Effect of NF-κB inhibition on TNF-α-induced apoptosis in human RPE cells

Ping Yang, Brian S Mckay, Janice B. Allen, Glenn J. Jaffe

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

PURPOSE. In many cell types, tumor necrosis factor (TNF)-α-induced apoptosis is prevented by production of TNF-α-induced antiapoptotic protein, a process mediated by nuclear transcription factor (NF)-κB. TNF-α is widely expressed in proliferative vitreoretinopathy (PVR) membranes and is present in the vitreous of eyes with PVR. To understand mechanisms responsible for RPE cell survival and death in this disease, this study was conducted to determine whether specific NF-κB blockade by mutant inhibitory (I)-κB (IκB) affects TNF-α-induced cell death. METHODS. Cultured human RPE cells and T-98G cells were infected with adenovirus encoding either β-galactosidase or mutant IκB and then treated with TNF-α or interleukin (IL)-1β. IκB, inhibitor of apoptosis protein (IAP)-1, and cellular Fas-associated death domain-like interleukin-1β-converting enzyme-like inhibitory protein (c-FLIP) expression was determined by Western blot. Functional NF-κB activation was examined by luciferase reporter assay. Cell viability was evaluated by trypan blue exclusion assay. Caspase-3 activity and DNA fragmentation was measured with an enzyme-linked immunosorbent assay. RESULTS. Mutant IκB expression blocked cytokine-induced IκB degradation and NF-κB transcriptional activity in RPE cells and T-98G cells. RPE cells were resistant to TNF-α-induced apoptosis, even after NF-κB activation was specifically blocked. In contrast, TNF-α dramatically induced apoptosis in T-98G cells after NF-κB inhibition. c-IAP1 expression was not affected by TNF-α or mutant IκB, and mutant IκB abolished TNF-α-induced c-FLIP induction in RPE cells. CONCLUSIONS. RPE cells are resistant to TNF-α-induced cell death, even after NF-κB activation is specifically blocked. RPE cell resistance to apoptotic signals present in eyes with PVR, mediated by survival factors such as c-FLIP and c-IAP1, may help to explain unwanted and unchecked cell proliferation in this disease.

Original languageEnglish (US)
Pages (from-to)2438-2446
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume45
Issue number7
DOIs
StatePublished - Jul 2004
Externally publishedYes

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Transcription Factors
Tumor Necrosis Factor-alpha
Apoptosis
Proliferative Vitreoretinopathy
Cell Death
Cell Survival
Galactosidases
Inhibitor of Apoptosis Proteins
Caspase 1
Trypan Blue
DNA Fragmentation
Luciferases
Interleukin-1
Adenoviridae
Caspase 3
Proteins
Western Blotting
Enzyme-Linked Immunosorbent Assay
Cell Proliferation
Cytokines

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Effect of NF-κB inhibition on TNF-α-induced apoptosis in human RPE cells. / Yang, Ping; Mckay, Brian S; Allen, Janice B.; Jaffe, Glenn J.

In: Investigative Ophthalmology and Visual Science, Vol. 45, No. 7, 07.2004, p. 2438-2446.

