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Inhibition of tristetraprolin expression by dexamethasone in activated macrophages
Abstract
Tristetraprolin (TTP) is a factor that regulates mRNA stability and the expression of certain inflammatory genes. In the present study, we found that TTP expression was increased in macrophages exposed to bacterial lipopolysaccharide (LPS). Dexamethasone and dissociated steroid RU24858 inhibited LPS-induced TTP protein and mRNA expression and the inhibitory effect was reversed by a glucocorticoid receptor antagonist mifepristone. Histone deacetylase inhibitors trichostatin A (TSA) and apicidin reduced the inhibitory effect of dexamethasone and RU24858 on TTP expression, but the glucocorticoids did not alter TTP mRNA half-life. These results suggest that anti-inflammatory steroids reduce TTP expression in activated macrophages by a glucocorticoid response element (GRE)-independent mechanism, possibly through histone deacetylation and transcriptional silencing.
... RNA extraction and quantitative real-time reverse transcriptase (RT)-PCR. The protocol for RNA extraction and quantitative real-time RT-PCR has been described in (Jalonen et al., 2005). Primers and TaqMan FAM-TAMRA probes (Table 1) for TTP, TNF-α, and for the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed using Primer Express Software (Applied Biosystems, Foster City, CA, USA) and supplied by Metabion (Martinsried, Germany). ...
... Western blotting. The protocol for Western blotting has been described in (Jalonen et al., 2005). When preparing cell lysates for ubiquitin Western blotting, lysis buffer also contained ubiquitin aldehyde (20 µg/ml) and MG132 (25 µM) to prevent deubiquitinylation of the sample. ...
... Dexamethasone has been shown to increase TTP mRNA and protein levels in resting human A549 lung epithelial cells and in rat lung tissue (Smoak and Cidlowski, 2006). On the other hand, dexamethasone reduced LPS-induced TTP expression in J774 macrophages (Jalonen et al., 2005). Interferons have been reported to up-regulate TTP expression, which in turn results in down-regulation of several proinflammatory genes (Sauer et al., 2006). ...
... RNA Extraction and Quantitative Real-Time Reverse Transcriptase-PCR. The protocol for RNA extraction and quantitative real-time reverse transcriptase (RT)-PCR has been described by Jalonen et al. (2005). Primers and TaqMan 6-carboxyfluorescein (Fam)-TAMRA probes (Table 1) for TTP, TNF-␣, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed using Primer Express Software (Applied Biosystems, Foster City, CA) and supplied by Metabion (Martinsried, Germany). ...
... Western Blotting. The protocol for Western blotting has been described by Jalonen et al. (2005). When preparing cell lysates for ubiquitin Western blotting, lysis buffer also contained ubiquitin aldehyde (20 g/ml) and MG132 (25 M) to prevent deubiquitinylation of the sample. ...
... Dexamethasone has been shown to increase TTP mRNA and protein levels in resting human A549 lung epithelial cells and in rat lung tissue (Smoak and Cidlowski, 2006). On the other hand, dexamethasone reduced LPS-induced TTP expression in J774 macrophages (Jalonen et al., 2005). Interferons have been reported to up-regulate TTP expression, which in turn results in down-regulation of several proinflammatory genes (Sauer et al., 2006). ...
Tristetraprolin (TTP) is a trans-acting factor that can regulate mRNA stability by binding to the cis-acting AU-rich element (ARE) in the 3'-untranslated region in mRNAs of certain transiently expressed genes. The best-studied target of TTP is tumor necrosis factor (TNF)-. By binding to ARE, TTP increases the degradation of TNF-alpha mRNA, thereby reducing the expression of TNF-alpha. We examined the effects of cAMP analogs and the cAMP-elevating agents forskolin and beta2-agonists on lipopolysaccharide (LPS)-induced TTP mRNA and protein expression by quantitative real-time reverse transcriptase-polymerase chain reaction and Western blotting in activated macrophages. All of these agents caused a slight increase in LPS-induced expression of TTP mRNA. However, TTP protein levels were significantly reduced when the cells were treated with the combination of LPS and cAMP-elevating agent compared with LPS alone. Proteasome inhibitors MG132 (N-[(phenylmethoxy)-carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide) and lactacystin increased TTP protein levels and abolished the effects of cAMP-enhancing compounds on TTP protein levels. The results suggest that mediators and drugs that enhance intracellular cAMP reduce TTP expression in macrophages exposed to inflammatory stimuli by increasing TTP degradation through the proteasome pathway.
... This transcript contained a single potential TTP family binding site, but two other highly up-regulated chemokine transcripts, Ccl7 (46fold increase) and Ccl8 (37fold increase) mRNAs, did not contain obvious binding sites, and may be examples of autocrine/paracrine secondary stimulation. Similarly, five transcripts from the Cxcl family of chemokines were increased, but only two of them, Cxcl1 and Cxcl2 mRNAs, are well-known TTP targets (Jalonen et al, 2005;Datta et al, 2008;Qiu et al, 2015), whereas the other three do not contain obvious TTP family binding sites. A final example is interferon β1 (Ifnb1) mRNA, whose levels were increased by about 10.5fold. ...
... Good examples of members of this group are Cxcl2 and Ccl2 mRNAs, as shown in Fig 9. Both mRNAs have been described previously as TTP targets (Jalonen et al, 2005;Sauer et al, 2006;Qiu et al, 2015), and both encode important chemokines involved in leukocyte chemotaxis and pathogenesis of inflammatory diseases (Sauer et al, 2006;Deshmane et al, 2009;De Filippo et al, 2013). ...
Members of the tristetraprolin (TTP) family of RNA-binding proteins can bind to and promote the decay of specific transcripts containing AU-rich motifs. ZFP36 (TTP) is best known for regulating pro-inflammatory cytokine expression in myeloid cells; however, its mammalian paralogues ZFP36L1 and ZFP36L2 have not been viewed as important in controlling inflammation. We knocked out these genes in myeloid cells in mice, singly and together. Single-gene myeloid-specific knockouts resulted in almost no spontaneous phenotypes. In contrast, mice with myeloid cell deficiency of all three genes developed severe inflammation, with a median survival of 8 wk. Macrophages from these mice expressed many more stabilized transcripts than cells from myeloid-specific TTP knockout mice; many of these encoded pro-inflammatory cytokines and chemokines. The failure of weight gain, arthritis, and early death could be prevented completely by two normal alleles of any of the three paralogues, and even one normal allele of Zfp36 or Zfp36l2 was enough to prevent the inflammatory phenotype. Our findings emphasize the importance of all three family members, acting in concert, in myeloid cell function.
... Dexamethasone has been reported to regulate the levels of HuR and TTP, thereby reducing the levels of infl ammatory gene mRNAs such as COX-2 and CCL11 (Lu and Cidlowski, 2005;Shim and Karin, 2002) through a p38-MAPK-mediated pathway subsequent to induction of MKP-1 (Bergmann et al., 2004;Lasa et al., 2001). However, signifi cant modulation of these genes often appears at 10 nM dexamethasone rather than the 1 nM concentrations associated with suppression of many infl ammatory genes, although again these effects may be cell selective (Jalonen et al., 2005). Intriguingly, dexamethasone has recently been reported to decrease TTP expression in LPS-stimulated murine macrophages (Jalonen et al., 2005). ...
... However, signifi cant modulation of these genes often appears at 10 nM dexamethasone rather than the 1 nM concentrations associated with suppression of many infl ammatory genes, although again these effects may be cell selective (Jalonen et al., 2005). Intriguingly, dexamethasone has recently been reported to decrease TTP expression in LPS-stimulated murine macrophages (Jalonen et al., 2005). ...
Glucocorticoids are the most effective therapy for the treatment of many chronic inflammatory diseases but they are ineffective in severe asthma and chronic obstructive pulmonary disease (COPD) for example. Glucocorticoids act by binding to and activating specific cytosolic receptors (GR). These receptors then translocate to the nucleus where they regulate gene expression. GR is able to selective repress specific inflammatory genes by differing actions on promoter-specific components of NF-κB and AP-1 activation complexes and also by effects on MAPK pathways. Importantly, these actions are mutually inhibitory. These pleitropic effects of GR may underlie there effectiveness in most patients with airways disease but suggests that abnormal activation of these pathways may result in glucocorticoid refractoriness. Understanding the molecular mechanisms of GR action may lead to the development of new anti-inflammatory drugs or enable clinicians to reverse the relative steroid-insensitivity that is characteristic of severe asthma and COPD for example.
... In addition to this role in feedback control, TTP expression is also reported to be up-regulated by glucocorticoids, and this may contribute to the post-transcriptional repression of inflammatory gene expression (Smoak and Cidlowski, 2006;Ishmael et al., 2008;Kaur et al., 2008). However, this point remains unclear because TTP expression induced by an inflammatory stimulus is reduced by dexamethasone (Jalonen et al., 2005). ...
... However, it is clear from our present study that the level of dexamethasoneinduced TTP protein is very modest compared with the level induced by IL-1 alone. Furthermore, our data indicate that in the presence of dexamethasone, IL-1-induced levels of TTP are considerably reduced, and this observation is consistent with the repression of TTP by dexamethasone in lipopolysaccharide-treated macrophages (Jalonen et al., 2005). Because, in the presence of IL-1 alone, TTP is a negative regulator of GM-CSF, and in the presence of IL-1 plus dexamethasone TTP expression is reduced, our data indicate that TTP is predominantly a feedback control gene rather than primarily an effector of glucocorticoid-dependent inhibition. ...
The mRNA-destabilizing protein tristetraprolin (TTP) negatively regulates adenine- and uridine-rich element (ARE)-containing mRNAs. In A549 pulmonary cells, TTP mRNA and both a approximately 40- and a approximately 45-kDa phosphorylated version of TTP protein were rapidly induced in response to interleukin (IL)-1beta. Analysis with IkappaBalphaDeltaN, a dominant version of inhibitor of kappaBalpha (IkappaBalpha), as well as dominant-negative and small-molecule IkappaB kinase (IKK) inhibitors demonstrated that IL-1beta-induced TTP is nuclear factor-kappaB (NF-kappaB)-dependent. Likewise, TTP expression and formation of the approximately 45-kDa phosphorylated form of TTP are blocked by the p38 mitogen-activated protein kinase (MAPK) inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580). By contrast, and despite a 3- to 4-fold induction of TTP mRNA, the anti-inflammatory glucocorticoid dexamethasone only modestly induced expression of the approximately 40-kDa form of TTP. In the context of IL-1beta, dexamethasone exerted a marginal repressive effect on TTP mRNA expression and more considerably reduced TTP protein. Given a requirement for p38 MAPK in the induction of TTP by IL-1beta, this repressive effect may be explained by repression of the p38 MAPK pathway by dexamethasone. Knockdown of TTP protein by siRNA elevated IL-1beta-induced expression of granulocyte macrophage-colony-stimulating factor (GM-CSF) and IL-8, demonstrating a role for TTP in feedback control. Likewise, knockdown of TTP increased GM-CSF expression in the presence of IL-1beta plus dexamethasone, suggesting that feedback control by TTP also occurs in the context of IL-1beta plus dexamethasone. Taken together, our data demonstrate that NF-kappaB and p38 MAPK are critical to the induction of TTP by IL-1beta and that TTP induction provides feedback control of the ARE-containing genes GM-CSF and IL-8.
... 80,81 However, significant modulation of these genes often appears at 10 nM dexamethasone rather than the 1 nM concentrations associated with suppression of many inflammatory genes, although again these effects might be cell selective. 82 Intriguingly, dexamethasone has recently been reported to decrease TTP expression in LPS-stimulated murine macrophages. 82 ...
... 82 Intriguingly, dexamethasone has recently been reported to decrease TTP expression in LPS-stimulated murine macrophages. 82 ...
Extensive development of inhaled and oral glucocorticoids has resulted in highly potent molecules that have been optimized to target activity to the lung and minimize systemic exposure. These have proved highly effective for most asthmatic subjects, but despite these developments, there are a number of subjects with asthma who fail to respond to even high doses of inhaled or even oral glucocorticoids. Advances in delineating the fundamental mechanisms of glucocorticoid pharmacology, especially the concepts of transactivation and transrepression and cofactor recruitment, have resulted in better understanding of the molecular mechanisms whereby glucocorticoids suppress inflammation. The existence of multiple mechanisms underlying glucocorticoid insensitivity raises the possibility that this might indeed reflect different diseases with a common phenotype, and studies examining the efficacy of potential new agents should be targeted toward subgroups of patients with severe corticosteroid-resistant asthma who clearly require effective new drugs and other approaches to improved asthma control.
... For Western blot shTTP and shNEG cells were plated on 6 well plates and grown to confluence. Cells were treated with or without LPS (100 ng/mL) for 6 h and proteins were extracted as described [23]. ...
... The protocol for Western blotting was described in [23]. The gels were loaded with 50 μg of protein. ...
