Protein kinase C regulation of corneal endothelial cell proliferation and cell cycle

Citation
Ma. Graham et al., Protein kinase C regulation of corneal endothelial cell proliferation and cell cycle, INV OPHTH V, 41(13), 2000, pp. 4124-4132
Citations number
38
Categorie Soggetti
da verificare
Journal title
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
ISSN journal
01460404 → ACNP
Volume
41
Issue
13
Year of publication
2000
Pages
4124 - 4132
Database
ISI
SICI code
0146-0404(200012)41:13<4124:PKCROC>2.0.ZU;2-X
Abstract
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.