Retinoid-binding proteins and retinol esterification in cultured retinal pigment epithelium cells

Abstract

The esterification of all-trans retinol and the occurrence of cytosolic retinoid-binding proteins was investigated in cultured bovine retinal pigment epithelium (RPE) cells. (3)H-labeled all-trans retinyl ester (mainly palmitate) was formed at an initial rate of 0.1 nmol.mg protein(?1).min(?1) when (3)H-labeled all-trans retinol was incubated with the 100,000 g pellet obtained from a homogenate of freshly-harvested cells. No esterification could be detected under the same conditions after 14 days in culture in defined medium (DM) or in medium containing 20% fetal bovine serum (CM). No enhancement or restoration of esterifying capacity was observed when the assay mixture was supplemented with palmitoyl CoA. As determined by specific, saturable binding of (3)H-labeled all-trans retinol and (3)H-labeled 11-cis retinal to proteins with mol. wts 16,000 and 33,000 dalton on calibrated Bio-Sil TSK 250 size-exclusion columns, the cytosol of freshly-harvested RPE cells contained cellular retinol-binding protein (CRBP) and cellular retinal-binding protein (CRAlBP). By comparison with the quantity of (3)H-labeled all-trans retinol bound under identical conditions to pure dog liver CRBP, it was estimated that fresh RPE cells contained 102 +/- 3 ng CRBP.?g cytosol protein(?1). In cultured and subcultured cells, CRBP was present at much lower levels (down to one-tenth of the initial amounts) and CRAlBP could not be detected. Since binding of (3)H-labeled all-trans retinoic acid to a protein with molecular weight of 17,000 dalton was not observed in the cytosols of fresh or cultured cells, it was concluded that cellular retinoic acid binding protein (CRABP) was either present at very low levels or absent altogether. An unidentified peak of specific (3)H-labeled all-trans-retinoic acid binding at mol. wt 61,000 dalton was prominent in subcultured cells. These results show that in RPE cells in culture the expression of differentiated phenotype with respect to retinoid utilization undergoes significant modification. It is postulated that changes in the composition of the extracellular matrix (e.g. absence of interstitial retinol-binding protein, IRBP) may be involved.