Purpose: 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.