Identification of Primary Equilibrative Nucleoside Transporter 1 mRNA Isoforms Resulting from Alternative Promoter Usage in Human Hepatocytes

Abstract

Human equilibrative nucleoside transporter 1 (hENT1) transports various nucleoside analogues into cells. Although the single hENT1 promoter region (P1) and the mRNA isoform (a1) have been characterized previously, we have recently identified additional promoter regions P2 and P3 (which primarily generate c1/2/3 mRNAs and d1/2/3/4 mRNAs, respectively) in the human liver. Therefore, this study aimed at identifying the primary hENT1 mRNA isoforms expressed in human hepatocytes, while simultaneously obtaining functional evidence of alternative hENT1 promoter usage. Our results showed that the expressions of hENT1c1, d3, and (to a lesser extent) c2 mRNAs were strikingly predominant over the other mRNA isoforms in human hepatocytes, that the abundant expression of these mRNAs was consistent with the high levels of P2 and P3 promoter activity, and that these promoters were significantly marked by transcriptionally active histone modification in hepatic cells. To summarize, our results demonstrate that, resulting from the manipulated alternative promoter usage, hENT1c1 and d3 (and c2) mRNAs are primarily expressed in human hepatocytes, which suggests that they may play important roles in controlling hENT1 expression levels in those cells. Our findings are expected to provide significant insights into the molecular machinery of hENT1 expression control.

Establishment and characterization of a new conditionally immortalized human astrocyte cell line

“…In addition, because we have recently demonstrated that alternative promoter usage of the ENT1 gene results in its multiple mRNA isoforms in human hepatocytes (Furihata et al . ), it was intriguing to see what ENT1 mRNA isoforms expressed in HASTR/ci35 cells. The qPCR results showed that, among the isoforms, ENT1c and ENT1d mRNA isoforms were primarily expressed in both HASTR/ci35 and prHASTR cells (Fig.…”

“…GIRK2, Gprotein-regulated inward-rectifier potassium channel 2; N-CAM1, neuronal cell adhesion molecule 1; NEFH, neurofilament, heavy polypeptide; NeuN, neuronal nuclei; EN1, engrailed 1; DAT, dopamine transporter; FOXA2, forkhead box protein A2; LMX1B, LIM homeobox transcription factor 1-beta. (Furihata et al 2014b), it was intriguing to see what ENT1 mRNA isoforms expressed in HASTR/ci35 cells. The qPCR results showed that, among the isoforms, ENT1c and ENT1d mRNA isoforms were primarily expressed in both HASTR/ ci35 and prHASTR cells (Fig.…”