Ribavirin-Induced Intracellular GTP Depletion Upregulates Factor VII Expression

Abstract 1113

In this study, we investigated the molecular basis of upregulation of factor VII (FVII) gene expression by ribavirin, and found that intracellular GTP depletion induced by ribavirin activated FVII gene transcription and modulated transcription elongation.

In 2006, Yamamoto et al. reported that anti-hepatitis C virus (HCV) agent ribavirin elevated the activity of FVII in HCV-infected hemophilia patients; however, the precise mechanisms were still unknown. In addition, the anti-HCV mechanisms of ribavirin were not yet fully elucidated, although the extended studies have been done. We investigated the effects of ribavirin in vitro and confirmed the approximately 4-fold upregulation of FVII mRNA by ribavirin treatment in HepG2 cells. FVII mRNA was increased in a dose-dependent manner up to 100μg/mL of ribavirin at a lower concentration than therapeutic concentration of 150μg/mL. FVII mRNA induction by ribavirin was also observed in a time-dependent manner from 24 h to 72 h after treatment.

Ribavirin metabolite ribavirin 5'-monophosphate is one of the IMP dehydrogenase (IMPDH) inhibitors, and the other IMPDH inhibitors mycophenolic acid (MPA) and 6-mercaptupurine (6-MP) also induced FVII upregulation. It is well known that inhibition of IMPDH causes intracellular GTP depletion, and guanosine supplementation to salvage GTP could reverse FVII mRNA increase in ribavirin-treated cells. These results indicated that cellular GTP reduction associated with FVII gene upregulation.

The mechanisms of gene upregulation by GTP depletion were not elucidated. The promoter activities and mRNA stability of FVII were analyzed under ribavirin treatment. The FVII gene promoter activity was enhanced up to 1.5-fold by ribavirin treatment; however the activation did not reach 4-fold induction of FVII mRNA increase. There was no significant change of FVII mRNA half-life in ribavirin-treated cells. Since the promoter activation might display transcription initiation capacity, the contribution of transcription elongation stage was further investigated.

Transcription elongation was regulated by phosphorylation of carbo-terminal domain (CTD) of RNA polymerase II (PolII). Transcription elongation factor P-TEFb (positive-transcription elongation factor b), which consists as a complex of CDK9 and cyclin T, phosphorylates Ser of PolII CTD. The kinase activity of P-TEFb could be inhibited by 5,6-dichlorobenzimidazole 1-b-D-ribofuranoside (DRB). In FVII gene upregulation, DRB completely canceled ribavirin-induced FVII mRNA increase. We also performed nuclear run-on assay to verify the potential transcription elongation capacity of paused PolII, and observed a dramatic increase of FVII mRNA in ribavirin-treated cells. These results suggested that ribavirin-induced FVII gene upregulation was caused not only by transcription initiation but also by accelerated transcription elongation rate.

There are various transcription factor associated with transcription elongation in addition to P-TEFb, such as elongin, ELL (eleven nineteen-lysine rich leukemia). We found that ELL3, a member of ELL family protein, was upregulated by ribavirin treatment. A ELL3 mRNA increase occurred prior to FVII mRNA upregulation, and the ELL3 upregulation was also canceled by guanosine supplementation. These results indicated ELL3 induction by ribavirin was also a response to cellular GTP depletion. To confirm the contribution of ELL3 protein to FVII gene transcription elongation, we used siRNAs specific to ELL3 and as expected, knockdown of ELL3 resulted in diminished FVII upregulation. A chromatin immunoprecipitation (ChIP) revealed ELL3 recruitment to the FVII gene, and the recruitments of PolII and CDK9 were also enhanced by ribavirin treatment.

Taken together, FVII gene upregulation by ribavirin was associated with intracellular GTP depletion. The GTP reduction mainly modulates transcription elongation rate rather than transcription initiation, though the relationships between cellular GTP depletion and enhanced transcription elongation must be investigated. This study uncovered candidate mechanisms of ribavirin and the other IMPDH inhibitors and highlights a development of novel pharmaceutical therapies for hemophilia.