazuko Shiraishi, Kallayanee Chawengsaksophak, Janet Rossant, Domenico Accili, Mihaela Skobe, Jan Kitajewski

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Figure 1

Notch upregulates VEGFR-3 in cultured endothelial cells.

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Notch upregulates VEGFR-3 in cultured endothelial cells.
(A) Diagram of ...
(A) Diagram of Notch protein structure. N1IC encodes the cytoplasmic domain of Notch1. N4/int-3 encodes 30 amino acids upstream of the transmembrane domain, the transmembrane domain, and the cytoplasmic domain of Notch4 fused to a HA tag. (B) Western blot analysis of Ad-LacZ or N4/int-3 endothelial cells using anti-HA antibody. (C) Activation of a CSL luciferase reporter in endothelial cells expressing N4/int-3, represented as fold induction in Ad–N4/int-3 cells relative to Ad-LacZ. Data are an average of 3 independent experiments. (D) qRT-PCR of Tie and VEGFRs in Ad-infected endothelial cells. Values were normalized to β-actin qRT-PCR. Data of 3 independent qRT-PCR analyses are represented as fold induction in Ad–N4/int-3 cells relative to Ad-LacZ. (E) Western blot analysis of cell surface proteins isolated from Ad-LacZ and Ad–N4/int-3 endothelial cells immunoblotted with anti–VEGFR-3 antibody and total cell lysates immunoblotted with anti–α-tubulin antibody. (F) Western blot analysis of cell surface proteins (CSPs) isolated from Ad-LacZ–, Ad–N4/int-3–, and Ad-N1IC–infected endothelial cells immunoblotted with antibodies against VEGFR-3 and α-tubulin. Total cell lysates (TCLs) immunoblotted with antibodies against the HA epitope of N4/int-3 (12CA5), N1IC, and α-tubulin. (G and H) VEGFR-3, Hey1, and Hey2 qRT-PCR of Jagged1/Notch4 (J1/N4) or Dll4/Notch4 (Dll4/N4) HUVEC cocultures, respectively. Values were normalized by β-actin qRT-PCR and are presented as relative values. (I) VEGFR-2 and VEGFR-3 FACS of Ad-LacZ, Ad-N1IC, and Ad–N4/int-3 BECs.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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