Protein kinase C alpha and gamma in N/N 1003A rabbit lens epithelial cell differentiation.
OBJECTIVE
To determine if Protein Kinase C (PKC) plays a role in the initiation of lens epithelial cell differentiation into a lens fiber cell.


METHODS
PKCalpha or PKCgamma was overexpressed in N/N 1003A lens epithelial cells for up to 7 days. Phase contrast microscopy was used to observe morphological changes associated with PKCalpha or PKCgamma overexpression. Cell cycle changes in cells overexpressing PKCalpha or PKCgamma were measured using acridine orange staining and flow cytometry. Crystallin levels in cells overexpressing PKCalpha or PKCgamma were measured using Western blots and RT-PCR.


RESULTS
Significant differences in cell cycling were observed between untransfected cells and those overexpressing PKCalpha or PKCgamma. Overexpression of PKCalpha and PKCgamma caused the cells to lose their epithelial-like appearance and elongate. alphaB-crystallin expression was detected in all the samples while alphaA-crystallin was detected only in cells after 7 days of PKCalpha or PKCgamma overexpression.


CONCLUSIONS
The observations that alphaA-crystallin is only found in N/N 1003A cells overexpressing PKCalpha or PKCgamma for 7 days along with the finding that a block in the G0/G1 phase of the cell cycle and the consequent morphological changes are observed, indicate that PKCalpha and PKCgamma may have a role in the initiation of differentiation in lens epithelial cells.
Department of Anatomy and Physiology, Kansas State University, Manhattan, KS 66506, USA.
L M
LM
Wagner
D J
DJ
Takemoto
D000165:Acridine Orange; D000818:Animals; D015153:Blotting, Western; D002453:Cell Cycle / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D003459:Crystallins / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D007527:Isoenzymes / Q000502:physiology*; D007908:Lens, Crystalline / Q000166:cytology* / Q000378:metabolism; D008858:Microscopy, Phase-Contrast; D011493:Protein Kinase C / Q000502:physiology*; D051571:Protein Kinase C-alpha; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D003459:Crystallins; D017931:DNA Primers; D007527:Isoenzymes; D012333:RNA, Messenger; C087857:protein kinase C gamma; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D000165:Acridine Orange