AUF1/hnRNP D represses expression of VEGF in macrophages.

Vascular endothelial growth factor (VEGF) is a regulator of vascularization in development and is a key growth factor in tissue repair. In disease, VEGF contributes to vascularization of solid tumors and arthritic joints. This study examines the role of the mRNA-binding protein AUF1/heterogeneous nuclear ribonucleoprotein D (AUF1) in VEGF gene expression. We show that overexpression of AUF1 in mouse macrophage-like RAW-264.7 cells suppresses endogenous VEGF protein levels. To study 3' untranslated region (UTR)-mediated regulation, we introduced the 3' UTR of VEGF mRNA into a luciferase reporter gene. Coexpression of AUF1 represses VEGF-3' UTR reporter expression in RAW-264.7 cells and in mouse bone marrow-derived macrophages. The C-terminus of AUF1 contains arginine-glycine-glycine (RGG) repeat motifs that are dimethylated. Deletion of the RGG domain of AUF1 eliminated the repressive effects of AUF1. Surprisingly, expression of an AUF1-RGG peptide reduced endogenous VEGF protein levels and repressed VEGF-3' UTR reporter activity in RAW-264.7 cells. These findings demonstrate that AUF1 regulates VEGF expression, and this study identifies an RGG peptide that suppresses VEGF gene expression.

Inhibition of arterial lesion progression in CD16-deficient mice: evidence for altered immunity and the role of IL-10.

AIMS: Given the importance of IgG Fc receptors in immune regulation, we hypothesized that Fcg receptor type III (FcgRIII, CD16) plays an important role in atherogenesis. We therefore analysed the formation of arterial lesions in LDL receptor-deficient (LDLR(-/-)) and FcgRIII(-/-)xLDLR(-/-) double knockout mice at three different points up to 24 weeks of exposure to a high-fat diet. METHODS AND RESULTS: Analysis of Oil Red-O-stained sections revealed no difference in lesion formation between strains after 6 weeks of a high-fat diet, and a modest decrease after 14 weeks in double knockouts relative to LDLR(-/-) controls. After 24 weeks, lesion formation was decreased in the aortic root (30%) and innominate artery (50%) in FcgRIII double knockouts relative to LDLR(-/-) controls. Analysis of peripheral CD4+ T-cells by intracellular flow cytometry from double knockouts after 24 weeks of a high-fat diet revealed statistically significant increases in the percentages of cells producing interferon-gamma, interleukin (IL)-10, and IL-4 relative to controls, differences that were also observed by analyses of whole aortas for cytokine mRNA levels. As determined by flow cytometry, FcgRIII deficiency resulted in an expansion of CD4+ cells and an increase in the CD4 to CD8 ratio. Analysis of plasma anti-oxidized LDL (OxLDL) antibodies by chemiluminescent assay revealed that IgG1 and IgG2c titers to OxLDL were increased in FcgRIII (-/-)xLDLR(-/-) double knockouts relative to LDLR(-/-) controls, while total IgG levels were similar. CONCLUSION: These results reveal altered immunity in FcgRIII(-/-)xLDLR(-/-) mice and a reduction in lesion formation associated with increased production of IL-10 by an expansion of CD4+ T-cells. The reduction in lesion formation was manifest well after evidence of an immune response to OxLDL, suggesting that FcgRIII contributes to lesion progression in murine atherosclerosis.

Post-transcriptional regulation of glucose transporter-1 by an AU-rich element in the 3'UTR and by hnRNP A2.

Glucose transporter-1 (GLUT1) mediates uptake of glucose and is up-regulated in some cancers. The amount of this membrane protein is regulated by a post-transcriptional mechanism in which mRNA binding proteins recognize cis-acting elements in the 3'-untranslated (3'UTR) of the mRNA. To identify cis elements in GLUT1 mRNA we introduced 3'UTR sequences into the 3'UTR of the luciferase gene in a reporter construct. A 30 nt adenosine-uridine-rich element ("GLUT1 AURE") inhibited luciferase activity in HEK-293 cells. This inhibitory effect was confirmed by deleting the GLUT1 AURE from a reporter containing the full-length 3'UTR. Deletion of the GLUT1 AURE caused reporter activity to increase. Deletion of a larger fragment ("Bsu" region) containing the GLUT1 AURE increased reporter activity still further, suggesting that there are additional cis elements in the GLUT1 mRNA. The GLUT1 AURE was also active in GBM-T98G glioblastoma cells. Next, we tested the action of a trans-acting factor, hnRNP A2, on GLUT1 gene expression. We show that a cytoplasmic-localizing isoform of hnRNP A2 binds human GLUT1 RNA by gel-shift assay and by UV-crosslinking. Finally, over-expression of the hnRNP A2 isoform inhibited GLUT1 reporter expression in GBM-T98G cells. These results identify the AURE cis element in human GLUT1 mRNA and show that hnRNP A2 acts on GLUT1 mRNA to inhibit expression of GLUT1 in a brain cancer cell line.