Klotho is associated with VEGF receptor-2 and the transient receptor potential canonical-1 Ca2+ channel to maintain endothelial integrity.
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.
Kusaba
Tetsuro
T
Department of Cardiovascular and Renal Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.
Okigaki
Mitsuhiko
M
Matui
Akihiro
A
Murakami
Manabu
M
Ishikawa
Kazuhiko
K
Kimura
Taikou
T
Sonomura
Kazuhiro
K
Adachi
Yasushi
Y
Shibuya
Masabumi
M
Shirayama
Takeshi
T
Tanda
Shuji
S
Hatta
Tsuguru
T
Sasaki
Susumu
S
Mori
Yasukiyo
Y
Matsubara
Hiroaki
H
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels; D005966:Glucuronidase / Q000172:deficiency / Q000378:metabolism*; D000090265:Klotho Proteins; D051379:Mice; D011485:Protein Binding; D050052:TRPC Cation Channels / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*
D015220:Calcium Channels; D050052:TRPC Cation Channels; D040301:Vascular Endothelial Growth Factor Receptor-2; D005966:Glucuronidase; D000090265:Klotho Proteins; D002118:Calcium