Regulation of reduced-folate transporter-1 in retinal pigment epithelial cells by folate.
OBJECTIVE
Reduced-folate transporter-1 (RFT-1), a typical transport protein with 12 membrane-spanning domains, transports reduced-folates, such as N5-methyltetrahydrofolate (MTF), the predominant circulating form of folate. RFT-1 is localized to the RPE apical membrane and transports folate from RPE to photoreceptor cells. We asked whether RFT-1 activity in RPE is altered under high folate conditions.


METHODS
ARPE-19 cells were cultured 24, 48, or 72 h in medium containing either 0.5 nM, 5.0 nM, or 2.26 microM MTF, and the activity of RFT-1 was assessed by determining the uptake of N5-MTF. Semiquantitative reverse transcription-polymerase chain reaction and Western blot analysis were used to study RFT-1 gene and protein expression.


RESULTS
Cells treated for 72 h with 2.26 microM MTF showed a significant (40%) decrease in MTF uptake compared to cells exposed to 0.5 nM or 5 nM MTF. The effect of high concentrations of folate on RFT-1 activity was specific. Kinetic analysis showed that folate-induced attenuation of RFT-1 activity was associated with a decrease in the maximal velocity of the transporter, but no change in the substrate affinity. Steady-state levels of RFT-1 mRNA and protein decreased significantly in the presence of excess folate.


CONCLUSIONS
Excess folate levels downregulate RFT-1 in RPE. This study represents the first molecular analysis of the regulation of RFT-1 by folate in RPE and reveals attenuation of the activity and expression of a folate transport protein under conditions of high levels of folate.
Naggar
Hany
H
Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia 30912-2000, USA.
Van Ells
Tracy K
TK
Ganapathy
Vadivel
V
Smith
Sylvia B
SB
D015153:Blotting, Western; D002478:Cells, Cultured; D015536:Down-Regulation; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013763:Tetrahydrofolates / Q000494:pharmacology*
D026901:Membrane Transport Proteins; D012333:RNA, Messenger; C120043:SLC19A2 protein, human; D013763:Tetrahydrofolates; C005984:5-methyltetrahydrofolate