Proteolytic activation defines distinct lymphangiogenic mechanisms for VEGFC and VEGFD
Hung M. Bui, … , Kari Alitalo, Mark L. Kahn
Hung M. Bui, … , Kari Alitalo, Mark L. Kahn
Published May 9, 2016
Citation Information: J Clin Invest. 2016;126(6):2167-2180. https://doi.org/10.1172/JCI83967.
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Research Article Vascular biology

Proteolytic activation defines distinct lymphangiogenic mechanisms for VEGFC and VEGFD

Abstract

Lymphangiogenesis is supported by 2 homologous VEGFR3 ligands, VEGFC and VEGFD. VEGFC is required for lymphatic development, while VEGFD is not. VEGFC and VEGFD are proteolytically cleaved after cell secretion in vitro, and recent studies have implicated the protease a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS3) and the secreted factor collagen and calcium binding EGF domains 1 (CCBE1) in this process. It is not well understood how ligand proteolysis is controlled at the molecular level or how this process regulates lymphangiogenesis, because these complex molecular interactions have been difficult to follow ex vivo and test in vivo. Here, we have developed and used biochemical and cellular tools to demonstrate that an ADAMTS3-CCBE1 complex can form independently of VEGFR3 and is required to convert VEGFC, but not VEGFD, into an active ligand. Consistent with these ex vivo findings, mouse genetic studies revealed that ADAMTS3 is required for lymphatic development in a manner that is identical to the requirement of VEGFC and CCBE1 for lymphatic development. Moreover, CCBE1 was required for in vivo lymphangiogenesis stimulated by VEGFC but not VEGFD. Together, these studies reveal that lymphangiogenesis is regulated by two distinct proteolytic mechanisms of ligand activation: one in which VEGFC activation by ADAMTS3 and CCBE1 spatially and temporally patterns developing lymphatics, and one in which VEGFD activation by a distinct proteolytic mechanism may be stimulated during inflammatory lymphatic growth.

Authors

Hung M. Bui, David Enis, Marius R. Robciuc, Harri J. Nurmi, Jennifer Cohen, Mei Chen, Yiqing Yang, Veerpal Dhillon, Kathy Johnson, Hong Zhang, Robert Kirkpatrick, Elizabeth Traxler, Andrey Anisimov, Kari Alitalo, Mark L. Kahn

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Figure 7

VEGFC, ADAMTS3, and CCBE1 form a molecular complex prior to VEGFC cleavage.

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VEGFC, ADAMTS3, and CCBE1 form a molecular complex prior to VEGFC cleava...
(A) VEGFC did not directly bind CCBE1. VEGFC VHD-FLAG and CCBE1-V5 conditioned media were mixed prior to immunoprecipitation with anti-V5 Abs and to immunoblot analysis with anti-FLAG Abs. Precipitated CCBE1-V5 is shown in the bottom panel. (B) ADAMTS3 coprecipitated with full-length, but not N-terminal, CCBE1. The indicated conditioned media were mixed, and CCBE1-V5 was immunoprecipitated. Coprecipitated ADAMTS3-HA is shown at the top, and precipitated CCBE1-V5 proteins are shown at the bottom. (C) VEGFC coprecipitated with ADAMTS3 in a CCBE1-dependent manner. Conditioned media containing ADAMTS3-HA and VEGFC-V5 were mixed alone and with media containing untagged CCBE1 prior to anti-V5 immunoprecipitation. (D) ADAMTS3-CCBE1 release of VEGFC required ADAMTS3 enzymatic activity. VEGFC coprecipitation with ADAMTS3 was performed in the presence of untagged CCBE1, as described in C, at 4°C and 37°C and in the presence of EDTA to inhibit metalloprotease activity. Note that VEGFC was retained at 4°C and in the presence of EDTA, conditions that block ADAMTS3 enzymatic activity. Data shown are representative of 3 separate experiments.