PKC alpha and PKC gamma overexpression causes lentoid body formation in the N/N 1003A rabbit lens epithelial cell line

PURPOSE: The overexpression of PKC alpha or PKC gamma 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 a re, we have looked for alphaA-, alphaB-, beta-, and gamma -crystallin level s in lentoid bodies after 4 and 8 weeks of lentoid body development. METHODS: Cells overexpressing PKC alpha or PKC gamma were plated in 6 well plates and were allowed to form lentoid bodies for up to 8 weeks. Lentoid b odies were fixed and stained with PKC alpha or PKC gamma antibodies along w ith either alphaA-, alphaB-, beta-, or gamma -crystallin antisera and viewe d under a confocal microscope. Lentoid bodies were harvested in lysis buffe r and homogenized. Fifty micrograms of protein per lane was loaded onto an SDS-PAGE gel and the bands transferred onto nitrocellulose. The blot was pr obed with either alphaA-, alphaB-, beta-, or gamma -crystallin antibodies f or 12 h. Total RNA from lentoid bodies was isolated and 5 mug 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 le ntoid bodies formed from PKC alpha and PKC gamma overexpression. alphaA-cry stallin was only detectable in lentoid bodies after PKC alpha or PKC gamma 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 the se lentoid bodies. CONCLUSIONS: Overexpression of PKC alpha or PKC gamma in the N/N 1003A cell line induced lentoid body formation. These lentoid bodies expressed not on ly alphaB-crystallin but alphaA- and beta -crystallin. These results sugges t a role for PKCs in lens epithelial cell differentiation to a fiber cell.