Abstract
Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), which reduces levels of misfolded proteins. However, if ER homeostasis is not restored and the UPR remains chronically activated, cells undergo apoptosis. The UPR regulator, PKR-like endoplasmic reticulum kinase (PERK), plays an important role in promoting cell death when persistently activated; however, the underlying mechanisms are poorly understood. Here, we profiled the microRNA (miRNA) transcriptome in human cells exposed to ER stress and identified miRNAs that are selectively induced by PERK signaling. We found that expression of a PERK-induced miRNA, miR-483, promotes apoptosis in human cells. miR-483 induction was mediated by a transcription factor downstream of PERK, activating transcription factor 4 (ATF4), but not by the CHOP transcription factor. We identified the creatine kinase brain-type (CKB) gene, encoding an enzyme that maintains cellular ATP reserves through phosphocreatine production, as being repressed during the UPR and targeted by miR-483. We found that ER stress, selective PERK activation, and CKB knockdown all decrease cellular ATP levels, leading to increased vulnerability to ER stress-induced cell death. Our findings identify miR-483 as a downstream target of the PERK branch of the UPR. We propose that disruption of cellular ATP homeostasis through miR-483-mediated CKB silencing promotes ER stress-induced apoptosis.
Highlights
Eukaryotic cells employ the endoplasmic reticulum (ER) organelle to fold secreted and membrane proteins, synthesize hydrophobic lipids and sterols; and store free calcium [1]
PKR-like endoplasmic reticulum kinase (PERK) responds to ER stress by oligomerizing to activate its cytosolic kinase domain, which phosphorylates a serine at position 51 of the alpha subunit of eukaryotic initiation factor 2 [4]. eIF2α partners with eIF2β and eIF2γ to form the heterotrimeric eukaryotic initiation factor 2 that is essential for canonical translational initiation at AUG start codons, but phosphorylation of eIF2α at serine 51 abrogates eIF2-dependent protein translation leading to global slowdown of protein synthesis and diminished protein folding demands on the ER [5,6]
We found that the gene encoding miR-616 lay within intron 2 of DDIT3/C/ebp homologous protein (Chop), a pro-apoptotic transcription factor strongly upregulated by ER stress
Summary
Eukaryotic cells employ the endoplasmic reticulum (ER) organelle to fold secreted and membrane proteins, synthesize hydrophobic lipids and sterols; and store free calcium [1]. The UPR is controlled by three ER-resident transmembrane sensors: inositol requiring enzyme 1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) [3] Each of these UPR regulators has ER stresssensing luminal domains coupled across the ER membrane to cytosolic effector domains that initiate distinct signal transduction cascades during the UPR [3]. UPR-induced transcription factors such as CHOP can directly upregulate specific miR genes [30,31] These findings suggest that miRs sculpt the UPR by regulating expression of UPR genes, and the UPR induces miRs as part of its transcriptional programs to influence cellular responses to ER stress. We provide evidence that miR-483 decreased cell viability by targeting expression of Creatine Kinase BrainType, leading to loss of cellular ATP stores
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