PKCalpha and PKCgamma overexpression causes lentoid body formation in the N/N 1003A rabbit lens epithelial cell line.
OBJECTIVE
The overexpression of PKCalpha or PKCgamma for extended periods of time causes the formation of lentoid bodies in the N/N 1003A rabbit lens epithelial cell line. To determine how differentiated the lentoid bodies are, we have looked for alphaA-, alphaB-, beta-, and gamma-crystallin levels in lentoid bodies after 4 and 8 weeks of lentoid body development.


METHODS
Cells overexpressing PKCalpha or PKCgamma were plated in 6 well plates and were allowed to form lentoid bodies for up to 8 weeks. Lentoid bodies were fixed and stained with PKCalpha or PKCgamma antibodies along with either alphaA-, alphaB-, beta-, or gamma-crystallin antisera and viewed under a confocal microscope. Lentoid bodies were harvested in lysis buffer and homogenized. Fifty micrograms of protein per lane was loaded onto an SDS-PAGE gel and the bands transferred onto nitrocellulose. The blot was probed with either alphaA-, alphaB-, beta-, or gamma-crystallin antibodies for 12 h. Total RNA from lentoid bodies was isolated and 5 microg of total RNA was transcribed to first-strand cDNA. The PCR products were analyzed by 2% agarose gel electrophoresis.


RESULTS
alphaB-crystallin was present in normal N/N 1003A cells and the lentoid bodies formed from PKCalpha and PKCgamma overexpression. alphaA-crystallin was only detectable in lentoid bodies after PKCalpha or PKCgamma overexpression. RT-PCR was able to detect beta-crystallin expression while the Western blot analysis and immunocytostaining detected small amounts of beta-crystallin protein. No gamma-crystallin expression was noted in these lentoid bodies.


CONCLUSIONS
Overexpression of PKCalpha or PKCgamma in the N/N 1003A cell line induced lentoid body formation. These lentoid bodies expressed not only alphaB-crystallin but alphaA- and beta-crystallin. These results suggest a role for PKCs in lens epithelial cell differentiation to a fiber cell.
Wagner
L M
LM
Department of Anatomy and Physiology, Kansas State University, Manhattan, KS 66506, USA. lynnw@ksu.edu
Takemoto
D J
DJ
D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D002460:Cell Line; D003459:Crystallins / Q000096:biosynthesis*; D017931:DNA Primers / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000378:metabolism*; D015870:Gene Expression; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D007908:Lens, Crystalline / Q000378:metabolism*; D018613:Microscopy, Confocal; D008858:Microscopy, Phase-Contrast; D011493:Protein Kinase C / Q000235:genetics* / Q000378:metabolism; D051571:Protein Kinase C-alpha; D012333:RNA, Messenger / Q000096:biosynthesis; 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