In inflammation, the post-transcriptional regulation of transiently expressed genes provides a potential therapeutic target. Tristetraprolin (TTP) is of the factors regulating decay of cytokine mRNAs. The aim of the present study was to identify cytokines whose expression is regulated by TTP. We established a TTP knock-down cell line by expressing shRNA against TTP (shTTP cell line). A cytokine antibody array was used to measure cytokine production in macrophages exposed to lipopolysaccharide (LPS). Cytokines IL-6, IL-12, TNF-α, and MIP-2 (a homologue to human IL-8) were expressed at higher
levels whereas MIP-3α was produced at lower levels in LPS-treated shTTP cells than in control cells suggesting that the expression of these cytokines is regulated by TTP. The present data provide IL-12, MIP-2, and MIP-3α as novel inflammatory cytokine targets for TTP-mediated mRNA decay and stress the role of TTP in the regulation of the inflammatory process.
... Tristetraprolin (TTP) is a 34-kDa protein characterized by the presence of two tandemly located CCCH-type zinc finger domains and three proline rich motifs [1][2][3][4]. In mammalian cells TTP is localized both in the nucleus and cytoplasm and its total protein expression and organelle-specific distribution are regulated by signal transduction pathways activated by interleukins, growth factors and glucocorticoids [5][6][7][8][9]. Functional analysis performed in animal models and cells in culture revealed that the cytoplasmic form of TTP uses its zinc finger domains to interact with AU-rich elements (AREs) present in the 3′-untranslated region of mRNAs [10][11][12][13]. ...
Tristetraprolin (TTP) is a 34-kDa, zinc finger-containing factor that in mammalian cells acts as a tumor suppressor protein through two different mechanisms. In the cytoplasm TTP promotes the decay of hundreds of mRNAs encoding cell factors involved in inflammation, tissue invasion, and metastasis. In the cell nucleus TTP has been identified as a transcriptional corepressor of the estrogen receptor alpha (ERα), which has been associated to the development and progression of the majority of breast cancer tumors. In this work we report that nuclear TTP modulates the transactivation activity of progesterone receptor (PR), glucocorticoid receptor (GR) and androgen receptor (AR). In recent years these steroid nuclear receptors have been shown to be of clinical and therapeutical relevance in breast cancer. The functional association between TTP and steroid nuclear receptors is supported by the finding that TTP physically interacts with ERα, PR, GR and AR in vivo. We also show that TTP overexpression attenuates the transactivation of all the steroid nuclear receptors tested. In contrast, siRNA-mediated reduction of endogenous TTP expression in MCF-7 cells produced an increase in the transcriptional activities of ERα, PR, GR and AR. Taken together, these results suggest that the function of nuclear TTP in breast cancer cells is to act as a corepressor of ERα, PR, GR and AR. We propose that the reduction of TTP expression observed in different types of breast cancer tumors may contribute to the development of this disease by producing a dysregulation of the transactivation activity of multiple steroid nuclear receptors.
... ZFP36 expression is rapidly induced by multiple pro-inflammatory stimuli, including IL1B or lipopolysaccharide (LPS) in various cells, including macrophage, fibroblasts and A549 pulmonary epithelial cells (14)(15)(16)(17). Given the ability to reduce the expression of ARE-containing mRNAs, this means that ZFP36 is a negative (incoherent) feedforward regulator of inflammatory gene expression (Fig. 1). ...
TNF is central to inflammation and may play a role in the pathogenesis of asthma. The 3′-untranslated region of the TNF transcript contains AU-rich elements (AREs) that are targeted by the RNA-binding protein, tristetraprolin (also known as zinc finger protein 36 (ZFP36)), which is itself up-regulated by inflammatory stimuli, to promote mRNA degradation. Using primary human bronchial epithelial and pulmonary epithelial A549 cells, we confirm that interleukin-1β (IL1B) induces expression of dual-specificity phosphatase 1 (DUSP1), ZFP36, and TNF. Whereas IL1B-induced DUSP1 is involved in feedback control of MAPK pathways, ZFP36 exerts negative (incoherent) feed-forward control of TNF mRNA and protein expression. DUSP1 silencing increased IL1B-induced ZFP36 expression at 2 h and profoundly repressed TNF mRNA at 6 h. This was partly due to increased TNF mRNA degradation, an effect that was reduced by ZFP36 silencing. This confirms a regulatory network, whereby DUSP1-dependent negative feedback control reduces feed-forward control by ZFP36. Conversely, whereas DUSP1 overexpression and inhibition of MAPKs prevented IL1B-induced expression of ZFP36, this was associated with increased TNF mRNA expression at 6 h, an effect that was predominantly due to elevated transcription. This points to MAPK-dependent feed-forward control of TNF involving ZFP36-dependent and -independent mechanisms. In terms of repression by dexamethasone, neither silencing of DUSP1, silencing of ZFP36, nor silencing of both together prevented the repression of IL1B-induced TNF expression, thereby demonstrating the need for further repressive mechanisms by anti-inflammatory glucocorticoids. In summary, these data illustrate why understanding the competing effects of feedback and feed-forward control is relevant to the development of novel anti-inflammatory therapies.
Background
ZFP36 negatively regulates AU-rich element-containing mRNAs.
Results
DUSP1 silencing increases ZFP36 expression and enhances negative feed-forward control of TNF.
Conclusion
MAPK inhibition initially attenuates TNF mRNA, but reduced feed-forward control subsequently produces an increase.
Significance
Understanding such networks is essential to develop novel anti-inflammatory therapies or understand TNF repression by glucocorticoids, which may not require DUSP1 or ZFP36 expression.
... 31 Furthermore, RU24858 inhibited the expression of tristetraprolin at the level of mRNA and protein expression in J774 mouse macrophages at concentrations used in the present study. 32 In the present study dexamethasone and RU2485 suppressed cytokine production in human neutrophils to a similar extent. A possible explanation for the differences in potency and the divergent dissociation abilities of this compound is that RU24858 works as a partial agonist at GR. ...
Glucocorticoids are widely used anti-inflammatory medication in diseases like asthma and chronic obstructive pulmonary disease. Glucocorticoids can either activate (transactivation) or inhibit (transrepression) transcription. RU24858 was introduced as a "dissociated" glucocorticoid and it has been reported to transrepress but not to transactivate. The aim of this study was to compare the effects of RU24858 and dexamethasone in human neutrophils. RU24858 delayed spontaneous neutrophil apoptosis and further enhanced GM-CSF- induced neutrophil survival to a similar extent as dexamethasone. Like dexamethasone RU24858 also reduced CXCL8 and MIP-1α. Unexpectedly however, RU24858 increased the expression of the glucocorticoid-inducible genes BLT-1, Annexin-1 and Grb-2 in neutrophils to a similar level as seen with dexamethasone. We have shown here that dexamethasone and RU24858 both increase Grb-2, BLT1 and Annexin-1 expression and inhibit CXCL8 and MIP-1α production. This suggests that RU24858 was not able to dissociate between transactivation and transrepression in human neutrophils but enhanced neutrophil survival.
... On the other hand, p38 signaling may play a more striking role at the 2 h stimulation of LPS; mRNA expression of TTP was reduced by 22 % by SB203580 treatment at the 30 min-induction of LPS, whereas the mRNA level of TTP was reduced by 79 % in the 2 h induction of LPS. Nevertheless, previous reports indicated that pharmacological inhibition of p38, JNK, and ERK pathways, as well as NF-jB, reduced the amount of TTP mRNA by 74, 43, 35, and 21 % in LPS stimulated J774 macrophages, respectively [32]. The differences between their results and ours may be due to their longer LPS induction (6 h) and that the cell line used was J774 macrophages, instead of RAW264.7 macrophages. ...
... 36 AU-rich elements (AREs) in the 3 0 -untranslated region (UTR) are known to regulate mRNA stability, and the expression of the ARE-binding protein, tristetraprolin, which promotes mRNA deadenylation and destabilization, has been shown to be inhibited in Dex-treated macrophages. 37,38 The interference with tristetraprolin-mediated mRNA destabilization is, thus, likely to be responsible for the Dex-dependent prolongation of MFG-E8 mRNA half-life, as an ARE is located in the MFG-E8 3 0 UTR at position þ 1857. ...
The phagocytic clearance of apoptotic cells is essential to prevent chronic inflammation and autoimmunity. The phosphatidylserine-binding protein milk fat globule-EGF factor 8 (MFG-E8) is a major opsonin for apoptotic cells, and MFG-E8(-/-) mice spontaneously develop a lupus-like disease. Similar to human systemic lupus erythematosus (SLE), the murine disease is associated with an impaired clearance of apoptotic cells. SLE is routinely treated with glucocorticoids (GCs), whose anti-inflammatory effects are consentaneously attributed to the transrepression of pro-inflammatory cytokines. Here, we show that the GC-mediated transactivation of MFG-E8 expression and the concomitantly enhanced elimination of apoptotic cells constitute a novel aspect in this context. Patients with chronic inflammation receiving high-dose prednisone therapy displayed substantially increased MFG-E8 mRNA levels in circulating monocytes. MFG-E8 induction was dependent on the GC receptor and several GC response elements within the MFG-E8 promoter. Most intriguingly, the inhibition of MFG-E8 induction by RNA interference or genetic knockout strongly reduced or completely abolished the phagocytosis-enhancing effect of GCs in vitro and in vivo. Thus, MFG-E8-dependent promotion of apoptotic cell clearance is a novel anti-inflammatory facet of GC treatment and renders MFG-E8 a prospective target for future therapeutic interventions in SLE.Cell Death and Differentiation advance online publication, 5 July 2013; doi:10.1038/cdd.2013.82.
... Increased transcription of small numbers of anti-inflammatory genes does not entirely explain the widespread anti-inflammatory actions of GCs. Furthermore, high concentrations of GCs are required to increase the transcriptions of anti-inflammatory genes (11)(12)(13). Switching off inflammatory genes through interactions with transcription factors, such as activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB), may be the major effect of corticosteroids. ...
Bronchial asthma is assumed to be the result of excessive inflammation driven by an aberrant T-helper-2 (Th2) response. Recently, it has begun to be recognized that asthma is a heterogeneous disorder. Glucocorticoids (GCs) are effective treatment for bronchial asthma; however, the inflammation in bronchial asthma cannot always be fully controlled. A recent study demonstrated a new underlying mechanism of glucocorticoid resistance that acts in a Th2-independent manner. Thus, responses to GCs are highly heterogeneous.
... On the other hand, p38 signaling may play a more striking role at the 2 h stimulation of LPS; mRNA expression of TTP was reduced by 22 % by SB203580 treatment at the 30 min-induction of LPS, whereas the mRNA level of TTP was reduced by 79 % in the 2 h induction of LPS. Nevertheless, previous reports indicated that pharmacological inhibition of p38, JNK, and ERK pathways, as well as NF-jB, reduced the amount of TTP mRNA by 74, 43, 35, and 21 % in LPS stimulated J774 macrophages, respectively [32]. The differences between their results and ours may be due to their longer LPS induction (6 h) and that the cell line used was J774 macrophages, instead of RAW264.7 macrophages. ...
Lipopolysaccharide (LPS) treatment causes the marked changes of gene expression in macrophages. Tristetraprolin (TTP), which is an mRNA-destabilizing protein that negatively regulates the expression of pro-inflammatory mediators, is induced by LPS. To delineate the molecular mechanism of LPS-stimulated TTP expression, several inhibitors blocking different signaling pathways were used initially. We observed that inhibitors of the NF-κB signaling pathway could down-regulate the TTP expression during LPS-induction. Consistently, TTP expression was increased upon recombinant TNFα stimulation which activates NF-κB signaling. The 5' regulatory region of zfp36 gene spanning from -2 k to +50 was isolated, which contained a putative NF-κB-binding site located in -1859 to -1850. Analysis of luciferase reporter activity driven by a serial 5'-deletion of TTP promoter showed that NF-κB inhibitor-mediated suppression of LPS or TNFα induced activity was through the predicted κB-binding sites. When the NF-κB-binding site was mutated, the TTP promoter decreased its response to the ectopic expression of NF-κB. Physical interaction analysis including oligonucleotides competition, gel shift and chromatin immunoprecipitation assays demonstrated that NF-κB activated by LPS or TNFα bound to the TTP promoter specifically. These results suggested that during LPS stimulation, NF-κB signaling were activated to regulate the transcription of TTP mRNA.
... In line with these results and in additional support of the potential relevance of this mechanism beyond the model we tested and in human biology, Smoak and Cidlowski report the partial loss of dexamethasone-induced downregulation of TNFα in human airway epithelial cell line A549 where TTP was suppressed by stable transfection of a TTP shRNA (35). Although induction of TTP after in vivo administration of GC has been documented in several mouse tissues (35), in vitro treatment of mouse macrophages with GC is reported to inhibit LPS-induced TTP expression (67), suggesting tissue-specificity of TTP regulation possibly linked to the protective effect of GC treatment on innate immune responses. ...
