Foxc1 and Foxc2 deletion causes abnormal lymphangiogenesis and correlates with ERK hyperactivation
Anees Fatima, … , Yoh-suke Mukouyama, Tsutomu Kume
Anees Fatima, … , Yoh-suke Mukouyama, Tsutomu Kume
Published May 23, 2016
Citation Information: J Clin Invest. 2016;126(7):2437-2451. https://doi.org/10.1172/JCI80465.
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Research Article Angiogenesis Vascular biology

Foxc1 and Foxc2 deletion causes abnormal lymphangiogenesis and correlates with ERK hyperactivation

Abstract

The lymphatic vasculature is essential for maintaining interstitial fluid homeostasis, and dysfunctional lymphangiogenesis contributes to various pathological processes, including inflammatory disease and tumor metastasis. Mutations in FOXC2 are dominantly associated with late-onset lymphedema; however, the precise role of FOXC2 and a closely related factor, FOXC1, in the lymphatic system remains largely unknown. Here we identified a molecular cascade by which FOXC1 and FOXC2 regulate ERK signaling in lymphatic vessel growth. In mice, lymphatic endothelial cell–specific (LEC-specific) deletion of Foxc1, Foxc2, or both resulted in increased LEC proliferation, enlarged lymphatic vessels, and abnormal lymphatic vessel morphogenesis. Compared with LECs from control animals, LECs from mice lacking both Foxc1 and Foxc2 exhibited aberrant expression of Ras regulators, and embryos with LEC-specific deletion of Foxc1 and Foxc2, alone or in combination, exhibited ERK hyperactivation. Pharmacological ERK inhibition in utero abolished the abnormally enlarged lymphatic vessels in FOXC-deficient embryos. Together, these results identify FOXC1 and FOXC2 as essential regulators of lymphangiogenesis and indicate a new potential mechanistic basis for lymphatic-associated diseases.

Authors

Anees Fatima, Ying Wang, Yutaka Uchida, Pieter Norden, Ting Liu, Austin Culver, William H. Dietz, Ford Culver, Meredith Millay, Yoh-suke Mukouyama, Tsutomu Kume

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Figure 5

FOXC1 and FOXC2 regulate lymphatic vessel development by controlling the Ras/ERK signaling pathway.

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FOXC1 and FOXC2 regulate lymphatic vessel development by controlling the...
(A) Reduced expression of Rasa4 and Rasal3 in Foxc1/c2-double mutant LECs isolated from the dorsal skin at E15.5. Graphs show RPKM values from RNA-seq analysis. (B and C) Putative FOXC-binding sites in the human RASA4 (B) and RASAL3 (C) loci, as viewed on the UCSC Human Genome Browser (http://genome.ucsc.edu; ref. 71). Red boxes indicate evolutionary conserved regions (ECRs) containing FOXC-binding sites between human and mouse. (D and E) ChIP showing specific binding of FOXC1 (D) and FOXC2 (E) to the consensus FOXC-binding sites within ECRs in RASA4 and RASAL3 in human LECs (HMVEC-dLyNeo). Asterisks indicate FOXC-specific binding. Note that specific antibodies against FOXC1 (left, Abcam; right Origene) and FOXC2 (left, Santa Cruz Biotechnology Inc.; right, Abcam) were used in C and D, respectively. (F) qPCR analysis of relative mRNA levels of FOXC2 in HUVECs transfected with negative control siRNA or siRNA targeting FOXC2. P values were obtained by 2-tailed Student’s t test. Data are reported as mean ± SEM. n = 3. **P < 0.01. (G) HUVECs transfected with negative control or FOXC2 siRNA were serum deprived and stimulated with VEGF where indicated prior to lysis and pull down of GTP-associated Ras. Detection of active Ras pull down was performed by Western blotting, and a representative image of 3 separate experiments is shown.