Expression of transthyretin and retinol binding protein mRNAs and secretion of transthyretin by cultured monkey retinal pigment epithelium.
OBJECTIVE
To document the expression of mRNA for transthyretin (TTR) and retinol binding protein (RBP) in native and cultured Rhesus monkey retinal pigmented epithelium (RPE); to compare mRNA transcripts for these two proteins expressed in RPE with those found in whole monkey liver and brain; to demonstrate the secretion of TTR by RPE during short-term maintenance in a protein-free, defined medium, as a manifestation of the differentiated state of these cells in vitro.


METHODS
Total RNA was isolated from cultured RPE in first passage, after incubation for eight days in defined, protein-free medium. Conditioned medium was collected for western analysis at this time. Total RNA was also extracted from RPE/choroid freshly dissected from monkey eyes. Using cDNA probes for human TTR and RBP, northern analysis was performed on the total RNA from fresh and cultured RPE samples, together with poly(A+) mRNA purified from monkey liver and brain.


RESULTS
Conditioned medium from RPE yielded TTR protein of the expected monomer subunit molecular size. The TTR secreted de novo from the cultured cells was detectable in the absence of biosynthetic labeling. With the exception of some extremely low abundance transcripts expressed in cultured RPE, all samples contained a single 900 bp transcript for TTR. Based on relative amounts of actual message, RPE ranks higher than liver in abundance of TTR mRNA. In contrast, both native monkey RPE and cultured RPE cells expressed comparatively low levels of mRNA for RBP. All samples displayed a single RBP mRNA transcript at 1100 bp.


CONCLUSIONS
Our results indicate that TTR is a significant gene product of the RPE, and may be considered as a marker for a differentiated phenotype for these cells in culture. There is increased recognition of various forms of ocular pathology associated with mutations or other malfunctions involving TTR and RBP, warranting a greater understanding of mechanisms of transcriptional and translational control for these two proteins.
Pfeffer
Bruce A
BA
Research, Bausch and Lomb, Inc., Rochester, NY 14609, USA. Bruce_Pfeffer@bausch.com
Becerra
S Patricia
SP
Borst
Diane E
DE
Wong
Paul
P
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation / Q000502:physiology*; D005819:Genetic Markers; D008099:Liver / Q000378:metabolism; D008253:Macaca mulatta; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D011228:Prealbumin / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
C020932:11-cis-retinal-binding protein; D002352:Carrier Proteins; D005819:Genetic Markers; D011228:Prealbumin; D012333:RNA, Messenger