title: Modulation of TNF-alpha-induced apoptosis in corneal fibroblasts by transcription factor NF-kappaB.
abstract: OBJECTIVE
  Previous studies have suggested no role for tumor necrosis factor (TNF)-alpha in the modulation of apoptosis in corneal fibroblasts. However, recent investigations have demonstrated that nuclear factor (NF)-kappaB activation by TNF-alpha mediates negative apoptotic effects that must be blocked to unmask the apoptotic effects of TNF-alpha in vitro. The purpose of this study was to investigate the role of transcription factor NF-kappaB in the suppression of TNF-alpha-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-alpha in presence or absence of the specific inhibitor of NF-kappaB 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-KB activation was monitored using electrophoretic gel shift assay.
  
  
  RESULTS
  TNF-alpha, 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-alpha mRNA was also detected in ex vivo corneal epithelium, corneal epithelial cells, and stromal fibroblasts with the RNase protection assay. TNF-alpha, TNFRI, and TNFRII proteins were detected by immunocytochemistry in all three major corneal cell types in human corneal tissue. TNF-alpha protein was also detected in ex vivo corneal epithelium, primary corneal epithelial cells, and primary stromal fibroblasts using immunoprecipitation and Western blot analysis. TNF-alpha stimulated corneal fibroblast cell death when NF-kappaB 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-KB by TNF-alpha and suppression of induction in the presence of actinomycin D or SN50, but not by the control peptide SN50M.
  
  
  CONCLUSIONS
  The TNF-alpha receptor system is expressed in the cornea, and NF-kappaB activation is an important regulator of TNF-alpha-mediated corneal fibroblast apoptosis. Nonsteroidal anti-inflammatory agents or corticosteroids may potentiate corneal fibroblast apoptosis in response to cytokine stimulation.
authors.affiliation: Department of Ophthalmology, University of Washington School of Medicine, Seattle, USA.
authors.forename: R R
authors.initials: RR
authors.lastname: Mohan
authors.forename: R R
authors.initials: RR
authors.lastname: Mohan
authors.forename: W J
authors.initials: WJ
authors.lastname: Kim
authors.forename: S E
authors.initials: SE
authors.lastname: Wilson
mesh_terms: D015703:Antigens, CD / Q000096:biosynthesis / Q000235:genetics; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D002675:Child, Preschool; D003315:Cornea / Q000378:metabolism / Q000473:pathology*; D004247:DNA / Q000032:analysis; D017931:DNA Primers / Q000737:chemistry; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology*; D015870:Gene Expression; D006801:Humans; D007124:Immunoenzyme Techniques; D007223:Infant; D007231:Infant, Newborn; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis / Q000235:genetics; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000494:pharmacology*
chemical_list: D015703:Antigens, CD; D017931:DNA Primers; D016328:NF-kappa B; D012333:RNA, Messenger; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D014409:Tumor Necrosis Factor-alpha; D004247:DNA; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases