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

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.

Keywords:

• KLF6
• ATF4
• apoptosis
• HLECs
• endoplasmic reticulum stress

Acknowledgments

This work was supported by a grant from the National Natural Science Foundation of China (NSFC:81570872), Tianjin Municipal Science and Technology Commission Grants (15JCYBJC24900), The Science & Technology Development Fund of Tianjin Education Commission for Higher Education (2016YD08), Tianjin Medical University Natural Science Foundation (2016KYZM14, 2015KYZM10), Tianjin Medical University Eye Institute Foundation (15YKYJS002).

We thank Liwen Bianji, Edanz Editing China, for editing the English text of a draft of this manuscript.

The use of gifted HLECs was approved by Tianjin Medical University Eye Hospital ethics committee.

Abbreviations

AARE, amino acid regulatory element; ATF3, activating transcription factor 4; ATF4, activating transcription factor 3; ATF6, activating transcription factor 6; ATF/CREB, ATF/cAMP response element binding; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; CHOP, C/EBP homologous protein; DAPI, 4’,6-diamidino-2-phenylindole; DR5, death receptor 5; ERS, endoplasmic reticulum stress; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IRE1, inositol-requiring enzyme 1; JNK, c-Jun N-terminal kinase; KLF6, Krüppel-like factor 6; LECs, lens epithelial cells; Nrf2, nuclear factor (erythroid-derived 2)-like 2; PERK, protein kinase R-like ER kinase; ROS, reactive oxygen species; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; UPR, unfolded protein response; UVB, ultraviolet radiation B.

Data Sharing Statement

The data used to support the findings of this study are included within the article.

Disclosure

The authors declare that they have no conflicts of interest.