PERK deficiency distorts the ER-mitochondrial interface and protects against ROS-induced intrinsic apoptosis

Any insult that disturbs ER homeostasis, like alteration of the cellular redox status or release of ER Ca2+, ultimately results in ER stress due to the accumulation of misfolded proteins. ER stress subsequently triggers the Unfolded Protein Response (UPR) which sustains both pro-survival and pro-apoptotic pathways. Interestingly, the hostile environment encountered by tumor cells also activates the UPR and has been shown to alter the chemosensitiviy of cancer cells. Therefore, a better understanding of the molecular mechanisms underlying the proapoptotic and prosurvival mechanisms governed by the UPR is of great importance for current and future cancer therapies. Real time PCR studies revealed an upregulation of several targets of the PERK branch of the UPR after perturbation of ER homeostasis through the generation of reactive oxygen species (ROS) by photodynamic therapy (PDT) with the ER localizing photosensitizer hypericin, in both MEFs and T24 bladder cancer cells. Cell death analysis, involving detection of PARP and caspases 3 and 9 cleavage, loss of mitochondrial transmembrane potential as well as clonogenic assays, revealed that PERK-KO MEFs display an increased resistance to ROS-mediated mitochondrial apoptosis, while being sensitized against thaspigargin. Moreover, our data indicate that the contact site between ER and mitochondria as well as Ca2+ signaling is deranged in PERK deficient cells. Interestingly, Ca2+ signaling in IRE1 deficient cells is not perturbed, thus indicating a PERK specific effect. Experiments assessing direct mitochondrial Ca2+ uptake with a mitochondrially targeted cameleon and ROS-mediated mitochondrial damage in WT and PERK deficient cells are ongoing to clarify the crucial role the ER-mitochondria interplay in ROS-mediated intrinsic apoptosis.status: accepte