Activation of senescence and aging characteristics by mitochondrially generated ROS

The elevated and sustained expression of stress-response signaling pathways (p38 MAPK, SAPK/JNK) is a major physiological characteristic of aged tissues.^1-3 This age-associated state of chronic stress is attributed to mitochondrial electron transport chain (mETC) dysfunction and is a key factor that promotes stress-induced-aging characteristics (SIAC), declining tissue function and age-associated diseases. Endogenous reactive oxygen species (ROS) are important factors whose modulation regulates the signaling pathway activities that control the development of aging and longevity phenotypes.^5-7 Mitochondria have been identified as a major source of age-associated ROS,^7-9 thus emphasizing the importance of understanding the mechanism that links mitochondrial dysfunction to ROS-sensitive signaling that promotes the characteristics of senescence, aging or longevity.

The fact that ∼90% of age-associated ROS originate from mitochondrial dysfunction raises the question of whether mETC-generated ROS play a role in the induction of SIAC via the activation of p38 MAPK. Our hypothesis linking mETC-generated ROS to the activation of SIAC proposes that the ROS-responsive ASK1-signalosome activates the ASK1 → p38 pathway, thus promoting its downstream targets of senescence and aging, e.g., p16^Ink4a and p19^Arf (Fig. 1). This suggests that ROS generated by mETC (CI, CII and CIII) dysfunction may activate SIAC via the ASK1-signalosome → p38 MAPK pathway.^10-15

Figure 1.

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)₂ Trx complexes with ASK1 to form (SH)₂Trx-ASK1 complex, a component of the inASK1-signalosome; (4) the (SH)₂Trx-ASK1 complex (inASK1-signalosome) inhibits p38 MaPK activity; (5 → 7) inhibition of p38 MAPK activity attenuates stress response gene expression and favors expression of longevity assurance genes and resistance to oxidtive stress. (8) Increased endogenous ROS stimulates 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 that promotes accelerated aging and sensitivity to oxidative stress. (13) The in vitro and in vivo attenuation of SIAC associated with extended lifespan or the activation of SIAC associated with aging and senescence.

The ASK1-signalosome is a ROS-sensitive, high molecular weight (HMW) protein complex (∼1,500 kDa) that serves as a center of distribution for ROS signals.¹¹ It consists of ROS-sensitive inhibitor and activator proteins that together regulate the activation or repression of the p38 MAPK pathway (Fig. 1). ASK1 (a 170 kDa protein) provides the critical binding domains that form the ASK1-signalosome, which includes an inhibitory N-terminal domain that binds (SH)₂Trx, a kinase domain and a C-terminal regulatory domain that activates responses to ROS. ASK1 activation involves dimerization and autophosphorylation at Thr⁸⁴⁵ (in the kinase domain activation loop). Activated ASK1 recruits and activates its downstream targets, MKK3/6 (p38 activation) and MKK4/7 (JNK activation).¹² The mechanism of regulation of p38 MAPK activity in response to mETC-generated ROS involves activation of the ASK1-signalosome, which is mediated by the dissociation of a reduced thioredoxin-ASK1complex.^10,13-15 By this mechanism the reduced thioredoxin-ASK1 complex [(SH)₂ Trx-ASK1] inhibitory form of the ASK1-signalosome (inASK1-signalo-some) serves as a downregulator of the ASK1 → p38 MAPK pathway^13,15 (Fig. 1). Thus, the inASK1-signalosome exists as an inactive cytoplasmic HMW complex in unstressed cells; the ROS-mediated oxidation of ASK1-bound Trx(SH)₂ stimulates dissociation of the complex, thereby forming the activating ASK1-signalosome (actASK1-signalosome) that activates the ASK1 → p38 MAPK pathway.^13,16 The ASK1 signalosome also includes 3 regulatory C-terminal domains. These are (a) 14-3-3, an inhibitory sequestering docking site;¹⁷ (b) AIP1, the ASK1-interacting protein that responds to ROS by facilitating the release of ASK1 from its 14-3-3 inhibitor;¹⁸ and (c) homodomain interacting protein kinase 1 (HIPK1), a nuclear sumoylated protein that is desumoylated in response to ROS and translocated to the cytoplasm, where it associates with AIP1-ASK1 and induces the release of Trx and 14-3-3 from ASK1.¹⁹ All of these regulatory proteins form the ROS-sensitive ASK1-signalosome, which is an ROS sensory center that distributes ROS signals to pathways that promote senescence and aging.

