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      KLF6 Induces Apoptosis in Human Lens Epithelial Cells Through the ATF4-ATF3-CHOP Axis

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          Abstract

          Background

          Many studies have confirmed that high myopia is related to the high prevalence of cataracts, which results from apoptosis of lens epithelial cells (LECs) due to endoplasmic reticulum stress. Krüppel-like factor 6 (KLF6) is a tumor suppressor that is involved in the regulation of cell proliferation and apoptosis.

          Purpose

          In this study, our purpose was to find the relationship between KLF6-induced apoptosis in LECs and ATF4 (activating transcription factor 4)-ATF3 (activating transcription factor 3)-CHOP (C/EBP homologous protein) signaling pathway.

          Methods

          KLF6, ATF4, ATF3, and CHOP were ectopically expressed using cDNAs subcloned into the pCDNA3.1+ vector. ATF4, ATF3, and CHOP knockdown were performed by small interfering RNA (siRNA). Expression of relative gene was tested using QT-PCR and western-blot. Then, accompanied by UVB stimulation, cell viability was measured by CCK-8 assay; The cell damage was examined by live & dead staining; The apoptotic markers Bax and Bcl-2 were detected by immunoblotting; Quantitative apoptotic levels were measured with the Apoptosis Detection Kit; The expression level of reactive oxygen-free radical (ROS) was analyzed by DCFH-DA` probe.

          Results

          Ectopically expressed ATF4, ATF3, and CHOP-induced apoptosis in cells, whereas ATF4, ATF3, and CHOP knockdown by small interfering RNA (siRNA) blocked KLF6-induced apoptosis. In addition, we determined that ATF4 regulates ATF3 and CHOP expression and that ATF3 silencing reduces CHOP upregulation without changing ATF4 levels; however, ATF4 and ATF3 expression was unaffected by blockade of CHOP, suggesting that KLF6 triggers endoplasmic reticulum stress in LECs by mediating the ATF4-ATF3/CHOP axis. Besides, KLF6 overexpression significantly induced LEC apoptosis under UV radiation, as demonstrated by the elevated Bax/Bcl-2 ratio.

          Conclusion

          The ATF4-ATF3-CHOP pathway plays an important role in KLF6-induced apoptosis in HLECs. Our results increase our understanding of the mechanisms that regulate LEC apoptosis and contribute to the development of a new preventative strategy for cataract.

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          Most cited references 66

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          The Role of the PERK/eIF2α/ATF4/CHOP Signaling Pathway in Tumor Progression During Endoplasmic Reticulum Stress.

          Hypoxia is a major hallmark of the tumor microenvironment that is strictly associated with rapid cancer progression and induction of metastasis. Hypoxia inhibits disulfide bond formation and impairs protein folding in the Endoplasmic Reticulum (ER). The stress in the ER induces the activation of Unfolded Protein Response (UPR) pathways via the induction of protein kinase RNA-like endoplasmic reticulum kinase (PERK). As a result, the level of phosphorylated Eukaryotic Initiation Factor 2 alpha (eIF2α) is markedly elevated, resulting in the promotion of a pro-adaptive signaling pathway by the inhibition of global protein synthesis and selective translation of Activating Transcription Factor 4 (ATF4). On the contrary, during conditions of prolonged ER stress, pro-adaptive responses fail and apoptotic cell death ensues. Interestingly, similar to the activity of the mitochondria, the ER may also directly activate the apoptotic pathway through ER stress-mediated leakage of calcium into the cytoplasm that leads to the activation of death effectors. Apoptotic cell death also ensues by ATF4-CHOP- mediated induction of several pro-apoptotic genes and suppression of the synthesis of anti-apoptotic Bcl-2 proteins. Advancing molecular insight into the transition of tumor cells from adaptation to apoptosis under hypoxia-induced ER stress may provide answers on how to overcome the limitations of current anti-tumor therapies. Targeting components of the UPR pathways may provide more effective elimination of tumor cells and as a result, contribute to the development of more promising anti-tumor therapeutic agents.
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            The PERK/eIF2alpha/ATF4 module of the UPR in hypoxia resistance and tumor growth.

            Hypoxia is a dynamic feature of the tumor microenvironment that contributes to cancer progression. In order to adapt and overcome hypoxic stress, tumor cells activate survival pathways that attempt to couple metabolic processes to reduced energy availability due to oxygen deprivation. While the hypoxia-inducible factors HIF-1 and HIF-2 are critical to the cellular response to hypoxia, HIF-independent processes are known to contribute to this adaptation. Recent evidence demonstrates that hypoxia activates components of the Unfolded Protein Response (UPR), a coordinated program that regulates cellular adaptation to increased levels of unfolded proteins in the endoplasmic reticulum (ER). Here we review the evidence implicating the ER kinase PERK, its downstream target translation initiation factor eIF2alpha, and the subsequent translational upregulation of the transcription factor ATF4 in this response. Not only are cells with compromised PERK-eIF2alpha-ATF4 signaling more sensitive to hypoxic stress in vitro but they also form tumors that grow slower in vivo with smaller hypoxic areas, indicating that the PERK-eIF2alpha-ATF4 pathway confers a survival advantage for tumor cells under hypoxia. These results, together with evidence for an involvement of other UPR pathways and ER stress proteins in hypoxia tolerance and tumor maintenance, point to a central role for UPR activation in tumor progression and suggest that this response may offer an attractive target for new anti-tumor modalities.
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              KLF6, a candidate tumor suppressor gene mutated in prostate cancer.

              Kruppel-like factor 6 (KLF6) is a zinc finger transcription factor of unknown function. Here, we show that the KLF6 gene is mutated in a subset of human prostate cancer. Loss-of-heterozygosity analysis revealed that one KLF6 allele is deleted in 77% (17 of 22) of primary prostate tumors. Sequence analysis of the retained KLF6 allele revealed mutations in 71% of these tumors. Functional studies confirm that whereas wild-type KLF6 up-regulates p21 (WAF1/CIP1) in a p53-independent manner and significantly reduces cell proliferation, tumor-derived KLF6 mutants do not. Our data suggest that KLF6 is a tumor suppressor gene involved in human prostate cancer.
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                Author and article information

                Journal
                Drug Des Devel Ther
                Drug Des Devel Ther
                DDDT
                dddt
                Drug Design, Development and Therapy
                Dove
                1177-8881
                09 March 2020
                2020
                : 14
                : 1041-1055
                Affiliations
                [1 ]Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Medical University Eye Hospital , Tianjin, People’s Republic of China
                Author notes
                Correspondence: Xiaorong Li; Lijie Dong Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Medical University Eye Hospital , No. 251 Fu Kang Road Nankai District, Tianjin300384, People’s Republic of ChinaTel +86 22 23346430; Tel +86 1405326-6077 Email xiaorli@163.com; aitaomubang@126.com
                [*]

                These authors contributed equally to this work

                Article
                218467
                10.2147/DDDT.S218467
                7069589
                32210535
                © 2020 Tian et al.

                This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License ( http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms ( https://www.dovepress.com/terms.php).

                Page count
                Figures: 7, Tables: 1, References: 75, Pages: 15
                Categories
                Original Research

                Pharmacology & Pharmaceutical medicine

                atf4, klf6, apoptosis, hlecs, endoplasmic reticulum stress

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