title: Role of cPKCalpha and nPKCepsilon in EGF-stimulated goblet cell proliferation.
abstract: OBJECTIVE
  The authors determined the role of the protein kinase C (PKC) isoforms cPKCalpha and nPKCepsilon in EGF-stimulated proliferation of cultured rat and human conjunctival goblet cells.
  
  
  METHODS
  Rat and human conjunctivas were minced, and goblet cells were allowed to grow. Passage 1 cells were serum starved for 24 to 48 hours and were incubated with the PKC inhibitors calphostin C and Gö 6983 (10(-10)-10(-7) M) for 20 minutes before stimulation with EGF (10(-7) M) for 24 hours. The presence and localization of PKC isoforms in cultured rat goblet cells were determined by Western blot analysis and immunofluorescence microscopy, respectively. Cultured rat goblet cells were serum starved and incubated with adenoviruses containing genes for dominant-negative cPKCalpha (Ad DNPKCalpha, 10(4) pfu), dominant-negative nPKCepsilon (Ad DNPKCepsilon, 10(4) pfu), and wild-type cPKCalpha (Ad WTPKCalpha, 10(7) pfu), and proliferation was measured.
  
  
  RESULTS
  In rat goblet cells, EGF-stimulated proliferation was completely inhibited by calphostin C, whereas Gö 6983 inhibited proliferation by 53%+/-15%. In human goblet cells, EGF-stimulated proliferation was completely inhibited by calphostin C. PKCalpha, -betaI, -betaII, -delta, -epsilon, -iota/lambda, -theta, -gamma, and -zeta were found in cultured rat goblet cells. Ad DNPKCalpha and Ad DNPKCepsilon inhibited EGF-stimulated proliferation in rat goblet cells by 78%+/-6% and 92%+/-8%, respectively. Incubation with Ad WTPKCalpha alone significantly increased proliferation.
  
  
  CONCLUSIONS
  cPKCalpha and nPKCepsilon play key roles in conjunctival goblet cell proliferation.
authors.affiliation: Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA.
authors.forename: Marie A
authors.initials: MA
authors.lastname: Shatos
authors.forename: Robin R
authors.initials: RR
authors.lastname: Hodges
authors.forename: Yoshia
authors.initials: Y
authors.lastname: Oshi
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Bair
authors.forename: Driss
authors.initials: D
authors.lastname: Zoukhri
authors.forename: Claire
authors.initials: C
authors.lastname: Kublin
authors.forename: Kameran
authors.initials: K
authors.lastname: Lashkari
authors.forename: Darlene A
authors.initials: DA
authors.lastname: Dartt
mesh_terms: D000368:Aged; D000818:Animals; D015153:Blotting, Western; D002227:Carbazoles / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology* / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005260:Female; D020397:Goblet Cells / Q000166:cytology* / Q000201:enzymology; D006801:Humans; D007211:Indoles; D007527:Isoenzymes; D008297:Male; D008301:Maleimides; D008856:Microscopy, Fluorescence; D009281:Naphthalenes / Q000494:pharmacology; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000502:physiology*; D051744:Protein Kinase C-epsilon / Q000037:antagonists & inhibitors / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: C465664:2-(1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl)-3-(1H-indol-3-yl)maleimide; D002227:Carbazoles; D004791:Enzyme Inhibitors; D007211:Indoles; D007527:Isoenzymes; D008301:Maleimides; D009281:Naphthalenes; D004815:Epidermal Growth Factor; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; C058819:calphostin C