抄録
Objectives: Fibroblast-like synoviocytes (FLS) in the synovial intimal linings play a key role by producing cytokines that perpetuate inflammation and increase the production of matrix-degrading enzymes such as matrix metalloproteinases that contribute to cartilage destruction in rheumatoid arthritis (RA). Therefore, therapeutic targets such as the activated FLS are now providing exciting possibilities for future treatment. Choline is an essential nutrient in all animal cells. It is used as a precursor of both the neurotransmitter acetylcholine (ACh) and S-adenosylmethionine (SAM), a methyl donor, and plays an important role in the formation of phosphatidylcholine and sphingomyelin. The intracellular accumulation of choline through choline transporters is the rate-limiting step in phospholipid metabolism and the synthesis of ACh and SAM, as well as a prerequisite for cell proliferation. In this study, we examined the functional characteristics of choline uptake and sought to identify the transporters in rheumatoid arthritis synovial fibroblasts (RASFs).
Methods: The expression of choline transporters was evaluated by quantitative real-time PCR, western blotting and immunocytochemistry. Time course, Na+-dependency and kinetics of [3H]choline uptake were investigated. Effects of cationic drugs on the uptake of [3H]choline, cell viability and caspase-3/7 activity were also examined. Finally, we investigated the influence of choline uptake inhibitor, hemicholinium-3 (HC-3), and choline deficiency on cell viability and caspase-3/7 activity.
Results: Choline transporter-like protein 1 (CTL1) and CTL2 mRNA and protein were highly expressed in RASFs and were localized to the plasma membrane. [3H]Choline uptake occurred via a Na+-independent and pH-dependent transport system. The cells have two different choline transport systems, high- and low-affinity. Various organic cations, HC-3 and choline deficiency inhibited both [3H]choline uptake and cell viability, and enhanced the caspase-3/7 activity. The functional inhibition of choline transporters could promote apoptotic cell death. In RASFs, [3H]choline uptake was significantly increased compared with that in osteoarthritis synovial fibroblasts without a change in gene expression.
Conclusions: These results suggest that CTL1 (high-affinity) and CTL2 (low-affinity) are highly expressed in RASFs and choline may be transported by a choline/H+ antiport system. Identification of this CTL1- and CTL2-mediated choline transport system should provide a potential new target for RA therapy.
