Ribavirin-induced intracellular GTP depletion activates transcription elongation in coagulation factor VII gene expression.

Coagulation FVII (Factor VII) is a vitamin K-dependent glycoprotein synthesized in hepatocytes. It was reported previously that FVII gene (F7) expression was up-regulated by ribavirin treatment in hepatitis C virus-infected haemophilia patients; however, its precise mechanism is still unknown. In the present study, we investigated the molecular mechanism of ribavirin-induced up-regulation of F7 expression in HepG2 (human hepatoma cell line). We found that intracellular GTP depletion by ribavirin as well as other IMPDH (inosine-5'-monophosphate dehydrogenase) inhibitors, such as mycophenolic acid and 6-mercaptopurine, up-regulated F7 expression. FVII mRNA transcription was mainly enhanced by accelerated transcription elongation, which was mediated by the P-TEFb (positive-transcription elongation factor b) complex, rather than by promoter activation. Ribavirin unregulated ELL (eleven-nineteen lysine-rich leukaemia) 3 mRNA expression before F7 up-regulation. We observed that ribavirin enhanced ELL3 recruitment to F7, whereas knockdown of ELL3 diminished ribavirin-induced FVII mRNA up-regulation. Ribavirin also enhanced recruitment of CDK9 (cyclin-dependent kinase 9) and AFF4 to F7. These data suggest that ribavirin-induced intracellular GTP depletion recruits a super elongation complex containing P-TEFb, AFF4 and ELL3, to F7, and modulates FVII mRNA transcription elongation. Collectively, we have elucidated a basal mechanism for ribavirin-induced FVII mRNA up-regulation by acceleration of transcription elongation, which may be crucial in understanding its pleiotropic functions in vivo.

Thrombosis from a prothrombin mutation conveying antithrombin resistance.

We identified a novel mechanism of hereditary thrombosis associated with antithrombin resistance, with a substitution of arginine for leucine at position 596 (p.Arg596Leu) in the gene encoding prothrombin (called prothrombin Yukuhashi). The mutant prothrombin had moderately lower activity than wild-type prothrombin in clotting assays, but the formation of thrombin-antithrombin complex was substantially impaired. A thrombin-generation assay revealed that the peak activity of the mutant prothrombin was fairly low, but its inactivation was extremely slow in reconstituted plasma. The Leu596 substitution caused a gain-of-function mutation in the prothrombin gene, resulting in resistance to antithrombin and susceptibility to thrombosis.

A novel ENG mutation causing impaired co-translational processing of endoglin associated with hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an inherited autosomal dominant vascular dysplasia caused by mutations in mainly the endoglin gene (ENG) or activin-like kinase receptor 1 (ALK1) gene (ACVRL1). We investigated the molecular basis of HHT in a Japanese patient, and identified a novel missense mutation in ENG (c.38T>A, p.Leu13Gln) located in the signal peptide's hydrophobic core, but not in ACVRL1. In experiments in COS-1 cells, the Leu13Gln (L13Q) mutant endoglin appeared to be expressed as a precursor form, probably due to impaired protein processing. Flow cytometry analyses of the COS-1 cells transiently expressing recombinant endoglins revealed that the wild-type endoglin was detected on the cell surface, but the L13Q mutant was not. We also analyzed expression patterns of the recombinant endoglins by immunofluorescent staining, and found that the wild-type co-localized with the endoplasmic reticulum (ER), but the L13Q mutant did not. These results implied that the L13Q mutant endoglin fails to insert into the ER, probably due to destruction of the hydrophobic core structure in the signal peptide to be recognized by signal recognition particles. Thus, the Leu13 in the signal peptide of endoglin might be essential for correct protein processing through the ER and cell-surface expression. Taken together, the novel c.38T>A mutation in ENG would impair co-translational processing of the endoglin, and could be responsible for HHT in this patient.

Down-regulation of PROS1 gene expression by 17beta-estradiol via estrogen receptor alpha (ERalpha)-Sp1 interaction recruiting receptor-interacting protein 140 and the corepressor-HDAC3 complex.

