β-Carotene conversion into vitamin A in human retinal pigment epithelial cells

PURPOSE. Vitamin A is essential for vision. The key step in the vitamin A biosynthetic pathway is the oxidative cleavage of beta-carotene into retinal by the enzyme beta,beta-carotene-15,15'-monooxygenase (BCO). The purpose of the study was to investigate beta-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 beta-carotene to retinoids was determined by measuring retinol levels in D407 cells on beta-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 beta-carotene application to D407 cells resulted in a concentration ( 75% at 0.5 mu M and 96% at 5 mu M; P < 0.05)- and time (127% at 2 hours and 97% at 4 hours in 5 mu M beta-carotene, P < 0.05)- dependent upregulation of BCO mRNA expression. Application of exogenous retinoic acid downregulated BCO mRNA levels at higher concentrations ( 1 mu M; - 96%, P < 0.0005) and upregulated it at a lower concentration (0.01 mu M; 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 beta-carotene in a time-dependent manner and converted it to retinol. CONCLUSIONS. The results suggest that BCO is expressed in the RPE and that beta-carotene can be metabolized into retinol. beta-Carotene cleavage in the RPE may be an alternative pathway that would ensure the retinoid supply of photoreceptor cells.