Previous reports suggested that rapamycin, the potent immunosuppressant, may be useful for the treatment of ER stress-related disorders.7
However, molecular mechanisms underlying its therapeutic utility are largely unknown. In the present report, we elucidated the potential of rapamycin to attenuate ER stress-induced apoptosis. Our results disclosed; (1) mTORC1 is activated under ER stress conditions, (2) inhibition of mTORC1 selectively suppresses the IRE1–JNK pathway, but not the PERK and ATF6 pathways, and (3) activation of mTORC1 triggers the IRE1–JNK pathway, but not other UPR branches. We also elucidated that mTORC1 causes inhibition of Akt and consequent activation of the IRE1–JNK pathway, leading to apoptosis. These results suggest that, under ER stress conditions, mTORC1 causes apoptosis through suppression of Akt and consequent induction of the particular pro-apoptotic pathway. The outline of our current findings was summarized in .
Some previous reports showed that activation of mTORC1 resulted in induction of ER stress. For example, Ozcan et al.
reported that loss of TSC1 or TSC2 and consequent activation of mTORC1 caused ER stress, leading to increased vulnerability of cells to apoptotic cell death.18
The authors showed that three major branches of the UPR were activated in TSC-deficient cells. In contrast, in the present report, we showed that activation of mTORC1 by knockdown of TSC2 triggered the IRE1–JNK pathway, but not PERK and ATF6 pathways. The discrepancy between our current results and the previous finding is possibly owing to the level of mTORC1 activation. That is, complete deletion of TSC results in strong, constitutive activation of mTORC1 and sustained overproduction and attenuated degradation of proteins,19
leading to activation of three branches of the UPR. In contrast, as we demonstrated, partial knockdown of TSC does not trigger substantial ER stress and causes activation of the IRE1 pathway without induction of PERK and ATF6 signaling. The fact that GRP78, the most popular ER stress marker, was not induced by the treatment with siTSC2 supports our speculation.
In the present report, we demonstrated that ER stress triggers activation of mTORC1. What is the event upstream of mTORC1 activation? One possible answer is Akt. As demonstrated in this report, ER stress rapidly induces phosphorylation of Akt. However, this early activation is only transient. In the later phase, the phosphorylation level of Akt is depressed below basal phosphorylation levels. It is caused by the negative feedback loop initiated by mTORC1, because rapamycin abolished the suppression of Akt in the late phase. It is known that mTORC1 is upregulated by Akt.12
The activation of mTORC1 by ER stress is possibly mediated by the early phosphorylation of Akt. Huang and Manning reported that mTORC2 activation was severely blunted in mTORC1-activated cells. On the other hand, suppression of mTORC1 increased mTORC2 kinase activity.20
mTORC2 is known to serve as an upstream activator of Akt via direct phosphorylation at S473.21
The ability of mTORC2 to phosphorylate Akt is negatively regulated by mTORC1-mediated activation of p70S6K.22
Based on these previous findings, activation of mTORC1 by ER stress may lead to suppression of mTORC2 and consequent inhibition of Akt.
Three major branches of the UPR are activated in response to ER stress. Chemical inhibitors of ER stress (e.g., chemical chaperones) usually block all three arms of the UPR. Currently, little is known about agents that selectively inhibit particular UPR branches. In the present report, we demonstrated the potential of rapamycin for selective inhibition of the IRE1 pathway. As rapamycin did not affect ER stress-induced activation of the PERK and ATF6 pathways, and downstream expression of CHOP
, its suppressive effect on the IRE1–JNK pathway is not via attenuation of ER stress per se
. If so, how does Akt suppress the IRE1 pathway selectively? Currently, the mechanism is not fully understood, but our current results indicate possible involvement of TRAF2, an essential component for IRE1-mediated ASK1–JNK activation.23
We found; (1) inhibition of mTORC1 by rapamycin upregulated Akt and downregulated TRAF2, (2) activation of mTORC1 by siTSC2 downregulated Akt and upregulated TRAF2, (3) activation of Akt decreased TRAF2, whereas inhibition of Akt increased TRAF2, and (4) under ER stress conditions, the kinetics of TRAF2 levels was closely correlated with the kinetics of JNK activity. These results indicate that suppression of TRAF2 is, at least in part, responsible for Akt-mediated inhibition of JNK activation.
The action of IRE1 as kinase requires TRAF2, whereas its action as endoribonuclease does not. The fact that Akt inhibited splicing of XBP1
mRNA implies additional mechanisms underlying the suppression of IRE1 by Akt. Previous studies suggested that Bcl-2 family proteins including Bax, Bak and Bcl-2 homology domain 3 (BH3)-only proteins directly interacted with IRE1 and regulated both its kinase and endoribonuclease activity.24
Akt is known to directly phosphorylate and inhibit Bax and BH3-only proteins.12, 25
The selective blockade of the IRE1 pathway by rapamycin might also be ascribed to the suppression of Bcl-2 family members via Akt.
In this report, we examined a role of mTORC1 in ER stress-induced apoptosis. However, mTORC1 may also be involved in cellular senescence.26
Rapamycin could inhibit ER stress-induced cellular death via intervention in the senescence program. We examined this possibility using senescence-associated β
-gal) as a marker.27
However, in our experimental setting, neither thapsigargin nor tunicamycin caused senescence of NRK-52E cells (Supplementary Figure S6
), excluding this possibility.
Individual UPR pathways possess both pro-apoptotic and antiapoptotic aspects. For example, the PERK pathway causes translational suppression that attenuates ER stress, whereas it also induces pro-apoptotic molecules including CHOP and GADD34 (growth arrest and DNA damage gene 34). The IRE1 pathway triggers apoptosis via activation of ASK1 and JNK, but this pathway also induces ER chaperones and ER stress-associated degradation factors, both of which attenuate ER stress.28
In general, however, the PERK and the ATF6 pathways are considered pro-survival, whereas the IRE1 pathway is regarded pro-apoptotic.29, 30, 31, 32
A selective inhibitor of the IRE1 pathway should, therefore, have an advantage for the treatment of ER stress-related disorders. Indeed, in the present report, we showed that in vivo
administration with rapamycin markedly suppressed ER stress-triggered activation of the IRE1–JNK pathway and consequent apoptosis in the kidney. Our current results raise a possibility that rapamycin may be useful for therapeutic intervention in a wide range of ER stress-related pathologies including infections, diabetes, ischemia, cancers and neurodegenerative disorders.