Carbohydrate-binding protein CLEC14A regulates VEGFR-2– and VEGFR-3–dependent signals during angiogenesis and lymphangiogenesis
Sungwoon Lee, … , Young-Myeong Kim, Young-Guen Kwon
Sungwoon Lee, … , Young-Myeong Kim, Young-Guen Kwon
Published December 19, 2016
Citation Information: J Clin Invest. 2017;127(2):457-471. https://doi.org/10.1172/JCI85145.
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Research Article Angiogenesis Vascular biology

Carbohydrate-binding protein CLEC14A regulates VEGFR-2– and VEGFR-3–dependent signals during angiogenesis and lymphangiogenesis

Abstract

Controlled angiogenesis and lymphangiogenesis are essential for tissue development, function, and repair. However, aberrant neovascularization is an essential pathogenic mechanism in many human diseases, including diseases involving tumor growth and survival. Here, we have demonstrated that mice deficient in C-type lectin family 14 member A (CLEC14A) display enhanced angiogenic sprouting and hemorrhage as well as enlarged jugular lymph sacs and lymphatic vessels. CLEC14A formed a complex with VEGFR-3 in endothelial cells (ECs), and CLEC14A KO resulted in a marked reduction in VEGFR-3 that was concomitant with increases in VEGFR-2 expression and downstream signaling. Implanted tumor growth was profoundly reduced in CLEC14A-KO mice compared with that seen in WT littermates, but tumor-bearing CLEC14A-KO mice died sooner. Tumors in CLEC14A-KO mice had increased numbers of nonfunctional blood vessels and severe hemorrhaging. Blockade of VEGFR-2 signaling suppressed these vascular abnormalities and enhanced the survival of tumor-bearing CLEC14A-KO mice. We conclude that CLEC14A acts in vascular homeostasis by fine-tuning VEGFR-2 and VEGFR-3 signaling in ECs, suggesting its relevance in the pathogenesis of angiogenesis-related human disorders.

Authors

Sungwoon Lee, Seung-Sik Rho, Hyojin Park, Jeong Ae Park, Jihye Kim, In-Kyu Lee, Gou Young Koh, Naoki Mochizuki, Young-Myeong Kim, Young-Guen Kwon

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Figure 3

CLEC14A deficiency attenuates VEGFR-3 expression, promotes VEGFR-2 expression, and forms a CLEC14A–VEGFR-3 complex via the CLEC14A cytosolic domain in ECs.

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CLEC14A deficiency attenuates VEGFR-3 expression, promotes VEGFR-2 expre...
(A–C) Relative GAPDH-normalized mRNA levels of human CLEC14A (hCLEC14A), hVEGFR-3, and hVEGFR-2 after CLEC14A silencing in HUVECs. (D) Reduced VEGFR-3 and increased VEGFR-2 protein expression in HUVECs after silencing of CLEC14A with 50 nM CLEC14A siRNA. (E and F) Confocal images and quantification of relative VEGFR-3 staining intensity in P5 retinae from WT and CLEC14A-KO mice, demonstrating reduced VEGFR-3 in CLEC14A-KO retinae (percentage of control). Scale bars: 500 μm. (G and H) Confocal images and quantification of relative VEGFR-2 intensity of P5 retinae from WT and CLEC14A-KO mice showing increased VEGFR-2 expression (percentage of control). n = 6 per group. Scale bars: 100 μm. (I and J) Decreased VEGFR-3 and increased VEGFR-2 protein expression in retinal and aortal lysates isolated from P5 WT and CLEC14A-KO pups. (K) VEGFR-3, CLEC14A, and VEGFR-2 protein expression in HUVECs after silencing of VEGFR-3 with 50 nM siRNA. VEGFR-3 silencing reduced CLEC14A expression and promoted VEGFR-2 expression. (L) Co-IP of endogenous CLEC14A with VEGFR-3 but not with VEGFR-2 in HUVECs. (M) IP assay showing that overexpressed GFP-tagged CLEC14A (GFP-CL14A) bound to VEGFR-3 but not to VEGFR-2 in HUVECs. Western blot shows transfection of GFP-Mock and GFP-CLEC14A in HUVECs. (N) Clec14a deletion series shows that VEGFR-3 interacted with the cytosolic domain of CLEC14A. IP analysis shows deletion mutants of GFP-tagged CLEC14A (GFP-Mock, GFP-CL14A-ΔLEC, GFP-CL14A-ΔEGF-like, GPF-CL14A-ΔCyto, and GFP-CL14A-FL) interacting with VEGFR-3. The deletion mutants were transfected into HUVECs and immunoprecipitated with anti-GFP antibody or IgG. Western blot analysis of cell lysates after transfection of each deletion mutant into HUVECs. All experiments were repeated at least 4 times with similar results. *P < 0.05, **P < 0.005, and ***P < 0.0001, by paired, 2-tailed Student’s t test. SC, scrambled siRNA; FL, full-length; WB, Western blot; WCL, whole-cell lysate. Error bars represent the mean ± SD.