Herein we show that mTOR is markedly activated in the setting of I/R and that activation is cardioprotective in vivo as well as in various in vitro models of I/R. Furthermore we delineate a novel signaling pathway regulated by the stress activated MAPK, p38, that leads to activation of mTOR. Key to this is p38-mediated inhibition of the mTOR regulator TSC2, which is due, at least in part, to down-regulation of the REDD1 protein. Akt also contributes to mTOR activation by stresses since we find that inhibiting Akt reduces mTOR activation. Our findings are summarized in .
Proposed model for p38 regulation of ROS mediated mTOR activation and cell survival
Based on the central role of p38 in regulating levels of inflammatory cytokines, p38 inhibitors were developed for use in patients with chronic inflammatory disorders such as rheumatoid arthritis. However liver toxicity was dose-limiting in animal models. Recent pre-clinical studies with SB239063 showed that endothelial dysfunction and atherosclerotic plaque inflammation were reduced. More recently, patients treated with a low dose of p38 inhibitor for 3 months had reduced levels of C-reactive protein (indicative of less inflammation) and improved vascular reactivity. Another p38 inhibitor, GW856553 has entered into a clinical trial in patients with acute coronary syndromes (http://clinicaltrials.gov/ct2/show/NCT00910962?term=solstice&rank=1
). While the evidence is mounting that p38 inhibition might be beneficial in reducing inflammation, it is obviously critical to be certain that p38 inhibition will not exacerbate I/R injury. Unfortunately, there is no clear answer to this question. In a recent review examining our understanding of the role of p38 in ischemic injury, the conclusion was reached that “dissecting the beneficial vs. detrimental aspects of p38 signaling will be a major issue to be addressed in the future.”[5
Herein, we believe we have identified one key beneficial role for p38 in the setting of I/R: activation of mTOR. Recently there has been an increase in interest in the role of mTOR in the heart. Activation of mTOR via over-expression of the wild-type protein was shown to protect against pressure overload induced by thoracic aortic constriction [20
]. Deletion of Raptor, a key component of the mTORC1 complex, leads to reduced cardiomyocyte growth and heart failure that is particularly pronounced in the setting of pressure overload. Furthermore, rapamycin was recently proposed as a therapeutic strategy to limit the pathologic hypertrophy seen in LEOPARD syndrome, one of the ras-opathies caused by a mutation in the tyrosine phosphatase SHP2 [21
]. Thus mTOR appears to be protective in the setting of excess hypertrophic signaling, possibly irrespective of cause.
The role of mTOR in regulating ischemic injury is less clear in that it has been examined in a number of settings with what appear to be disparate conclusions. [22
]. Several studies have examined the role of mTOR in insulin cardioprotection and have shown that mTOR inhibition with rapamycin or its analogs negate the beneficial effects of insulin infusion. Rapamycin also reduces the protection provided by ischemic pre-conditioning (IPC) [25
]. More recently, Buss et al. examined the role of mTOR in a chronic MI model and reported that inhibition of mTOR with everolimus reduced infarct size. In contrast, Lajoie et al.[27
] used a similar model to that of Buss et al. [26
]and concluded that rapamycin increased infarct size in female rats.
The only other report of which we are aware that addressed the role of mTOR in “standard” I/R (i.e. no IPC, no insulin infusion, etc.) stated that rapamycin decreased infarct size in a Langendorf model. Our findings clearly differ from those of Khan et al. [24
]since we demonstrate that rapamycin increased I/R injury. The disparate conclusions underline the confusion in this area and, we believe, in order to understand the true roles of mTOR in the heart, it is key to identify molecular mechanisms regulating mTOR in the stressed heart.
Therefore, we set out to identify the signaling pathways that regulate mTOR activity in the heart exposed to I/R. Our central finding is that the mTOR pathway is positively regulated by p38 MAPK and inability to activate mTOR via this mechanism increases cell death in models of I/R injury. We have also delineated the pathway upstream of mTOR activation in response to H/R and oxidant stress, defining p38 and Akt as two essential activators of the mTOR pathway in these situations. Importantly, we were able to reproduce this p38-dependent mTOR activation by ROS in different cell types, confirming that this is not a cell-type-specific phenomenon.
The fact that stress activates mTOR is a relatively new concept, and in fact the vast majority of work has suggested that stress inactivates mTOR. This inactivation was reported to depend on AMPK-mediated TSC2 phosphorylation in the setting of energy stress and hypoxia, and by HIF-1 inducible REDD1-mediated displacement of inhibitory 14-3-3 from TSC2 in the setting of hypoxic stress ([28
] and reviewed in [31
]. In contrast, we found that with oxidative stress, despite AMPK being activated, AMPK was unable to inactivate mTOR or to block its activation. Regulation of TSC2 by AMPK is known to be a complex event that requires phosphorylation of TSC2 at several residues, in which a priming phosphorylation by AMPK is followed by additional phosphorylation events by GSK3β. We speculate that inactivation of GSK3β by Akt in cells exposed to ROS () may impair the GSK3β-dependent phosphorylation of TSC2. Experimental confirmation of this hypothesis will depend on the availability of antibodies specific to GSK3β-dependent phosphorylation sites of TSC2.
We also find that REDD-1 is down-regulated by reactive oxygen species and this likely accounts for the inactivation of TSC2 and activation of mTOR in this setting. p38 MAPK, but not Akt, is necessary for this downregulation. Overall, this shows that p38 stimulates the mTOR pathway in a specific and an unexpected manner.
To our knowledge, there have been only two reports prior to this showing that p38 can activate mTOR. Li et al [32
], have proposed a model whereby the p38 substrate MK2 phosphorylates TSC2 at Ser1210, thereby inducing its binding to 14-3-3.We however were unable to detect phosphoSer1210 TSC2 after H2
treatment using commercially available antibodies (data not shown). However, we do see a clear downregulation of REDD1 after H2
treatment that is dependent on p38. Since TSC2 is inactive in hypoxia mainly due to high REDD1 levels, the mechanism we describe here is likely to be generally important in ischemic syndromes. The second publication was from Cully and coworkers who recently identified p38 as an upstream regulator of TORC1 activity, at least in Drosophila melanogaster cells and in a transformed human cell line, but the functional significance of this remains unclear. The effects of p38 described by Cully et al. are independent of TSC2, at least in Drosophila, and, as such, the mechanism differs significantly from our findings [6
]. The existence of several possible routes by which p38 can modulate the mTOR pathway suggests that this may be a central mechanism of mTOR regulation.
In summary, mTOR activation protects cardiomyocytes from I/R injury and cell death. Mechanistically, ROS-induced activation of mTOR is mediated by p38-driven TSC2 inactivation which occurs despite activation of AMPK. These data underline the fact that regulation of downstream targets by p38 is complex as it is regulating both pro-survival and pro-death pathways, sometimes simultaneously. Our findings raises some concerns over the use of small molecule inhibitors of p38, some of which are advancing through clinical trials. The findings also suggest that the development of effective treatments based on inhibition of the p38 pathway might be better-targeted at factors downstream of p38 itself.