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

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.

Figure 1.. Klotho ablation activates the ASK1-signalosome - p38 MAPK pathway.

(A) Klotho^(-/-) exhibits low levels of the inhibitory (SH)₂Trx-ASK1 complex. Western blot analysis of levels of Trx co-immunoprecipitated with ASK1 (IP: ASK1) in liver extracts of Klotho^(-/-) (129) and WT (129) mice. (B) MKK3/MKK6 activity is elevated in Klotho^(-/-). Western blot analysis of levels of MKK3/6 catalytic site amino acids, P-Ser189/207 in livers of Klotho-/- and WT 129 mice. (C) p38 activity is elevated in Klotho^(-/-). Western blot analyses of levels of the p38 MAPK catalytic site amino acids, P-Thr180/Tyr182, in livers of Klotho (-/-) (129) and WT (129) mice. The bar graphs depict the mean +/- SE of samples from Klotho^(-/-) (129; #20, 21, 22) and from WT (129; #23, 24, 25).

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-Ser^189/207 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).

Figure 3.. Binding of 14-3-3 and Trx to ASK1 is part of the inhibitory ASK1-signalosome complex.

Assembly of the inhibitory ASK1-signalosome involves the binding of ASK1 to 14-3-3 [56]. Using anti-ASK1 for co-immunoprecipitation analyses we demonstrated (A, C) 14-3-3 and (B, D) Trx are complexed with ASK1. Furthermore, the data show that the ASK1-Trx-14-3-3 complex, a characteristic of the inhibitory ASK1-signalosome, is significantly elevated in the Klotho overexpressing model. (E) Treatment of the liver extracts with the thiol-reacting reagent shows a higher level of reduced Trx in the Klotho overexpressing liver.

Figure 7.. The oxidative stress-Chronic stress cycle of aging.

Integration of the Role of the ASK1-Signalosome in ROS-Mediated Regulation of the p38 MAPK Pathway and Physiological Characteristics of Aging. [1] ROS generated by mitochondrial dysfunction; [2] the ASK1-signalosome responds to changes in levels of oxidative stress (ROS); [3] (SH)₂Trx complexes with ASK1 to form (SH)₂Trx-ASK1 complex, a component of the inASK1-signalosome; [4] the (SH)₂Trx-ASK1 complex (inASK1-siganlosome) inhibits p38 MAPK activity; (5 → 7) inhibition of p38 MAPK activity attenuates stress response gene expression and favors expression of longevity assurance genes. This is the predominating pathway of the long-lived Klotho overexpressing, Snell and Ames mice that favors resistance to oxidative stress. [8] Klotho ablation causes increased endogenous ROS, dissociation of the (SH)₂Trx-ASK1 complex to form the actASK1-signalosome. [9] ASK1 activates the p38 MAPK pathway and [10] p38 targeted genes that promote aging. (8 → 12) This is the predominant pathway of Klotho ^(-/-) that promotes accelerated aging and sensitivity to oxidative stress; [13] The activation and nuclear localization of Nrf2 in the Klotho overexpressing mice and decreased Nrf2 activity in Klotho^(-/-).

Figure 4.. The effects of Klotho^(-/-) and Klotho overexpression on the nuclear and cytoplasmic localization of Nrf2.

(A) Cytoplasmic and (B) nuclear levels of Nrf2 in the KL^(-/-) and EFmKL46 Klotho overexpressing mice.

Figure 5.. Western blot analysis of pool level of ASK1 in the livers of Klotho^(-/-) and Klotho overexpressing mice.

Western blot analyses of levels of ASK1 in the livers of (A) Klotho^(-/-)(129)and WT (129) mice and (B) the EFmKL46 Klotho overexpressing and WT mice. The bar graphs depict the mean +/- SE of samples from WT, Klotho^(-/-) and EFmKL 46 Klotho overexpressing mice.

Figure 6.. Western blot analysis of the phosphorylation of the p46- and p54-JNK.

The bar graphs and immuno blots show levels of phosphorylation of the catalytic site amino acids, P-Thr¹⁸³/Tyr¹⁸⁵, of p46- and p54-JNK in livers of Klotho ^(-/-) (129) and WT (129) mice; and (B) in EFmKL 46 Klotho overexpressing and WT mice.