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Depletion of eukaryotic initiation factor 5B (eIF5B) reprograms the cellular transcriptome and leads to activation of endoplasmic reticulum (ER) stress and c-Jun N-terminal kinase (JNK).

ABSTRACT: During the integrated stress response (ISR), global translation initiation is attenuated; however, noncanonical mechanisms allow for the continued translation of specific transcripts. Eukaryotic initiation factor 5B (eIF5B) has been shown to play a critical role in canonical translation as well as in noncanonical mechanisms involving internal ribosome entry site (IRES) and upstream open reading frame (uORF) elements. The uORF-mediated translation regulation of activating transcription factor 4 (ATF4) mRNA plays a pivotal role in the cellular ISR. Our recent study confirmed that eIF5B depletion removes uORF2-mediated repression of ATF4 translation, which results in the upregulation of growth arrest and DNA damage-inducible protein 34 (GADD34) transcription. Accordingly, we hypothesized that eIF5B depletion may reprogram the transcriptome profile of the cell. Here, we employed genome-wide transcriptional analysis on eIF5B-depleted cells. Further, we validate the up- and downregulation of several transcripts from our RNA-seq data using RT-qPCR. We identified upregulated pathways including cellular response to endoplasmic reticulum (ER) stress, and mucin-type O-glycan biosynthesis, as well as downregulated pathways of transcriptional misregulation in cancer and T cell receptor signaling. We also confirm that depletion of eIF5B leads to activation of the c-Jun N-terminal kinase (JNK) arm of the mitogen-activated protein kinase (MAPK) pathway. This data suggests that depletion of eIF5B reprograms the cellular transcriptome and influences critical cellular processes such as ER stress and ISR.

SUBMITTER: Bressler KR

PROVIDER: S-EPMC7736443 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundXanthoangelol (XAG) was reported to exhibit antitumor properties in several cancer. However, the specific anti-tumor activity of XAG in human hepatocellular carcinoma (HCC) and the relevant mechanisms are not known.MethodsThe effects of XAG on HCC cell proliferation and apoptosis were respectively examined by CCK-8 assay and Annexin V-FITC/PI apoptosis kit. Western blotting was conducted to detect the expression of proteins. The effect of XAG on the development of acidic vesicle organelles was assessed using acridine orange staining. mRFP-GFP-LC3 adenovirus was used to transfect HCC cells and the formation of autolysosome was detected using a confocal microscope.ResultsMechanistically, XAG promotes HCC cell death through triggering intrinsic apoptosis pathway, not extrinsic apoptotic pathway. Furthermore, XAG treatment induced autophagy in Bel 7402 and SMMC 7721 cells, as evidenced by an increase in autophagy-associated proteins, including LC3B-II, Beclin-1, and Atg5. Interestingly, inhibition of autophagy with 3-MA, Bafilomycin A1 (Baf A1), or siRNA targeting Atg5 effectively enhanced the apoptotic cell ratio in XAG-treated cells, indicating that protective effect of autophagy induced by XAG in HCC. Moreover, autophagy induced by XAG was mediated by activating endoplasmic reticulum stress (ERS), along with administration of XAG, the expression levels of ERS-associated proteins, including CHOP, GRP78, ATF6, p-eIF2α, IRE1α, and cleaved caspase-12 were significantly increased in HCC cells. Meanwhile, suppressing ERS with chemical chaperones (TUDCA) or CHOP shRNA could effectively abrogate the autophagy-inducing effect of XAG, and increase the apoptotic cell death. Further mechanistic studies showed that ERS-induced autophagy in XAG-treated cells was mediated by activation of JNK/c-jun pathway. XAG treatment resulted in the increase of p-JNK and p-c-jun, while suppressing ERS with TUDCA or CHOP shRNA could effectively reverse it. Meanwhile, SP600125, a JNK inhibitor, effectively reversed XAG-induced protective autophagy and enhanced cell apoptosis in XAG-treated HCC cells. In vivo results demonstrated that XAG exerts potent antitumor properties with low toxicity.ConclusionsCollectively, these results suggested that XAG could be served as a promising candidate for the treatment and prevention of HCC.

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Depletion of eukaryotic initiation factor 5B (eIF5B) reprograms the cellular transcriptome and leads to activation of endoplasmic reticulum (ER) stress and c-Jun N-terminal kinase (JNK).

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