Research output: Contribution to journalArticle

Yang, Ping ; Mckay, Brian S ; Allen, Janice B. ; Jaffe, Glenn J. / Effect of NF-κB inhibition on TNF-α-induced apoptosis in human RPE cells. In: Investigative Ophthalmology and Visual Science. 2004 ; Vol. 45, No. 7. pp. 2438-2446.
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abstract = "PURPOSE. In many cell types, tumor necrosis factor (TNF)-α-induced apoptosis is prevented by production of TNF-α-induced antiapoptotic protein, a process mediated by nuclear transcription factor (NF)-κB. TNF-α is widely expressed in proliferative vitreoretinopathy (PVR) membranes and is present in the vitreous of eyes with PVR. To understand mechanisms responsible for RPE cell survival and death in this disease, this study was conducted to determine whether specific NF-κB blockade by mutant inhibitory (I)-κB (IκB) affects TNF-α-induced cell death. METHODS. Cultured human RPE cells and T-98G cells were infected with adenovirus encoding either β-galactosidase or mutant IκB and then treated with TNF-α or interleukin (IL)-1β. IκB, inhibitor of apoptosis protein (IAP)-1, and cellular Fas-associated death domain-like interleukin-1β-converting enzyme-like inhibitory protein (c-FLIP) expression was determined by Western blot. Functional NF-κB activation was examined by luciferase reporter assay. Cell viability was evaluated by trypan blue exclusion assay. Caspase-3 activity and DNA fragmentation was measured with an enzyme-linked immunosorbent assay. RESULTS. Mutant IκB expression blocked cytokine-induced IκB degradation and NF-κB transcriptional activity in RPE cells and T-98G cells. RPE cells were resistant to TNF-α-induced apoptosis, even after NF-κB activation was specifically blocked. In contrast, TNF-α dramatically induced apoptosis in T-98G cells after NF-κB inhibition. c-IAP1 expression was not affected by TNF-α or mutant IκB, and mutant IκB abolished TNF-α-induced c-FLIP induction in RPE cells. CONCLUSIONS. RPE cells are resistant to TNF-α-induced cell death, even after NF-κB activation is specifically blocked. RPE cell resistance to apoptotic signals present in eyes with PVR, mediated by survival factors such as c-FLIP and c-IAP1, may help to explain unwanted and unchecked cell proliferation in this disease.",
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N2 - PURPOSE. In many cell types, tumor necrosis factor (TNF)-α-induced apoptosis is prevented by production of TNF-α-induced antiapoptotic protein, a process mediated by nuclear transcription factor (NF)-κB. TNF-α is widely expressed in proliferative vitreoretinopathy (PVR) membranes and is present in the vitreous of eyes with PVR. To understand mechanisms responsible for RPE cell survival and death in this disease, this study was conducted to determine whether specific NF-κB blockade by mutant inhibitory (I)-κB (IκB) affects TNF-α-induced cell death. METHODS. Cultured human RPE cells and T-98G cells were infected with adenovirus encoding either β-galactosidase or mutant IκB and then treated with TNF-α or interleukin (IL)-1β. IκB, inhibitor of apoptosis protein (IAP)-1, and cellular Fas-associated death domain-like interleukin-1β-converting enzyme-like inhibitory protein (c-FLIP) expression was determined by Western blot. Functional NF-κB activation was examined by luciferase reporter assay. Cell viability was evaluated by trypan blue exclusion assay. Caspase-3 activity and DNA fragmentation was measured with an enzyme-linked immunosorbent assay. RESULTS. Mutant IκB expression blocked cytokine-induced IκB degradation and NF-κB transcriptional activity in RPE cells and T-98G cells. RPE cells were resistant to TNF-α-induced apoptosis, even after NF-κB activation was specifically blocked. In contrast, TNF-α dramatically induced apoptosis in T-98G cells after NF-κB inhibition. c-IAP1 expression was not affected by TNF-α or mutant IκB, and mutant IκB abolished TNF-α-induced c-FLIP induction in RPE cells. CONCLUSIONS. RPE cells are resistant to TNF-α-induced cell death, even after NF-κB activation is specifically blocked. RPE cell resistance to apoptotic signals present in eyes with PVR, mediated by survival factors such as c-FLIP and c-IAP1, may help to explain unwanted and unchecked cell proliferation in this disease.

AB - PURPOSE. In many cell types, tumor necrosis factor (TNF)-α-induced apoptosis is prevented by production of TNF-α-induced antiapoptotic protein, a process mediated by nuclear transcription factor (NF)-κB. TNF-α is widely expressed in proliferative vitreoretinopathy (PVR) membranes and is present in the vitreous of eyes with PVR. To understand mechanisms responsible for RPE cell survival and death in this disease, this study was conducted to determine whether specific NF-κB blockade by mutant inhibitory (I)-κB (IκB) affects TNF-α-induced cell death. METHODS. Cultured human RPE cells and T-98G cells were infected with adenovirus encoding either β-galactosidase or mutant IκB and then treated with TNF-α or interleukin (IL)-1β. IκB, inhibitor of apoptosis protein (IAP)-1, and cellular Fas-associated death domain-like interleukin-1β-converting enzyme-like inhibitory protein (c-FLIP) expression was determined by Western blot. Functional NF-κB activation was examined by luciferase reporter assay. Cell viability was evaluated by trypan blue exclusion assay. Caspase-3 activity and DNA fragmentation was measured with an enzyme-linked immunosorbent assay. RESULTS. Mutant IκB expression blocked cytokine-induced IκB degradation and NF-κB transcriptional activity in RPE cells and T-98G cells. RPE cells were resistant to TNF-α-induced apoptosis, even after NF-κB activation was specifically blocked. In contrast, TNF-α dramatically induced apoptosis in T-98G cells after NF-κB inhibition. c-IAP1 expression was not affected by TNF-α or mutant IκB, and mutant IκB abolished TNF-α-induced c-FLIP induction in RPE cells. CONCLUSIONS. RPE cells are resistant to TNF-α-induced cell death, even after NF-κB activation is specifically blocked. RPE cell resistance to apoptotic signals present in eyes with PVR, mediated by survival factors such as c-FLIP and c-IAP1, may help to explain unwanted and unchecked cell proliferation in this disease.

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