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1.  Differential Expression and Functional Analysis of the Tristetraprolin Family during Early Differentiation of 3T3-L1 Preadipocytes 
The tristetraprolin (TTP) family comprises zinc finger-containing AU-rich element (ARE)-binding proteins consisting of three major members: TTP, ZFP36L1, and ZFP36L2. The present study generated specific antibodies against each TTP member to evaluate its expression during differentiation of 3T3-L1 preadipocytes. In contrast to the inducible expression of TTP, results indicated constitutive expression of ZFP36L1 and ZFP36L2 in 3T3-L1 preadipocytes and their phosphorylation in response to differentiation signals. Physical RNA pull-down and functional luciferase assays revealed that ZFP36L1 and ZFP36L2 bound to the 3′ untranslated region (UTR) of MAPK phosphatase-1 (MKP-1) mRNA and downregulated Mkp-1 3′UTR-mediated luciferase activity. Mkp-1 is an immediate early gene for which the mRNA is transiently expressed in response to differentiation signals. The half-life of Mkp-1 mRNA was longer at 30 min of induction than at 1 h and 2 h of induction. Knockdown of TTP or ZFP36L2 increased the Mkp-1 mRNA half-life at 1 h of induction. Knockdown of ZFP36L1, but not ZFP36L2, increased Mkp-1 mRNA basal levels via mRNA stabilization and downregulated ERK activation. Differentiation induced phosphorylation of ZFP36L1 through ERK and AKT signals. Phosphorylated ZFP36L1 then interacted with 14-3-3, which might decrease its mRNA destabilizing activity. Inhibition of adipogenesis also occurred in ZFP36L1 and TTP knockdown cells. The findings indicate that the differential expression of TTP family members regulates immediate early gene expression and modulates adipogenesis.
PMCID: PMC3371571  PMID: 22701344
tristetraprolin; 3T3-L1; ZFP36L1; ZFP36L2; MKP-1; AU-rich element.
2.  Green tea increases anti-inflammatory tristetraprolin and decreases pro-inflammatory tumor necrosis factor mRNA levels in rats 
Tristetraprolin (TTP/ZFP36) family proteins have anti-inflammatory activity by binding to and destabilizing pro-inflammatory mRNAs such as Tnf mRNA, and represent a potential therapeutic target for inflammation-related diseases. Tea has anti-inflammatory properties but the molecular mechanisms have not been completely elucidated. We hypothesized that TTP and/or its homologues might contribute to the beneficial effects of tea as an anti-inflammatory product.
Quantitative real-time PCR was used to investigate the effects of green tea (0, 1, and 2 g solid extract/kg diet) on the expression of Ttp family genes (Ttp/Tis11/Zfp36, Zfp36l1/Tis11b, Zfp36l2/Tis11d, Zfp36l3), pro-inflammatory genes (Tnf, Csf2/Gm-csf, Ptgs2/Cox2), and Elavl1/Hua/Hur and Vegf genes in liver and muscle of rats fed a high-fructose diet known to induce insulin resistance, oxidative stress, inflammation, and TNF-alpha levels.
Ttp and Zfp36l1 mRNAs were the major forms in both liver and skeletal muscle. Ttp, Zfp36l1, and Zfp36l2 mRNA levels were more abundant in the liver than those in the muscle. Csf2/Gm-csf and Zfp36l3 mRNAs were undetectable in both tissues. Tea (1 g solid extract/kg diet) increased Ttp mRNA levels by 50–140% but Tnf mRNA levels decreased by 30% in both tissues, and Ptgs2/Cox2 mRNA levels decreased by 40% in the muscle. Tea (2 g solid extract/kg diet) increased Elavl1/Hua/Hur mRNA levels by 40% in the liver but did not affect any of the other mRNA levels in liver or muscle.
These results show that tea can modulate Ttp mRNA levels in animals and suggest that a post-transcriptional mechanism through TTP could partially account for tea's anti-inflammatory properties. The results also suggest that drinking adequate amounts of green tea may play a role in the prevention of inflammation-related diseases.
PMCID: PMC1783848  PMID: 17207279
3.  The RNA Binding Zinc Finger Protein Tristetraprolin Regulates AU-Rich mRNAs Involved in Breast Cancer-Related Processes 
Oncogene  2010;29(29):4205-4215.
