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ß-Carotene Conversion into Vitamin A in Human Retinal Pigment Epithelial Cells

¹From the Institute of Biological Chemistry and Nutrition, University of Hohenheim, Stuttgart, Germany; and the ⁴Institute of Biology I, Animal Physiology and Neurobiology, University of Freiburg, Freiburg, Germany.

PURPOSE. Vitamin A is essential for vision. The key step in the vitamin A biosynthetic pathway is the oxidative cleavage of ß-carotene into retinal by the enzyme ß,ß-carotene-15,15'-monooxygenase (BCO). The purpose of the study was to investigate ß-carotene metabolism and its effects on BCO expression in the human retinal pigment epithelial (RPE) cell line D407.

METHODS. BCO mRNA and protein expression were analyzed by real-time quantitative PCR and Western blot analysis, respectively. BCO activity was assayed in protein extracts isolated from D407 cells. The conversion of ß-carotene to retinoids was determined by measuring retinol levels in D407 cells on ß-carotene supplementation.

RESULTS. By RT-PCR, BCO mRNA was detected in D407 cells, bovine RPE, and retina. Western blot analyses revealed the presence of BCO at the protein level in D407 cells. Exogenous ß-carotene application to D407 cells resulted in a concentration (75% at 0.5 µM and 96% at 5 µM; P < 0.05)- and time (127% at 2 hours and 97% at 4 hours in 5 µM ß-carotene, P < 0.05)-dependent upregulation of BCO mRNA expression. Application of exogenous retinoic acid downregulated BCO mRNA levels at higher concentrations (1 µM; –96%, P < 0.0005) and upregulated it at a lower concentration (0.01 µM; 399%, P < 0.005). The RAR-a-specific antagonist upregulated BCO expression by sixfold (P < 0.005). Tests for enzymatic activity demonstrated that the mRNA upregulation resulted in enzymatically active BCO protein (7.3 ng all-trans-retinal/h per milligram of protein). Furthermore, D407 cells took up ß-carotene in a time-dependent manner and converted it to retinol.

CONCLUSIONS. The results suggest that BCO is expressed in the RPE and that ß-carotene can be metabolized into retinol. ß-Carotene cleavage in the RPE may be an alternative pathway that would ensure the retinoid supply of photoreceptor cells.

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