Expression of transthyretin and retinol binding protein mRNAs andsecretion of transthyretin by cultured monkey retinal pigmentepithelium

[摘要] Purpose: To document the expression of mRNA for transthyretin (TTR)and retinol binding protein (RBP) in native and cultured Rhesus monkeyretinal pigmented epithelium (RPE); to compare mRNA transcripts forthese two proteins expressed in RPE with those found in whole monkeyliver and brain; to demonstrate the secretion of TTR by RPE duringshort-term maintenance in a protein-free, defined medium, as amanifestation 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 frommonkey eyes. Using cDNA probes for human TTR and RBP, northern analysiswas 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 theexpected monomer subunit molecular size. The TTR secreted de novo fromthe cultured cells was detectable in the absence of biosyntheticlabeling. With the exception of some extremely low abundance transcriptsexpressed in cultured RPE, all samples contained a single 900 bptranscript for TTR. Based on relative amounts of actual message, RPEranks higher than liver in abundance of TTR mRNA. In contrast, bothnative monkey RPE and cultured RPE cells expressed comparatively lowlevels of mRNA for RBP. All samples displayed a single RBP mRNAtranscript at 1100 bp.Conclusions: Our results indicate that TTR is a significant geneproduct of the RPE, and may be considered as a marker for adifferentiated phenotype for these cells in culture. There is increasedrecognition of various forms of ocular pathology associated withmutations or other malfunctions involving TTR and RBP, warranting agreater understanding of mechanisms of transcriptional and translationalcontrol for these two proteins.

[学科分类] 生物化学/生物物理