Role of cPKCalpha and nPKCepsilon in EGF-stimulated goblet cell proliferation.
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.
Shatos
Marie A
MA
Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA.
Hodges
Robin R
RR
Oshi
Yoshia
Y
Bair
Jeffrey A
JA
Zoukhri
Driss
D
Kublin
Claire
C
Lashkari
Kameran
K
Dartt
Darlene A
DA
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
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