title: Induction of intercellular adhesion molecule-1 by tumor necrosis factor-alpha through the 55-kDa receptor is dependent on protein kinase C in human retinal pigment epithelial cells.
abstract: OBJECTIVE
  To determine second messenger signaling pathways associated with tumor necrosis factor-alpha (TNF)-mediated induction of intercellular adhesion molecule (ICAM)-1 expression on human retinal pigment epithelial (HRPE) cells, a cell type known to express only the 55-kDa TNF receptor (TNFR p55).
  
  
  METHODS
  SV 40-immortalized HRPE (SVRPE) cells were exposed to TNF with and without pretreatment with the protein kinase C (PKC) inhibitor calphostin C or the protein kinase A (PKA) inhibitor H8. SV40-immortalized HRPE cells also were treated with the PKC activator phorbol 12-myristate 13-acetate (PMA) or with the PKA activators forskolin plus 3-isobutyl-1-methyl-xanthine or dibutyryl cyclic adenosine monophosphate (cAMP) alone. Membrane fractions from untreated and treated SVRPE cells were assayed for PKC activity, and whole cell lysates were assayed for cAMP accumulation and PKA activity. Flow cytometry was performed on SVRPE cells using a monoclonal antibody specific to ICAM-1.
  
  
  RESULTS
  Activation of TNFR p55 on SVRPE cells with TNF resulted in a rapid increase of PKC activity at 1 minute, with a subsequent downregulation to baseline. There was no increase in intracellular cAMP accumulation or PKA activity within the first 10 minutes; however, both increased within 30 minutes and returned to baseline within 1 hour. SV40-immortalized HRPE cells treated with TNF for 1 hour showed maximal induction of ICAM-1 expression at 18 hours. ICAM-1 induction by TNF treatment was inhibited by calphostin C pretreatment and not by H8 pretreatment. Protein kinase C activation with PMA for 3 hours was sufficient to induce ICAM-1 on SVRPE cells at 18 hours, whereas treatment with the PKA activators forskolin or dibutyryl cAMP did not induce ICAM-1 expression.
  
  
  CONCLUSIONS
  Tumor necrosis factor sequentially activates the PKC and PKA pathways in SVRPE cells by way of the TNFR p55. The PKC pathway in necessary for TNF-mediated ICAM-1 upregulation, and specific activation of the PKC pathway with PMA is sufficient to induce ICAM-1 on these cells. SV40-immortalized HRPE cells may serve as a model in which to study further the functional signaling pathways associated with TNFR p55.
authors.affiliation: Department of Pathology, University of Southern California School of Medicine, Los Angeles, 90033, USA.
authors.forename: B D
authors.initials: BD
authors.lastname: Sippy
authors.forename: F M
authors.initials: FM
authors.lastname: Hofman
authors.forename: A D
authors.initials: AD
authors.lastname: Wright
authors.forename: J L
authors.initials: JL
authors.lastname: Wang
authors.forename: R
authors.initials: R
authors.lastname: Gopalakrishna
authors.forename: U
authors.initials: U
authors.lastname: Gundimeda
authors.forename: S
authors.initials: S
authors.lastname: He
authors.forename: S J
authors.initials: SJ
authors.lastname: Ryan
authors.forename: D R
authors.initials: DR
authors.lastname: Hinton
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology; D002461:Cell Line, Transformed; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis*; D007546:Isoquinolines / Q000494:pharmacology; D009281:Naphthalenes / Q000494:pharmacology; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism*; D015290:Second Messenger Systems; D015398:Signal Transduction; D013539:Simian virus 40; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D002273:Carcinogens; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D009281:Naphthalenes; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; C053177:N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate; D015056:1-Methyl-3-isobutylxanthine