Our previous studies have shown that the (SH)₂Trx-ASK1 complex in the in ASK1-signalosme is dissociated by ETC-generated ROS, i.e., rotenone (ROT), an inhibitor of complex I (CI); 3-nitro-propionic acid (3-NPA), an inhibitor of complex II (CII); and antimycin A (AA), an inhibitor of complex III (CIII), thus linking mitochondrially generated ROS to the activation of ASK1 and its downstream substrates MKK3, MKK6 and p38 MAPK.^10,15 By linking mitochondrially generated ROS to p38 MAPK activation, we have presented a potential mechanism that would link ROS-mediated activation of senescence pathways (p16^Ink4a and p19^Arf) via p38 MAPK (Fig. 1), and which explains the physiological processes that sustain elevated p38 MAPK activity in aged tissues thereby promoting aging characteristics.¹⁰

We have proposed that the mechanism of age-associated activity of many of the stress response genes may be a consequence of sustained elevated p38 MAPK activity.^1,2,15 The physiological environment caused by oxidative stress is favorable for the maintenance of high levels of the actASK1-signalosome and elevated and sustained levels of p38 MAPK activity, thereby promoting characteristics of senescence in vitro and aging in vivo. Thus we propose that the consequences of sustained elevated p38 MAPK activity enforce a chronic stimulation of the downstream signals that induce senescence and aging.

The endogenous levels of the inASK1-sigalosome in resting, unchallenged cells are activated in response to mETC-generated ROS by dissociation of the (SH)₂ Trx-ASK1 complex at the N-terminus. Our studies indicate that this may be part of the molecular mechanism that either elevates or decreases the endogenous activity of p38 MAPK pathway in aged mice. Our studies with Snell and Ames long-lived mice have shown that the endogenous level of the inASK1-signalosome is significantly higher in these oxidative stress-resistant long-lived models and in Ames dwarf fibroblasts in culture.^10,15 Our current studies have shown a similar phenomenon in the Klotho-overexpressing model, an example of resistance to oxidative stress and extended lifespan and a dramatically increased level of the actASK1-signalosome in the oxidatively stressed Klotho(-/-) model, an example of decreased lifespan due to mitochondrial dysfunction (Hsieh CC, et al. unpublished data). These data are consistent with our proposal that lower ASK1 → p38 MAPK activity is a mechanistic characteristic of the resistance to oxidative stress seen in mouse models of longevity.

Ink4a and Arf (components of the Cdkn2a tumor suppressor locus) are cell cycle inhibitors and promoters of senescence and aging.²⁰ The activities of these genes increases markedly with aging in several murine tissues and are modulated by anti-aging factors such as caloric restriction.^21,22 We propose that by sustaining the elevated level of p38 MAPK activity, this chronic stimulation promotes the downstream signals of senescence and aging.

