Expression of type I and type III procollagen by lens epithelial cells.
OBJECTIVE
To identify the types of procollagen that are synthesized by cultured bovine lens epithelial cells and to determine what types of post-translational modifications are involved in procollagen biosynthesis and what patterns of procollagen gene expression occur in these cells.


METHODS
The epithelial nature of the cultured cells was confirmed with transmission electron and phase contrast microscopy. To label collagen, primary or secondary monolayer cultures were pulsed with [3H] proline for various periods of time. Procollagens were identified by immunoprecipitation with specific antibodies, pepsin digestion, and electrophoretic separation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Procollagen messenger RNA was identified by Northern blot analysis using specific DNA probes.


RESULTS
In the normal lens epithelium, cells are attached to each other by tight junctions punctuated with desmosomes. After the proteins were labeled, they were immunoprecipitated with antibodies specific to collagen precursors and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to further confirm their identification as types I and III procollagen or processed forms of these proteins. The secretion of procollagen was inhibited by treatment with 2,2'-dipyridyl and bands of underhydroxylated procollagen were observed in the cell layer after electrophoresis. The sizes of messenger RNA observed were 6.9 kb and 5.4 kb for type alpha 1(I), 5.0 kb for type alpha 2(I), and 5.3 kb for the type III transcripts.


CONCLUSIONS
This is the first report that lens epithelial cells express fibrillar procollagen genes, making the lens capsule a unique ocular basement membrane.
Department of Medicine and Biochemistry, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612.
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Sawhney
D000818:Animals; D015152:Blotting, Northern; D002417:Cattle; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis / Q000235:genetics; D015342:DNA Probes; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000378:metabolism / Q000648:ultrastructure; D007908:Lens, Crystalline / Q000378:metabolism* / Q000648:ultrastructure; D008858:Microscopy, Phase-Contrast; D011233:Precipitin Tests; D011347:Procollagen / Q000096:biosynthesis* / Q000235:genetics; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000096:biosynthesis
D015342:DNA Probes; D011347:Procollagen; D012333:RNA, Messenger; D003094:Collagen