Glucocorticoids (GCs) are the mainstay of anti-inflammatory therapy. Modulation of posttranscriptional regulation (PTR) of gene expression by GCs is a relevant yet poorly characterized mechanism of their action. The RNA-binding protein tristetraprolin (TTP) plays a central role in PTR by binding to AU-rich elements in the 3'-untranslated region of proinflammatory transcripts and accelerating their decay. We found that GCs induce TTP expression in primary and immortalized human bronchial epithelial cells. To investigate the importance of PTR and the role of TTP in GC function, we compared the effect of GC treatment on genome-wide gene expression using mouse embryonic fibroblasts (MEFs) obtained from wild-type and TTP(-/-) mice. We confirmed that GCs induce TTP in MEFs and observed in TTP(-/-) MEFs a striking loss of up to 85% of GC-mediated gene expression. Gene regulation by TNF-alpha was similarly affected, as was the antagonistic effect of GC on TNF-alpha-induced response. Inflammatory genes, including cytokines and chemokines, were among the genes whose sensitivity to GCs was affected by lack of TTP. Silencing of TTP in WT MEFs by small interfering RNA confirmed loss of GC response in selected targets. Immunoprecipitation of ribonucleoprotein complexes revealed binding of TTP to several validated transcripts. Changes in the rate of transcript degradation studied by actinomycin D were documented for only a subset of transcripts bound to TTP. These results reveal a strong and previously unrecognized contribution of PTR to the anti-inflammatory action of GCs and point at TTP as a key factor mediating this process through a complex mechanism of action.
... Corticosteroids may have inhibitory effects on the proteins that stabilise mRNA, leading to more rapid breakdown and thus a reduction in inflammatory protein expression (Newton et al., 1998b;Bergmann et al., 2000;Newton et al., 2001). Corticosteroids do not appear to have any effect on HuR or TTP expression; however, (Bergmann et al., 2004), although a recent report indicate that corticosteroids may suppress TTP gene expression through a nongenomic mechanism, potentially destabilising certain inflammatory gene mRNAs (Jalonen et al., 2005). ...
Corticosteroids are the most effective anti-inflammatory therapy for many chronic inflammatory diseases, such as asthma but are relatively ineffective in other diseases such as chronic obstructive pulmonary disease (COPD). Chronic inflammation is characterised by the increased expression of multiple inflammatory genes that are regulated by proinflammatory transcription factors, such as nuclear factor-kappaB and activator protein-1, that bind to and activate coactivator molecules, which then acetylate core histones to switch on gene transcription. Corticosteroids suppress the multiple inflammatory genes that are activated in chronic inflammatory diseases, such as asthma, mainly by reversing histone acetylation of activated inflammatory genes through binding of liganded glucocorticoid receptors (GR) to coactivators and recruitment of histone deacetylase-2 (HDAC2) to the activated transcription complex. At higher concentrations of corticosteroids GR homodimers also interact with DNA recognition sites to active transcription of anti-inflammatory genes and to inhibit transcription of several genes linked to corticosteroid side effects. In patients with COPD and severe asthma and in asthmatic patients who smoke HDAC2 is markedly reduced in activity and expression as a result of oxidative/nitrative stress so that inflammation becomes resistant to the anti-inflammatory actions of corticosteroids. Theophylline, by activating HDAC, may reverse this corticosteroid resistance. This research may lead to the development of novel anti-inflammatory approaches to manage severe inflammatory diseases.
British Journal of Pharmacology (2006) 148, 245–254. doi:10.1038/sj.bjp.0706736
... In the present study, we report that RU24858 was equipotent with classical glucocorticoids at inducing transactivation but was less potent at inducing transrepression. We have recently reported that RU24858 inhibits the expression of tristetraprolin at the level of mRNA and protein expression in J774 mouse macrophages at concentrations used in the present study.[35] In addition, we have shown that RU24858 inhibits LPS-induced inducible nitric oxide (iNOS) expression and nitric oxide production in mouse J774 macrophages and the effect of RU24858 was qualitatively and quantitatively similar to that of dexamethasone at similar drug concentrations.[36] ...
Glucocorticoids are used to treat chronic inflammatory diseases such as asthma. Induction of eosinophil apoptosis is considered to be one of the main mechanisms behind the anti-asthmatic effect of glucocorticoids. Glucocorticoid binding to its receptor (GR) can have a dual effect on gene transcription. Activated GR can activate transcription (transactivation), or by interacting with other transcription factors such as NF-kappaB suppress transcription (transrepression). RU24858 has been reported to transrepress but to have little or no transactivation capability in other cell types. The dissociated properties of RU24858 have not been previously studied in non-malignant human cells. As the eosinophils have a very short lifetime and many of the modern molecular biological methods cannot be used, a "dissociated steroid" would be a valuable tool to evaluate the mechanism of action of glucocorticoids in human eosinophils. The aim of this study was to elucidate the ability of RU24858 to activate and repress gene expression in human eosinophils in order to see whether it is a dissociated steroid in human eosinophils.
Human peripheral blood eosinophils were isolated under sterile conditions and cultured in the presence and/or absence RU24858. For comparison, dexamethasone and mometasone were used. We measured chemokine receptor-4 (CXCR4) and Annexin 1 expression by flow cytometry and cytokine production by ELISA. Apoptosis was measured by DNA fragmentation and confirmed by morphological analysis.
RU24858 (1 microM) increased CXCR4 and Annexin 1 expression on eosinophils to a similar extent as mometasone (1 microM) and dexamethasone (1 microM). Like dexamethasone and mometasone, RU24858 did suppress IL-8 and MCP-1 production in eosinophils. RU24858 also increased spontaneous eosinophil apoptosis to a similar degree as dexamethasone and mometasone, but unlike dexamethasone and mometasone it did not reverse IL-5- or GM-CSF-induced eosinophil survival.
Our results suggest that in human eosinophils RU24858 acts as transactivator and transrepressor like classical glucocorticoids. Thus, RU24858 seems not to be a "dissociated steroid" in primary human eosinophils in contrast to that reported in animal cells. In addition, functionally RU24858 seems to be a less potent glucocorticoid as it did not reverse IL-5- and GM-CSF-afforded eosinophil survival similarly to dexamethasone and mometasone.
... In this regard, TTP promoter reporters (30), generously provided by Yan Chen (Indiana University School of Medicine and the Walther Cancer Institute, Indianapolis, Ind.), were unresponsive to dexamethasone treatment (data not shown), suggesting either that the native conformation of the gene within chromatin is critical for this regulation or that the 3 UTR binding site is also necessary. Supporting this hypothesis, a recent report investigating dexamethasone inhibition of LPS-induced murine TTP suggested that histone deacetylation is critical for negative regulation of TTP through transcriptional silencing (18). ...
Glucocorticoids are used to treat various inflammatory disorders, but the mechanisms underlying these actions are incompletely understood. The zinc finger protein tristetraprolin (TTP) destabilizes several proinflammatory cytokine mRNAs by binding to AU-rich elements within their 3' untranslated regions, targeting them for degradation. Here we report that glucocorticoids induce the synthesis of TTP mRNA and protein in A549 lung epithelial cells and in rat tissues. Dexamethasone treatment leads to a sustained induction of TTP mRNA expression that is abrogated by RU486. Glucocorticoid induction of TTP mRNA is also blocked by actinomycin D but not by cycloheximide, suggesting a transcriptional mechanism which has been confirmed by transcription run-on experiments. The most widely characterized TTP-regulated gene is the AU-rich tumor necrosis factor alpha (TNF-alpha) gene. Dexamethasone represses TNF-alpha mRNA in A549 cells and decreases luciferase expression of a TNF-alpha 3' untranslated region reporter plasmid in an orientation-dependent manner. Small interfering RNAs to TTP significantly prevent this effect, and a cell line stably expressing a short-hairpin RNA to TTP conclusively establishes that TTP is critical for dexamethasone inhibition of TNF-alpha mRNA expression. These studies provide the molecular evidence for glucocorticoid regulation of human TTP and reflect a novel inductive anti-inflammatory signaling pathway for glucocorticoids that acts via posttranscriptional mechanisms.
... Indeed, glucocorticoids can induce the transcription of TTP in the A549 lung epithelial cell line; small interfering RNA to TTP prevented dexamethasone-induced reduction of TNF-␣ mRNA stability and activity of reporter fused with TNF-␣ 3ЈUTR [91]. On the contrary, this action may be tissuetype-specific, as dexamethasone may inhibit rather than induce TTP in activated macrophages [92]. ...
There have been recent, significant advances about the role of mRNA turnover in controlling gene expression in immune cells. Post-transcriptional regulation of gene expression contributes to the characteristics of many of the processes underlying the immune response by ensuring early, rapid, and transient action. The emphasis of this review is on current work that deals with the regulation of mRNA decay during innate immunity against microbes and T cell activation as a model of the adaptive response.
... The induction of TTP may contribute to the post-transcriptional inhibitory effects of GCs described above. However, it should be noted that dex inhibited the LPS-induced expression of TTP mRNA and protein in macrophages (Jalonen et al., 2005), and had no effect on PMA/PHA-induced TTP protein expression in human peripheral blood mononuclear cells (Bergmann et al., 2004). The control of TTP expression by pro-inflammatory stimuli and GCs needs further investigation. ...
There is a broad consensus that glucocorticoids (GCs) exert anti-inflammatory effects largely by inhibiting the function of nuclear factor kappaB (NFkappaB) and consequently the transcription of pro-inflammatory genes. In contrast, side effects are thought to be largely dependent on GC-induced gene expression. Biochemical and genetic evidence suggests that the positive and negative effects of GCs on transcription can be uncoupled from one another. Hence, novel GC-related drugs that mediate inhibition of NFkappaB but do not activate gene expression are predicted to retain therapeutic effects but cause fewer or less severe side effects. Here, we critically re-examine the evidence in favor of the consensus, binary model of GC action and discuss conflicting evidence, which suggests that anti-inflammatory actions of GCs depend on the induction of anti-inflammatory mediators. We propose an alternative model, in which GCs exert anti-inflammatory effects at both transcriptional and post-transcriptional levels, both by activating and inhibiting expression of target genes. The implications of such a model in the search for safer anti-inflammatory drugs are discussed.
... Indeed AU-rich elements (AREs), in the 3Ј untranslated regions of unstable mRNAs can mediate both signal-induced mRNA stabilization and destablization by glucocorticoids (Peppel et al., 1991;Lasa et al., 2001;Fan et al., 2005). Furthermore, the ARE binding protein tristetraprolin, which promotes mRNA deadenylation and destabilization (Lai et al., 1999), may be induced by dexamethasone to post-transcriptionally repress inflammatory gene expression (Smoak and Cidlowski, 2006) (but see Jalonen et al., 2005). ...
Glucocorticoids (corticosteroids) are highly effective in combating inflammation in the context of a variety of diseases. However, clinical utility can be compromised by the development of side effects, many of which are attributed to the ability of the glucocorticoid receptor (GR) to induce the transcription of, or transactivate, certain genes. By contrast, the anti-inflammatory effects of glucocorticoids are due largely to their ability to reduce the expression of pro-inflammatory genes. This effect has been predominantly attributed to the repression of key inflammatory transcription factors, including AP-1 and NF-kappaB, and is termed transrepression. The ability to functionally separate these transcriptional functions of GR has prompted a search for dissociated GR ligands that can differentially induce transrepression but not transactivation. In this review, we present evidence that post-transcriptional mechanisms of action are highly important to the anti-inflammatory actions of glucocorticoids. Furthermore, we present the case that mechanistically distinct forms of glucocorticoid-inducible gene expression are critical to the development of anti-inflammatory effects by repressing inflammatory signaling pathways and inflammatory gene expression at multiple levels. Considerable care is therefore required to avoid loss of anti-inflammatory effectiveness in the development of novel transactivation-defective ligands of GR.
Inflammatory signals induce complex feedback and feedforward systems that allow tight temporal control. Whereas glucocorticoids generally repress inflammatory gene expression, GR recruitment increases expression of negative feedback and feedforward regulators, including DUSP1, IRAK3, NFKBIA, TNFAIP3 or ZFP36. Moreover, GR cooperativity with factors, such as NF-κB, can enhance regulator expression to promote repression. Conversely, MAPKs, which are inhibited by glucocorticoids, limit expression of the transcription factor IRF1, and the host defense gene, CXCL10, through a feedforward control mechanism. We propose that the balance of feedback and feedforward control determines repression or resistance of inflammatory genes in response to glucocorticoid.
Glucocorticoids (GCs) are corticosteroid hormones critically involved in the homeostatic control of stress and immune responses. Endogenous GCs generally tend to support the innate immune responses while inhibiting overexpressed adaptive immune responses. Synthetic GCs, given systemically or topically, are the first-line anti-inflammatory drug class prescribed in the treatment of the majority of chronic inflammatory and immune-mediated diseases. Regulation of gene transcription through the ligand-activated glucocorticoid receptor (GR) is the core mechanism of their anti-inflammatory action; however their regulatory function spans from early signaling—through non-genomic actions—to downstream post-translational modifications. The anti-inflammatory action of GCs is achieved also through post-transcriptional regulation (PTR) of gene expression, which modulates the rates of mRNA transport, decay and translation. The molecular mechanisms by which GC act on PTR are still ill-defined but studies aimed at their discovery have been increasing in the last decade, subsequent to definitive experimental evidences of the important pathogenic role of PTR in inflammatory responses. In this chapter we will review the growing data indicating that control of deregulated post-transcriptional pathways is a critical, yet underappreciated component of GCs’ efficient anti-inflammatory action. A more detailed understanding of the molecular species and pathways by which GC regulate inflammation by post-transcriptional mechanisms may lead to novel anti-inflammatory strategies that could be pursued as a GC-independent therapeutic approach.
