beta-Carotene conversion into vitamin A in human retinal pigment epithelial cells.
OBJECTIVE
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 microM and 96% at 5 microM; P < 0.05)- and time (127% at 2 hours and 97% at 4 hours in 5 microM beta-carotene, P < 0.05)-dependent upregulation of BCO mRNA expression. Application of exogenous retinoic acid downregulated BCO mRNA levels at higher concentrations (1 microM; -96%, P < 0.0005) and upregulated it at a lower concentration (0.01 microM; 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.
Chichili
Gurunadh Reddy
GR
Institute of Biological Chemistry and Nutrition, University of Hohenheim, Stuttgart, Germany.
Nohr
Donatus
D
Schäffer
Michael
M
von Lintig
Johannes
J
Biesalski
Hans K
HK
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D018929:Cell Culture Techniques; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D000072516:Retinoic Acid 4-Hydroxylase; D000072482:Retinoic Acid Receptor alpha; D047490:Retinoid X Receptor alpha / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014212:Tretinoin / Q000494:pharmacology; D015854:Up-Regulation; D014801:Vitamin A / Q000378:metabolism*; D019207:beta Carotene / Q000378:metabolism* / Q000494:pharmacology
C000607071:RARA protein, human; D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D047490:Retinoid X Receptor alpha; D019207:beta Carotene; D014801:Vitamin A; D014212:Tretinoin; D003577:Cytochrome P-450 Enzyme System; D000072516:Retinoic Acid 4-Hydroxylase