Recent investigations have established a mechanism for the regulation of inflammatory responses at the post-transcriptional level by tristetraprolin (TTP) family of CCCH tandem zinc finger proteins (ZFP). TTP family consists of three known members in mammals (ZFP36 or TTP, ZFP36L1 or TIS11B, and ZFP36L2 or TIS11D) and the fourth member in mouse and rat but not in humans (ZFP36L3) 12. TTP (TIS11, G0S24, or NUP475), the best-studied family member, is the product of the immediate-early response gene Zfp36 in the mouse (ZFP36 in humans, refer to "Abbreviations" for the nomenclature of genes, mRNAs, and proteins) 345. TTP binds to AU-rich elements (AREs) in some mRNAs and destabilizes those transcripts encoding proteins such as tumor necrosis factor-α (TNF-α) 6789, granulocyte-macrophage colony-stimulating factor (GM-CSF) 1011, and cyclooxygenase 2 (COX-2) 12. The mRNAs encoding TNF-α and GM-CSF are stabilized in TTP knockout mice and in cells derived from them 810. Excessive secretion of these cytokines in TTP knockout mice results in a severe systemic inflammatory response including arthritis, autoimmunity, and myeloid hyperplasia 1314. In contrast, up-regulation of TTP reduces inflammatory responses in macrophages 15. These lines of evidence support the conclusion that TTP is an anti-inflammatory protein or arthritis suppressor.

Agents that induce Ttp gene expression may have potential therapeutic value for the prevention and/or treatment of inflammation-related diseases. A number of agents have been shown to increase Ttp mRNA and/or protein levels in mammalian cells. The inducible agents include growth factors (insulin, insulin-like growth factor I, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, and fetal calf serum) 34, cytokines (TNF-α, GM-CSF, and interferon-gamma) 45815 and zinc 16. However, Ttp gene expression is also induced by tumor promoters (phorbol 12-myristate 13-acetate and tetradecanoyl phorbol acetate) 35 and bacterial endotoxin lipopolysaccharide (LPS) 81718. Finally, Ttp gene expression is induced by viral infection 19. The fact that most of these agents also increase the expression levels of pro-inflammatory cytokines such as TNF-α in the same cells and/or tissues 820 may limit the therapeutic potential of these agents. Therefore, it is important to search for other agents with the potential to up-regulate anti-inflammatory but down-regulate pro-inflammatory gene expression.

Tea (Camellia sinensis) is a popular beverage worldwide. Recent studies indicate that tea has a wide range of effects on animal and human health. A number of studies have indicated that green tea has anti-inflammatory properties 21. Tea has been reported to have beneficial effects in conditions such as collagen-induced arthritis 22, inflammatory bowel disease 23 and carrageenan-induced paw edema 24. However, the molecular mechanism of tea's anti-inflammatory property has not been completely elucidated. We hypothesized that TTP and/or its homologues might be involved in the beneficial effects of tea as an anti-inflammatory product.

A high fructose diet has been used as a model for the study of insulin resistance and oxidative stress in rats 25. Overproduction of free radicals under oxidative stress is associated with inflammation. TNF-α levels are increased in rat muscle by high fructose diet, which is one of the determinants of insulin resistance in skeletal muscle 2627. A high fructose diet was also shown to activate inflammatory pathways in the liver 28. Since liver and muscle are two of the most insulin-responsive organs and Ttp gene expression is induced by insulin 3, we hypothesized that TTP and/or its homologues might be involved in the inflammatory response induced by a high-fructose diet in rat liver and muscle, and that green tea might possess beneficial effects in high fructose-fed rats. Therefore, the demonstration of the effects of tea on inflammation-related gene expression in this model is important and complementary of its known antioxidant effects.

In this study, quantitative real-time PCR was used to investigate the expression profiles and the effects of green tea on the expression levels of the anti-inflammatory Ttp family mRNAs and some of the pro-inflammatory mRNAs known to be regulated by TTP family proteins. Our results showed that liver had more abundant Ttp family mRNA levels than skeletal muscle, and that Ttp mRNA levels were increased significantly and Tnf mRNA levels were reduced in the liver and skeletal muscle of rats fed a high-fructose diet and given 1 g of green tea extract/kg in the diet.

TTP is an anti-inflammatory protein with the potential as a therapeutic target for the prevention and/or treatment of inflammation-related diseases. Previous studies have shown that Ttp mRNA and/or TTP protein levels are increased in mammalian cells by a wide range of agents. However, the fact that most of the same agents also increase the expression levels of pro-inflammatory cytokines in the same cells and/or tissues 820 limits these agents' therapeutic potential. In this study, we determined that green tea possessed the ability to increase Ttp but decrease Tnf gene expression in rats fed a high fructose diet known to induce insulin resistance, oxidative stress, and inflammation.