Tristetraprolin (TTP) is an acute phase protein, and its expression is rapidly up-regulated by inflammatory signals, such as lipopolysaccharide (LPS) and cytokines. TTP regulates gene expression by governing the mRNA stability of its target genes, which include cytokines and growth factors. MAP kinase phosphatase-1 (MKP-1) is a nuclear phosphatase that inhibits p38 mitogen-activated protein kinase (MAPK) signaling. This study investigated the role of MKP-1 in TTP expression in A549 human lung epithelial cells, THP-1 human macrophages, J774 mouse macrophages, and primary mouse macrophages. TTP and MKP-1 expression was increased by cytokines or LPS. Silencing of MKP-1 by siRNA enhanced TTP expression in response to LPS, and LPS-induced TTP expression was increased in macrophages from MKP-1 (-/-) mice as compared with that in macrophages from wild-type animals. The inhibition of p38 MAPK by SB202190 reduced TTP expression. In conclusion, MKP-1 suppressed TTP expression by inhibiting p38 MAPK pathway.
Since the discovery of the therapeutic utility of glucocorticoids in the 1950's they have been the mainstay of treatment for chronic inflammatory diseases. However, their debilitating side effects have tempered their use. Both the beneficial and detrimental effects of glucocorticoids are mediated through the same receptor (glucocorticoid receptor, GR). The improved understanding of the molecular mechanisms of GR action along with the description of the GR-ligand crystal structure has enabled significant progress to be made toward minimizing side effects through the use of increasingly selective molecules. These discoveries are likely to generate significant new opportunities for the enhancement of the therapeutic index by dissociating the ability of GR to mediate its beneficial effects from its detrimental side-effects.
Thrombomodulin (TM) is expressed on the surface of monocytes and is a key regulator of actual immune capacity. Propofol is an anesthetic agent that exerts anti-inflammatory effects. The objective of this study was to determine whether propofol could modulate TM in TNF-α-stimulated monocytes. THP-1 cells and male New Zealand rabbits were used in this study. The results showed that TNF-α decreases the TM expression by mediating posttranscriptional modification, and this inhibition may be repressed by treatment with propofol. Immunofluorescence, immunoprecipitation, and pull-down assays were used to demonstrate that Rac1-dependent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, Cdc42, and p38 mitogen-activated protein kinase activation, as well as tristetraprolin (TTP) expression, all contributed to the downregulation of TM in TNF-α-treated cells. Propofol reversed the effects of TNF-α on TM downregulation. Propofol mediated the expression of intracellular TTP and the distribution of cytosolic human antigen R (HuR) and changed their interactions with the 3'-untranslated region of TM mRNA regulating by Cdc42 and Rac1. In addition, the animal study showed that propofol regulates TM, TTP, and HuR expression on monocytes in TNF-α-treated rabbits. In conclusion, the inhibition of TM expression in TNF-α-treated monocytes was mediated by the activation of NADPH oxidase and the expression of TTP. Propofol may inhibit the downregulation of TM by mediating NADPH oxidase and TTP inactivation and through the activation of HuR in vitro and in vivo. Utilizing TTP and HuR to control TM expression may be a promising approach for controlling systemic inflammation, and propofol may possess potential implications for the clinical immunity of monocytes after anesthesia or surgery.
Inhaled glucocorticoids, also know as corticosteroids (ICS), revolutionized the treatment of asthma by suppressing airways inflammation and ICS therapy now forms the basis of treatment of asthma of all severities. More recently and usually in combination with a long-acting β-agonist (LABA), ICS use has been established in the treatment of chronic obstructive pulmonary disease (COPD). In asthma, ICS improves asthma control, lung function and prevents exacerbations, including hospital admissions and probably decreases mortality. Similar effects are seen in COPD but to a much lesser degree, however, an improvement in symptoms such as breathlessness and reduction in exacerbations occur particularly in more advanced disease with ICS. Chronic inflammation is a feature of both asthma and COPD, although there are differences in the site and characteristics of the inflammatory response. ICS have proven to be less effective in patients with severe asthma, smoking asthmatics and in patients with COPD. ICS act by binding to and activating specific cytosolic receptors (GR), which then translocate to the nucleus where they regulate gene expression by either binding to DNA and inducing anti-inflammatory genes or by repressing the induction of pro-inflammatory mediators. GR is able to selective repress specific inflammatory genes by differing actions on specific intracellular signalling pathways and transcription factors such as nuclear factor κB and on kinases pathways. Abnormal activation of these pathways may result in glucocorticoid resistance. Although, ICS/LABA combinations will remain the main focus of treatment of airways diseases in the near future; other combinations that improve the efficacy of ICS by reducing the abnormal activation of pathways that cause glucocorticoid resistance will be developed.
Keratinocyte-derived growth factors and cytokines play an important role in epidermal homeostasis and particularly in cutaneous wound repair. Thus, we analyzed a potential role of the ZFP36/tristetraprolin family of zinc finger proteins, which are targets of these factors, but also regulate their production, in keratinocytes. We show that expression of ZFP36, ZFP36L1, and ZFP36L2 is induced by a broad variety of growth factors and cytokines, and by scratch wounding. Since ZFP36L1 is a modulator of vascular endothelium growth factor (VEGF) mRNA stability, we subsequently used siRNA technology to inhibit ZFP36L1 gene expression. Notably, this treatment resulted in prolonged maintenance of elevated VEGF levels in HaCaT keratinocytes upon epidermal growth factor stimulation of these cells. Taken together, our results suggest an important role of ZFP36L1 in wound healing.
Glucocorticoids are widely used to treat various inflammatory lung diseases. Acting via the glucocorticoid receptor (GR), they exert clinical effects predominantly by modulating gene transcription. This may be to either induce (transactivate) or repress (transrepress) gene transcription. However, certain individuals, including those who smoke, have certain asthma phenotypes, chronic obstructive pulmonary disease (COPD) or some interstitial diseases may respond poorly to the beneficial effects of glucocorticoids. In these cases, high dose, often oral or parental, glucocorticoids are typically prescribed. This generally leads to adverse effects that compromise clinical utility. There is, therefore, a need to enhance the clinical efficacy of glucocorticoids while minimizing adverse effects. In this context, a long-acting beta(2)-adrenoceptor agonist (LABA) can enhance the clinical efficacy of an inhaled corticosteroid (ICS) in asthma and COPD. Furthermore, LABAs can augment glucocorticoid-dependent gene expression and this action may account for some of the benefits of LABA/ICS combination therapies when compared to ICS given as a monotherapy. In addition to metabolic genes and other adverse effects that are induced by glucocorticoids, there are many other glucocorticoid-inducible genes that have significant anti-inflammatory potential. We therefore advocate a move away from the search for ligands of GR that dissociate transactivation from transrepression. Instead, we submit that ligands should be functionally screened by virtue of their ability to induce or repress biologically-relevant genes in target tissues. In this review, we discuss pharmacological methods by which selective GR modulators and "add-on" therapies may be exploited to improve the clinical efficacy of glucocorticoids while reducing potential adverse effects.
Toll-like receptor 4 (TLR4) plays a major role in regulating the innate immune response, which is related to postoperative complications. Although inflammatory capacity and TNF-alpha synthesis were altered on monocytes after cardiopulmonary bypass (CPB), whether the CPB and the CPB-induced TNF-alpha affect TLR4 expression on monocytes have not yet clarified. We speculate that the changing of TNF-alpha level during CPB may be involved in monocytic TLR4 expression. As previous report, our enzyme-linked immunosorbent assay showed that CPB elevated the plasma level of TNF-alpha, whereas off-pump cardiac surgery does not. Flow cytometry reported decreased levels of monocytic TLR4 in patients undergoing CPB but not undergoing off-pump cardiac surgery. To elucidate whether the CPB-induced TNF-alpha is related to TLR4 down-regulation, we used human monocytic THP-1 cells. Actinomycin D chase experiments demonstrated that TNF-alpha decreased TLR4 expression and TLR4 mRNA stability on THP-1. Confocal microscopy and real-time polymerase chain reaction showed that TNF-alpha induced intracellular tristetraprolin (TTP) expression. Transfection with TTP siRNA reversed the down-regulation of TLR4 in TNF-alpha-stimulated THP-1. Treatment with ERK1/2 inhibitor and SAPK/JNK inhibitor decreased TNF-alpha-induced TTP expression. Immunoprecipitation and Western blot analysis showed that the TNF-alpha-mediated activation of TTP might be inhibited by p38 mitogen-activated protein kinase inhibitor and by PD98059. We also demonstrated in clinical samples with confocal microscopy and flow cytometry that CPB led to an elevation of TTP in monocytes. In conclusion, CPB and TNF-alpha decrease TLR4 expression on monocytes; TTP expression and mitogen-activated protein kinase-signaling pathways play critical roles in CPB- and TNF-alpha-mediated decreases of TLR4 on monocytes. Our results suggest that using TTP to control cytokine message decay rate may be a promising approach for controlling system inflammation and preventing post-CPB complications.
Glucocorticoids down regulate the inducible nitric oxide synthase (iNOS) gene both transcriptionally and post-transcriptionally. The post-transcriptional events are suggested to involve destabilization of the iNOS transcript although the molecular mechanisms for this effect are not known. Recently, our laboratory demonstrated a lipopolysaccharide (LPS)/interferon-gamma (IFNgamma)-induction-dependent interaction of heterogeneous nuclear ribonucleoprotein (hnRNP) I and hnRNPL with a destabilizing element contained in the 3'untranslated region (UTR) of iNOS mRNA. The aim of this study was to investigate if dexamethasone, which down regulates iNOS, is able to modulate this protein-mRNA interaction. As expected, dexamethasone inhibited the induction of iNOS by LPS and IFNgamma in RAW 264.7 cells, and destabilized the iNOS mRNA. Dexamethasone also counteracted the LPS/IFNgamma-induced disappearance of a gel shifted iNOS mRNA-protein complex containing hnRNPI and hnRNPL. UV cross-linking and Western blot analyses revealed that the RNA-binding and levels of hnRNPI, which decreased by LPS/IFNgamma treatment, were restored by dexamethasone. The results support our hypothesis that hnRNPI is pivotal in the post-transcriptional regulation of iNOS and strongly suggest that hnRNPI is one of the trans-acting factors mediating the post-transcriptional effects of dexamethasone.
We have previously shown that the E2A-encoded transcription factor E47, which regulates class switch in splenic B cells, is down-regulated in old B cells, due to increased E47 mRNA decay. At least part of the decreased stability of E47 mRNA seen in aged B cells is mediated by proteins. We have herein looked at the specific proteins responsible for the degradation of the E47 mRNA and found that tristetraprolin (TTP), a physiological regulator of mRNA expression and stability, is involved in the degradation of the E47 mRNA. Although many studies have characterized TTP expression and function in macrophages, monocytes, mast cells, and T cells, little is known about the expression and function of TTP in primary B cells. We show herein that TTP mRNA and protein expression are induced by LPS in B cells from young and old mice, the levels of TTP in old B cells always being higher than those in young B cells. Although TTP mRNA is degraded at a significantly higher rate in old B cells, TTP mRNA expression is higher in old than in young, likely due to its increased transcription. Like in macrophages, TTP protein expression and function in B cells are dependent upon p38 MAPK. We found that there is less phospho-TTP (inactive form), as well as phospho-p38, in old than in young splenic-activated B cells. This is the first report showing that TTP is involved in the degradation of the E47 mRNA and is up-regulated in old B cells.
In inflammation, nitric oxide (NO) is produced by inducible nitric oxide synthase (iNOS) induced by bacterial products and cytokines, and NO acts as a regulatory and pro-inflammatory mediator. Glucocorticoids are powerful anti-inflammatory agents that inhibit the expression of iNOS and various other inflammatory factors. Histone deacetylation has been recently described as a novel mechanism how glucocorticoids down-regulate transcriptional activation of some inflammatory genes. The aim of the present study was to investigate the effects of inhibitors of histone deacetylation on the suppressive effects of glucocorticoids on NO production and iNOS expression. Dexamethasone and a dissociated glucocorticoid RU24858 inhibited NO production, and iNOS protein and mRNA expression in macrophages exposed to bacterial lipopolysaccharide (LPS). In the presence of a glucocorticoid receptor (GR) antagonist mifepristone, dexamethasone and RU24858 had no effect on NO production. The role of histone deacetylation in the glucocorticoid effect was studied by using three structurally different inhibitors of histone deacetylases (HDACs): trichostatin A, apicidin and MC1293. HDAC inhibitors reversed the effects of dexamethasone and RU24858 on iNOS expression and NO production. Stably transfected A549/8 cells containing luciferase gene under the control of human iNOS promoter were used in promoter-activity studies. iNOS promoter activity induced by IL-1beta was inhibited by dexamethasone and the inhibitory effect was reversed by HDAC inhibitor trichostatin A. The results suggest that glucocorticoids inhibit iNOS expression and NO production by a GR-mediated and GRE-independent manner through histone deacetylation and transcriptional silencing.
