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The ASK1-Signalosome regulates p38 MAPK activity in response to levels of endogenous oxidative stress in the Klotho mouse models of aging.

Hsieh CC, Kuro-o M, Rosenblatt KP, Brobey R, Papaconstantinou J - Aging (Albany NY) (2010)

Bottom Line: Reactive oxygen species (ROS) and elevated levels of p38 MAPK activity accelerate physiological aging.Our results suggest that increased endogenous ROS generated by Klotho(-/-) and resistance to oxidative stress in Klotho overexpression are linked to the regulation of ASK1-signalosome -> p38 activity.We conclude that the Klotho suppressor-of-aging activity is linked to the ASK1-signalsome, a physiological ROS-sensitive signaling center.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.

ABSTRACT
Reactive oxygen species (ROS) and elevated levels of p38 MAPK activity accelerate physiological aging. This emphasizes the importance of understanding the molecular mechanism(s) that link ROS production to activation of the p38 mediated promotion of aging, longevity, and resistance to oxidative stress. We examined Klotho(-/-) (elevated ROS) and Klotho overexpressing mice (low ROS and resistance to ROS) to determine whether the ROS-sensitive apoptosis signal-regulating kinase (ASK1)-signalosome -> p38 MAPK pathway plays a role in the accelerated aging of Klotho(-/-), and resistance to oxidative stress and extended lifespan in the Klotho overexpressing models. Our results suggest that increased endogenous ROS generated by Klotho(-/-) and resistance to oxidative stress in Klotho overexpression are linked to the regulation of ASK1-signalosome -> p38 activity. We propose that (a) the ASK1-signalosome -> p38 MAPK pathway is activated by oxidative stress due to ablation of the Klotho gene; (b) increased longevity by Klotho overexpression is linked to suppression of the ASK1-signalosome-p38 MAPK activity; (c) the ROS-responsive ASK1-signalosome regulates physiological aging via its regulation of p38 MAPK, through a mechanism that balances the levels of inhibitory vs. activating ASK1-signalosomes. We conclude that the Klotho suppressor-of-aging activity is linked to the ASK1-signalsome, a physiological ROS-sensitive signaling center.

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Klotho overexpression attenuates the ASK1-signalosome - p38 pathway.(A) Overexpression of Klotho mediates increased levels of the inhibitory (SH)2Trx-ASK1 complex. Western blot analysis of the levels of Trx co-immunoprecipitated with anti-ASK1 antibody in liver extracts of Klotho overexpressing (EFmKL46) and wild-type (WT Hyb) mice. (B, C) MKK3/MKK6 activity is downregulated in the Klotho overexpressing models. Western blot analysis of levels of the MKK3/6 P-Ser189/207 in livers of (B) Klotho overexpressing EFmKL46 (#29, 30, 31) and wild-type (WT Hyb; #26, 27, 28) mice and (C) Klotho overexpressing EFmKL48 (#652, 653, 654, 655); and (D) p38 MAPK activity is downregulated by Klotho overexpression. Western blot analysis of levels of p38 MAPK catalytic site amino acids (P-Thr180/Tyr182 ) in livers of WT Hyb (#26,27,28) Klotho over-expressing mice (EFmKL46 ; #29,30,31).
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Figure 2: Klotho overexpression attenuates the ASK1-signalosome - p38 pathway.(A) Overexpression of Klotho mediates increased levels of the inhibitory (SH)2Trx-ASK1 complex. Western blot analysis of the levels of Trx co-immunoprecipitated with anti-ASK1 antibody in liver extracts of Klotho overexpressing (EFmKL46) and wild-type (WT Hyb) mice. (B, C) MKK3/MKK6 activity is downregulated in the Klotho overexpressing models. Western blot analysis of levels of the MKK3/6 P-Ser189/207 in livers of (B) Klotho overexpressing EFmKL46 (#29, 30, 31) and wild-type (WT Hyb; #26, 27, 28) mice and (C) Klotho overexpressing EFmKL48 (#652, 653, 654, 655); and (D) p38 MAPK activity is downregulated by Klotho overexpression. Western blot analysis of levels of p38 MAPK catalytic site amino acids (P-Thr180/Tyr182 ) in livers of WT Hyb (#26,27,28) Klotho over-expressing mice (EFmKL46 ; #29,30,31).

