Endothelial ERK signaling controls lymphatic fate specification
Yong Deng, … , Anne Eichmann, Michael Simons
Yong Deng, … , Anne Eichmann, Michael Simons
Published February 8, 2013
Citation Information: J Clin Invest. 2013;123(3):1202-1215. https://doi.org/10.1172/JCI63034.
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Research Article Vascular biology

Endothelial ERK signaling controls lymphatic fate specification

Abstract

Lymphatic vessels are thought to arise from PROX1-positive endothelial cells (ECs) in the cardinal vein in response to induction of SOX18 expression; however, the molecular event responsible for increased SOX18 expression has not been established. We generated mice with endothelial-specific, inducible expression of an RAF1 gene with a gain-of-function mutation (RAF1S259A) that is associated with Noonan syndrome. Expression of mutant RAF1S259A in ECs activated ERK and induced SOX18 and PROX1 expression, leading to increased commitment of venous ECs to the lymphatic fate. Excessive production of lymphatic ECs resulted in lymphangiectasia that was highly reminiscent of abnormal lymphatics seen in Noonan syndrome and similar “RASopathies.” Inhibition of ERK signaling during development abrogated the lymphatic differentiation program and rescued the lymphatic phenotypes induced by expression of RAF1S259A. These data suggest that ERK activation plays a key role in lymphatic EC fate specification and that excessive ERK activation is the basis of lymphatic abnormalities seen in Noonan syndrome and related diseases.

Authors

Yong Deng, Deepak Atri, Anne Eichmann, Michael Simons

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Figure 1

Endothelial-specific expression of RAF1S259A blocks RAF1-AKT crosstalk and activates ERK.

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Endothelial-specific expression of RAF1S259A blocks RAF1-AKT crosstalk a...
(A) RAF1-AKT crosstalk. Upon extracellular signal stimulation, AKT phosphorylates RAF1 at Ser259 and inhibits RAF1 activation. (B) Scheme of RAF1 phosphorylation sites. (C) Western blot demonstrates ERK1/2 activation by RAF1S259A. Serum-starved HUVECs transduced with empty control, wild-type HA-RAF1 (WT), or HA-RAF1S259A (S259A) lentiviruses were stimulated with 50 ng/ml VEGF-A164 for the indicated times. (D) ERK activation shown in (C) was quantified by densitometry and is represented as a ratio of pERK1/2 to total ERK1/2. (E) Scheme of construct for TRE-RAF1S259A transgenic mice. (F) Western blot showing RAF1 expression in purified primary lung ECs. Densitometry of RAF1 levels compared with those of β-tubulin is shown on the right. Data represent the mean ± SEM of 3 independent experiments. (G) X-gal staining of E9.5 and E10.5 embryos. Scale bars: 200 μm (E9.5) and 2 mm (E10.5).