Several lines of evidence support a role for p38 MAPK in regulating the expression of p16^Ink4a and p19^Arf. This includes the activation of p16^Ink4a by overexpress-ing MKK3/6.²³ The role of p38 in activating these cell cycle inhibitors has also been convincingly demonstrated by the development of a dominant-negative allele (p38^AF) in which the phosphorylation sites Thr¹⁸⁰ and Tyr¹⁸² are mutated.²⁴ The heterozygous p38^AF/+ mice show a marked attenuation of p38-dependent signaling and age-induced expression of these cell cycle inhibitors (p16^Ink4a and p19^Arf) in different organs, as well as in mouse embryonic fibroblasts (MEFs) in culture. Furthermore, aged p38^AF/+ mice show enhanced proliferation and regeneration of pancreatic islet cells when compared to their wild-type littermates, an indication of the attenuation of p16^Ink4a and p19^Arf in this mutant. Additional support of this mechanism is provided by the demonstration that decreased expression of Wip1, a specific phosphatase and negative regulator of p38 MAPK, in aged tissues and in Wip1-deficient mice upregulates phospho-p38, p16^Ink4a and p19^Arf expression,²⁵ and results in decreased islet proliferation, while Wip1 overexpression, which attenuates p38 MAPK, rescues the age-related decline in proliferation and regenerative capacity. Quantitative PCR reveals an increase in p16^Ink4a and p19^Arf mRNAs in multiple organs of aged WT mice. These markers remain low in p38^AF/+ mice. Thus, p38 activity is required for full Ink4a and Arf induction in aged tissues. These data clearly link the activity of p38 MAPK to physiological characteristics of senescence and aging. Our model expands this mechanism by linking mitochondrially generated ROS to activation of the ASK1-signalosome → p38 MAPK → senescence and aging.

Recent characterization of the ASK1-signalosome has provided further evidence of its multiple functions. In resting cells the HMW signalosome is bound to 14-3-3 and thioredoxin (our unpublished data) and MKK6, which are activated in response to H₂O₂.¹¹ Resistance to oxidative stress in the long-lived Snell and Ames dwarf mice and the overexpressing Klotho model is associated with increased levels of (SH)₂Trx-ASK1 complex. Thus a decrease in activated ASK1 accounts for the decreased activity of downstream targets and may be indicative of their resistance to oxidative stress. This occurs in Snell and Ames mice, suggesting a sustained lower level of ASK1 activity associated with the lower level of endogenous oxidative stress in both young and aged dwarf mice.^10,15 Furthermore, the fact that the level of reduced thioredoxin [Trx(SH)₂] is significantly higher in dwarf cells is consistent with their higher inASK1-signalosome complex levels, lower stress signaling activity and resistance to oxidative stress.

The ASK1-signalosome and p38 MAPK are master regulators of the activity of many stress-response genes associated with aging. Our proposed mechanism suggests that regulation of the ASK1-signalosome → p38 MAPK activity should target genes associated with resistance and sensitivity to oxidative stress. Both the up and downregulation of this pathway by the ROS-mediated regulation of the level of (SH)₂Trx-ASK1 complex suggest an integrated regulation of genes whose combined activity levels define the physiological status that confers resistance to oxidative stress and longevity.

p38 MAPK may also play an important role in the induction of cellular senescence by a diverse set of non-mitochondrial stimuli, i.e., Ras-induced senescence; replicative senescence, oxidative stress induced senescence and culture shock.^3,26,27 For example, the small GTPases are a major source of endogenous ROS that activate p38 MAPK.²⁸ Future studies will assess whether the small GTPase-generated ROS enhancement of senescence and aging is mediated via the ASK1-signalosome → p38 MAPK pathway.

Our mechanism suggests that the promotion of premature aging phenotypes may depend upon ASK1-signalosome → p38 MAPK activity. Thus attenuation of this pathway, either at the level of ASK1 or p38 MAPK, could be beneficial in ameliorating such conditions. A recent novel high-throughput screening of small molecule libraries has identified benzodiazepine as a potent inhibitor of ASK1 by stimulating phosphorylation of Ser⁹⁶⁷, which enhances its binding to 14-3-3.²⁹ Pharmacological intervention is therefore feasible via the identification of small molecule inhibitors of either ASK1 or p38 kinase activity. This is a fruitful future approach to understanding the molecular mechanism of mammalian aging.