| Role of Fragile X-related protein 1 in controlling the expression of TNF and other pro-inflammatory cytokines and chemokines | |
Abstract/OtherAbstract
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Tumor necrosis factor (TNF) is a key inflammatory cytokine that plays a major role in combating infections. Its dysregulation, however, can lead to uncontrolled inflammation which may cause deleterious conditions such as septic shock. Therefore, the expression of TNF is tightly regulated at both the transcriptional and post-transcriptional level. AU-rich elements, present in the 3'-untranslated region of TNF mRNA, are primarily known for their ability to stimulate rapid mRNA decay. ARE-binding proteins such as TTP and AUF-1 mediate this process. We have identified a new protein, Fragile X-related protein 1 (FXR1P), which binds to the ARE of TNF mRNA. The generation of a macrophage cell line, in which the Fxr1 gene is ablated, resulted in higher expression of TNF protein without affecting TNF mRNA steady state level or stability. Analysis of polysomes showed that TNF mRNA was associated preferentially with heavy polysome fractions in macrophages cell lines derived from FXR1P knockout mice, indicating a translational repressor role for FXR1P. We next defined the role of FXR1P in TNF induction by Toll-like receptor (TLR) ligands other than LPS (a TLR4 ligand). The effects of S28463, a synthetic TLR7 ligand, and CpG, a TLR9 ligand, were tested as they have potential clinical therapeutic properties. FXR1P ablation in macrophages did not alter TNF expression compared with wildtype macrophages stimulated with S28463. However, FXR1P-deficient macrophages stimulated with CpG had significantly higher TNF secretion than wildtype cells. Although CpG-stimulated FXR1P-KO cells had higher TNF mRNA steady state levels, no difference in TNF mRNA stability was observed compared with wildtype cells. These results therefore suggest that transcriptional events induced by TLR9 agonists are controlled by FXR1P. Additionally, FXR1P was shown to be involved in the induction of IL-6, CCL2, and CCL5 in macrophages following stimulation with either a TLR4 or TLR9 ligand. Our laboratory previously reported lower levels of several cytokines in fenretinide-treated animals. Therefore, we postulated that fenretinide, a vitamin A derivative, might be able to normalize the excessive production of inflammatory cytokines resulting from the ablation of the Fxr1 gene. We found that fenretinide treatment inhibited excessive production of TNF, IL-6, CCL2, and CCL5. We also found that fenretinide was able to selectively inhibit phosphorylation of ERK1/2, which was found to be higher in FXR1-KO versus WT macrophages. Furthermore, our results identified a possible mechanism of the over-phosphorylation of ERK1/2 proteins. Specifically, we found that macrophages with an ablated Fxr1 gene displayed diminished levels of DHA, an important phospholipid mediator. LPS stimulation led to a significant increase in AA levels in both FXR1-KO and WT macrophages, whereas DHA levels were significantly decreased only in FXR1-KO macrophages. Fenretinide-induced inhibition of inflammatory cytokine production was also associated with a dramatic reduction in AA/DHA ratios in both cell lines, which might explain at least in part the protective anti-inflammatory effects associated with fenretinide. Taken together, these results show that FXR1P plays an important role in the regulation of the expression of TNF and other important inflammatory mediators. We also showed that fenretinide has a capacity to regulate the levels of inflammatory cytokines even under conditions of excessive production caused by the ablation of an important regulatory protein. These data significantly contribute to a better understanding of the regulation of inflammatory cytokines and may help delineate the pathological consequences of inflammation. |
Authors
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Lachance, Claude |
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Contributors
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Danuta Radzioch (Supervisor) |
Publication Detail
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Publisher : McGill University Type : Electronic Thesis or Dissertation Format : application/pdf |
Date Detail
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2009 |
Subject
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Biology - Molecular |
Coverage
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Relation
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Source
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Copyright Information
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© Claude Lachance, 2009 |
Other Details
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Languages : en |
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