Tristetraprolin (TTP or ZFP36) is a tandem CCCH zinc finger RNA binding protein that regulates the stability of certain AU-rich mRNAs. Recent work suggests that TTP is deficient in cancer cells when compared to normal cell types. Here we found that TTP expression was lower in invasive breast cancer cells (MDA-MB-231) compared to normal breast cell lines, MCF12A and MCF-10. TTP targets were probed using a novel approach by expressing the C124R zinc finger TTP mutant that act as dominant negative and increase target mRNA expression. In contrast to wt TTP, C124R TTP was able to increase certain ARE-mRNA expression in serum-stimulated breast cancer cells. Using an ARE-gene microarray, novel targets of TTP regulation were identified; urokinase plasminogen activator (uPA), uPA receptor, and matrix metallo-proteinase-1 (MMP1), all known to play prominent roles in breast cancer invasion and metastasis. Expression of these targets was upregulated in the tumorigenic types, particularly, the highly invasive MDA-MB-231. The mRNA half lives of these TTP-regulated genes were increased in TTP-knockout embryonic mouse fibroblasts as assessed by real time PCR while forced restoration of TTP by transfection led to a reduction of their mRNA levels. RNA immunoprecipitation confirmed an association of TTP, but not C124R, with these target transcripts. Moreover, TTP reduced, while the mutant C124R TTP increased, the activity of reporter constructs fused to target ARE. As a result of TTP regulation, invasiveness of MDAMB231 cells was reduced. The data suggest that TTP, in a 3′UTR- and ARE-dependent manner, regulates an important subset of cancer-related genes that are involved in cellular growth, invasion, and metastasis.
PMCID: PMC3422647  PMID: 20498646
Post-transcriptional control; AU-rich elements; RNA binding proteins; RNA stability; Tristetraprolin
4.  Myeloid ZFP36L1 Does Not Regulate Inflammation or Host Defense in Mouse Models of Acute Bacterial Infection 
PLoS ONE  2014;9(10):e109072.
Zinc finger protein 36, C3H type-like 1 (ZFP36L1) is one of several Zinc Finger Protein 36 (Zfp36) family members, which bind AU rich elements within 3′ untranslated regions (UTRs) to negatively regulate the post-transcriptional expression of targeted mRNAs. The prototypical member of the family, Tristetraprolin (TTP or ZFP36), has been well-studied in the context of inflammation and plays an important role in repressing pro-inflammatory transcripts such as TNF-α. Much less is known about the other family members, and none have been studied in the context of infection. Using macrophage cell lines and primary alveolar macrophages we demonstrated that, like ZFP36, ZFP36L1 is prominently induced by infection. To test our hypothesis that macrophage production of ZFP36L1 is necessary for regulation of the inflammatory response of the lung during pneumonia, we generated mice with a myeloid-specific deficiency of ZFP36L1. Surprisingly, we found that myeloid deficiency of ZFP36L1 did not result in alteration of lung cytokine production after infection, altered clearance of bacteria, or increased inflammatory lung injury. Although alveolar macrophages are critical components of the innate defense against respiratory pathogens, we concluded that myeloid ZFP36L1 is not essential for appropriate responses to bacteria in the lungs. Based on studies conducted with myeloid-deficient ZFP36 mice, our data indicate that, of the Zfp36 family, ZFP36 is the predominant negative regulator of cytokine expression in macrophages. In conclusion, these results imply that myeloid ZFP36 may fully compensate for loss of ZFP36L1 or that Zfp36l1-dependent mRNA expression does not play an integral role in the host defense against bacterial pneumonia.
PMCID: PMC4192124  PMID: 25299049
5.  Transcriptional Regulation of EGR1 by EGF and the ERK Signaling Pathway in Prostate Cancer Cells 
Genes & Cancer  2011;2(9):900-909.
