Posttranslational regulation of type I collagen in corneal endothelial cells.
OBJECTIVE
Type I collagen synthesis in corneal endothelial cells does not correlate with steady state collagen RNA levels; although substantial amounts of alpha 2(I) collagen RNA are present in these cells, type I collagen is not detected. This allowed the authors to investigate the possibility of posttranscriptional control of type I collagen in corneal endothelial cells.


METHODS
The alpha 2(I) collagen RNA structures of normal and modulated corneal endothelial cells were analyzed by S1 nuclease protection analysis, whereas the nucleotide sequences were obtained by rapid amplification of cDNA ends technique. In situ hybridization of type I collagen was demonstrated with immunofluorescence; synthesis and degradation of the molecule were analyzed by pulse-chase experiments and then by immunoprecipitation with antiprocollagen I antibody.


RESULTS
The cDNA covering the 5'-untranslated region (UTR) of alpha 2(I) collagen RNA obtained from normal corneal endothelial cells and from modulated corneal endothelial cells that predominantly produce type I collagen demonstrate identical sequences in their 5' untranslated and coding sequences. In both mRNA, the length of the 5'-untranslated segment is 127 nucleotides. There were also two AUG codons; the second AUG codon, which is 17 nucleotides upstream from the translation initiation codon, is conserved, as observed in human and chicken alpha 2(I) mRNA. When the sequence covering the 3'-UTR of corneal endothelial alpha 2(I) mRNA was compared with that of alpha 2(I) mRNA obtained from the modulated cells, there were differences in only two nucleotides. The length of the 3'-untranslated segment of each mRNA is 297 nucleotides up to the consensus polyadenylation recognition site (AAUAAAAUAAA), which both cells use. Immunofluorescent staining of corneal tissue in vivo demonstrated that the corneal endothelium stains with anti-type I collagen antibodies, but there is no staining in the underlying Descemet's membrane. In pulse-chase experiments, the newly synthesized type I procollagen, composed of pro alpha 1(I) and pro alpha 2(I) chains as determined by V8 protease peptide mapping, reached the highest intracellular level at 45 minutes, after which its detection decreased. Cells chased for 120 minutes demonstrated no trace of type I procollagen in the cell layer; medium fractions showed no detectable type I procollagen during the entire 120-minute chase.


CONCLUSIONS
These results suggest that type I collagen is synthesized in corneal endothelial cells and that such undesired expression is regulated at the posttranslational level, perhaps by intracellular degradation.
Department of Ophthalmology, University of Southern California School of Medicine, Los Angeles 90033, USA.
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Kay
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Choi
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Ninomiya
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002478:Cells, Cultured; D002645:Chickens; D003001:Cloning, Molecular; D003094:Collagen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004728:Endothelium, Corneal / Q000166:cytology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D016133:Polymerase Chain Reaction; D011347:Procollagen / Q000096:biosynthesis; D011499:Protein Processing, Post-Translational; D012313:RNA / Q000032:analysis / Q000737:chemistry; D012333:RNA, Messenger / Q000096:biosynthesis / Q000737:chemistry; D011817:Rabbits
D017931:DNA Primers; D011347:Procollagen; D012333:RNA, Messenger; D012313:RNA; D003094:Collagen