Stimulatory role of PKCalpha in extracellular regulated kinase 1/2 pathway in conjunctival goblet cell proliferation.
OBJECTIVE
To determine whether a constitutively active protein kinase C (PKC)-alpha stimulates rat and human conjunctival goblet cell proliferation through activation of ERK 1/2.


METHODS
Conjunctivas from rat and human were minced and goblet cells were allowed to grow. Goblet cells were serum starved and incubated with an adenovirus containing a constitutively active form of PKCalpha (Ad-myr-PKCalpha, 1 x 10(7) pfu), EGF (10(-7) M), or both. The location of myrPKCalpha was determined by immunofluorescence microscopy. Cultured goblet cells were preincubated with the PKC inhibitor calphostin C (10(-10)-10(-7) M) or the ERK 1/2 inhibitor U0126 (10(-9)-10(-6) M) before incubation with Ad-myr-PKCalpha. Cell proliferation was measured.


RESULTS
Transduction of rat goblet cells with Ad-myr-PKCalpha did not change PKC location compared with nontransduced cells. Incubation with Ad-myr-PKCalpha caused an increase in cell proliferation by 2.5+/-0.3-fold, whereas EGF increased proliferation by 2.1+/-0.2-fold. Simultaneous addition of Ad-myr-PKCalpha and EGF did not further increase proliferation. U0126 inhibited Ad-myr-PKCalpha-stimulated proliferation a maximum of 70%. In human goblet cells, incubation with Ad-myr-PKCalpha caused an increase in cell proliferation by 2.3+/-0.3-fold, whereas EGF increased proliferation by 3.1+/-0.4-fold. Simultaneous addition of Ad-myr-PKCalpha and EGF decreased proliferation compared with either compound alone. Ad-myr-PKCalpha caused ERK 1/2 to translocate to the nucleus in rat and human cells, but the translocation was blocked by U0126.


CONCLUSIONS
Activation of PKCalpha alone by inducing phosphorylation of ERK 1/2 and translocating it to the nucleus is necessary and sufficient to cause conjunctival cell proliferation in rat, and probably human, goblet cells.
Shatos
Marie A
MA
Schepens Eye Research Institute, Boston, Massachusetts 02114, USA.
Hodges
Robin R
RR
Bair
Jeffrey A
JA
Lashkari
Kameran
K
Dartt
Darlene A
DA
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology; D049109:Cell Proliferation; D003228:Conjunctiva / Q000166:cytology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005260:Female; D020397:Goblet Cells / Q000166:cytology*; D006801:Humans; D008297:Male; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D009924:Organ Culture Techniques; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology
D002070:Butadienes; D004791:Enzyme Inhibitors; D009570:Nitriles; C113580:U 0126; D004815:Epidermal Growth Factor; D051571:Protein Kinase C-alpha; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3