Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Apr 9;12(4):386.
doi: 10.1038/s41419-021-03683-y.

Cellular FLICE-like inhibitory protein (cFLIP) critically maintains apoptotic resistance in human lens epithelial cells

Jingru Huangfu  1   2 , Caili Hao  1 , Zongbo Wei  1 , I Michael Wormstone  3 , Hong Yan  2   4 , Xingjun Fan  5
Affiliations

Cellular FLICE-like inhibitory protein (cFLIP) critically maintains apoptotic resistance in human lens epithelial cells

Jingru Huangfu et al. Cell Death Dis. .

Abstract

The present study aims to understand the mechanism of the lens epithelial cell's strong anti-apoptotic capacity and survival in the mature human lens that, on the one hand, maintains lens transparency over several decades, while on the other hand, increases the risk of posterior capsule opacification (PCO). Here we compared FHL124 cells and HeLa cells, spontaneously immortalized epithelial cell lines derived from the human lens and cervical cancer cells, respectively, of their resistance to TNFα-mediated cell death. TNFα plus cycloheximide (CHX) triggered almost all of HeLa cell death. FHL124 cells, however, were unaffected and able to block caspase-8 activation as well as prevent caspase-3 and PARP-1 cleavage. Interestingly, despite spontaneous NFκB and AP-1 activation and upregulation of multiple cell survival/anti-apoptotic genes in both cell types, only FHL124 cells were able to survive the TNFα challenge. After screening and comparing the cell survival genes, cFLIP was found to be highly expressed in FHL124 cells and substantially upregulated by TNFα stimulation. FHL124 cells with a mild cFLIP knockdown manifested a profound apoptotic response to TNFα stimulus similar to HeLa cells. Most importantly, we confirmed these findings in an ex vivo lens capsular bag culture system. In conclusion, our results show that cFLIP is a critical gene that is regulating lens epithelial cell survival.

PubMed Disclaimer

Conflict of interest statement

J.H., C.H., Z.W., M.W., and X.F. declare they have no conflict of interest.

