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Glucose Oxidase Induces Cellular Senescence in Immortal Renal Cells through ILK by Downregulating Klotho Gene Expression.

Troyano-Suárez N, del Nogal-Avila M, Mora I, Sosa P, López-Ongil S, Rodriguez-Puyol D, Olmos G, Ruíz-Torres MP - Oxid Med Cell Longev (2015)

Bottom Line: Additionally, GOx reduced Klotho gene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition.We demonstrated a direct link between ILK and Klotho since silencing ILK expression in cells and mice increases Klotho expression and reduces p53 and p16 expression in renal cortex.In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reduces Klotho expression.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología de Sistemas, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain.

ABSTRACT
Cellular senescence can be prematurely induced by oxidative stress involved in aging. In this work, we were searching for novel intermediaries in oxidative stress-induced senescence, focusing our interest on integrin-linked kinase (ILK), a scaffold protein at cell-extracellular matrix (ECM) adhesion sites, and on the Klotho gene. Cultured renal cells were treated with glucose oxidase (GOx) for long time periods. GOx induced senescence, increasing senescence associated β-galactosidase activity and the expression of p16. In parallel, GOx increased ILK protein expression and activity. Ectopic overexpression of ILK in cells increased p16 expression, even in the absence of GOx, whereas downregulation of ILK inhibited the increase in p16 due to oxidative stress. Additionally, GOx reduced Klotho gene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition. We demonstrated a direct link between ILK and Klotho since silencing ILK expression in cells and mice increases Klotho expression and reduces p53 and p16 expression in renal cortex. In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reduces Klotho expression. We hereby present ILK as a novel downregulator of Klotho gene expression.

No MeSH data available.


Related in: MedlinePlus

Increased ILK expression by GOx induced decrease in Klotho expression. (a, b) MCT cells were transfected with specific siRNA for ILK (siILK) or unspecific scramble siRNA (scRNA) as control. ILK (a) and Klotho (b) mRNA expression was evaluated by RT and real time PCR. Bar graph represents the mean ± s.e.m. of three different experiments, and the results are expressed as densitometric units. (c) A conditional ILK knockout mouse model (cKO-ILK) was used and, after treatment with TX for several days, ILK deletion was confirmed versus VH treatment in those mice by PCR of genomic DNA isolated from kidney. ILK (d) and Klotho (e) mRNA expression was analyzed in kidney from WT and cKO-ILK mice by RT and real time PCR. Bar graph represents the mean ± s.e.m. of 10 animals per group. (f) p53 and p16 protein expression was analyzed in kidney from WT and cKO-ILK mice by western blot. A representative blot is shown. Bar graphs represent the densitometric analysis of the bands. The results are expressed as densitometric units and are the mean ± s.e.m. of 10 animals per group. ∗p < 0.05 versus control (scRNA in panels (a)-(b) and WT in the rest panels).
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fig5: Increased ILK expression by GOx induced decrease in Klotho expression. (a, b) MCT cells were transfected with specific siRNA for ILK (siILK) or unspecific scramble siRNA (scRNA) as control. ILK (a) and Klotho (b) mRNA expression was evaluated by RT and real time PCR. Bar graph represents the mean ± s.e.m. of three different experiments, and the results are expressed as densitometric units. (c) A conditional ILK knockout mouse model (cKO-ILK) was used and, after treatment with TX for several days, ILK deletion was confirmed versus VH treatment in those mice by PCR of genomic DNA isolated from kidney. ILK (d) and Klotho (e) mRNA expression was analyzed in kidney from WT and cKO-ILK mice by RT and real time PCR. Bar graph represents the mean ± s.e.m. of 10 animals per group. (f) p53 and p16 protein expression was analyzed in kidney from WT and cKO-ILK mice by western blot. A representative blot is shown. Bar graphs represent the densitometric analysis of the bands. The results are expressed as densitometric units and are the mean ± s.e.m. of 10 animals per group. ∗p < 0.05 versus control (scRNA in panels (a)-(b) and WT in the rest panels).

Mentions: To analyze whether there was a relationship between the reduction of Klotho and the increase in ILK expression, we analyzed Klotho expression in MCT cells transfected with specific siRNA against ILK. Cells transfected with siILK showed a stronger reduction in ILK mRNA expression (Figure 5(a)) and a stronger increase in Klotho mRNA expression (Figure 5(b)) than cells transfected with the unspecific siRNA (scRNA), which were evaluated by RT-qPCR.


