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Published on: November 28, 2012
KLF6 Induces Apoptosis in Human Lens Epithelial Cells Through the ATF4-ATF3-CHOP Axis
Fang Tian1, Jinzhi Zhao1, Shaochong Bu1
1Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Medical University Eye Hospital, Tianjin, People's Republic of China.
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.
Insights
Krüppel-like factor 6 (KLF6) induces lens epithelial cell apoptosis via the ATF4-ATF3-CHOP pathway, a key mechanism in cataract development. Understanding this pathway offers new strategies for preventing cataracts.
Area of Science:
- Ophthalmology
- Cell Biology
- Molecular Biology
Background:
- High myopia is linked to cataracts, often caused by apoptosis of lens epithelial cells (LECs) due to endoplasmic reticulum stress.
- Krüppel-like factor 6 (KLF6), a tumor suppressor, regulates cell proliferation and apoptosis.
Purpose of the Study:
- To investigate the relationship between KLF6-induced apoptosis in LECs and the ATF4-ATF3-CHOP signaling pathway.
- To elucidate the role of KLF6 in endoplasmic reticulum stress within LECs.
Main Methods:
- Ectopic expression of KLF6, ATF4, ATF3, and CHOP using pCDNA3.1+ vector.
- Knockdown of ATF4, ATF3, and CHOP using small interfering RNA (siRNA).
- Gene expression analysis via QT-PCR and Western blot; cell viability, damage, apoptosis markers (Bax, Bcl-2), and reactive oxygen species (ROS) were assessed.
Main Results:
- ATF4, ATF3, and CHOP expression induced apoptosis, while their knockdown inhibited KLF6-induced apoptosis.
- ATF4 regulates ATF3 and CHOP expression; ATF3 silencing reduced CHOP upregulation without affecting ATF4.
- KLF6 triggers endoplasmic reticulum stress in LECs via the ATF4-ATF3/CHOP axis and enhances UV-induced apoptosis, indicated by an elevated Bax/Bcl-2 ratio.
Conclusions:
- The ATF4-ATF3-CHOP pathway is crucial in KLF6-induced apoptosis of human lens epithelial cells (HLECs).
- Findings enhance understanding of LEC apoptosis mechanisms.
- Results contribute to developing novel preventative strategies for cataracts.
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