1. Klotho is associated with VEGF receptor-2 and the transient receptor potential canonical-1 Ca 2+ channel to maintain endothelial integrity
- Author
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Mitsuhiko Okigaki, Taikou Kimura, Hiroaki Matsubara, Kazuhiro Sonomura, Yasukiyo Mori, Tetsuro Kusaba, Takeshi Shirayama, Yasushi Adachi, Susumu Sasaki, Kazuhiko Ishikawa, Shuji Tanda, Masabumi Shibuya, Tsuguru Hatta, Akihiro Matui, and Manabu Murakami
- Subjects
Proteases ,medicine.medical_specialty ,media_common.quotation_subject ,Biology ,urologic and male genital diseases ,Mice ,Transient receptor potential channel ,Internal medicine ,Extracellular ,medicine ,Animals ,Internalization ,Klotho Proteins ,Klotho ,Glucuronidase ,TRPC Cation Channels ,media_common ,Binding Sites ,Multidisciplinary ,Voltage-dependent calcium channel ,Kinase insert domain receptor ,Biological Sciences ,Vascular Endothelial Growth Factor Receptor-2 ,female genital diseases and pregnancy complications ,Cell biology ,Endocrinology ,Apoptosis ,Calcium ,Calcium Channels ,Protein Binding - 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.
- Published
- 2010