Tristetraprolin mediates anti-inflammatory effects of carbon monoxide on lipopolysaccharide-induced acute lung injury.

Low-dose inhaled carbon monoxide is reported to suppress inflammatory responses and exhibit a therapeutic effect in models of lipopolysaccharide (LPS)-induced acute lung injury (ALI). However, the precise mechanism by which carbon monoxide confers protection against ALI is not clear. Tristetraprolin (TTP; official name ZFP36) exerts anti-inflammatory effects by enhancing decay of proinflammatory cytokine mRNAs. With the use of TTP knockout mice, we demonstrate here that the protection by carbon monoxide against LPS-induced ALI is mediated by TTP. Inhalation of carbon monoxide substantially increased the pulmonary expression of TTP. carbon monoxide markedly enhanced the decay of mRNA-encoding inflammatory cytokines, blocked the expression of inflammatory cytokines, and decreased tissue damage in LPS-treated lung tissue. Moreover, knockout of TTP abrogated the anti-inflammatory and tissue-protective effects of carbon monoxide in LPS-induced ALI. These results suggest that carbon monoxide-induced TTP mediates the protective effect of carbon monoxide against LPS-induced ALI by enhancing the decay of mRNA encoding proinflammatory cytokines.

Natural compound cudraflavone B shows promising anti-inflammatory properties in vitro.

Cudraflavone B (1) is a prenylated flavonoid found in large amounts in the roots of Morus alba, a plant used as a herbal remedy for its reputed anti-inflammatory properties. The present study shows that this compound causes a significant inhibition of inflammatory mediators in selected in vitro models. Thus, 1 was identified as a potent inhibitor of tumor necrosis factor α (TNFα) gene expression and secretion by blocking the translocation of nuclear factor κB (NF-κB) from the cytoplasm to the nucleus in macrophages derived from a THP-1 human monocyte cell line. The NF-κB activity reduction resulted in the inhibition of cyclooxygenase 2 (COX-2) gene expression. Compound 1 acts as a COX-2 and COX-1 inhibitor with higher selectivity toward COX-2 than indomethacin. Pretreatment of cells by 1 shifted the peak in an regulatory gene zinc-finger protein 36 (ZFP36) expression assay. This natural product has noticeable anti-inflammatory properties, suggesting that 1 potentially could be used for development as a nonsteroidal anti-inflammatory drug lead.

Inhibition of protein kinase C beta II downregulates tristetraprolin expression in activated macrophages.

OBJECTIVE AND DESIGN: Tristetraprolin (TTP) is a 3'-UTR-binding protein known to destabilize mRNAs of TNFalpha and some other cytokines and to act as an anti-inflammatory factor. The aim of this study was to investigate the role of classical protein kinase C isoenzymes (cPKC) in the regulation of TTP expression in activated macrophages. MATERIALS AND METHODS: The expression of TTP in J774 macrophages was induced by a combination of LPS and phorbol myristate acetate (PMA). The effects of cPKC inhibitors and the effects of cPKC activation and downregulation by PMA on TTP protein and mRNA expression were determined by Western blotting and quantitative RT-PCR, respectively. Also, the effect of PKC beta II inhibitor CGP53353 on the activation of transcription factors AP-2, NF-kappaB, EGR1 and Sp1 was assessed. RESULTS: cPKC inhibitors RO318220, GO6976, LY333531 and CGP53353 inhibited LPS and PMA-induced expression of TTP protein and mRNA. Similar effects were obtained when cPKC isoenzymes were downregulated by PMA. In addition, CGP53353 decreased the activation of transcription factor AP-2. CONCLUSIONS: The results suggest that cPKCs, most likely PKC beta II, upregulate TTP expression in activated macrophages. This regulation is possibly mediated through the activation of transcription factor AP-2, and serves as an additional mechanism how PKC beta regulates the inflammatory process.

Salbutamol increases tristetraprolin expression in macrophages.

