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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

ILK mediates cellular senescence induced by GOx. (a) MCT cells were transfected with a plasmid coding for wild-type ILK protein (WT-ILK) at several doses (0, 1, or 2 μg). ILK and p16 proteins were evaluated by western blot. (b, c) MCT were transfected with the specific siRNA for ILK (siILK) or unspecific scramble siRNA (scRNA) as control and then treated with 2.5 mU/mL of GOx for 48 h. In those conditions, mRNA ILK was evaluated by RT and real time PCR (b) and ILK and p16 protein expression was evaluated by western blot (c). A representative blot is shown in each case. Bar graphs represent the densitometric analysis of the bands. The results are expressed as densitometric units and are the mean ± s.e.m. of the mean from five different experiments. ∗p < 0.05 versus control; #p < 0.05 versus scRNA.
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fig3: ILK mediates cellular senescence induced by GOx. (a) MCT cells were transfected with a plasmid coding for wild-type ILK protein (WT-ILK) at several doses (0, 1, or 2 μg). ILK and p16 proteins were evaluated by western blot. (b, c) MCT were transfected with the specific siRNA for ILK (siILK) or unspecific scramble siRNA (scRNA) as control and then treated with 2.5 mU/mL of GOx for 48 h. In those conditions, mRNA ILK was evaluated by RT and real time PCR (b) and ILK and p16 protein expression was evaluated by western blot (c). A representative blot is shown in each case. Bar graphs represent the densitometric analysis of the bands. The results are expressed as densitometric units and are the mean ± s.e.m. of the mean from five different experiments. ∗p < 0.05 versus control; #p < 0.05 versus scRNA.

Mentions: Next, to analyze whether there was a link between the increase in ILK and the induction of senescence, we overexpressed ILK on MCT cells by transfection with a plasmid containing a wild-type ILK protein (WT-ILK). Figure 3(a) shows how the expression of ILK increases in MCT cells transfected with WT-ILK in a dose-dependent way. MCT cells overexpressing ILK also had a strong expression of senescence gene p16, even in the absence of oxidant stress (Figure 3(a)). Moreover, ILK expression was knocked down by transfecting cells with specific siILK and then treated with 2.5 mU/mL GOx for 48 h. Transfection significantly reduced ILK mRNA expression, measured by RT-qPCR (Figure 3(b)), and also the protein content, measured by western blot (Figure 3(c)). Cells transfected with siILK did not show the expected increase in p16 expression after GOx treatment compared to cells transfected with scRNA (Figure 3(c)). Both results allow establishing a direct link between the increase in ILK protein content and increased senescence gene p16 expression.


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)

ILK mediates cellular senescence induced by GOx. (a) MCT cells were transfected with a plasmid coding for wild-type ILK protein (WT-ILK) at several doses (0, 1, or 2 μg). ILK and p16 proteins were evaluated by western blot. (b, c) MCT were transfected with the specific siRNA for ILK (siILK) or unspecific scramble siRNA (scRNA) as control and then treated with 2.5 mU/mL of GOx for 48 h. In those conditions, mRNA ILK was evaluated by RT and real time PCR (b) and ILK and p16 protein expression was evaluated by western blot (c). A representative blot is shown in each case. Bar graphs represent the densitometric analysis of the bands. The results are expressed as densitometric units and are the mean ± s.e.m. of the mean from five different experiments. ∗p < 0.05 versus control; #p < 0.05 versus scRNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4637093&req=5

fig3: ILK mediates cellular senescence induced by GOx. (a) MCT cells were transfected with a plasmid coding for wild-type ILK protein (WT-ILK) at several doses (0, 1, or 2 μg). ILK and p16 proteins were evaluated by western blot. (b, c) MCT were transfected with the specific siRNA for ILK (siILK) or unspecific scramble siRNA (scRNA) as control and then treated with 2.5 mU/mL of GOx for 48 h. In those conditions, mRNA ILK was evaluated by RT and real time PCR (b) and ILK and p16 protein expression was evaluated by western blot (c). A representative blot is shown in each case. Bar graphs represent the densitometric analysis of the bands. The results are expressed as densitometric units and are the mean ± s.e.m. of the mean from five different experiments. ∗p < 0.05 versus control; #p < 0.05 versus scRNA.
Mentions: Next, to analyze whether there was a link between the increase in ILK and the induction of senescence, we overexpressed ILK on MCT cells by transfection with a plasmid containing a wild-type ILK protein (WT-ILK). Figure 3(a) shows how the expression of ILK increases in MCT cells transfected with WT-ILK in a dose-dependent way. MCT cells overexpressing ILK also had a strong expression of senescence gene p16, even in the absence of oxidant stress (Figure 3(a)). Moreover, ILK expression was knocked down by transfecting cells with specific siILK and then treated with 2.5 mU/mL GOx for 48 h. Transfection significantly reduced ILK mRNA expression, measured by RT-qPCR (Figure 3(b)), and also the protein content, measured by western blot (Figure 3(c)). Cells transfected with siILK did not show the expected increase in p16 expression after GOx treatment compared to cells transfected with scRNA (Figure 3(c)). Both results allow establishing a direct link between the increase in ILK protein content and increased senescence gene p16 expression.

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