Glucose Oxidase Induces Cellular Senescence in Immortal Renal Cells through ILK by Downregulating Klotho Gene Expression.

Troyano-Suárez N, del Nogal-Avila M, Mora I, Sosa P, López-Ongil S, Rodriguez-Puyol D, Olmos G, Ruíz-Torres MP - Oxid Med Cell Longev (2015)

Increased ILK expression by GOx induced decrease in Klotho expression. (a, b) MCT cells were transfected with specific siRNA for ILK (siILK) or unspecific scramble siRNA (scRNA) as control. ILK (a) and Klotho (b) mRNA expression was evaluated by RT and real time PCR. Bar graph represents the mean ± s.e.m. of three different experiments, and the results are expressed as densitometric units. (c) A conditional ILK knockout mouse model (cKO-ILK) was used and, after treatment with TX for several days, ILK deletion was confirmed versus VH treatment in those mice by PCR of genomic DNA isolated from kidney. ILK (d) and Klotho (e) mRNA expression was analyzed in kidney from WT and cKO-ILK mice by RT and real time PCR. Bar graph represents the mean ± s.e.m. of 10 animals per group. (f) p53 and p16 protein expression was analyzed in kidney from WT and cKO-ILK mice by western blot. A representative blot is shown. Bar graphs represent the densitometric analysis of the bands. The results are expressed as densitometric units and are the mean ± s.e.m. of 10 animals per group. ∗p < 0.05 versus control (scRNA in panels (a)-(b) and WT in the rest panels).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4637093&req=5

fig5: Increased ILK expression by GOx induced decrease in Klotho expression. (a, b) MCT cells were transfected with specific siRNA for ILK (siILK) or unspecific scramble siRNA (scRNA) as control. ILK (a) and Klotho (b) mRNA expression was evaluated by RT and real time PCR. Bar graph represents the mean ± s.e.m. of three different experiments, and the results are expressed as densitometric units. (c) A conditional ILK knockout mouse model (cKO-ILK) was used and, after treatment with TX for several days, ILK deletion was confirmed versus VH treatment in those mice by PCR of genomic DNA isolated from kidney. ILK (d) and Klotho (e) mRNA expression was analyzed in kidney from WT and cKO-ILK mice by RT and real time PCR. Bar graph represents the mean ± s.e.m. of 10 animals per group. (f) p53 and p16 protein expression was analyzed in kidney from WT and cKO-ILK mice by western blot. A representative blot is shown. Bar graphs represent the densitometric analysis of the bands. The results are expressed as densitometric units and are the mean ± s.e.m. of 10 animals per group. ∗p < 0.05 versus control (scRNA in panels (a)-(b) and WT in the rest panels).
Mentions: To analyze whether there was a relationship between the reduction of Klotho and the increase in ILK expression, we analyzed Klotho expression in MCT cells transfected with specific siRNA against ILK. Cells transfected with siILK showed a stronger reduction in ILK mRNA expression (Figure 5(a)) and a stronger increase in Klotho mRNA expression (Figure 5(b)) than cells transfected with the unspecific siRNA (scRNA), which were evaluated by RT-qPCR.

Bottom Line: Additionally, GOx reduced Klotho gene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition.We demonstrated a direct link between ILK and Klotho since silencing ILK expression in cells and mice increases Klotho expression and reduces p53 and p16 expression in renal cortex.In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reduces Klotho expression.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología de Sistemas, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain.

ABSTRACT
Cellular senescence can be prematurely induced by oxidative stress involved in aging. In this work, we were searching for novel intermediaries in oxidative stress-induced senescence, focusing our interest on integrin-linked kinase (ILK), a scaffold protein at cell-extracellular matrix (ECM) adhesion sites, and on the Klotho gene. Cultured renal cells were treated with glucose oxidase (GOx) for long time periods. GOx induced senescence, increasing senescence associated β-galactosidase activity and the expression of p16. In parallel, GOx increased ILK protein expression and activity. Ectopic overexpression of ILK in cells increased p16 expression, even in the absence of GOx, whereas downregulation of ILK inhibited the increase in p16 due to oxidative stress. Additionally, GOx reduced Klotho gene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition. We demonstrated a direct link between ILK and Klotho since silencing ILK expression in cells and mice increases Klotho expression and reduces p53 and p16 expression in renal cortex. In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reduces Klotho expression. We hereby present ILK as a novel downregulator of Klotho gene expression.

No MeSH data available.


Related in: MedlinePlus