The early growth response gene 1, EGR1, is an important transcriptional regulator and acts as the convergent point between a variety of extracellular stimuli and activation of target genes. Unlike other tumor types, prostate tumors express high levels of EGR1 relative to normal tissues. However, the mechanism of EGR1 regulation in prostate tumor cells is unknown. As EGR1 expression and epidermal growth factor (EGF) signaling are frequently upregulated in prostate tumors, we tested the hypothesis that EGF induces EGR1 expression in prostate cancer cells. Using RT-PCR to quantify EGR1 transcripts, we found that EGF induced EGR1 expression in a dose- and time-dependent manner and the ERK pathway inhibitor, PD98059, abrogated the EGF-mediated EGR1 response in LNCaP and PC3 cells. Analysis of the EGR1 promoter using deletion constructs identified an EGF-responsive region in the proximal promoter (–771 to −245 bp) containing 3 potential serum response element (SRE) sites. In vivo chromatin immunoprecipitation assays demonstrated that Elk-1 binding at the SRE sites of the EGR1 promoter was enhanced by EGF treatment in PC3 cells. Overexpression of Elk-1 was sufficient to activate the EGF-responsive region of EGR1 promoter in PC3 cells and, similarly, a dominant-negative Elk-1 suppressed EGR1 promoter activity. Taken together, these results demonstrate for the first time that EGR1 expression in PC3 cells is mediated through an EGF-ERK-Elk-1 signaling cascade.
PMCID: PMC3352154  PMID: 22593802
prostate cancer; EGF; EGR1; Elk-1
6.  Role of the RNA-binding Protein Tristetraprolin in Glucocorticoid-mediated Gene Regulation1 
Glucocorticoids (GCs) are the mainstay of anti-inflammatory therapy. Modulation of post-transcriptional 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-α was similarly affected, as was the antagonistic effect of GC on TNF-α-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 siRNAconfirmed 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.
PMCID: PMC2505276  PMID: 18523301
Inflammation; glucocorticoids; posttranscriptional gene regulation; chemokines
7.  Myeloid-Specific Tristetraprolin Deficiency in Mice Results in Extreme Lipopolysaccharide Sensitivity in an Otherwise Minimal Phenotype1 
Tristetraprolin (TTP) is an mRNA destabilizing protein that binds to AU-rich elements in labile transcripts, such as the mRNA encoding tumor necrosis factor alpha (TNF), and promotes their deadenylation and degradation. TTP-deficient (KO) mice exhibit an early-onset, severe inflammatory phenotype, with cachexia, erosive arthritis, left-sided cardiac valvulitis, myeloid hyperplasia, and autoimmunity, which can be prevented by injections of anti-TNF antibodies, or interbreeding with TNF receptor-deficient mice. To determine whether the excess TNF that causes the TTP KO phenotype is produced by myeloid cells, we performed myeloid-specific disruption of Zfp36, the gene encoding TTP. We documented the lack of TTP expression in lipopolysaccharide-stimulated bone marrow-derived macrophages from the mice, whereas fibroblasts expressed TTP mRNA and protein normally in response to serum. The mice exhibited a minimal phenotype, characterized by slight slowing of weight gain late in the first year of life, compared to the early onset, severe weight loss and inflammation seen in the TTP KO mice. Instead, the myeloid-specific TTP KO mice were highly and abnormally susceptible to a low dose lipopolysaccharide challenge, with rapid development of typical endotoxemia signs and extensive organ damage, and elevations of serum TNF levels to 110-fold greater than control. We conclude that myeloid-specific TTP deficiency does not phenocopy complete TTP deficiency in C57BL/6 mice under normal laboratory conditions, implying contributions from other cell types to the complete phenotype. However, myeloid cell TTP plays a critical role in protecting mice against LPS-induced septic shock, primarily through its post-transcriptional regulation of TNF mRNA stability.
PMCID: PMC3345041  PMID: 22491258
Tristetraprolin; Tumor necrosis factor-alpha; FLOXed TTP mouse; LysMcre; Myeloid-specific TTP deficiency; Bone marrow-derived macrophages; Endotoxemia
8.  Production and Characterization of ZFP36L1 Antiserum against Recombinant Protein from Escherichia coli 
Biotechnology progress  2008;24(2):326-333.
Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) family proteins are anti-inflammatory. They bind and destabilize some AU-rich element-containing mRNAs such as tumor necrosis factor mRNA. In this study, recombinant ZFP36L1/TIS11B (a TTP homologue) was over-expressed in E. coli, purified, and used for polyclonal antibody production in rabbits. The antiserum recognized nanograms of the antigen on immunoblots. This antiserum and another antiserum developed against recombinant mouse TTP were used to detect ZFP36L1 and TTP in mouse 3T3-L1 adipocytes and RAW264.7 macrophages. Immunoblotting showed that ZFP36L1 was stably expressed with a size corresponding to the lower mass size of ZFP36L1 expressed in transfected human embryonic kidney 293 cells, but TTP was induced by cinnamon extract and not by lipopolysaccharide (LPS) in adipocytes. In contrast, ZFP36L1 was undetectable but TTP was strongly induced in LPS-stimulated RAW cells. Quantitative real-time polymerase chain reaction confirmed the higher levels of ZFP36L1 mRNA in adipocytes and TTP mRNA in RAW cells. Low levels of ZFP36L1 expression were also confirmed by northern blotting in mouse embryonic fibroblasts. These results demonstrate that ZFP36L1 antiserum is useful in the detection of this protein and that TTP and ZFP36L1 are differentially expressed and regulated at the mRNA and protein levels in mouse adipocytes and macrophages.
PMCID: PMC2674335  PMID: 18302406
antibody production; cinnamon; insulin; lipopolysaccharide; mouse cell; protein expression and purification; real-time PCR; tristetraprolin; ZFP36L1
9.  Identification of a Major Phosphopeptide in Human Tristetraprolin by Phosphopeptide Mapping and Mass Spectrometry 
PLoS ONE  2014;9(7):e100977.
Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) binds and destabilizes some pro-inflammatory cytokine mRNAs. TTP-deficient mice develop a profound inflammatory syndrome due to excessive production of pro-inflammatory cytokines. TTP expression is induced by various factors including insulin and extracts from cinnamon and green tea. TTP is highly phosphorylated in vivo and is a substrate for several protein kinases. Multiple phosphorylation sites are identified in human TTP, but it is difficult to assign major vs. minor phosphorylation sites. This study aimed to generate additional information on TTP phosphorylation using phosphopeptide mapping and mass spectrometry (MS). Wild-type and site-directed mutant TTP proteins were expressed in transfected human cells followed by in vivo radiolabeling with [32P]-orthophosphate. Histidine-tagged TTP proteins were purified with Ni-NTA affinity beads and digested with trypsin and lysyl endopeptidase. The digested peptides were separated by C18 column with high performance liquid chromatography. Wild-type and all mutant TTP proteins were localized in the cytosol, phosphorylated extensively in vivo and capable of binding to ARE-containing RNA probes. Mutant TTP with S90 and S93 mutations resulted in the disappearance of a major phosphopeptide peak. Mutant TTP with an S197 mutation resulted in another major phosphopeptide peak being eluted earlier than the wild-type. Additional mutations at S186, S296 and T271 exhibited little effect on phosphopeptide profiles. MS analysis identified the peptide that was missing in the S90 and S93 mutant protein as LGPELSPSPTSPTATSTTPSR (corresponding to amino acid residues 83–103 of human TTP). MS also identified a major phosphopeptide associated with the first zinc-finger region. These analyses suggest that the tryptic peptide containing S90 and S93 is a major phosphopeptide in human TTP.
PMCID: PMC4091943  PMID: 25010646
10.  CREB Targets Define the Gene Expression Signature of Malignancies Having Reduced Levels of the Tumor Suppressor Tristetraprolin 
PLoS ONE  2014;9(12):e115517.