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.
More and more evidence reveals that the transcription factor NF-kappaB plays a critical role in tumor development and progression and that it may constitute the missing link between inflammation and cancer. It turned out that many of the well known cancer drugs exert their anti-tumoral effect at least in part through modulating the activity of NF-kappaB. The potential of nuclear receptors to modulate the activity of this widespread transcription factor has repeatedly been reported and illustrates their enormous therapeutic potential. However, the efficacy of these liganded receptors is overshadowed by the occurrence of unwanted effects owing to their broad range of actions. Accordingly, researchers pursue the ambition to improve the specificity of nuclear receptor modulators. In this review we have explored the molecular mechanisms by which nuclear receptors interfere with NF-kappaB signalling and quoted the therapeutic implications of their cross-coupling. Strategies that are explored at the moment and that may hold great potential for the future are extensively reviewed.
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.
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.
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.
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.
PD 098059 has been shown previously to inhibit the dephosphorylated form of mitogen-activated protein kinase kinase-1 (MAPKK1)
and a mutant MAPKK1(S217E,S221E), which has low levels of constitutive activity (Dudley, D. T., Pang, L., Decker, S. J., Bridges,
A. J., and Saltiel, A. R.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7686-7689). Here we report that PD 098059 does not inhibit Raf-activated MAPKK1 but that it prevents the activation of
MAPKK1 by Raf or MEK kinase in vitro at concentrations (IC = 2-7 μM) similar to those concentrations that inhibit dephosphorylated MAPKK1 or MAPKK1(S217E,S221E). PD 098059 inhibited
the activation of MAPKK2 by Raf with a much higher IC value (50 μM) and did not inhibit the phosphorylation of other Raf or MEK kinase substrates, indicating that it exerts its
effect by binding to the inactive form of MAPKK1. PD 098059 also acts as a specific inhibitor of the activation of MAPKK in
Swiss 3T3 cells, suppressing by 80-90% its activation by a variety of agonists. The high degree of specificity of PD 098059
in vitro and in vivo is indicated by its failure to inhibit 18 protein Ser/Thr kinases (including two other MAPKK homologues) in vitro by its failure to inhibit the in vivo activation of MAPKK and MAP kinase homologues that participate in stress and interleukin-1-stimulated kinase cascades in
KB and PC12 cells, and by lack of inhibition of the activation of p70 S6 kinase by insulin or epidermal growth factor in Swiss
3T3 cells. PD 098059 (50 μM) inhibited the activation of p42 and isoforms of MAP kinase-activated protein kinase-1 in Swiss 3T3 cells, but the extent of inhibition depended on how potently
c-Raf and MAPKK were activated by any particular agonist and demonstrated the enormous amplification potential of this kinase
cascade. PD 098059 not only failed to inhibit the activation of Raf by platelet-derived growth factor, serum, insulin, and
phorbol esters in Swiss 3T3 cells but actually enhanced Raf activity. The rate of activation of Raf by platelet-derived growth
factor was increased 3-fold, and the subsequent inactivation that occurred after 10 min was prevented. These results indicate
that the activation of Raf is suppressed and that its inactivation is accelerated by a downstream component(s) of the MAP
kinase pathway.
Production of interleukin-1 and tumour necrosis factor from stimulated
human monocytes is inhibited by a new series of pyridinyl-imidazole
compounds. Using radiolabelled and radio-photoaffinity-labelled chemical
probes, the target of these compounds was identified as a pair of
closely related mitogen-activated protein kinase homologues, termed
CSBPs. Binding of the pyridinyl-imidazole compounds inhibited CSBP
kinase activity and could be directly correlated with their ability to
inhibit cytokine production, suggesting that the CSBPs are critical for
cytokine production.
Growth factors rapidly induce transcription of a set of genes that encode regulatory proteins, many of which have been identified by cDNA cloning. Here we report the analysis of a cDNA corresponding to a gene induced in mouse 3T3 cells by growth factors and a variety of other extracellular signaling agents. The cDNA encodes a proline-, serine-, and glycine-rich nuclear protein designated Nup475 of 319 amino acids that contains two tandemly repeated cysteine- and histidine-containing sequences (CX8CX5CX3H) suggestive of a novel heavy metal-binding domain. Nup475 produced in Escherichia coli binds zinc. Its mRNA is present in a number of mouse tissues and cell lines, being especially abundant in intestine, thymus, and regenerating liver and in a macrophage cell line stimulated by gamma-interferon. We hypothesize that Nup475 is a regulatory protein with a novel zinc finger structure.
The TIS11 primary response gene is rapidly and transiently induced by both 12-O-tetradecanoylphorbol-13-acetate and growth
factors. The predicted TIS11 protein contains a 6-amino-acid repeat, YKTELC. We cloned two additional cDNAs, TIS11b and TIS11d,
that contain the YKTELC sequence. TIS11, TIS11b, and TIS11d proteins share a 67-amino-acid region of sequence similarity that
includes the YKTELC repeat and two cysteine-histidine containing repeats. TIS11 gene family members are not coordinately expressed:
(i) unlike TIS11, the TIS11b and TIS11d mRNAs are detectable in quiescent Swiss 3T3 cells and are not dramatically induced
by 12-O-tetradecanoylphorbol-13-acetate; (ii) cycloheximide superinduction does not occur for TIS11b and TIS11d; and (iii)
unlike TIS11, TIS11b expression is extinguished in PC12 pheochromocytoma cells.
The mouse tristetraprolin (TTP) protein is a basic proline-nch protein of Mr 33,600 that contains three PPPPG repeats (1). Its mRNA levels increase dramatically after stimulation with various mitogens. Also called Nup 475 (2) and TISH (3), it has been localised to the cell nucleus and is thought to contain an unusual zinc-finger structure (2). A distinct protein (cMG1) with an analogous zinc-finger stn.icture was cloned recently (4). We report here the sequence of human TTP, which is 87% identical to the mouse protein; the putative zinc-finger structure is conserved among human and mouse TTP and rat cMG1 (Figure 1).
The human gene encoding TTP was localized to chromosome 19 using panels of rodent/human hybrid cell lines (5, 6). The mouse gene was localized to chromosome 7 using an interspecific cross; in 114 out of 114 meiotic events, it cosegregated with the D19F11S1h locus, which maps approximately 6.0 cM from the proximal end of mouse chromosome 7 (7). This is in the middle of a linkage group conserved with a segment of human chromosome 19 (7); the mouse mapping suggests that the human gene is at band q13.l on human chromosome 19. This was confirmed by in situ hybridization of the human cDNA probe to metaphase chromosome spreads.
In recognition of its probable zinc-finger structure (2), the gene encoding the mouse TTP protein has been labeled Zfp-36 by the Committee on Standardized Genetic Nomenclature for Mice. The human gene is ZFP36.
By differential hybridization screening of a cDNA library derived from insulin-stimulated cells, we selected a clone which hybridized to an mRNA species that rapidly accumulated in response to insulin. The insert from this clone encoded a putative polypeptide of Mr 33,600, pI 11.2; because the protein was enriched in proline residues (14.4 mol %) and contained three Pro-Pro-Pro-Pro repeats, we have tentatively labeled it tris-tetraprolin (TTP). The function of this protein is not known, but it contains two regions very rich in proline (30-40 mol %); similar proline-rich regions have been shown to be involved in transcriptional activation by other proteins. The mRNA (2.0 kilobases) encoding the TTP protein was essentially undetectable in serum-deprived HIR 3.5 cells, but accumulated dramatically within 10 min of stimulation by insulin. This effect appeared to be due to insulin acting through the intrinsic protein-tyrosine kinase activity of its own receptor. Insulin induction of TTP mRNA accumulation was prevented by actinomycin D and superinduced by cycloheximide. Accumulation of TTP mRNA was also stimulated by a variety of growth factors and active phorbol esters; however, the insulin effect was virtually normal in cells depleted of protein kinase C. A single TTP gene appeared to be present in the mouse genome. This gene joins the group of genes whose members are rapidly transcribed in response to insulin and other mitogens.
Induction of early-response genes (tetradecanoyl phorbol acetate [TPA]-induced sequences, or TIS genes; R.W. Lim, B.C. Varnum,
and H.R. Herschman, Oncogene 1:263-270, 1987) by granulocyte-macrophage colony-stimulating factor (GM-CSF) and TPA was examined
both in a factor-dependent murine cell line, 32D clone 3, and in mature human neutrophils. When GM-CSF-deprived 32D clone
3 cells were exposed to GM-CSF or to TPA, four TIS mRNAs (TIS7, TIS8, TIS10, and TIS11) were rapidly and transiently induced.
However, neither GM-CSF nor TPA could induce accumulation of TIS1 mRNA in 32D clone 3 cells, even under superinducing conditions.
Both GM-CSF and TPA also elicited rapid, transient expression of TIS8 and TIS11 mRNA in postmitotic human neutrophils. However,
neither agent could induce accumulation of TIS1 mRNA in human neutrophils. TIS1 is a member of the nuclear receptor supergene
family that codes for ligand-dependent transcription factors. Cell-type restriction of inducible transcription factors may
contribute to developmental specification.
We have previously identified by cDNA cloning 5 mRNAs that appear in resting BALB/c 3T3 cells soon after growth stimulation by serum or platelet-derived growth factor. Five additional mRNAs of this class are described in this report. The mRNAs reached peak levels between 40 and 120 min after serum addition and rapidly decayed thereafter. All 10 RNAs were superinduced in the presence of cycloheximide. Nuclear run-on experiments indicated that the increase in the mRNAs is the result of rapid transcriptional activation of their genes on stimulation by serum or platelet-derived growth factor. Superinducibility by cycloheximide is due to two effects: prolonged transcription and stabilization of mRNAs. This overall pattern of regulation is similar to that of the c-fos or c-myc protooncogenes reported previously. We hypothesize that these newly identified "immediate early" genes play a role in the proliferative response induced by growth factors.
RU-486 is an anti-fertility steroid which also has anti-glucocorticosteroid effects. RU-486 is shown to be a strong antagonist of the glucocorticosteroid-induced cytolytic response of the murine thymoma lines W7TB and T1M1b , and of the induction of mouse mammary tumor virus (MMTV) mRNA in T1M1b cells. The glucocorticosteroid receptor of W7 cells has high affinity for RU-486 (Kd = 3 X 10(-9) M) but the complex formed has low nuclear transfer capacity. Binding of RU-486, as compared with the glucocorticosteroid agonist triamcinolone acetonide, to mouse receptor results in a decreased affinity for DNA in general and a reduced specific recognition of a site in the promoter region of MMTV proviral DNA. The RU-486 complex formed with rat liver receptor exhibits the same behavior; in addition, it is shown that only a fraction of these complexes are activated by temperature and these form highly salt-sensitive interactions with DNA. These results indicate that the binding of RU-486 to glucocorticosteroid receptors mimics pharmacologically the properties of a class of receptor variants (nt-) which are non-functional and have reduced nuclear transfer and altered DNA binding capacity. These results substantiate the importance of DNA binding in receptor function.
Some of the most potent antiinflammatory and immunosuppressive agents are synthetic glucocorticoids. However, major side effects severely limit their therapeutic use. The development of improved glucocorticoid-based drugs will require the separation of beneficial from deleterious effects. One possibility toward this goal is to try to dissociate two main activities of glucocorticoids, i.e. transactivation and transrepression. Screening of a library of compounds using transactivation and AP-1 transrepression models in transiently transfected cells identified dissociated glucocorticoids, which exert strong AP-1 inhibition but little or no transactivation. Importantly, despite high ligand binding affinity, the prototypic dissociated compound, RU24858, acted as a weak agonist and did not efficiently antagonize dexamethasone-induced transcription in transfected cells. Similar results were obtained in hepatic HTC cells for the transactivation of the endogenous tyrosine amino transferase gene (TAT), which encodes one of the enzymes involved in the glucocorticoid-dependent stimulation of neoglucogenesis. To investigate whether dissociated glucocorticoids retained the antiinflammatory and immunosuppressive potential of classic glucocorticoids, several in vitro and in vivo models were used. Indeed, secretion of the proinflammatory lymphokine interleukin-1beta was severely inhibited by dissociated glucocorticoids in human monocytic THP 1 cells. Moreover, in two in vivo models, these compounds exerted an antiinflammatory and immunosuppressive activity as potent as that of the classic glucocorticoid prednisolone. These results may lead to an improvement of antiinflammatory and immunosuppressive therapies and provide a novel concept for drug discovery.
Zfp-36, the gene encoding the putative zinc finger protein tristetraprolin (TTP), is rapidly induced in fibroblasts by a variety
of growth factors. Recent gene knockout experiments have shown that TTP-deficient mice developed arthritis, cachexia, and
autoimmunity, all apparently mediated by an excess of tumor necrosis factor α. We recently showed that full serum inducibility
of Zfp-36 requires elements in the promoter; in addition, removal of the single intron strikingly inhibited serum-induced TTP expression.
We show here that replacement of the intron with unrelated sequences, or removal of 95% of the intron but retention of the
splice sites, each resulted in the maintenance of approximately 45 and 19%, respectively, of full serum-induced expression.
