Klotho is associated with VEGF receptor-2 and the transient receptor potential canonical-1 Ca2+ channel to maintain endothelial integrity

Proc Natl Acad Sci U S A. 2010 Nov 9;107(45):19308-13. doi: 10.1073/pnas.1008544107. Epub 2010 Oct 21.

Abstract

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 ([Ca(2+)]i) influx and hyperactivation of Ca(2+)-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 Ca(2+) 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 [Ca(2+)]i was sustained at higher levels in an extracellular Ca(2+)-dependent manner, and normalization of TRCP-1 expression restored the abnormal [Ca(2+)]i handling. These findings provide evidence that Klotho protein is associated with VEGFR-2/TRPC-1 in causing cointernalization, thus regulating TRPC-1-mediated Ca(2+) entry to maintain endothelial integrity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Calcium / metabolism
  • Calcium Channels
  • Glucuronidase / deficiency
  • Glucuronidase / metabolism*
  • Klotho Proteins
  • Mice
  • Protein Binding
  • TRPC Cation Channels / metabolism*
  • Vascular Endothelial Growth Factor Receptor-2 / metabolism*

Substances

  • Calcium Channels
  • TRPC Cation Channels
  • Vascular Endothelial Growth Factor Receptor-2
  • Glucuronidase
  • Klotho Proteins
  • Calcium