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Rajiv R. Mohan, Rahul R. Mohan, Woo-Jung Kim, Steven E. Wilson; Modulation of TNF-α–Induced Apoptosis in Corneal Fibroblasts by Transcription Factor NF-κB. Invest. Ophthalmol. Vis. Sci. 2000;41(6):1327-1336.
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purpose. Previous studies have suggested no role for tumor necrosis factor
(TNF)-α in the modulation of apoptosis in corneal fibroblasts.
However, recent investigations have demonstrated that nuclear factor
(NF)-κB activation by TNF-α mediates negative apoptotic effects
that must be blocked to unmask the apoptotic effects of TNF-α in
vitro. The purpose of this study was to investigate the role of
transcription factor NF-κB in the suppression of TNF-α–induced
apoptosis of corneal fibroblasts.
methods. mRNA was detected by reverse transcription–polymerase chain reaction
(RT-PCR) and RNase protection assay. Proteins were detected by
immunocytochemistry and immunoprecipitation with Western blot analysis.
Cell death was evaluated by trypan blue exclusion assay in corneal
fibroblasts treated with TNF-α in presence or absence of the specific
inhibitor of NF-κB activation, SN50, actinomycin D, or actinomycin D
with dexamethasone, ketorolac tromethamine, or diclofenac sodium.
Apoptosis was monitored by trypan blue exclusion, colorimetric cell
assay, CPP32 activation assay, DNA fragmentation assay, and
transmission electron microscopy. NF-κB activation was monitored
using electrophoretic gel shift assay.
results. TNF-α, TNF receptor (R)I, and TNFRII mRNAs were detected in all three
cultured corneal cell types and in ex vivo corneal epithelium using
RT-PCR. TNF-α mRNA was also detected in ex vivo corneal epithelium,
corneal epithelial cells, and stromal fibroblasts with the RNase
protection assay. TNF-α, TNFRI, and TNFRII proteins were detected by
immunocytochemistry in all three major corneal cell types in human
corneal tissue. TNF-α protein was also detected in ex vivo corneal
epithelium, primary corneal epithelial cells, and primary stromal
fibroblasts using immunoprecipitation and Western blot analysis.
TNF-α stimulated corneal fibroblast cell death when NF-κB
activation was blocked with actinomycin D or SN50. Enhanced cell death
was noted with dexamethasone, ketorolac tromethamine, or diclofenac
sodium when used in the presence, but not in the absence, of
actinomycin D. A gel shift assay revealed induction of NF-κB by
TNF-α and suppression of induction in the presence of actinomycin D
or SN50, but not by the control peptide SN50M.
conclusions. The TNF-α receptor system is expressed in the cornea, and NF-κB
activation is an important regulator of TNF-α–mediated corneal
fibroblast apoptosis. Nonsteroidal anti-inflammatory agents or
corticosteroids may potentiate corneal fibroblast apoptosis in response
to cytokine stimulation.
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