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Depletion of SLC4A11 causes cell death by apoptosis in an immortalized human corneal endothelial cell line.

ABSTRACT: To investigate the effects of SLC4A11 gene depletion in human corneal endothelial cells.To achieve stable downregulation of SLC4A11 gene expression in immortalized human corneal endothelial cells (HCECs), short-hairpin RNA (shRNA) targeted against SLC4A11 was used. Cell growth and viability were determined using the real-time cell analyzer and trypan blue staining respectively. Apoptosis was investigated by Annexin V and TUNEL assays. Alterations in apoptotic gene expression following SLC4A11 silencing were determined using the RT(2)Profiler PCR array for human apoptosis while activation of the apoptotic pathway was ascertained by western analysis.SLC4A11 silencing in HCECs could be achieved by stable expression of shRNA targeted against SLC4A11. SLC4A11 knockdown suppressed HCEC growth and reduced HCEC viability compared to the control. This reduction in cell growth is associated with increased apoptosis in SLC4A11-silenced cells.Our data suggest that the reduction of cell number with time in SLC4A11-depleted HCECs is due to an increase in cell death by apoptosis. This suggests that SLC4A11 is necessary for cell survival and may explain the pathologic corneal endothelial cell loss in endotheliopathies due to SLC4A11 mutations.

SUBMITTER: Liu J

PROVIDER: S-EPMC4007482 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Depletion of SLC4A11 causes cell death by apoptosis in an immortalized human corneal endothelial cell line.

Liu Jun J Seet Li-Fong LF Koh Li Wei LW Venkatraman Anandalakshmi A Venkataraman Divya D Mohan Rajiv R RR Praetorius Jeppe J Bonanno Joseph A JA Aung Tin T Vithana Eranga N EN

Investigative ophthalmology & visual science 20120605 7

<h4>Purpose</h4>To investigate the effects of SLC4A11 gene depletion in human corneal endothelial cells.<h4>Methods</h4>To achieve stable downregulation of SLC4A11 gene expression in immortalized human corneal endothelial cells (HCECs), short-hairpin RNA (shRNA) targeted against SLC4A11 was used. Cell growth and viability were determined using the real-time cell analyzer and trypan blue staining respectively. Apoptosis was investigated by Annexin V and TUNEL assays. Alterations in apoptotic gene ...[more]

PMID: 22447871

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Project description:PurposeEndoplasmic reticulum (ER) and mitochondrial stress are independently associated with corneal endothelial cell (CEnC) loss in many corneal diseases, including Fuchs' endothelial corneal dystrophy (FECD). However, the role of ER stress in mitochondrial dysfunction contributing to CEnC apoptosis is unknown. The purpose of this study is to explore the crosstalk between ER and mitochondrial stress in CEnC.MethodsHuman corneal endothelial cell line (HCEnC-21T) and human corneal endothelial tissues were treated with ER stressor tunicamycin. ER stress-reducing chemical 4-phenyl butyric acid (4-PBA) was used in HCEnC-21T after tunicamycin. Fuchs' corneal endothelial cell line (F35T) was used to determine differential activation of ER stress with respect to HCEnC-21T at the baseline. ER stress, mitochondrial-mediated intrinsic apoptotic, mitochondrial fission, and fusion proteins were determined using immunoblotting and immunohistochemistry. Mitochondrial bioenergetics were assessed by mitochondrial membrane potential (MMP) loss and ATP production at 48 hours after tunicamycin. Mitochondria dynamics (shape, area, perimeter) were also analyzed at 24 hours using transmission electron microscopy.ResultsTreatment of HCEnC-21T cell line with tunicamycin activated three ER stress pathways (PERK-eIF2α-CHOP, IRE1α-XBP1, and ATF6), reduced cell viability, upregulated mitochondrial-mediated intrinsic apoptotic molecules (cleaved caspase 9, caspase 3, PARP, Bax, cytochrome C), downregulated anti-apoptotic Bcl-2 protein, initiated mitochondrial dysfunction by loss of MMP and lowering of ATP production, and caused mitochondrial swelling and fragmentation with increased expression of mitochondrial fission proteins (Fis1 and p-Drp1). Fuchs' CEnC (F35T) cell line also showed activation of the ER stress-related proteins (p-eIF2α, GRP78, CHOP, XBP1) compared to HCEnC-21T at the baseline. The 4-PBA ameliorated cell loss and reduced cleaved caspase 3 and 9, thereby rescuing tunicamycin-induced cell death but not mitochondrial bioenergetics in HCEnC-21T cell line.ConclusionsTunicamycin-induced ER stress disrupts mitochondrial bioenegetics, dynamics and contributes to the loss of CEnC viability. This novel study highlights the importance of ER-mitochondria crosstalk and its contribution to CEnCs apoptosis, seen in many corneal diseases, including FECD.

Dataset Information

Depletion of SLC4A11 causes cell death by apoptosis in an immortalized human corneal endothelial cell line.

Publications

Depletion of SLC4A11 causes cell death by apoptosis in an immortalized human corneal endothelial cell line.

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