The RNA-binding protein Tristetraprolin (TTP, ZFP36) functions as a tumor suppressor that impairs the development and disables the maintenance of MYC-driven lymphoma. In addition, other human cancers expressed reduced levels of TTP, suggesting that it may function as a tumor suppressor in several malignancies. To identify genes that may be associated with TTP tumor suppressor functions in human cancer, we analyzed The Cancer Genome Atlas (TCGA) breast cancer, lung adenocarcinoma, lung squamous cell carcinoma, and colon adenocarcinoma datasets. These analyses defined a signature of 50 genes differentially regulated between high and low TTP-expressing tumors. Notably, patients with low TTP-expressing breast cancer and lung adenocarcinoma had decreased survival rates and more aggressive tumors with increased necrosis. In addition, analysis across non-TCGA tumor gene expression databases identified a broad spectrum of human cancers having similarities with the TTP-low tumor gene signature, including pancreatic, bladder, and prostate cancer. TTP has documented roles in regulating mRNAs encoding inflammatory proteins, and pathway analysis identified several inflammatory pathways that are altered in tumors with low TTP expression. Surprisingly, the TTP-low tumor gene signature includes a core component of 20 under-expressed CREB target genes, suggesting that the regulation of CREB activity may be related to the tumor suppressor function of TTP. Thus, reduced levels of TTP are a potential biomarker for human cancers with poor outcome, and targeting the CREB pathway may be a therapeutic route for treating aggressive TTP-low tumors.
PMCID: PMC4277357  PMID: 25541715
11.  Deletion of Tristetraprolin (TTP) caused spontaneous reactive granulopoiesis by a non-cell autonomous mechanism without disturbing LT-HSC quiescence1 
Tristetraprolin (TTP, Zfp36, Nup475, Tis11) dramatically reduces the stability of target mRNAs by binding to AU-rich elements (AREs) in their 3′UTRs. Through this mechanism, TTP functions as a rheostatic, temporal regulator of gene expression. TTP KO mice exhibit completely penetrant granulocytic hyperplasia. We have shown that the hematopoietic stem-progenitor cell (HSPC) compartment in TTP KO mice is also altered. Although no change was detected in long-term HSC (LT-HSC) frequency or function, as assayed by immunophenotypic markers or limiting dilution transplants, we observed increases in the frequencies and numbers of short-term HSCs (ST-HSCs), multipotent progenitors (MPPs) and granulocyte-monocyte progenitors (GMPs). This pattern is consistent with ‘reactive granulopoiesis,’ in which committed myeloid progenitors and more primitive progenitors cycle more actively to increase production of mature granulocytes in response to infection or adjuvant. We created reverse chimeras by transplanting WT bone marrow (BM) into TTP KO mice and found the ‘reactive granulopoiesis’ phenocopied, indicating a non-HSPC-autonomous mechanism. Correspondingly, we found elevated levels of the granulopoietic TTP targets IL-1β, TNFα and IL-6 in the plasma of TTP KO mice. Consistent with the non-cell autonomous nature of the phenotype, we found elevated levels of IL-1β, TNFα and IL-6 transcripts in the livers of TTP KO mice and no detectable difference in the BMs. These findings demonstrate the importance of TTP in inflammatory homeostasis and highlight the ability of the hematopoietic system to respond to stress without significant numbers of quiescent HSCs entering the cell cycle.
PMCID: PMC3114656  PMID: 21270394
12.  Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications†,‡ 
Biochemistry  2004;43(43):13724-13738.
Tristetraprolin (TTP) is a hyperphosphorylated protein that destabilizes mRNA by binding to an AU-rich element (ARE). Mice deficient in TTP develop a severe inflammatory syndrome. The biochemical properties of TTP have not been adequately characterized, due to the difficulties in protein purification and lack of a high-titer antiserum. Full-length human TTP was expressed in human HEK293 cells and purified to at least 70% homogeneity. The purified protein was free of endogenous ARE binding activity, and was used for investigating its size, zinc dependency, and binding kinetics for tumor necrosis factor α mRNA ARE. A high-titer rabbit antiserum was raised against the MBP–hTTP fusion protein expressed in Escherichia coli. Cellular localization studies of the transfected cells indicated that approximately 80% of the expressed TTP was in the cytosol, with 20% in the nuclei. TTP from both locations bound to the ARE and formed similar complexes. The purified TTP was shown to be intact by N-terminal His-tag purification, C-terminal peptide sequencing, and mass spectrometry analysis. Results from size exclusion chromatography are consistent with the predominant form of active TTP being a tetramer. TTP's ARE binding activity was increased by 10 μM Zn2+. The half-maximal binding of TTP from HEK293 cells was approximately 30 nM in assays containing 10 nM ARE. This value was about twice that of TTP from E. coli. TTP from HEK293 cells was highly phosphorylated, and its electrophoretic mobility was increased by alkaline phosphatase treatment and somewhat by T271A mutation, but not by PNGase F or S186A mutation. The gel mobility of TTP from E. coli was decreased by in vitro phosphorylation with p42/ERK2 and p38 mitogen-activated protein kinases. These results suggest that TTP's zinc-dependent ARE binding affinity is reduced by half by posttranslational modifications, mainly by phosphorylation but not by glycosylation, in mammalian cells. The results support a model in which each subunit of the TTP tetramer binds to one of the five overlapping UUAUUUAUU sequences of the ARE, resulting in a stable TTP–ARE complex.
