The SA increase in MMP-1 has been reported in a number of cellular aging models as well as in cells isolated from patients with premature ageing syndromes (Chen and Ames, 1994
). The current findings indicate that SA MMP-1 is attenuated by conditions that decrease steady state H2
production including limiting metabolic O2
flux and antioxidant treatments (Figs. and ). SA shifts in steady state [H2
] lead to redox-dependent activation of upstream MAP Kinases and depletion of MKP-1 that maintains JNK signaling and drives the SA increase in MMP-1 expression as outlined in .
Replicative senescence can be delayed by low oxygen conditions (3% O2
) or by treatment with glutathione-precursor, NAC (Haendeler et al, 2004
) (Dooley et al, 2004
). Our studies indicate that limiting metabolic O2
consumption by lowering O2
tension restricts intracellular ROS levels as determined both biochemically and using redox-sensing GFP constructs. We observed modest but significant differences in intracellular roGFP1 oxidation between aged cells maintained at 3% O2
relative those at 21% O2
(). This is in contrast to observations by Dooley et al
(Takahashi and Zeydel, 1982
) where cytosolic roGFP1 oxidation was insensitive to variations in O2
tension. In the current study we have restricted our focus to aged primary cells as compared to tumor cell lines that may possess distinct mechanisms for regulating redox homeostasis. Senescing IMR90 cells are known to decrease levels of the primary H2
-detoxifying antioxidant, glutathione (Vincenti and Brinckerhoff, 2002
). This likely accounts for the ability of the glutathione precursor, NAC, to restrict SA MMP-1 expression () Decreases in glutathione may limit the ability to scavenge ROS in senescent primary fibroblasts at 21% O2
. Our biochemical analysis has revealed that increased steady state H2
production is intrinsic to senescent cells. While under low oxygen tension both young and old cells produce similar steady quantities of H2
. However, upon acute transition of both young and old cells to ambient air, senescent cell cultures show a much more robust increase in oxidant production than presenescent cells. It is likely that shifts in both oxidant-generating and -scavenging systems contribute to enhanced steady state H2
generating capacity of aged cells.
The SA redox-dependent induction of MMP-1 was observed at the level of both protein and RNA (). MMP-1 is predominantly regulated at the level of transcription (Ranganathan et al, 2001
) and can involve the activation of a myriad of signaling networks. Our findings also indicate that inhibition of JNK, PI-3-K, Erk and p38 individually, can impair SA MMP-1 expression (). Phosphorylation of Akt is age-dependent but not redox-responsive () suggesting that this pathway might play redox-independent role in regulating SA
MMP-1 expression. The redox-insensitive nature of Akt may be related to upstream signals that drive age-related PI3K signaling. MEK/Erk inhibition also attenuated SA MMP-1 () and has been shown to be contributory to redox-dependent MMP-1 regulation in tumor cell lines (Brauchle et al, 2000
) (Brauchle et al, 2000
). Our current findings suggest that Erk phosphorylation is unaffected by age or oxygen tension () though inhibition of the pathway did bring about a decrease in MMP-1 levels suggesting that Erk may also contribute to maximal SA MMP-1 expression. Similarly, p38 inhibition also reduced MMP-1 expression and has been reported to play a role in regulating MMP-1 levels by stabilizing the MMP-1 mRNA via its 3′ untranslated region (Reunanen et al, 2002
) (Fleming et al, 2000
). Whether MMP-1 mRNA stability is altered with age or is redox-dependent requires examination.
The regulation of MAPK signaling is largely dependent on phosphorylation-dependent activation by upstream MAPK Kinases and negative regulation by phosphatases including MAP Kinase Phosphatases (MKPs). JNK is activated by MKK-4 and MKK-7 that are known to phosphorylate it on Thr 185 and Tyr-183 respectively (Sundarrajan et al, 2003
). MKK-4 and MKK-7 form a complex with JNK (Wang et al, 2007
) and genetic ablation of either MKK indicates that they are mutually exclusive in their function and that synergy is needed for complete JNK activation (Moriguchi et al, 1997
;Foltz et al, 1998
). The degree of activation of the MKKs can be dictated by the activating stimulus. MKK-7 is more sensitive to activation by inflammatory cytokines, environmental stress and physiological stimuli (Wada et al, 2001
). Wada et al
report that while MKK-4 and MKK-7 are both needed for JNK activation, loss of MKK-4 inhibits MKK-7 mediated phosphorylation of JNK (Kondoh and Nishida, 2007
). This suggests that MKK-4 mediated phosphorylation of JNK precedes that of MKK-7. We have also established that MKK-4 phosphorylation is increased in a redox-dependent manner in the senescent cells () and likely contributes to the SA associated expression of MMP-1.
In addition to phosphorylation-dependent activation, MAPK signaling is tempered by a large family of inhibitory phosphatases including the protein phosphatases and the dual specificity phosphatase or MAPK phosphatase (MKP) family members (Kamata et al, 2005
). MKPs show cross-reactivity in their dephosphorylating substrates with some degree of specificity and affinity. MKP-1, 2, 5, 7 and M3/6 are the primary JNK-specific phosphatases that are activated in response to oxidative stress. The current work suggest that the SA attenuation of MKP-1 is associated with its enhanced ubiquitination that is tempered by restricting exposure to 21% O2
( ). Oxidative stress can drive oxidative inactivation and degradation of active site cysteines leading to the formation of high molecular weight aggregates that are degraded by proteasome (Chen et al, 2001
). Thus, increases in steady state ROS production, as observed in senescent IMR90 fibroblast under ambient air (21% O2
) (), may drive MKP turnover through oxidation and subsequent ubiquitin-mediated degradation () leading to the low steady state immunoreactive MKP-1 in these cells (). Moreover, ubiquitinated MKP-1 was only observed in the presence of proteasome inhibitor and was decreased by lowering oxygen tension. These findings suggest that increases in SA steady state [H2
] may restrict regulatory phosphatases thereby enhancing overall kinase signaling. Overall a decrease in MKP-1 and a concomitant increase in MKK-4 activity as seen in the old cells under 21% O2
would potentially sustain JNK phosphorylation. Restoring MKP-1 levels in the senescent cells under 21% O2
by lentiviral infection impairs SA MMP-1 production (). A similar mechanism has been reported for the phosphatase M3/6 leading to JNK activation by H2
(Zhao et al, 2008
). It is also possible that other redox-sensitive phosphatases participate in JNK activation by modulating the activity of upstream kinases such as MKK-4 (Fisher et al, 2009
). Reconstitution studies with the distinct MKPs will be necessary to evaluate the contribution of any particular MKP in the SA redox-dependent expression of MMP-1.
In summary, these studies demonstrate that cellular aging is associated with an increase in MMP-1 expression that involves alterations in steady state ROS production leading to JNK activation. JNK activation is mediated by redox-alterations in the activation state of regulatory kinases and inhibitory phosphatases. Fisher et al. recently demonstrated that the levels of the JNK target, c-Jun, are increased in human dermal fibroblasts from aged (>80 years) relative to young individuals. Our findings extend those of Fisher et al. and further elaborate the redox-sensitive signaling networks that likely drive age-associated increases in MMP-1 expression. It is intriguing to speculate that other MMPs may also show similar age-associated and oxidant-dependent control as many of their transcriptional regulatory elements are positionally conserved. These findings also further provide a mechanistic link between the free radical theory of aging and degenerative disease processes associated with aberrant MMP production. We are excited by the possibility that antioxidant-based therapies, by their ability to limit MMP expression, may prove useful in the treatment of many age-related disease processes that are associated with excessive matrix turnover.