Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 107, Issue 45, Pages 19308-19313Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1008544107
Keywords
endothelial cell; vascular calcification
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Funding
- Ministry of Education, Culture, Sports, Science and Technology of Japan [15590778, 18590822]
- Grants-in-Aid for Scientific Research [15590778, 18590822] Funding Source: KAKEN
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Klotho is a circulating protein, and Klotho deficiency disturbs endothelial integrity, but the molecular mechanism is not fully clarified. We report that vascular endothelium in Klotho-deficient mice showed hyperpermeability with increased apoptosis and down-regulation of vascular endothelial (VE)-cadherin because of an increase in VEGF-mediated internal calcium concentration ([Ca2+] i) influx and hyperactivation of Ca2+-dependent proteases. Immunohistochemical analysis, the pull-down assay using Klotho-fixed agarose, and FRET confocal imaging confirmed that Klotho protein binds directly to VEGF receptor 2 (VEGFR-2) and endothelial, transient-receptor potential canonical Ca2+ channel 1 (TRPC-1) and strengthens the association to promote their cointernalization. An in vitro mutagenesis study revealed that the second hydrolase domain of Klotho interacts with sixth and seventh Ig domains of VEGFR-2 and the third extracellular loop of TRPC-1. In Klotho-deficient endothelial cells, VEGF-mediated internalization of the VEGFR-2/TRPC-1 complex was impaired, and surface TRPC-1 expression increased 2.2-fold; these effects were reversed by supplementation of Klotho protein. VEGF-mediated elevation of [Ca2+] i was sustained at higher levels in an extracellular Ca2+-dependent manner, and normalization of TRCP-1 expression restored the abnormal [Ca2+] i handling. These findings provide evidence that Klotho protein is associated with VEGFR-2/TRPC-1 in causing cointernalization, thus regulating TRPC-1-mediated Ca2+ entry to maintain endothelial integrity.
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