PURPOSE. The purpose of this study was to determine the role of protein kin
ase C (PKC) in corneal endothelial cell proliferation.
METHODS. Immunochemistry and Western blotting were used to define the PKC i
soforms expressed in primary cultures of rat corneal endothelial cells. For
proliferation studies, primary cultures of rat corneal endothelial cells w
ere serum-starved for 48 hours and incubated for 2 hours with the PKC inhib
itors staurosporine (10(-9) to 10(-7) M), chelerythrine (10(-9) to 5 x 10(-
8) M), or calphostin C (10(-9) to 10(-7) M). Individual PKC isoforms were i
nhibited using PKC alpha antisense oligonucleotide transfection or exposure
for 1 hour to myristoylated, pseudosubstrate-derived peptide inhibitors ag
ainst PKC alpha, -alpha beta gamma, -epsilon, and -delta (10(-8) to 10(-6)
M). Cells were then stimulated with 2.5% serum for 24 hours. Cell prolifera
tion was measured with bromodeoxyuridine (BrDU) and Ki67 immunocytochemistr
y. Protein level of cyclin E was determined by Western blotting.
RESULTS. PKC alpha, -beta II, -delta, -epsilon, -iota, -eta, -gamma, and -t
heta were detected in corneal endothelial cells. Maximum inhibition of PKC
with staurosporine, calphostin C, and chelerythrine reduced cell proliferat
ion to 7%, 31%, and 48% of control, respectively. Myristoylated peptide inh
ibition of PKC alpha and -epsilon reduced cell proliferation to 57% and 59%
of control, respectively. PKC alpha antisense oligonucleotide reduced cell
proliferation to 35% of control. Cyclin E protein level was decreased to 7
0%, 38%, 57%, and 43% of control in cells treated with calphostin C, stauro
sporine, chelerythrine, and PKC alpha antisense, respectively.
CONCLUSIONS. PKC activity, in particular PKC alpha and -epsilon activity, i
s important in promoting corneal endothelial cell proliferation. Inhibition
of PKC activity prohibits G1/S-phase progression and reduces cyclin E prot
ein levels.