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Melanie A. Graham, Ian Rawe, Darlene A. Dartt, Nancy C. Joyce; Protein Kinase C Regulation of Corneal Endothelial Cell Proliferation and Cell Cycle. Invest. Ophthalmol. Vis. Sci. 2000;41(13):4124-4132.
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purpose. The purpose of this study was to determine the role of protein kinase C
(PKC) in corneal endothelial cell proliferation.
methods. Immunocytochemistry and Western blotting were used to define the PKC
isoforms expressed in primary cultures of rat corneal endothelial
cells. For proliferation studies, primary cultures of rat corneal
endothelial cells were serum-starved for 48 hours and incubated for 2
hours with the PKC inhibitors staurosporine (10−9 to
10−7 M), chelerythrine (10−9 to 5 ×
10−8 M), or calphostin C (10−9 to
10−7 M). Individual PKC isoforms were inhibited using
PKCα antisense oligonucleotide transfection or exposure for 1 hour to
myristoylated, pseudosubstrate-derived peptide inhibitors against
PKCα, -αβγ, -ε, and -δ (10−8 to
10−6 M). Cells were then stimulated with 2.5% serum for
24 hours. Cell proliferation was measured with bromodeoxyuridine (BrDU)
and Ki67 immunocytochemistry. Protein level of cyclin E was determined
by Western blotting.
results. PKCα, -βΙΙ, -δ, -ε, -ι, -η, -γ, and -θ were
detected in corneal endothelial cells. Maximum inhibition of PKC with
staurosporine, calphostin C, and chelerythrine reduced cell
proliferation to 7%, 31%, and 48% of control, respectively.
Myristoylated peptide inhibition of PKCα and -ε reduced cell
proliferation to 57% and 59% of control, respectively. PKCα
antisense oligonucleotide reduced cell proliferation to 35% of
control. Cyclin E protein level was decreased to 70%, 38%, 57%, and
43% of control in cells treated with calphostin C, staurosporine,
chelerythrine, and PKCα antisense, respectively.
conclusions. PKC activity, in particular PKCα and -ε activity, is important in
promoting corneal endothelial cell proliferation. Inhibition of PKC
activity prohibits G1/S-phase progression and reduces cyclin E protein
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