Protein kinase C regulation of corneal endothelial cell proliferation and cell cycle.
OBJECTIVE
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 PKCalpha antisense oligonucleotide transfection or exposure for 1 hour to myristoylated, pseudosubstrate-derived peptide inhibitors against PKCalpha, -alphassgamma, -epsilon, and -delta (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
PKCalpha, -ssII, -delta, -epsilon, -iota, -eta, -gamma, and -theta 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 PKCalpha and -epsilon reduced cell proliferation to 57% and 59% of control, respectively. PKCalpha 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 PKCalpha antisense, respectively.


CONCLUSIONS
PKC activity, in particular PKCalpha and -epsilon activity, is important in promoting corneal endothelial cell proliferation. Inhibition of PKC activity prohibits G1/S-phase progression and reduces cyclin E protein levels.
Graham
M A
MA
Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA.
Rawe
I
I
Dartt
D A
DA
Joyce
N C
NC
D000818:Animals; D015153:Blotting, Western; D001973:Bromodeoxyuridine / Q000378:metabolism; D002455:Cell Division / Q000502:physiology*; D002478:Cells, Cultured; D019927:Cyclin E / Q000378:metabolism; D004261:DNA Replication; D004728:Endothelium, Corneal / Q000166:cytology* / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016193:G1 Phase / Q000502:physiology*; D007124:Immunoenzyme Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000502:physiology; D008297:Male; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016196:S Phase / Q000502:physiology*
D019927:Cyclin E; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011493:Protein Kinase C; D001973:Bromodeoxyuridine