title: beta-Carotene conversion into vitamin A in human retinal pigment epithelial cells.
abstract: 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.
authors.affiliation: Institute of Biological Chemistry and Nutrition, University of Hohenheim, Stuttgart, Germany.
authors.forename: Gurunadh Reddy
authors.initials: GR
authors.lastname: Chichili
authors.forename: Donatus
authors.initials: D
authors.lastname: Nohr
authors.forename: Michael
authors.initials: M
authors.lastname: Schäffer
authors.forename: Johannes
authors.initials: J
authors.lastname: von Lintig
authors.forename: Hans K
authors.initials: HK
authors.lastname: Biesalski
mesh_terms: 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
chemical_list: 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