Mentions: Our previous studies demonstrated that dissociation of the inhibitory (SH)2Trx-ASK1 complex is stimulated by mitochondrial dysfunction due to rotenone-mediated inhibition of CI [21]. In addition we demonstrated that the levels of the complex are significantly decreased in aged (24-mo) compared to young (3-4-mo) C57BL/6 mice and that the levels of the inhibitory complex are significantly higher in livers of young (3-4 mo) and aged (24 mo) Snell dwarf mice at all ages. Furthermore, we demonstrated that the inhibitory complex is significantly higher in dermal fibroblasts derived from young (3-4 mo), middle aged (12-14 mo) and aged (20-24 mo) compared to fibroblasts derived from wild-type mice of the same ages and genetic background [31]. These data support our hypothesis that the ASK1-signalosome may serve as a ROS sensory distribution center that communicates changes in endogenous ROS levels to signaling pathways regulating physiological characteristics of aging. Based on these studies we propose that the levels of the inhibitory (SH)2Trx-ASK1 complex are indicative of the levels of endogenous oxidative stress. Thus, since the Klotho gene regulates endogenous oxidative stress we conducted experiments to determine whether the overexpression of Klotho would alter the pool levels of this inhibitory complex. To conduct these experiments we used two independent transgenic mouse lines, EFmKL46 and EFmKL48 that overexpress Klotho under the control of the human elongation factor 1α promoter. The data in Figure 2A clearly show that the level of the inhibitory (SH)2Trx-ASK1 complex is significantly elevated (~4.5-fold) in the EFmKL46 and ~6-fold in the EFmKL48 overexpressing models (Figure 3B). These data are consistent with our results showing that the complex levels in Klotho(-/-) are decreased and support our hypothesis that the complex may serve as a major ROS-sensitive center of distribution of signals of oxidative stress.


The ASK1-Signalosome regulates p38 MAPK activity in response to levels of endogenous oxidative stress in the Klotho mouse models of aging.

Hsieh CC, Kuro-o M, Rosenblatt KP, Brobey R, Papaconstantinou J - Aging (Albany NY) (2010)

