Protein kinase C promotes choline transporter-like protein 1 function via improved cell surface expression in immortalized human hepatic cells

Choline is used to synthesize phospholipids and a lack of choline induces a number of liver-related diseases, including non-alcoholic steatohepatitis. The current study characterized the choline uptake system, at molecular and functional levels, in the immortalized human hepatic cell line, Fa2N-4, to identify the specific choline transporter involved in choline uptake. The present study also assesed whether choline deficiency or the inhibited choline uptake affected cell viability and apoptosis. Reverse transcription-quantitative polymerase chain reaction (PCR) revealed choline transporter-like protein 1 (CTL1) and CTL2 mRNA and protein expression in Fa2N-4 cells. [Methyl-H-3]choline studies revealed choline uptake was saturable and mediated by a single transport system that functioned in a Na+-independent but pH-dependent manner, which was similar to CTL1. Hemicholinium-3 (HC-3), which is a choline uptake inhibitor, and choline deficiency inhibited cell viability, increased caspase-3 and -7 activities, and increased fluorescein isothiocyanate-Annexin V immunofluorescent staining indicated apoptosis. Immunofluorescent staining also revealed CTL1 and CTL2 localized in plasma and mitochondrial membranes, respectively. [Methyl-H-3]choline uptake was enhanced by a protein kinase C (PKC) activator, phorbol-12-myristate 13-acetate (PMA). Immunofluorescence staining and western blot analysis demonstrated increased CTL1 expression on the cell membrane following PMA treatment. The results of current study indicated that extracellular choline is primarily transported via CTL1, relying on a direct H+ gradient that functions as a driving force in Fa2N-4 cells. Furthermore, it was hypothesized that CTL1 and the choline uptake system are strongly associated with cell survival, and that the choline uptake system is modulated by PKC signaling via increased CTL1 expression on the cell surface. These findings provide further insights into the pathogenesis of liver disease involving choline metabolism.