In addition, deletion of intron sequences base pairs 601–655 decreased the serum-induced expression of TTP by 65%. Sequence
base pairs 618–626 bound specifically to the transcription factor Sp1; mutation of this binding motif decreased TTP expression
by 70%, suggesting that Sp1 binding to this motif contributes to serum induction of Zfp-36. We conclude that full serum-induced expression of Zfp-36 depends on the activation of conventional promoter elements as well as elements in the single intron, and that the presence
per se of the intron in its natural location also contributes significantly to the regulated expression of this gene.
Control of mRNA stability is critical for expression of short-lived transcripts from cytokines and proto-oncogenes. Regulation
involves an AU-rich element (ARE) in the 3′ untranslated region (3′UTR) and cognatetrans-acting factors thought to promote either degradation or stabilization of the mRNA. In this study we present a novel approach
using somatic cell genetics designed to identify regulators of interleukin-3 (IL-3) mRNA turnover. Mutant cell lines were
generated from diploid HT1080 cells transfected with a reporter construct containing green fluorescent protein (GFP) linked
to the IL-3 3′UTR. GFP was expressed at low levels due to rapid decay of the mRNA. Following chemical mutagenesis and selection
of GFP-overexpressing cells, we could isolate three mutant clones (slowA, slowB, and slowC) with a specific, trans-acting defect in IL-3 mRNA degradation, while the stability of IL-2 and tumor necrosis factor alpha reporter transcripts
was not affected. Somatic cell fusion experiments revealed that the mutants are genetically recessive and form two complementation
groups. Expression of the tristetraprolin gene in both groups led to reversion of the mutant phenotype, thereby linking this
gene to the IL-3 mRNA degradation pathway. The genetic approach described here should allow identification of the defective
functions by gene transfer and is also applicable to the study of other mRNA turnover pathways.
Inflammatory diseases such as asthma and rheumatoid arthritis are characterised at the molecular level by chronically increased expression of multiple cytokines, chemokines, kinins and their receptors, adhesion molecules, and inflammatory enzymes such as inducible nitric oxide synthase (iNOS) and the inducible cyclooxygenase (COX-2).1 At the cellular level, inflamed regions show a substantial influx of various inflammatory cells, arterial dilation, increased blood flow, plasma protein leakage, and oedema whilst, in the case of chronic asthma, substantial remodelling of the airways is observed involving excessive smooth muscle proliferation. However, these parameters of inflammation are effectively reduced by treatment with glucocorticoids by both direct and indirect mechanisms.2 3 For example, the reduced eosinophilia following glucocorticoid treatment in asthmatic subjects arises by direct promotion of eosinophil apoptosis and indirectly by suppressing receptor expression and production of cytokines or growth factors.4 These include factors such as interleukin (IL)-3, IL-5, granulocyte-macrophage colony stimulating factor (GM-CSF), and eotaxin which are involved in eosinophil maturation, recruitment, and survival. Similarly, glucocorticoids reduce T cell proliferation and increase T cell apoptosis via mechanisms that are at least partly the result of inhibition of the T cell growth factor, IL-2.5-8 Likewise, monocyte apoptosis is increased and influx of other infiltrating inflammatory cells is also repressed.2 9 Again, this is partly caused by reduced expression of adhesion molecules, both on migrating and target cells, as well as reduced expression of cytokines and chemokines from sites of inflammation.
Therapeutically, the ability to suppress a number of inflammatory indices makes glucocorticoids among the most potent anti-inflammatory agents currently available for the treatment of chronic inflammatory diseases such as asthma.2 3 The clinical efficacy of synthetic glucocorticoids such as prednisolone or dexamethasone stems from their ability to mimic natural glucocorticosteroids. Bodily insults, including inflammation, pain, infection or even mental …
We have investigated the ability of dexamethasone to regulate interleukin-1beta (IL-1beta)-induced gene expression, histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity. Low concentrations of dexamethasone (10(-10) M) repress IL-1beta-stimulated granulocyte-macrophage colony-stimulating factor (GM-CSF) expression and fail to stimulate secretory leukocyte proteinase inhibitor expression. Dexamethasone (10(-7) M) and IL-1beta (1 ng/ml) both stimulated HAT activity but showed a different pattern of histone H4 acetylation. Dexamethasone targeted lysines K5 and K16, whereas IL-1beta targeted K8 and K12. Low concentrations of dexamethasone (10(-10) M), which do not transactivate, repressed IL-1beta-stimulated K8 and K12 acetylation. Using chromatin immunoprecipitation assays, we show that dexamethasone inhibits IL-1beta-enhanced acetylated K8-associated GM-CSF promoter enrichment in a concentration-dependent manner. Neither IL-1beta nor dexamethasone elicited any GM-CSF promoter association at acetylated K5 residues. Furthermore, we show that GR acts both as a direct inhibitor of CREB binding protein (CBP)-associated HAT activity and also by recruiting HDAC2 to the p65-CBP HAT complex. This action does not involve de novo synthesis of HDAC protein or altered expression of CBP or p300/CBP-associated factor. This mechanism for glucocorticoid repression is novel and establishes that inhibition of histone acetylation is an additional level of control of inflammatory gene expression. This further suggests that pharmacological manipulation of of specific histone acetylation status is a potentially useful approach for the treatment of inflammatory diseases.
Signal transduction pathways regulate gene expression in part by modulating the stability of specific mRNAs. For example,
the mitogen-activated protein kinase (MAPK) p38 pathway mediates stabilization of tumor necrosis factor alpha (TNF-α) mRNA
in myeloid cells stimulated with bacterial lipopolysaccharide (LPS). The zinc finger protein tristetraprolin (TTP) is expressed
in response to LPS and regulates the stability of TNF-α mRNA. We show that stimulation of RAW264.7 mouse macrophages with
LPS induces the binding of TTP to the TNF-α 3′ untranslated region. The p38 pathway is required for the induction of TNF-α
RNA-binding activity and for the expression of TTP protein and mRNA. Following stimulation with LPS, TTP is expressed in multiple,
differentially phosphorylated forms. We present evidence that phosphorylation of TTP is mediated by the p38-regulated kinase
MAPKAPK2 (MAPK-activated protein kinase 2). Our findings demonstrate a direct link between a specific signal transduction
pathway and a specific RNA-binding protein, both of which are known to regulate TNF-α gene expression at a posttranscriptional
level.
Jun N-terminal kinase (JNK) is a stress-activated protein kinase that can be induced by inflammatory cytokines, bacterial endotoxin, osmotic shock, UV radiation, and hypoxia. We report the identification of an anthrapyrazolone series with significant inhibition of JNK1, -2, and -3 (K(i) = 0.19 microM). SP600125 is a reversible ATP-competitive inhibitor with >20-fold selectivity vs. a range of kinases and enzymes tested. In cells, SP600125 dose dependently inhibited the phosphorylation of c-Jun, the expression of inflammatory genes COX-2, IL-2, IFN-gamma, TNF-alpha, and prevented the activation and differentiation of primary human CD4 cell cultures. In animal studies, SP600125 blocked (bacterial) lipopolysaccharide-induced expression of tumor necrosis factor-alpha and inhibited anti-CD3-induced apoptosis of CD4(+) CD8(+) thymocytes. Our study supports targeting JNK as an important strategy in inflammatory disease, apoptotic cell death, and cancer.
Tristetraprolin (TTP), the prototype of a small family of CCCH tandem zinc finger (TZF) domain proteins, is a physiological stimulator of instability of the mRNAs encoding tumor necrosis factor-alpha and granulocyte/macrophage colony-stimulating factor in certain cell types. TTP stimulates mRNA turnover after binding to class II AU-rich elements (AREs) within the 3'-untranslated regions of both mRNAs. In turn, this binding is dependent upon the key CCCH residues in the TZF domain. To evaluate other primary sequence requirements for ARE binding in this novel mRNA-binding domain, we mutated many of the conserved residues within the TZF domain of human TTP and evaluated the effects of these mutations on RNA binding in a cell-free system and TTP-induced mRNA instability in cell transfection experiments. These mutations revealed a number of conserved amino acids that were required for binding and begin to define the primary protein sequence requirements for this novel mRNA-binding motif. Unexpectedly, all of the point mutations that prevented TTP binding to RNA also caused an increase in steady-state levels of ARE-containing mRNAs in cell transfection experiments. Actinomycin D experiments suggested that this effect was due to inhibition of mRNA turnover. Although expression of the mutant form of TTP could also inhibit the destruction of tumor necrosis factor-alpha mRNA by wild-type TTP, the primary mechanism did not involve heterodimerization with wild-type TTP because the 293 cells used in these studies express no detectable endogenous TTP. These data suggest that TTP may act, at least in part, by physically interacting with an enzyme activity or protein complex and functionally stimulating its ability to deadenylate class II ARE-containing mRNAs.
Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved enzymes which connect cell-surface receptors to regulatory targets within cells and convert receptor signals into various outputs. In mammalian cells, four distinct MAPKs have been identified: the extracellular signal-related kinases (ERK)-1/2, the c-jun N-terminal kinases or stress-activated protein kinases 1 (JNK1/2/3, or SAPK1s), the p38 MAPKs (p38 alpha/beta/gamma/delta, or SAPK2s), and the ERK5 or big MAP kinase 1 (BMK1). The p38 MAPK cascade is activated by stress or cytokines and leads to phosphorylation of its central elements, the p38 MAPKs. Downstream of p38 MAPKs there is a diversification and extensive branching of signalling pathways. For that reason, we will focus in this review on the different signalling events that are triggered by p38 activity, and analyse how these events contribute to specific gene expression and cellular responses.
In human colorectal adenocarcinoma cell lines, we found two major transcripts of cyclooxygenase-2, the full-length mRNA and a short polyadenylation variant (2577 kb) lacking the distal segment of the 3'-untranslated region. Tristetraprolin, an mRNA-binding protein that promotes message instability, was shown to bind the cyclooxygenase-2 mRNA in the region of the 3'-untranslated region between nucleotides 3125 and 3432 and to reduce levels of the full-length mRNA. During cell growth and confluence, the expression of tristetraprolin mRNA was inversely correlated with that of the full-length cyclooxygenase-2 transcript, and transfection of tristetraprolin into HCA-7 cells reduced the level of full-length cyclooxygenase-2 mRNA. However, the truncated transcript escaped tristetraprolin binding and downregulation.
Tristetraprolin (TTP) is a regulator of TNF-alpha mRNA stability and is the only trans-acting factor shown to be capable of regulating AU-rich element-dependent mRNA turnover at the level of the intact animal. Using the THP-1 myelomonocytic cell line, we demonstrated for the first time that TTP is encoded by an mRNA with a short half-life under resting conditions. Using pharmacologic inhibitors of the mitogen-activated protein kinase pathways, we show that the induction of TTP by LPS activation is mediated through changes in transcription, mRNA stability, and translation. A coordinate increase in both TTP and TNF-alpha mRNA stability occurs within 15 min of LPS treatment, but is transduced through different mitogen-activated protein kinase pathways. This regulation of TTP and TNF-alpha mRNA stability is associated with the finding that TTP binds these mRNA under both resting and LPS-activated conditions in vivo. Finally, we demonstrate that TTP can regulate reporter gene expression in a TTP 3' untranslated region-dependent manner and identify three distinct AU-rich elements necessary to mediate this effect. Thus, TTP regulates its own expression in a manner identical to that seen with the TNF-alpha 3' untranslated region, indicating that this autoregulation is mediated at the level of mRNA stability. In this manner, TTP is able to limit the production of its own proteins as well as that of TNF-alpha and thus limit the response of the cell to LPS.
Tristetraprolin (TTP) is an mRNA-destabilizing protein that negatively regulates the expression of proinflammatory mediators such as tumor necrosis factor alpha, granulocyte/macrophage colony-stimulating factor, and cyclooxygenase 2. Here we investigate the regulation of TTP expression in the mouse macrophage cell line RAW264.7. We show that TTP mRNA is expressed in a biphasic manner following stimulation of cells with lipopolysaccharide and that the second phase of expression, like the first, is dependent on mitogen-activated protein kinase (MAPK) p38. MAPK p38 acts through a downstream kinase to stabilize TTP mRNA, and this stabilization is mediated by an adenosine/uridine-rich region at the 3'-end of the TTP 3'-untranslated region. Hence TTP is post-transcriptionally regulated in a similar manner to several proinflammatory genes. We also demonstrate that TTP is able to bind to its own 3'-untranslated region and negatively regulate its own expression, forming a feedback loop to limit expression levels.
