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Protein Kinase C Regulation of Corneal Endothelial Cell Proliferation and Cell Cycle

¹ From the Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts; and the ² Massachusetts Eye and Ear Infirmary, Boston, Massachusetts.

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 x 10^-8 M), or calphostin C (10^-9 to 10^-7 M). Individual PKC isoforms were inhibited using PKC antisense oligonucleotide transfection or exposure for 1 hour to myristoylated, pseudosubstrate-derived peptide inhibitors against PKC, -ß, -, and - (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. PKC, -ßII, -, -, -, -, -, and - 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 PKC and - reduced cell proliferation to 57% and 59% of control, respectively. PKC 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 PKC antisense, respectively.

CONCLUSIONS. PKC activity, in particular PKC and - activity, is important in promoting corneal endothelial cell proliferation. Inhibition of PKC activity prohibits G1/S-phase progression and reduces cyclin E protein levels.

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