Klotho overexpression attenuates the ASK1-signalosome - p38 pathway.(A) Overexpression of Klotho mediates increased levels of the inhibitory (SH)2Trx-ASK1 complex. Western blot analysis of the levels of Trx co-immunoprecipitated with anti-ASK1 antibody in liver extracts of Klotho overexpressing (EFmKL46) and wild-type (WT Hyb) mice. (B, C) MKK3/MKK6 activity is downregulated in the Klotho overexpressing models. Western blot analysis of levels of the MKK3/6 P-Ser189/207 in livers of (B) Klotho overexpressing EFmKL46 (#29, 30, 31) and wild-type (WT Hyb; #26, 27, 28) mice and (C) Klotho overexpressing EFmKL48 (#652, 653, 654, 655); and (D) p38 MAPK activity is downregulated by Klotho overexpression. Western blot analysis of levels of p38 MAPK catalytic site amino acids (P-Thr180/Tyr182 ) in livers of WT Hyb (#26,27,28) Klotho over-expressing mice (EFmKL46 ; #29,30,31).
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Figure 2: Klotho overexpression attenuates the ASK1-signalosome - p38 pathway.(A) Overexpression of Klotho mediates increased levels of the inhibitory (SH)2Trx-ASK1 complex. Western blot analysis of the levels of Trx co-immunoprecipitated with anti-ASK1 antibody in liver extracts of Klotho overexpressing (EFmKL46) and wild-type (WT Hyb) mice. (B, C) MKK3/MKK6 activity is downregulated in the Klotho overexpressing models. Western blot analysis of levels of the MKK3/6 P-Ser189/207 in livers of (B) Klotho overexpressing EFmKL46 (#29, 30, 31) and wild-type (WT Hyb; #26, 27, 28) mice and (C) Klotho overexpressing EFmKL48 (#652, 653, 654, 655); and (D) p38 MAPK activity is downregulated by Klotho overexpression. Western blot analysis of levels of p38 MAPK catalytic site amino acids (P-Thr180/Tyr182 ) in livers of WT Hyb (#26,27,28) Klotho over-expressing mice (EFmKL46 ; #29,30,31).
Mentions: Our previous studies demonstrated that dissociation of the inhibitory (SH)2Trx-ASK1 complex is stimulated by mitochondrial dysfunction due to rotenone-mediated inhibition of CI [21]. In addition we demonstrated that the levels of the complex are significantly decreased in aged (24-mo) compared to young (3-4-mo) C57BL/6 mice and that the levels of the inhibitory complex are significantly higher in livers of young (3-4 mo) and aged (24 mo) Snell dwarf mice at all ages. Furthermore, we demonstrated that the inhibitory complex is significantly higher in dermal fibroblasts derived from young (3-4 mo), middle aged (12-14 mo) and aged (20-24 mo) compared to fibroblasts derived from wild-type mice of the same ages and genetic background [31]. These data support our hypothesis that the ASK1-signalosome may serve as a ROS sensory distribution center that communicates changes in endogenous ROS levels to signaling pathways regulating physiological characteristics of aging. Based on these studies we propose that the levels of the inhibitory (SH)2Trx-ASK1 complex are indicative of the levels of endogenous oxidative stress. Thus, since the Klotho gene regulates endogenous oxidative stress we conducted experiments to determine whether the overexpression of Klotho would alter the pool levels of this inhibitory complex. To conduct these experiments we used two independent transgenic mouse lines, EFmKL46 and EFmKL48 that overexpress Klotho under the control of the human elongation factor 1α promoter. The data in Figure 2A clearly show that the level of the inhibitory (SH)2Trx-ASK1 complex is significantly elevated (~4.5-fold) in the EFmKL46 and ~6-fold in the EFmKL48 overexpressing models (Figure 3B). These data are consistent with our results showing that the complex levels in Klotho(-/-) are decreased and support our hypothesis that the complex may serve as a major ROS-sensitive center of distribution of signals of oxidative stress.

Bottom Line: Reactive oxygen species (ROS) and elevated levels of p38 MAPK activity accelerate physiological aging.Our results suggest that increased endogenous ROS generated by Klotho(-/-) and resistance to oxidative stress in Klotho overexpression are linked to the regulation of ASK1-signalosome -> p38 activity.We conclude that the Klotho suppressor-of-aging activity is linked to the ASK1-signalsome, a physiological ROS-sensitive signaling center.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.

ABSTRACT
Reactive oxygen species (ROS) and elevated levels of p38 MAPK activity accelerate physiological aging. This emphasizes the importance of understanding the molecular mechanism(s) that link ROS production to activation of the p38 mediated promotion of aging, longevity, and resistance to oxidative stress. We examined Klotho(-/-) (elevated ROS) and Klotho overexpressing mice (low ROS and resistance to ROS) to determine whether the ROS-sensitive apoptosis signal-regulating kinase (ASK1)-signalosome -> p38 MAPK pathway plays a role in the accelerated aging of Klotho(-/-), and resistance to oxidative stress and extended lifespan in the Klotho overexpressing models. Our results suggest that increased endogenous ROS generated by Klotho(-/-) and resistance to oxidative stress in Klotho overexpression are linked to the regulation of ASK1-signalosome -> p38 activity. We propose that (a) the ASK1-signalosome -> p38 MAPK pathway is activated by oxidative stress due to ablation of the Klotho gene; (b) increased longevity by Klotho overexpression is linked to suppression of the ASK1-signalosome-p38 MAPK activity; (c) the ROS-responsive ASK1-signalosome regulates physiological aging via its regulation of p38 MAPK, through a mechanism that balances the levels of inhibitory vs. activating ASK1-signalosomes. We conclude that the Klotho suppressor-of-aging activity is linked to the ASK1-signalsome, a physiological ROS-sensitive signaling center.

Show MeSH
Related in: MedlinePlus