Figures

Fig. 1
Fig. 1. FHL124 but not HeLa cells are resistant to TNFα-induced cell death.
A FHL124 cells were treated by 0, 10, 30, and 60 ng/ml TNFα for 5, 12, and 24 h. The cell viability was determined by CCK8 assay (n = 8). No cell viability loss was seen at any of the TNFα concentrations and time points. B HeLa cells were treated with the same levels of TNFα concentrations and time points. Cell viability loss was seen at 12 h and significantly elevated at 24 h. C FHL124 and HeLa cells were treated with 30 ng/ml TNFα and 10 μg/ml CHX for multiple time points from 3 h to 24 h (n = 8). Profound cell viability loss was seen in HeLa cells 7 h after treatment, but less than 50% cell viability loss was observed in FHL124 cells even after 24 h of treatment. D Cell morphology recorded by the phase-contrast imaging showed normal FHL124 cell morphology at 12 h and 24 h time point, but shrunken cell bodies of HeLa cells at 5 h and 7 h time point after TNFα and CHX treatment compared to non-treated cells. One-way ANOVA with Tukey’s Honest post-hoc analysis was used to compare between groups, and only p < 0.05 is considered significant. *<0.05, **<0.01, ***<0.001, ****<0.0001.
Fig. 2
Fig. 2. TNFα induces apoptosis in HeLa cells but is resisted by FHL124 cells.
A–D Cell nuclear morphology imaged via Hoechst 33342 stain showed no remarkable changes in FHL124 cells 24 h after TNFα and CHX stimulation. E–H Nuclear chromatin packaging into apoptotic bodies manifested as condensed nuclei with super bright appearance were seen in HeLa cells 7 h after treatment compared to no treated cells. J Active apoptotic cells measured by flow cytometry probed by FITC-Annexin V and propidium iodide (PI) demonstrated significantly increased apoptosis in HeLa cells 7 h after TNFα and CHX stimulation. On the contrary, less than 20% active apoptotic cells were detected 24 h after TNFα and CHX treatment in FHL124 cells. The Student’s t test was used to compare treated and no treated cells in each cell type, and only p < 0.05 was considered significant. *<0.05, **<0.01, ***<0.001, ****<0.0001.
Fig. 3
Fig. 3. FHL124 cells block caspase 8, 9, and 3 activation and PARP-1 cleavage.
A Activated caspase-8, caspase-9, and caspase-3 were detected in HeLa cells starting at 3 h and further increased at 5 h and 7 h after TNFα and CHX stimulation. No activated caspase-8, caspase-9, and caspase-3 were detectable in FHL124 cells spanning 24 h of treatment with TNFα and CHX. A higher pro-caspase-9 quantity (around 2-fold) was seen in HeLa cells than in FHL124 cells, but a higher pro-caspase-3 quantity (around 3-fold) was seen in FHL124 compared to that in HeLa cells. B Cleavage PARP-1 was detected only in HeLa cells but not in FHL124 cells after TNFα and CHX stimulation.
Fig. 4
Fig. 4. cFLIP is highly expressed in FHL124 cells and also significantly upregulated after TNFα stimulation.
A Anti-apoptotic protein expression in FHL124 and HeLa cell with and without TNFα and TNFα plus CHX stimulation. B Relative cFLIP mRNA expression in FHL124 and HeLa cells with and without TNFα and CHX stimulation. FHL 124 cells demonstrated a more robust response to TNFα and CHX stimulation compared to HeLa cells. C The endogenous cFLIP, Caspase-8, caspase-9, and caspase-3 mRNA levels in FHL124 cells vs. HeLa cells. A much higher cFLIP mRNA expression was seen in FHL124 cells compared to HeLa cells. D,E cFLIP protein expression after TNFα and CHX stimulation in FHL124 and HeLa cells. cFLIP protein level was increased around 3-fold in FHL124 cells at 5 h after TNFα and CHX stimulation, while only a mild increase was seen in HeLa cells after stimulation. F,G FHL124 cells had 6-fold endogenous cFLIP protein levels vs. HeLa cells. KD3 shRNA was able to knock down 50% of cFLIP expression in FHL124 cells. Each assay was repeated at least three times. One-way ANOVA with Tukey’s Honest post-hoc analysis was used to compare between groups, and only p < 0.05 is considered significant. *<0.05, **<0.01, ***<0.001, ****<0.0001.
Fig. 5
Fig. 5. A mild cFLIP knocking down of FHL124 cells manifested with an increased apoptotic phenotype.
A,B Scrambled control shRNA FHL124 cells showed normal nuclear morphology 24 h after TNFα and CHX treatment. C,D KD3 cFLIP knockdown FHL124 cells demonstrated scattered apoptotic cells after 7 h of treatment. E,F KD3 cFLIP knockdown FHL124 cells demonstrated a large volume of apoptotic cells after 24 h of treatment. G,H Active apoptotic cells were analyzed by APC-Annexin V and 7-AAD stain and flow cytometry in scrambled shRNA FHL124 control cells treated with or without 24 h TNFα and CHX stimulation. J,K Active apoptotic cells were analyzed by APC-Annexin V and 7-AAD stain and flow cytometry in KD3 cFLIP knockdown FHL124 cells treated with or without 24 h TNFα and CHX stimulation. L Quantitative data of the ratio of apoptosis in control and cFLIP knockdown FHL124 cells. One-way ANOVA with Tukey’s Honest post-hoc analysis was used to compare between groups, and only p < 0.05 is considered significant. *<0.05, **<0.01, ***<0.001, ****<0.0001.
Fig. 6
Fig. 6. TNFα and CHX trigger an apoptotic response in cFLIP knockdown FHL124 cells by activating caspase-8, caspase-9, and caspase-3.
A Activated caspase-8 was detected in cFLIP KD3 cFLIP knockdown FHL124 cells from 5 h to 24 h TNFα and CHX treatment. Similar results were seen in activated caspase-9 and caspase-3. PARP-1 cleavage was detected at 7 h and 24 h time point after the stimulation. B–E The semi-quantitative densitometric measurement. One-way ANOVA was used to compare between groups, and only p < 0.05 is considered significant. *<0.05, **<0.01, ***<0.001, ****<0.0001.
Fig. 7
Fig. 7. TNFα and CHX trigger an apoptotic response in cFlip knockdown 17EM15 cells and ex vivo cultured mouse lens capsular bag by activating caspase-8, caspase-9, and caspase-3.
A cFlip knocking down in 17EM15 mouse LECs determined by immunoblot-assay. B The semi-quantitative densitometric measurement of 17EM15 cFlip knocking down from immunoblot-assay. C cFlip knocking down in ex vivo cultured mouse lens capsular bags. D The semi-quantitative densitometric measurement of cFlip knocking down mouse lens capsular bags from immunoblot-assay. E Pro-caspase-8, cleaved caspase-8, pro-caspase-3, and cleaved caspase-3 in cFlip knockdown and scrambled shRNA control (SC) 17EM15 cells with and without 30 ng/ml TNFα and 10μg/ml CHX stimulation for 7 h. F Pro-caspase-8, cleaved caspase-8, pro-caspase-3, and cleaved caspase-3 in cFlip knockdown and scrambled shRNA control (SC) ex vivo cultured mouse lens capsular bags with and without 60 ng/ml TNFα and 10 μg/ml CHX treatment for 24 h. The cFlip KD 17EM15 treated cell lysate was used as a positive control. Only 1/10th amount of protein relative to capsular bag lysate was loaded. One-way ANOVA was used to compare between groups, and only p < 0.05 is considered significant. *<0.05, **<0.01, ***<0.001, ****<0.0001.
Fig. 8
Fig. 8. Phase-contrast images of mouse lens capsular bags with and without TNFα and CHX stimulation.
(Left panel) Non-treated capsular bags. (Right panel) 60 ng/ml TNFα and 10 μg/ml CHX-treated capsular bags. (Upper parentheses) 11 h treatment with medium (control) or TNFα plus CHX. (Lower parentheses) 24 h treatment with medium (control) or TNFα plus CHX. 10x objective was used for image capture, and n = 6/group.
Fig. 9
Fig. 9. A model of cFLIP-mediated LEC’s survival.
A proposed model is highlighting cFLIP regulation of lens epithelial cells’ survival and anti-apoptotic function.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Asbell PA, et al. Age-related cataract. Lancet. 2005;365:599–609. doi: 10.1016/S0140-6736(05)70803-5. - DOI - PubMed
    1. Lou MF. Redox regulation in the lens. Prog. Retin. Eye Res. 2003;22:657–682. doi: 10.1016/S1350-9462(03)00050-8. - DOI - PubMed
    1. Augusteyn RC. On the growth and internal structure of the human lens. Exp. Eye Res. 2010;90:643–654. doi: 10.1016/j.exer.2010.01.013. - DOI - PMC - PubMed
    1. McAvoy JW, Chamberlain CG, de Iongh RU, Hales AM, Lovicu FJ. Lens development. Eye. 1999;13(Pt 3b):425–437. doi: 10.1038/eye.1999.117. - DOI - PubMed
    1. Fan X, et al. Vitamin C mediates chemical aging of lens crystallins by the Maillard reaction in a humanized mouse model. Proc. Natl Acad. Sci. USA. 2006;103:16912–16917. doi: 10.1073/pnas.0605101103. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances

-