PMCID: PMC1351390  PMID: 15504035
13.  Regulation of p21/CIP1/WAF-1 mediated cell-cycle arrest by RNase L and tristetraprolin, and involvement of AU-rich elements 
Nucleic Acids Research  2012;40(16):7739-7752.
The p21Cip1/WAF1 plays an important role in cell-cycle arrest. Here, we find that RNase L regulates p21-mediated G1 growth arrest in AU-rich elements-dependent manner. We found a significant loss of p21 mRNA expression in RNASEL−/− MEFs and that the overexpression of RNase L in HeLa cells induces p21 mRNA expression. The p21 mRNA half-life significantly changes as a result of RNase L modulation, indicating a post-transcriptional effect. Indeed, we found that RNase L promotes tristetraprolin (TTP/ZFP36) mRNA decay. This activity was not seen with dimerization- and nuclease-deficient RNase L mutants. Deficiency in TTP led to increases in p21 mRNA and protein. With induced ablation of RNase L, TTP mRNA and protein expressions were higher, while p21 expression became reduced. We further establish that TTP, but not C124R TTP mutant, binds to, and accelerates the decay of p21 mRNA. The p21 mRNA half-life was prolonged in TTP−/− MEFs. The TTP regulation of p21 mRNA decay required functional AU-rich elements. Thus, we demonstrate a novel mechanism of regulating G1 growth arrest by an RNase L-TTP-p21 axis.
PMCID: PMC3439922  PMID: 22718976
14.  IL-10-Mediated Tristetraprolin Induction is part of a feedback loop that controls Macrophage STAT3 activation and cytokine production1 
In activated macrophages, the anti-inflammatory cytokine IL-10 inhibits expression of molecules that propagate inflammation in a manner that depends on transcription factor STAT3. Expression of IL-10 is regulated post-transcriptionally by the RNA-binding protein tristetraprolin (TTP), which destabilizes IL-10 mRNA in activated macrophages. Using LPS-activated bone marrow-derived murine macrophages, we demonstrate that TTP is a negative regulator of the IL-10/STAT3 anti-inflammatory response. LPS-stimulated TTP-deficient macrophages overproduced IL-10, contained increased amounts of activated STAT3, and showed reduced expression of inflammatory cytokines, including cytokines encoded by TTP-target mRNAs. Thus, in LPS-stimulated TTP-deficient macrophages, increased IL-10/STAT3 anti-inflammatory control was dominant over the mRNA-stabilization of specific TTP targets. The TTP gene promoter contains a conserved STAT3 binding site and IL-10 induces STAT3 recruitment to this site. Correspondingly, STAT3 was required for efficient IL-10-induced TTP expression. Hence, by inducing TTP expression, STAT3 activates a negative regulatory loop that controls the IL-10/STAT3 anti-inflammatory response.
PMCID: PMC3424405  PMID: 22865915
15.  Posttranslational Regulation of Tristetraprolin Subcellular Localization and Protein Stability by p38 Mitogen-Activated Protein Kinase and Extracellular Signal-Regulated Kinase Pathways 
Molecular and Cellular Biology  2006;26(6):2408-2418.
