Non-proteolytic calpain-6 interacts with VEGFA and promotes angiogenesis by increasing VEGF secretion.

Angiogenesis is involved in both normal physiological and pathological conditions. Vascular endothelial growth factor (VEGF) is a major factor for promoting angiogenesis. The current anti-VEGF therapies have limited efficacy and significant adverse effects. To find novel targets of VEGFA for angiogenesis inhibition, we performed yeast two-hybrid screening and identified calpain-6 as a novel VEGFA-interaction partner and confirmed the endogenous VEGFA-calpain-6 interaction in mammalian placenta. A domain mapping study revealed that the Gly321-Asp500 domain in calpain-6 is required for the interaction with the C-terminus of the VEGFA protein. The functional significance of the VEGFA-calpain-6 interaction was explored by assessing its effect on angiogenesis in vitro. Whereas forced overexpression of calpain-6 increased the secretion of the VEGF protein and tube formation, knockdown of calpain-6 expression abrogated the calpain-6-mediated VEGF secretion and tube formation in HUVECs. Consistent with the domain mapping result, overexpressing calpain-6 without the VEGFA-interacting domain III (Gly321-Asp500) failed to increase the secretion of VEGF protein. Our results identify calpain-6, an unconventional non-proteolytic calpain, as a novel VEGFA-interacting protein and demonstrate that their interaction is necessary to enhance VEGF secretion. Thus, calpain-6 might be a potential molecular target for angiogenesis inhibition in many diseases.