Efficient activation of the lymphangiogenic growth factor VEGF-C requires the C-terminal domain of VEGF-C and the N-terminal domain of CCBE1

Sawan Kumar Jha,Michael Jeltsch,Khushbu Rauniyar,Pascal Brouillard,Miikka Vikkula,Terhi Karpanen,Kari Alitalo,Veli-Matti Leppänen

doi:10.1038/s41598-017-04982-1

Abstract

The collagen- and calcium-binding EGF domains 1 (CCBE1) protein is necessary for lymphangiogenesis. Its C-terminal collagen-like domain was shown to be required for the activation of the major lymphangiogenic growth factor VEGF-C (Vascular Endothelial Growth Factor-C) along with the ADAMTS3 (A Disintegrin And Metalloproteinase with Thrombospondin Motifs-3) protease. However, it remained unclear how the N-terminal domain of CCBE1 contributed to lymphangiogenic signaling. Here, we show that efficient activation of VEGF-C requires its C-terminal domain both in vitro and in a transgenic mouse model. The N-terminal EGF-like domain of CCBE1 increased VEGFR-3 signaling by colocalizing pro-VEGF-C with its activating protease to the lymphatic endothelial cell surface. When the ADAMTS3 amounts were limited, proteolytic activation of pro-VEGF-C was supported by the N-terminal domain of CCBE1, but not by its C-terminal domain. A single amino acid substitution in ADAMTS3, identified from a lymphedema patient, was associated with abnormal CCBE1 localization. These results show that CCBE1 promotes VEGFR-3 signaling and lymphangiogenesis by different mechanisms, which are mediated independently by the two domains of CCBE1: by enhancing the cleavage activity of ADAMTS3 and by facilitating the colocalization of VEGF-C and ADAMTS3. These new insights should be valuable in developing new strategies to therapeutically target VEGF-C/VEGFR-3-induced lymphangiogenesis.

Highlights

Vascular endothelial growth factor C (VEGF-C), the major effector of lymphangiogenesis, is indispensable for lymphatic development in mouse embryos and essential for most lymphangiogenic processes in adults[1,2,3]
In solid-phase binding assays, we found that calcium-binding EGF domains 1 (CCBE1) binds to VEGFR-3 and that this binding was dependent on the presence of the first N-terminal Ig-like domains that form the VEGF-C/ VEGF-D binding part of VEGFR-3 (Fig. 4d)
Our findings show that pro-VEGF-C becomes localized by virtue of its C-terminal domain to the extracellular matrix (ECM), notably fibronectin, and cell surfaces

Summary

Introduction

Vascular endothelial growth factor C (VEGF-C), the major effector of lymphangiogenesis, is indispensable for lymphatic development in mouse embryos and essential for most lymphangiogenic processes in adults[1,2,3]. VEGF-C is synthesized as a precursor molecule, in which the central VEGF homology domain (VHD) is flanked by amino (N)-terminal and carboxy (C)-terminal propeptides Both propeptides are proteolytically removed during the generation of the active (“mature”) form of VEGF-C4. Mutations in the CCBE1 (collagen- and calcium-binding epidermal growth factor domains 1) gene were found in a subset of patients with Hennekam lymphangiectasia-lymphedema syndrome[10]. Consistent with these data, deletion of either Ccbe[1] or Vegfc in mice completely halts lymphatic vasculature development at embryonic day (E) 10.511–13. Both Ccbe[1] null mice and mice devoid of the Ccbe[1] C-terminal collagen-like domain showed a complete lack of lymphatic structures, whereas mice devoid of the Ccbe[1] N-terminal EGF-like domain showed some clusters of lymphatic endothelial cells (LECs), which were unable to form contiguous structures, suggesting that the CCBE1 N-terminal domain is involved in the organization and migration of LECs15

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Results

Conclusion