Tea is the most consumed beverage in the world and there are numerous reports of the health benefits of tea. One of the effects of tea is its anti-inflammatory property. In this study, we explored the relationship between green tea and the mRNA levels of some pro-inflammatory and anti-inflammatory genes in the liver and muscle of rats with metabolic syndrome induced by a high-fructose diet. We demonstrated that a low dose of green tea (1 g solid extract/kg diet) significantly increased the mRNA levels of the anti-inflammatory protein TTP and decreased those of the pro-inflammatory TNF-α in both liver and muscle of rats and also decreased Ptgs2 mRNA in the muscle. The induction of Ttp and reduction of Tnf mRNAs by green tea in rats suggests that at least part of the mechanisms of tea's anti-inflammatory effects may be involved in TTP at the post-transcriptional level.

Previous investigations have suggested that the mechanism of tea's anti-inflammatory effects also involves the regulation of gene transcription. Tea affects a number of important molecular targets including TNF-α 36, interleukin 2 (IL-2) 23, signal transducer and activator of transcription-1α (STAT-1α) 37, inducible nitric-oxide synthase (iNOS) 38, nuclear factor-kappa B (NF-κB), insulin activity 394041, and affects DNA and RNA directly 42. TNF-α plays a crucial role in inflammation. NF-κB, an oxidative stress-sensitive nuclear transcription factor, controls the expression of Tnf and other genes. It was shown that green tea polyphenol EGCG inhibited LPS-mediated Tnf mRNA levels by blocking NF-κB activation in macrophage RAW264.7 cells 36. Therefore, it was proposed that the anti-inflammatory mechanism of green tea polyphenols is mediated at least in part through down-regulation of Tnf gene expression by blocking NF-κB activation 36. Taken together, the molecular mechanism of tea's anti-inflammatory effects may be involved in the down-regulation of pro-inflammatory cytokines such as TNF-α at both transcriptional and post-transcriptional levels 81718.

We also found that a higher dose of green tea extract at 2 g in the high-fructose diet significantly increased the mRNA levels of Elavl1/Hua/Hur in rat liver. ELAVL1 is known to be an RNA binding protein 333435. Previous studies suggest that ELAVL1 stabilizes ARE-containing mRNAs 34, but a recent study suggests that ELAVL1 in murine innate compartments suppresses inflammatory responses in vivo by inducing the translational silencing of specific cytokine mRNAs 33. This study suggests that a higher dose of green tea might also have beneficial effects on reducing inflammation via ELAVL1 in these tissues. Vegfa and Vegfb mRNAs are known to be destabilized by TIS11B protein 32. Our study showed that Tis11b and Vegf mRNA levels in rat liver or muscle were not affected by either tea dose. These results suggest that Vegf mRNAs may be the physiological targets of TIS11B rather than TTP in these tissues.

There are two known TTP-related proteins in mammalian cells, ZFP36L1 (TIS11B, cMG1, ERF1, BRF1, or Berg36) and ZFP36L2 (TIS11D, ERF2, or BRF2) 1. A third related protein (ZFP36L3) was recently identified that seems to be expressed only in rodents as a placenta-specific protein 2, and the expression in other species has not been reported. These four proteins are all capable of binding to and destabilizing ARE-containing mRNAs in vitro and in transfected cells 1143. However, gene knockout studies have provided evidence for their unique roles in vivo. TTP knockout mice develop a severe inflammatory syndrome 1314. Mice deficient in ZFP36L1 develop chorioallantoic fusion defects and embryonic lethality 44. Mice with decreased levels of an amino-terminal truncated form of ZFP36L2 exhibit female infertility and disrupted early embryonic development 45. Despite their unique contributions, information is limited on the expression profiles of Ttp family mRNAs in the same tissue and/or in different tissues.

We therefore described the expression profiles of the four Ttp family mRNAs in rat liver and muscle. Real-time PCR analyses showed that Ttp and Tis11b mRNAs were the two major forms in the liver and muscle and that Ttp, Tis11b, and Tis11d mRNA levels were more abundant in liver than those in muscle. The PCR results are in agreement with immunoblotting results reported previously that TTP protein levels are more abundant in mouse liver than muscle 17. Zfp36l3 mRNA levels in the liver or muscle were below the detection limit even after 50 cycles of PCR amplification. These PCR results are in agreement with those of the Northern blot results reported previously to be specific to placenta and extra-embryonic tissues in mouse 2. However, these expression profiles of Ttp family mRNAs in the rat liver and muscle are somewhat different from those in cultured human monocytes, in which the mRNA levels of these three forms are similar in un-induced monocytes 46.