G0S24 is a member of a set of genes (putative G0/G1 switch regulatory genes) that are expressed transiently within 1-2 hr of the addition of lectin or cycloheximide to human blood mononuclear cells. Comparison of a full-length cDNA sequence with the corresponding genomic sequence reveals an open reading frame of 326 amino acids, distributed across two exons. Potential phosphorylation sites include the sequence PSPTSPT, which resembles an RNA polymerase II repeat reported to be a target of the cell cycle control kinase cdc2. Comparison of the derived protein sequence with those of rodent homologs allows classification into three groups. Group 1 contains G0S24 and the rat and mouse TIS11 genes (also known as TTP, Nup475, and Zfp36). Members of this group have three tetraproline repeats. Groups 1 and 2 have a serine-rich region and an ''arginine element'' (RRLPIF) at the carboxyl terminus. All groups contain cysteine- and histidine-rich putative zinc finger domains and a serine-phenylalanine ''SFS'' domain similar to part of the large subunit of eukaryotic RNA polymerase II. Comparison of group 1 human and mouse genomic sequences shows high conservation in the 5' flank and exons. A CpG island suggests expression in the germ line. G0S24 has potential sites for transcription factors in the 5' flank and intron; these include a serum response element. Protein and genomic sequences show similarities with those of a variety of proteins involved in transcription, suggesting that the G0S24 product has a similar role.
Many primary response genes, including cyclooxygenase-2 (COX-2), exhibit mRNA superinduction following agonist stimulation in the presence of translational blockers such as cycloheximide. This is widely assumed to result from mRNA stabilisation. However, superinduction of IL-1β-induced COX-2 mRNA levels by cycloheximide in pulmonary type II A549 cells occurred by increased transcription and not by mRNA stabilisation. Furthermore, equivalent effects were observed on NF-κB binding to COX-2 promoter κB sites and activation of the Jun N-terminal kinases (JNK), p54 and p46. These signalling pathways play important roles in COX-2 induction and may therefore account for the observed increases in COX-2 transcription. These data are consistent with negative feed-back involving down-regulation of NF-κB by de novo IκBα synthesis and suggest that JNK activation may also be down-regulated by a cycloheximide sensitive process.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
We previously reported the sequence of a cDNA, TIS11, cloned from TPA-treated Swiss 3T3 cells. Two laboratories have reported sequences for cDNAs that have a region of identity with the TIS11 cDNA, but differ in their 5' and 3' flanking regions. We now report that the original TIS11 cDNA is likely to be a chimeric molecule, and find that the TIS11 gene encodes a protein identical to the TTP and Nup475 genes.
Accumulation of TIS1 and TIS11 (Lim et al.: Oncogene 1:263–270, 1987) mRNAs in secondary cultures of rat neocortical astrocytes was much greater in response to tetradecanoyl phorbol acetate (TPA) than in response to either epidermal growth factor (EGF) or fibroblast growth factor (FGF). In contrast, EGF, FGF, and TPA were equally effective in inducing accumulation of TIS8 and TIS28/c‐fos mRNAs. These data suggested that TPA and the polypeptide mitogens might induce TIS gene expression by distinct pathways. When maximally inducing concentrations of EGF and FGF were co‐administered to astrocyte cultures, TIS mRNA accumulations were no greater than those observed for the individual growth factors, suggesting that EGF and FGF saturate a common, limiting step in their induction pathways. In contrast, when either EGF or FGF was presented to astrocytes in combination with maximally inducing levels of TPA, the resulting levels of accumulation of TIS mRNAs were at least as great as the sum of the levels induced by the individual mitogens. Stimulation of [ ³ H]‐thymidine incorporation demonstrated an identical pattern of interaction; EGF and FGF co‐administration was no more effective than either polypeptide mitogen alone, but, when presented to astrocyte cultures along with maximally inducing concentrations of TPA, either EGF or FGF was able to increase incorporation of [ ³ H]‐thymidine.
Superinduction of all the TIS genes occurred if cycloheximide (CHX) was present during TPA exposure. Once again, two distinet classes of responses of the various TIS genes occurred; superinduction of TIS1, TIS7, TIS11, and TIS28/c‐fos mRNA accumulation ranged from 10‐ to 20‐fold, while CHX superinduction of TIS8 and TIS10 was far more modest, ranging from 2‐ to 3‐fold. Differential superinduction of TIS gene expression also occurred when astrocytes were challenged with TPA in the presence of the peripherally active benzodiazepine (BZD) Ro5‐4864, ranging from a high of fivefold (TIS28/c‐fos) to no superinduction effect (TIS8). The temporal patterns and relative efficacies of BZD and CHX superinduction of the various TIS genes were quite distinct.
Induction of TIS gene accumulation occurs in response to a wide variety of ligands by several independent pathways and can be secondarily modulated by a number of additional factors. The differential expression of this set of transient primary‐response gene products may play a major role in integrating information that impinges on cells and in directing their physiological responses.
The biological potencies of four antiglucocorticoids, RU486 (RU), dexamethasone-oxetanone (DOX), R5020, and progesterone have been studied with respect to dexamethasone induction of tyrosine aminotransferase (TAT) in rat hepatoma tissue culture (HTC) cells. Their inhibitory effects in whole-cell competition binding studies (at 37 degrees C) and in TAT induction studies were analyzed by Dixon plots and Schild plots, respectively. We show that: In both cases, there is an actual competition of each antiglucocorticoid with the agonist dexamethasone for the same binding site; the two Kd values derived from the two plots are almost identical for each antiglucocorticoid; RU486 can be distinguished from the three other antiglucocorticoids by its high biological efficacy and its high affinity for the glucocorticoid receptor in whole cells at 37 degrees C (identical to its affinity in cytosol at 0 degree C). These results imply that: There is a linear correlation between the antagonist efficacies of antiglucocorticoids and their affinities for the glucocorticoid receptor in whole cells at 37 degrees C; the antagonistic action is solely mediated by competition with the agonist for the receptor binding site; this is verified by the fact that in all cases, in the presence or absence of antiglucocorticoids, a specific TAT induction level was always related to the same level of receptor saturation by the agonist in whole cells; the phenomena responsible for the high antagonist efficacy of RU486 are also responsible for its high affinity in whole cells at 37 degrees C.
We report here the nucleic acid sequence and deduced amino acid sequence of a cDNA for TIS11, a gene induced in 3T3 cells by tetradecanoyl phorbol acetate.
PD 098059 has been shown previously to inhibit the dephosphorylated form of mitogen-activated protein kinase kinase-1 (MAPKK1) and a mutant MAPKK1(S217E,S221E), which has low levels of constitutive activity (Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 7686-7689). Here we report that PD 098059 does not inhibit Raf-activated MAPKK1 but that it prevents the activation of MAPKK1 by Raf or MEK kinase in vitro at concentrations (IC50 = 2-7 microM) similar to those concentrations that inhibit dephosphorylated MAPKK1 or MAPKK1(S217E,S221E). PD 098059 inhibited the activation of MAPKK2 by Raf with a much higher IC50 value (50 microM) and did not inhibit the phosphorylation of other Raf or MEK kinase substrates, indicating that it exerts its effect by binding to the inactive form of MAPKK1. PD 098059 also acts as a specific inhibitor of the activation of MAPKK in Swiss 3T3 cells, suppressing by 80-90% its activation by a variety of agonists. The high degree of specificity of PD 098059 in vitro and in vivo is indicated by its failure to inhibit 18 protein Ser/Thr kinases (including two other MAPKK homologues) in vitro by its failure to inhibit the in vivo activation of MAPKK and MAP kinase homologues that participate in stress and interleukin-1-stimulated kinase cascades in KB and PC12 cells, and by lack of inhibition of the activation of p70 S6 kinase by insulin or epidermal growth factor in Swiss 3T3 cells. PD 098059 (50 microM) inhibited the activation of p42MAPK and isoforms of MAP kinase-activated protein kinase-1 in Swiss 3T3 cells, but the extent of inhibition depended on how potently c-Raf and MAPKK were activated by any particular agonist and demonstrated the enormous amplification potential of this kinase cascade. PD 098059 not only failed to inhibit the activation of Raf by platelet-derived growth factor, serum, insulin, and phorbol esters in Swiss 3T3 cells but actually enhanced Raf activity. The rate of activation of Raf by platelet-derived growth factor was increased 3-fold, and the subsequent inactivation that occurred after 10 min was prevented. These results indicate that the activation of Raf is suppressed and that its inactivation is accelerated by a downstream component(s) of the MAP kinase pathway.
Production of interleukin-1 and tumour necrosis factor from stimulated human monocytes is inhibited by a new series of pyridinyl-imidazole compounds. Using radiolabelled and radio-photoaffinity-labelled chemical probes, the target of these compounds was identified as a pair of closely related mitogen-activated protein kinase homologues, termed CSBPs. Binding of the pyridinyl-imidazole compounds inhibited CSBP kinase activity and could be directly correlated with their ability to inhibit cytokine production, suggesting that the CSBPs are critical for cytokine production.
The hepatic acute-phase response is the result of reprogramming of gene expression in the liver. Similar acute-phase responses occur in regenerating liver after partial hepatectomy and are preceded by increases in the expression of a set of transcriptional regulatory proteins that are encoded by "immediate-early" genes. The purpose of this study was to determine whether acute systemic inflammation after lipopolysaccharide injection induces hepatic immediate-early genes that are induced by partial hepatectomy.
Two- to 4-month-old Balb/c mice received intraperitoneal Escherichia coli lipopolysaccharide (0111:B4; 100 micrograms), and total liver RNA, nuclear protein extracts, or total liver protein lysates were obtained at 0, 1, 3, 12, and 24 hours. RNA blot hybridization analysis was used to determine steady-state messenger RNA levels for c-jun, jun-B, jun-D, c-fos, fos-B, fra-1, nup475, and zif268. Specific nuclear protein-binding activity was determined by gel mobility shift assay. The protein c-Jun was detected by antibody-blocking experiments, and Jun-B was detected by gel supershift assay of the activating protein (AP-1) complex. Steady-state Jun-B levels were determined by immunoblot analysis.
Intraperitoneal injection of lipopolysaccharide is followed by induction (from fivefold to 13-fold) of c-jun, jun-B, c-fos, zif268, and nup475 messenger RNAs in the liver. Lipopolysaccharide induced increases in AP-1 and Zif268 consensus DNA-binding activity in mouse liver. The proteins c-Jun and Jun-B are detected in the AP-1 complex after administration of lipopolysaccharide.
The induction of hepatic immediate-early genes after lipopolysaccharide is similar to that that follows partial hepatectomy. These transcription factors likely have important roles in the reprogramming of gene expression that leads to the acute-phase response.
G0S24 is a member of a set of genes (putative G0/G1 switch regulatory genes) that are expressed transiently within 1-2 hr of the addition of lectin or cycloheximide to human blood mononuclear cells. Comparison of a full-length cDNA sequence with the corresponding genomic sequence reveals an open reading frame of 326 amino acids, distributed across two exons. Potential phosphorylation sites include the sequence PSPTSPT, which resembles an RNA polymerase II repeat reported to be a target of the cell cycle control kinase cdc2. Comparison of the derived protein sequence with those of rodent homologs allows classification into three groups. Group 1 contains G0S24 and the rat and mouse TIS11 genes (also known as TTP, Nup475, and Zfp36). Members of this group have three tetraproline repeats. Groups 1 and 2 have a serine-rich region and an "arginine element" (RRLPIF) at the carboxyl terminus. All groups contain cysteine- and histidine-rich putative zinc finger domains and a serine-phenylalanine "SFS" domain similar to part of the large subunit of eukaryotic RNA polymerase II. Comparison of group 1 human and mouse genomic sequences shows high conservation in the 5' flank and exons. A CpG island suggests expression in the germ line. G0S24 has potential sites for transcription factors in the 5' flank and intron; these include a serum response element. Protein and genomic sequences show similarities with those of a variety of proteins involved in transcription, suggesting that the G0S24 product has a similar role.
Tristetraprolin (TTP) is a widely expressed potential transcription factor that contains two unusual CCCH zinc fingers and is encoded by the immediate-early response gene, Zfp-36. Mice made deficient in TTP by gene targeting appeared normal at birth, but soon manifested marked medullary and extramedullary myeloid hyperplasia associated with cachexia, erosive arthritis, dermatitis, conjunctivitis, glomerular mesangial thickening, and high titers of anti-DNA and antinuclear antibodies. Myeloid progenitors from these mice showed no increase in sensitivity to growth factors. Treatment of young TTP-deficient mice with antibodies to tumor necrosis factor alpha (TNF alpha) prevented the development of essentially all aspects of the phenotype. These results indicate a role for TTP in regulating TNF alpha synthesis, secretion, turnover, or action. TTP-deficient mice may serve as useful models of the autoimmune inflammatory state resulting from chronic effective TNF alpha excess.
Tristetraprolin-deficient [TTP (-/-)] mice exhibit a complex syndrome of myeloid hyperplasia, cachexia, dermatitis, autoimmunity, and erosive arthritis. Virtually the entire syndrome can be prevented by the repeated injection of anti-TNFalpha antibodies (Taylor, G.A., E. Carballo, D.M. Lee, W.S. Lai, M.J. Thompson, D.D. Patel, D.I. Schenkman, G.S. Gilkeson, H.E. Broxmeyer, B.F. Haynes, and P.J. Blackshear. 1996. Immunity. 4:445-454). In the present study, we transplanted bone marrow from TTP (-/-) and (+/+) mice into recombination activating gene-2 (-/-) mice. After a lag period of several months, marrow transplantation from the (-/-) but not the (+/+) mice resulted in the full syndrome associated with TTP deficiency, suggesting that hematopoietic progenitors are responsible for the development of the syndrome. Western blot analysis of supernatants from cultured TTP-deficient macrophages derived from the peritoneal cavity or bone marrow of adult TTP (-/-) mice, or from fetal liver, demonstrated an increased accumulation of TNFalpha after stimulation with LPS compared to control cells, and also increased accumulation of TNFalpha mRNA. This difference was not observed with cultured fibroblasts or T and B lymphocytes. These data suggest that macrophages are among the cells responsible for the effective excess of TNFalpha that leads to the pathology reported in TTP (-/-) animals, and that macrophage progenitors may be involved in the transplantability of this syndrome.
