Direct transcriptional regulation of neuropilin-2 by COUP-TFII modulates multiple steps in murine lymphatic vessel development
Fu-Jung Lin, … , Ming-Jer Tsai, Sophia Y. Tsai
Fu-Jung Lin, … , Ming-Jer Tsai, Sophia Y. Tsai
Published April 1, 2010
Citation Information: J Clin Invest. 2010;120(5):1694-1707. https://doi.org/10.1172/JCI40101.
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Direct transcriptional regulation of neuropilin-2 by COUP-TFII modulates multiple steps in murine lymphatic vessel development

Abstract

The lymphatic system plays a key role in tissue fluid homeostasis. Lymphatic dysfunction contributes to the pathogenesis of many human diseases, including lymphedema and tumor metastasis. However, the mechanisms regulating lymphangiogenesis remain largely unknown. Here, we show that COUP-TFII (also known as Nr2f2), an orphan member of the nuclear receptor superfamily, mediates both developmental and pathological lymphangiogenesis in mice. Conditional ablation of COUP-TFII at an early embryonic stage resulted in failed formation of pre-lymphatic ECs (pre-LECs) and lymphatic vessels. COUP-TFII deficiency at a late developmental stage resulted in loss of LEC identity, gain of blood EC fate, and impaired lymphatic vessel sprouting. siRNA-mediated downregulation of COUP-TFII in cultured primary human LECs demonstrated that the maintenance of lymphatic identity and VEGF-C–induced lymphangiogenic activity, including cell proliferation and migration, are COUP-TFII–dependent and cell-autonomous processes. COUP-TFII enhanced the pro-lymphangiogenic actions of VEGF-C, at least in part by directly stimulating expression of neuropilin-2, a coreceptor for VEGF-C. In addition, COUP-TFII inactivation in a mammary gland mouse tumor model resulted in inhibition of tumor lymphangiogenesis, suggesting that COUP-TFII also regulates neo-lymphangiogenesis in the adult. Thus, COUP-TFII is a critical factor that controls lymphangiogenesis in embryonic development and tumorigenesis in adults.

Authors

Fu-Jung Lin, Xinpu Chen, Jun Qin, Young-Kwon Hong, Ming-Jer Tsai, Sophia Y. Tsai

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Figure 6

COUP-TFII knock down impairs in vitro lymphangiogenesis, VEGF-C–induced cell proliferation, and migration of LECs.

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COUP-TFII knock down impairs in vitro lymphangiogenesis, VEGF-C–induced...
(A) Human primary LECs were transfected with control or COUP-TFII–pooled or 2 different COUP-TFII siRNAs. Cell lysates were subjected to Western blotting with antibodies against COUP-TFII and β-actin. (B) Representative images and statistical summary of 3-dimensional in vitro lymphangiogenesis assays, with spheroids generated from control- or COUP-TFII–pooled siRNA– or 2 different COUP-TFII siRNA–treated LECs. The cumulative sprout length was quantified after 24 hours. Extensive endothelial outgrowth can be observed in control spheroids but not in COUP-TFII–silenced spheroids. Scale bars: 100 μm. Error bars indicate SD. ***P < 0.01. (C) Cell proliferation assay measuring LEC response to increasing concentrations (0, 58, 250 ng/ml) of VEGF-C. Stimulation of scrambled siRNA-transfected but not COUP-TFII–pooled siRNA–transfected LECs induces a dose-dependent increase in cell proliferation. Error bars indicate SD. *P < 0.05, ***P < 0.01. (D) Wound scratch assay of LEC response to 250 ng/ml VEGF-C. The edges of the wound scratch are shown by dashed lines.