The induction of Ttp and reduction of Tnf mRNAs in rat liver and muscle by green tea (1 g solid extract/kg high fructose diet) suggests that TTP may be involved at the post-transcriptional level in the mechanisms of tea's anti-inflammatory effects. However, some issues need to be investigated in future experiments. First, the reason(s) why tea at 2 g solid extract/kg diet did not increase Ttp mRNA levels and decreased Tnf mRNA levels in the same tissues requires further studies to determine the optimum dose that demonstrates green tea effects. It is possible that Ttp gene expression is regulated by a narrow dose of tea for certain period, since its gene expression is transiently induced by growth factors such as insulin, i.e., Ttp mRNA levels peaked at 45 min but declined to normal level in 2 h in a cell culture system 3. Furthermore, the effect of green tea on insulin levels in the plasma showed a similar pattern, i.e., 2 g tea treatment was less effective than 1 g tea treatment. The insulin levels were 412 ± 100 pmol/l, 113 ± 28, and 197 ± 43 for the diet control, 1 g tea, and 2 g tea treatments for 6 weeks 47. Therefore, it is important to conduct more extensive analysis of the dosage effect and time course of tea's effect on Ttp and Tnf gene expression. We believe cell culture systems could be a much more effective way to address this area of research in the future. Second, confirmation of TTP protein levels in rats with tea treatments requires future development of high-titer rat antibodies, since TTP protein is only detectable with the mouse TTP antibodies in the spleen of rats after LPS stimulation 81718 or in rats following dexamethasone induction 48. Third, it's of interest to determine if the change of Tnf mRNA level correlates with the change of TNF-α protein level. However, since TNF-α is a secreted protein, a cell culture system may be more suitable to do detailed analyses of the TNF-α protein levels and would allow a detailed time course of the TNF-α protein levels. Fourth, it will be interesting to investigate if green tea has any beneficial effects in rats fed a normal diet. However, we speculate that there might be large differences in gene expression in rats treated with a high-fructose diet and a normal diet (no fructose), since the dietary environment between rats fed a normal diet and those fed a high fructose diet is dramatically different. Finally, what is the mechanism(s) of green tea activation of Ttp gene expression? Our working hypothesis is that Ttp gene expression is increased by catechins, the major polyphenols in green tea. We reported that polyphenolic compounds in cinnamon increased TTP protein levels in mouse 3T3-L1 adipocytes 4950. Recently, it was demonstrated that tea catechins directly bind to DNA and preferentially bind to poly(A), poly(U), and poly(AU) in RNA 42. It will be interesting to investigate if green tea polyphenols could bind to the regulatory elements of Ttp gene and/or bind to the AU-rich elements in the 3'-untranslated region of Tnf mRNA, which are also the preferred binding sites for TTP protein 5152.

This study describes profiles of the anti-inflammatory Ttp family mRNA levels and green tea effects on these and some of the pro-inflammatory mRNAs in the liver and muscle of rats with metabolic syndrome induced by a high-fructose diet. Our results suggest that the molecular mechanism of the anti-inflammatory effects of green tea may be partially due to its ability to increase mRNA levels encoding anti-inflammatory factors such as TTP/TIS11/ZFP36 and/or ELAVL1/HuA/HuR and to decrease mRNA levels encoding pro-inflammatory factors such as TNF-α and/or PTGS2/COX-2. To our knowledge, this is the first report to show that a plant nutritional product such as green tea can modulate Ttp mRNA levels in a biological system and the results provide a novel post-transcriptional mechanism for green tea's anti-inflammatory properties. These results suggest that drinking adequate amounts of green tea may play a role in the prevention of inflammation-related diseases.

ANOVA: one way analysis of variance; ARE: AU-rich element; COX-2/PTGS2: cyclooxgenase-2/prostaglandin-endoperoxide synthase 2; EGCG: Epigallocatechin-3-gallate; GM-CSF/CSF2: granulocyte-macrophage colony-stimulating factor; HuR(HuA)/ELAVL1: Hu antigen R/embryonic lethal, abnormal vision-like 1; LPS: lipopolysaccharide; NF-κB: nuclear factor-kappa B; RPL32: ribosomal protein L32; TNF: tumor necrosis factor; TTP: tristetraprolin; VEGF: vascular endothelial growth factor; ZFP36: zinc finger protein 36; ZFP36L1: ZFP36-like 1; ZFP36L2: ZFP36-like 2; ZFP36L3: ZFP36-like 3. The nomenclature of genes, mRNAs, and proteins was according to reference 1 and was accepted by the Mouse Genome Database. For example, Zfp36, Zfp36, and ZFP36 represent Zfp36 gene, mRNA, and protein, respectively.

The author(s) declare that they have no competing interests.

HC designed the experiment, designed PCR primers and probes, performed PCR assays, analyzed PCR data and wrote the manuscript. MAK performed RNA isolation and cDNA synthesis. FK and HDD provided advice on RNA isolation, cDNA synthesis, and PCR assay. JFU analyzed the data and revised the manuscript. SC provided the green tea solid extract. AMR designed the animal study and provided the animal tissues. RAA designed the animal study, analyzed the data, and revised the manuscript.