Tumor necrosis factor–α (TNF-α) is a major mediator of both acute and chronic inflammatory responses in many diseases. Tristetraprolin
(TTP), the prototype of a class of Cys-Cys-Cys-His (CCCH) zinc finger proteins, inhibited TNF-α production from macrophages
by destabilizing its messenger RNA. This effect appeared to result from direct TTP binding to the AU-rich element of the TNF-α
messenger RNA. TTP is a cytosolic protein in these cells, and its biosynthesis was induced by the same agents that stimulate
TNF-α production, including TNF-α itself. These findings identify TTP as a component of a negative feedback loop that interferes
with TNF-α production by destabilizing its messenger RNA. This pathway represents a potential target for anti–TNF-α therapies.
Mice deficient in tristetraprolin (TTP), the prototype of a family of CCCH zinc finger proteins, develop an inflammatory syndrome
mediated by excess tumor necrosis factor alpha (TNF-α). Macrophages derived from these mice oversecrete TNF-α, by a mechanism
that involves stabilization of TNF-α mRNA, and TTP can bind directly to the AU-rich element (ARE) in TNF-α mRNA (E. Carballo,
W. S. Lai, and P. J. Blackshear, Science 281:1001–1005, 1998). We show here that TTP binding to the TNF-α ARE is dependent
upon the integrity of both zinc fingers, since mutation of a single cysteine residue in either zinc finger to arginine severely
attenuated the binding of TTP to the TNF-α ARE. In intact cells, TTP at low expression levels promoted a decrease in size
of the TNF-α mRNA as well as a decrease in its amount; at higher expression levels, the shift to a smaller TNF-α mRNA size
persisted, while the accumulation of this smaller species increased. RNase H experiments indicated that the shift to a smaller
size was due to TTP-promoted deadenylation of TNF-α mRNA. This CCCH protein is likely to be important in the deadenylation
and degradation of TNF-α mRNA and perhaps other ARE-containing mRNAs, both in normal physiology and in certain pathological
conditions.
Synthetic glucocorticoids (GCs) remain among the most effective agents for the management of chronic inflammatory diseases. However, major side effects severely limit their therapeutic use. Physiologic and therapeutic activities of GCs are mediated by a nuclear receptor belonging to a superfamily of ligand-inducible transcription factors that, in addition to directly regulating their cognate gene programs, can also mutually interfere with other signaling pathways. We recently identified selective ligands of the glucocorticoid receptor that dissociate transactivation from activator protein 1 transrepression, and most importantly retain in vivo anti-inflammatory activity. To further document the mechanisms of action sustaining the observed in vivo activity, we report here on the interference of dissociated GCs with nuclear factor kappaB (NF-kappaB)-driven gene activation. We show that dissociated GCs repress tumor necrosis factor-induced interleukin-6 gene expression by an NF-kappaB-dependent mechanism, without changing the expression level of inhibitor kappaB. The DNA-binding activity of induced NF-kappaB also remained unchanged after stimulation of cells with the various compounds. Evidence for a direct nuclear mechanism of action was obtained by analysis of cell lines constitutively expressing a fusion protein between the DNA-binding domain of the yeast Gal4 protein and the transactivating p65 subunit of NF-kappaB, which was able to efficiently repress a Gal4-dependent luciferase reporter gene upon addition of the dissociated compounds. We therefore conclude that, in addition to dissociating transactivation from activator protein 1 transrepression, dissociated GCs mediate inhibition of NF-kappaB signaling by a mechanism that is independent of inhibitor kappaB induction.
Using digoxigenin (DIG)-based differential hybridization, a series of immediate early genes (IEG) was identified following the adipogenic stimulation in 3T3-L1 preadipocyte cells. Most of the known IEGs were identified as well as new members such as zf9 and Stra13. To delineate possible signaling pathways accounting for these gene expression, a subset of specific kinase inhibitors, SB203580, PD98059, rapamycin, LY294002, and Ro-32-0432, which inhibit p38 (HOG), MEK (MAPKK), S6 kinase, PI3 kinase, and protein kinase C (PKC), respectively, were employed. The IEGs were classified into three categories according to their susceptibility to the inhibitors. Expression of the first group (c-fos, jun-B, egr-1, tis11, tis21, thrombospondin-1, erp, thyroid hormone receptor [N-10], cyr61, and zf9) was mainly dependent on PKC and MEK pathways, while that of the second class (gene33 and tis10) exhibited an additional dependence on PI3 kinase pathways. The third one (Id-3, gly96, and Stra13) was characterized in that none of these inhibitors interfered with gene expression. Our results suggest that the induction of IEGs by the adipogenic stimuli is mediated by common as well as distinct signaling pathways.
Deficiency of tristetraprolin (TTP), the prototype of the CCCH zinc finger proteins, results in a complex inflammatory syndrome in mice. Most aspects of the syndrome are secondary to excess circulating tumor necrosis factor (TNF)-alpha, a consequence of increased stability of TNF-alpha messenger RNA (mRNA) in TTP-deficient macrophages. TTP can bind directly to the AU-rich element in TNF-alpha mRNA, increasing its lability. Here we show that TTP deficiency also results in increased cellular production of granulocyte-macrophage colony-stimulating factor (GM-CSF) and increased stability of its mRNA, apparently secondary to decreased deadenylation. Similar findings were observed in mice also lacking both types of TNF-alpha receptors, excluding excess TNF-alpha production as a cause of the increased GM-CSF mRNA levels and stability. TTP appears to be a physiological regulator of GM-CSF mRNA deadenylation and stability. (Blood. 2000;95:1891-1899)
Macrophages derived from tristetraprolin (TTP)-deficient mice exhibited increased tumor necrosis factor alpha (TNFalpha) release as a consequence of increased stability of TNFalpha mRNA. TTP was then shown to destabilize TNFalpha mRNA after binding directly to the AU-rich region (ARE) of the 3'-untranslated region of the TNFalpha mRNA. In mammals and in Xenopus, TTP is the prototype of a small family of three known zinc finger proteins containing two CCCH zinc fingers spaced 18 amino acids apart; a fourth more distantly related family member has been identified in Xenopus and fish. We show here that representatives of all four family members were able to bind to the TNFalpha ARE in a cell-free system and, in most cases, promote the breakdown of TNFalpha mRNA in intact cells. Because the primary sequences of these CCCH proteins are most closely related in their tandem zinc finger domains, we tested whether various fragments of TTP that contained both zinc fingers resembled the intact protein in these assays. We found that amino- and carboxyl-terminal truncated forms of TTP, as well as a 77 amino acid fragment that contained both zinc fingers, could bind to the TNFalpha ARE in cell-free cross-linking and gel shift assays. In addition, these truncated forms of TTP could also stimulate the apparent deadenylation and/or breakdown of TNFalpha mRNA in intact cells. Alignments of the tandem zinc finger domains from all four groups of homologous proteins have identified invariant residues as well as group-specific signature amino acids that presumably contribute to ARE binding and protein-specific activities, respectively.
The number of chromatin modifying and remodeling complexes implicated in genome control is growing faster than our understanding of the functional roles they play. We discuss recent in vitro experiments with biochemically defined chromatin templates that illuminate new aspects of action by histone acetyltransferases and ATP-dependent chromatin remodeling engines in facilitating transcription. We review a number of studies that present an 'ordered recruitment' view of transcriptional activation, according to which various complexes enter and exit their target promoter in a set sequence, and at specific times, such that action by one complex sets the stage for the arrival of the next one. A consensus emerging from all these experiments is that the joint action by several types of chromatin remodeling machines can lead to a more profound alteration of the infrastructure of chromatin over a target promoter than could be obtained by these enzymes acting independently. In addition, it appears that in specific cases one type of chromatin structure alteration (e.g., histone hyperacetylation) is contingent upon prior alterations of a different sort (i.e., ATP-dependent remodeling of histone-DNA contacts). The striking differences between the precise sequence of action by various cofactors observed in these studies may be - at least in part - due to differences between the specific promoters studied, and distinct requirements exhibited by specific loci for chromatin remodeling based on their pre-existing nucleoprotein architecture.
Tristetraprolin (TTP) is a zinc finger protein that has been implicated in the control of tumor necrosis factor (TNF) mRNA stability. We show here that TTP protein has a suppressive effect on promoter elements from TNF-alpha and interleukin-8 and that lipopolysaccharide (LPS) stimulation can release this suppression. The release in LPS-stimulated cells was found to be primarily mediated by the p38 pathway because activation of p38 is sufficient to remove the suppressive effect of TTP. Indeed, TTP seems to be a direct substrate of p38 in vivo since it is an excellent substrate of p38 in vitro, and mutation of potential phosphorylation sites in TTP prevents release of the suppression imposed on TNF transcription. We found TTP protein to be present at low levels in the resting macrophage cell line RAW 264.7 and to be quickly induced after LPS stimulation. The kinetics of TTP induction suggests a potential role of TTP as an important player in switching off LPS-induced genes after induction. In conclusion, TTP plays an important role in maintaining gene quiescence, and this quenching effect on transcription can be released by p38 phosphorylation of TTP.
The tristetraprolin (TTP) family of CCCH tandem zinc-finger proteins is composed of three known members in mammals, with a fourth member recently identified in frogs and fish. Although TTP was first cloned more than 10 years ago as a growth factor-induced gene, a physiological function for the protein has been discovered only within the last few years. TTP is now known to bind to so-called class II AU-rich elements within the mRNAs that encode tumour necrosis factor-alpha and granulocyte/macrophage colony-stimulating factor. In both cases, this binding results in destabilization of the mRNA and decreased secretion of the protein. Recent evidence suggests that TTP can accomplish this accelerated mRNA degradation by first promoting removal of the polyadenylated tail from the mRNA (deadenylation). In functional assays in cells, the other family members have similar activities, but are expressed differently in tissues and in response to stimuli, suggesting that they may control the stability of mRNAs under different circumstances from those in which TTP affects mRNA. All of these proteins are phosphoproteins and nucleocytoplasmic shuttling proteins, suggesting that their activities can be regulated in ways other than regulating gene transcription. Together, the TTP family members should be capable of complex regulation of short-lived mRNAs containing this type of AU-rich instability motif.
Tristetraprolin (TTP) is an mRNA-binding protein, but studies of this interaction have been difficult due to problems with the purification of recombinant TTP. In the present study, we expressed human and mouse TTP as glutathione S-transferase and maltose-binding protein (MBP) fusion proteins in Escherichia coli, and purified them by affinity resins and Mono Q chromatography. TTP cleaved from the fusion protein was identified by immunoblotting, MALDI-MS, and protein sequencing, and was further purified to homogeneity by continuous-elution SDS-gel electrophoresis. Purified recombinant TTP bound to the AU-rich element of tumor necrosis factor-alpha (TNFalpha) mRNA and this binding was dependent on Zn(2+). Results from sizing columns suggested that the active species might be in the form of an oligomer of MBP-TTP. Recombinant TTP was phosphorylated by three members of the mitogen-activated protein (MAP) kinase family, p42, p38, and JNK, with half-maximal phosphorylation occurring at approximately 0.5, 0.25, and 0.25 microM protein, respectively. Phosphorylation by these kinases did not appear to affect the ability of TTP to bind to TNFalpha mRNA under the assay conditions. This study describes a procedure for purifying nonfusion protein TTP to homogeneity, demonstrates that TTP's RNA-binding activity is zinc dependent, and that TTP can be phosphorylated by JNK as well as by the other members of the greater MAP kinase family.
Glucocorticoids are widely used to treat inflammatory and immune diseases. The most common use of glucocorticoids today is in the treatment of asthma. Inhaled glucocorticoids are first-line treatment in adults and children with persistent asthma, the most common chronic airway inflammatory disease. Our knowledge of how glucocorticoids suppress inflammation is based on recent developments in understanding the fundamental mechanisms of gene transcription, namely recruitment of histone-modifying co-factors. The determination of the crystal structure of the ligand-binding domain of the human glucocorticoid receptor (GR) has advanced our understanding of how ligands interact with GR and provide a glimpse of a future of rational drug design based on "space-filling" structures with dissociated properties. This might have important clinical implications, leading to a better understanding of the inflammatory mechanisms of many diseases and might signal the development of new anti-inflammatory treatments in the future.
A pathogenetic role for TNF alpha in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency
- Ga Taylor
- E Carballo
- Dm Lee
- Ws Lai
- Mj Thompson
- Dd Patel
Taylor GA, Carballo E, Lee DM, Lai WS, Thompson MJ, Patel DD, et
al. A pathogenetic role for TNF alpha in the syndrome of cachexia,
arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency. Immunity 1996;4:445–54.