Polyamine-dependent regulation of spermidine-spermine N-1-acetyltransferase mRNA translation

Spermidine-spermine N-1-acetyltransferase (SSAT) is induced in response to an elevation in intracellular polyamine pools. The increased enzyme activity is the result of an increase in gene transcription, mRNA translation, and protein stability. Induction of SSAT by polyamine analogues can lead to intracellular polyamine depletion and apoptosis. The mechanism by which polyamines alter the translational efficiency of SSAT mRNA is not well understood. In this study, we investigated the regulation of SSAT translation by the polyamine analogue N-1,N-11-diethylnorspermine ( DENSPM). DENSPM induced expression of both FLAG-tagged SSAT and SSAT fused to Renilla luciferase in a time- and concentration-dependent manner. This effect was not inhibited by actinomycin D indicating that changes in gene transcription did not explain the enhanced expression in the presence of DENSPM. Furthermore, because FLAG-SSAT did not contain the 5'- or 3'-untranslated regions of SSAT, translational regulation involved the coding sequence only. By contrast, cycloheximide completely inhibited induction by DENSPM, indicating a requirement for new protein synthesis. Deletion constructs identified two regions of the SSAT protein-coding RNA sequence that conferred polyamine responsiveness. Using these regions as probes in RNA electrophoretic mobility shift assays, we observed specific binding of a cytoplasmic protein. In addition, we found that the interaction between the RNA probes and the binding protein could be inhibited by DENSPM in a concentration-dependent manner. These results suggest that polyamines regulate SSAT mRNA translational efficiency by inhibiting a repressor protein from binding to regions of the coding sequence of the SSAT transcript

HCV+ Hepatocytes Induce Human Regulatory CD4+ T Cells through the Production of TGF-β

Background: Hepatitis C Virus (HCV) is remarkably efficient at establishing persistent infection and is associated with the development of chronic liver disease. Impaired T cell responses facilitate and maintain persistent HCV infection. Importantly, CD4 + regulatory T cells (Tregs) act by dampening antiviral T cell responses in HCV infection. The mechanism for induction and/or expansion of Tregs in HCV is unknown. Methodology/Principal Findings: HCV-expressing hepatocytes were used to determine if hepatocytes are able to induce Tregs. The infected liver environment was modeled by establishing the co-culture of the human hepatoma cell line, Huh7.5, containing the full-length genome of HCV genotype 1a (Huh7.5-FL) with activated CD4 + T cells. The production of IFN-c was diminished following co-culture with Huh7.5-FL as compared to controls. Notably, CD4 + T cells in contact with Huh7.5-FL expressed an increased level of the Treg markers, CD25, Foxp3, CTLA-4 and LAP, and were able to suppress the proliferation of effector T cells. Importantly, HCV + hepatocytes upregulated the production of TGF-b and blockade of TGF-b abrogated Treg phenotype and function. Conclusions/Significance: These results demonstrate that HCV infected hepatocytes are capable of directly inducing Tregs development and may contribute to impaired host T cell responses

Expression of kinin B1 and B2 receptors in immature, monocyte-derived dendritic cells and bradykinin-mediated increase in intracellular Ca2+ and cell migration

The kinins, bradykinin (BK) and Lysdes[ Arg9]-BK, are important inflammatory mediators that act via two specific G protein-coupled kinins, B1 and B2 receptors (B2R). Kinins influence the activity of immune cells by stimulating the synthesis of cytokines