Pregnant women show a low level of protein S (PS) in plasma, which is known to be a risk for deep venous thrombosis. 17Beta-estradiol (E(2)), an estrogen that increases in concentration in the late stages of pregnancy, regulates the expression of various genes via the estrogen receptor (ER). Here, we investigated the molecular mechanisms behind the reduction in PS levels caused by E(2) in HepG2-ERalpha cells, which stably express ERalpha, and also the genomic ER signaling pathway, which modulates the ligand-dependent repression of the PSalpha gene (PROS1). We observed that E(2) repressed the production of mRNA and antigen of PS. A luciferase reporter assay revealed that E(2) down-regulated PROS1 promoter activity and that this E(2)-dependent repression disappeared upon the deletion or mutation of two adjacent GC-rich motifs in the promoter. An electrophoretic mobility shift assay and DNA pulldown assay revealed that the GC-rich motifs were associated with Sp1, Sp3, and ERalpha. In a chromatin immunoprecipitation assay, we found ERalpha-Sp protein-promoter interaction involved in the E(2)-dependent repression of PROS1 transcription. Furthermore, we demonstrated that E(2) treatment recruited RIP140 and the NCoR-SMRT-HDAC3 complex to the PROS1 promoter, which hypoacetylated chromatin. Taken together, this suggested that E(2) might repress PROS1 transcription depending upon ERalpha-Sp1 recruiting transcriptional repressors in HepG2-ERalpha cells and, consequently, that high levels of E(2) leading to reduced levels of plasma PS would be a risk for deep venous thrombosis in pregnant women.

Impaired secretion of carboxyl-terminal truncated factor VII due to an F7 nonsense mutation associated with FVII deficiency.

INTRODUCTION: Factor VII (FVII) is a vitamin K-dependent glycoprotein secreted into the blood circulation from hepatic cells. We investigated the molecular basis of the congenital FVII deficiency found in a Japanese patient. MATERIALS AND METHODS: We analyzed the F7 gene of the patient, who was diagnosed with a FVII deficiency at pregnancy. We expressed a carboxyl-terminal truncated FVII (Arg462X FVII) corresponding to the identified mutation in CHO-K1 cells. To study roles of the carboxyl-terminus in the secretion of FVII, we also expressed a series of recombinant FVIIs deleted of limited numbers of carboxyl-terminal amino acids (462Arg-466Pro). RESULTS: We identified a nonsense mutation (c.1384C>T: p.Arg462X) in F7, leading to a lack of five amino acids in the carboxyl-terminus. In expression experiments, Arg462X FVII was undetectable not only by Western blotting, but also by ELISA. A Western blot analysis of the truncated FVIIs revealed that all mutants were expressed in the cells the same as the wild type, but were secreted into the culture medium in lesser amounts than the wild type depending on the length of the deletion, which was confirmed by ELISA. Arg462X FVII did not colocalize with the Golgi on immunofluorescence staining, suggesting that it might be retained in the ER and degraded in the cell. CONCLUSION: The carboxyl-terminal amino acids of FVII play an important role in its secretion, and the p.Arg462X mutation was likely to have caused the FVII deficiency in this patient.

A novel missense mutation causing abnormal LMAN1 in a Japanese patient with combined deficiency of factor V and factor VIII.

Combined deficiency of coagulation factor V (FV) and factor VIII (FVIII) (F5F8D) is an inherited bleeding disorder characterized by a reduction in plasma concentrations of FV and FVIII. F5F8D is genetically linked to mutations in either LMAN1 or MCFD2. Here, we investigated the molecular basis of F5F8D in a Japanese patient, and identified a novel missense mutation (p.Trp67Ser, c.200G>C) in the LMAN1, but no mutation in the MCFD2. The amount of LMAN1 in Epstein-Barr virus-immortalized lymphoblasts from the patient was found to be almost the same as that in cells from a normal individual. Interestingly, an anti-MCFD2 antibody did not co-immunoprecipitate the mutant LMAN1 with MCFD2 in lymphoblasts from the patient, suggesting the affinity of MCFD2 for the mutant LMAN1 is weak or abolished by the binding of the anti-MCFD2 antibody. In addition, a Myc/6xHis-tagged recombinant form of wild-type LMAN1 could bind to D-mannose, but that of the mutant could not. The p.Trp67Ser mutation was located in the carbohydrate recognition domain (CRD), which is thought to participate in the selective binding of LMAN1 to the D-mannose of glycoproteins as well as the EF-motif of MCFD2. Taken together, it was suggested that the p.Trp67Ser mutation might affect the molecular chaperone function of LMAN1, impairing affinity for D-mannose as well as for MCFD2, which may be responsible for F5F8D in the patient. This is the first report of F5F8D caused by a qualitative defect of LMAN1 due to a missense mutation in LMAN1. Am. J. Hematol. 2009. (c) 2009 Wiley-Liss, Inc.