Tristetraprolin (TTP) is a tandem zinc finger protein that can bind to AU-rich elements (AREs) in the 3'-untranslated regions (3'-UTR) in mRNAs of transiently expressed genes, e.g. tumor necrosis factor-alpha (TNF-alpha) and granulocyte macrophage colony-stimulating factor (GM-CSF). TTP increases the turnover rate of the target mRNAs, thereby reducing, for example, the expression of TNF-alpha and GM-CSF. We examined the role of beta(2)-agonists, cAMP analogs, and forskolin (an activator of adenylate cyclase) on TTP mRNA and protein expression by quantitative real-time RT-PCR and Western blotting in J774 murine macrophages and THP-1 human macrophages. All of these agents increased TTP expression. A nonspecific inhibitor of phosphodiesterases (PDEs) 3-isobutyl-1-methylxanthine (IBMX) and type IV PDE-inhibitor rolipram further enhanced the increase in TTP expression levels, suggesting a cAMP-mediated effect. A possible mediator of these effects is transcription factor activator protein 2 (AP-2), whereas nuclear factor kappaB (NF-kappaB) seemed not to play any role. This mechanism may, at least in part, explain the anti-inflammatory effects which beta(2)-agonists have been reported to have in macrophages.

A retroviral expression system based on tetracycline-regulated tricistronic transactivator/repressor vectors for functional analyses of antiproliferative and toxic genes.

Establishment of stably transfected mammalian cells with conditional expression of antiproliferative or proapoptotic proteins is often hampered by varying expression within bulk-selected cells and high background in the absence of the inducing drug. To overcome such limitations, we designed a gene expression system that transcribes the tetracycline-dependent rtTA2-M2-activator, TRSID-silencer, and selection marker as a tricistronic mRNA from a single retroviral vector. More than 92% of bulk-selected cells expressed enhanced green fluorescent protein or luciferase over more than three orders of magnitude in an almost linear, dose-dependent manner. To functionally test this system, we studied how dose-dependent expression of p27(Kip1) affects proliferation and viability of SH-EP neuroblastoma cells. Low to moderate p27(Kip1) expression caused transient G(0)-G(1) accumulation without reduced viability, whereas high p27(Kip1) levels induced significant apoptosis after 72 hours. This proves that this expression system allows concentration-dependent analysis of gene function and implicates p27(Kip1) as a critical regulator of both proliferation and apoptosis in SH-EP neuroblastoma cells.

Posttranslational regulation of tristetraprolin subcellular localization and protein stability by p38 mitogen-activated protein kinase and extracellular signal-regulated kinase pathways.

The p38 mitogen-activated protein kinase (MAPK) signaling pathway, acting through the downstream kinase MK2, regulates the stability of many proinflammatory mRNAs that contain adenosine/uridine-rich elements (AREs). It is thought to do this by modulating the expression or activity of ARE-binding proteins that regulate mRNA turnover. MK2 phosphorylates the ARE-binding and mRNA-destabilizing protein tristetraprolin (TTP) at serines 52 and 178. Here we show that the p38 MAPK pathway regulates the subcellular localization and stability of TTP protein. A p38 MAPK inhibitor causes rapid dephosphorylation of TTP, relocalization from the cytoplasm to the nucleus, and degradation by the 20S/26S proteasome. Hence, continuous activity of the p38 MAPK pathway is required to maintain the phosphorylation status, cytoplasmic localization, and stability of TTP protein. The regulation of both subcellular localization and protein stability is dependent on MK2 and on the integrity of serines 52 and 178. Furthermore, the extracellular signal-regulated kinase (ERK) pathway synergizes with the p38 MAPK pathway to regulate both stability and localization of TTP. This effect is independent of kinases that are known to be synergistically activated by ERK and p38 MAPK. We present a model for the actions of TTP and the p38 MAPK pathway during distinct phases of the inflammatory response.