Anti-apoptotic and anti-senescence effects of Klotho on vascular endothelial cells

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Abstract

Klotho-mutated mice manifest multiple age-related disorders that are observed in humans. A recent study suggested that Klotho protein might function as an anti-aging hormone in mammals. Because it has been reported that apoptosis and senescence in vascular endothelial cells are closely related to the progression of atherosclerosis, we investigated Klotho’s ability to interfere with apoptosis and cellular senescence in human umbilical vascular endothelial cells (HUVEC). Klotho overexpression decreased H2O2-induced apoptosis in COS-1 cells and Jurkat cells. Klotho protein also reduced H2O2- and etoposide-induced apoptosis in HUVEC. Caspase-3 and caspase-9 activity was lower in Klotho-treated HUVEC than in control cells. Senescence-associated β-gal staining showed that Klotho protein interferes with H2O2-induced premature cellular senescence. The expression of p53 and p21 was lower in Klotho-treated cells. Our study suggests that Klotho acts as a humoral factor to reduce H2O2-induced apoptosis and cellular senescence in vascular cells.

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Methods

Materials. A mouse membrane-form Klotho cDNA was kindly donated by Professor Yoichi Nabeshima (Department of Pathology and Tumor Biology, Kyoto University Graduate School of Medicine). The PCAGGS plasmid was kindly donated by Professor Jun-ichi Miyazaki (Division of Stem Cell Regulation, Osaka University Graduate School of Medicine) [22]. Anti-Klotho rat monoclonal antibody (KM2076) was kindly donated by Kyowa Hakko Kogyo (Tokyo, Japan) [23]. All other materials were commercial products of the

Transfection efficiency and Klotho expression in COS-1 cells

To investigate the efficiency of COS-1 cell transfection, we examined the expression of Klotho protein in cell lysates from COS-1 cells. As reported recently [26], Klotho was expressed abundantly in COS-1 cells without transfection, as was demonstrated in IMCD cells (data not shown). In Klotho-transfected COS-1 cells, the expression of Klotho protein was dramatically increased (data not shown).

Klotho overexpression reduced apoptosis in COS-1 cells

It was reported that apoptotic cells were detected in the hippocampus in KL−/− mice at the age of 7

Discussion

This is the first report that purified Klotho recombinant protein has anti-apoptotic and anti-senescent effects in endothelial cells. Kuro-o et al.[1] reported that Klotho mutant mice manifest multiple age-related disorders that are also observed in humans, including atherosclerosis. It is well known that apoptosis and senescence in vascular endothelial cells initiate atherosclerosis. It has also been reported that apoptotic cells can be detected in the hippocampus in Klotho mutant mice at the

Acknowledgments

We thank Ms. Seiko Kaji for her excellent technical assistance. This work was supported by the Osaka Medical Research Foundation for Incurable Diseases (2000, 2005), NOVARTIS Foundation for Gerontological Research (2001), and Grants-in-Aid for scientific research from the Ministry of Education, Science, Sports, Culture and Technology of Japan (2003, 2004).

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