The p38 mitogen-activated protein kinase (MAPK) signaling pathway, acting through the downstream kinase MK2, regulates the stability of many proinflammatory mRNAs that contain adenosine/uridine-rich elements (AREs). It is thought to do this by modulating the expression or activity of ARE-binding proteins that regulate mRNA turnover. MK2 phosphorylates the ARE-binding and mRNA-destabilizing protein tristetraprolin (TTP) at serines 52 and 178. Here we show that the p38 MAPK pathway regulates the subcellular localization and stability of TTP protein. A p38 MAPK inhibitor causes rapid dephosphorylation of TTP, relocalization from the cytoplasm to the nucleus, and degradation by the 20S/26S proteasome. Hence, continuous activity of the p38 MAPK pathway is required to maintain the phosphorylation status, cytoplasmic localization, and stability of TTP protein. The regulation of both subcellular localization and protein stability is dependent on MK2 and on the integrity of serines 52 and 178. Furthermore, the extracellular signal-regulated kinase (ERK) pathway synergizes with the p38 MAPK pathway to regulate both stability and localization of TTP. This effect is independent of kinases that are known to be synergistically activated by ERK and p38 MAPK. We present a model for the actions of TTP and the p38 MAPK pathway during distinct phases of the inflammatory response.
PMCID: PMC1430283  PMID: 16508015
16.  Inactivation or loss of TTP promotes invasion in head and neck cancer via transcript stabilization and secretion of MMP9, MMP2 and IL-6 
Invasion is the critical step in progression of a pre-cancerous lesion to squamous cell carcinoma of the head and neck (SCCHN). Invasion is regulated by multiple pro-inflammatory mediators. Tristetraprolin (TTP) is an mRNA degrading protein that regulates multiple pro-inflammatory mediators. TTP may serve as an excellent treatment target. Rap1 is a ras-like oncoprotein that induces critical signaling pathways. In this study, the role of rap1 in TTP-mediated invasion was investigated.
Experimental Design
Using complementary approaches we modulated TTP and altered expression of IL-6 and MMP2/9, which were quantified by ELISA and zymogram. Invasion was evaluated in vitro using the Oral-Cancer-Equivalent (OCE) 3D model and in vivo in the chick chorioallantoic membrane (CAM). The role of rap1 and p38 were established using knockdown strategies.
Downregulation of TTP significantly increased invasion via secretion of MMP9/2 and IL-6. In the novel OCE and CAM invasion models of SCCHN, cells with downregulated TTP destroyed the basement membrane to invade the underlying connective tissue. Rap1 induces p38 mitogen activated protein kinase (p38)-mediated inactivation of TTP. Inactive TTP enhances transcript stability via binding to the 3′-UTR. High IL-6 and MMP9 are prognostic for poor clinical outcomes in SCCHN patients.
Targeting the rap1-p38-TTP cascade is an attractive novel treatment strategy in SCCHN to concurrently suppress multiple mediators of invasion.
PMCID: PMC3594656  PMID: 23349315
cytokines; post-transcriptional regulation; 3′UTR; chick chorioallantoic membrane assay (CAM); oral cancer equivalent (OCE)
17.  Tristetraprolin expression and microRNA-mediated regulation during simian immunodeficiency virus infection of the central nervous system 
Molecular Brain  2013;6:40.
The RNA-binding protein tristetraprolin (TTP) participates in normal post-transcriptional control of cytokine and chemokine gene expression, dysregulation of which contributes to the HIV-associated neurocognitive disorders. Transcriptional and post-transcriptional regulation of TTP has been described, including regulation by microRNA-29a. In the simian immunodeficiency virus (SIV) model of HIV CNS disease, control of cytokine/chemokine expression coincides with the end of acute phase infection. This control is lost during progression to disease. In this study, we assessed TTP regulation and association with cytokine regulation in the brain during SIV infection.
Quantitation of TTP expression over the course of SIV infection revealed downregulation of TTP during acute infection, maintenance of relatively low levels during asymptomatic phase, and increased expression only during late-stage CNS disease, particularly in association with severe disease. The ability of miR-29a to regulate TTP was confirmed, and evidence for additional miRNA targeters of TTP was found. However, increased miR-29a expression in brain was not found to be significantly negatively correlated with TTP. Similarly, increased TTP during late-stage disease was not associated with lower cytokine expression.
TTP expression is regulated during SIV infection of the CNS. The lack of significant negative correlation of miR-29a and TTP expression levels suggests that while miR-29a may contribute to TTP regulation, additional factors are involved. Reduced TTP expression during acute infection is consistent with increased cytokine production during this phase of infection, but the increases in TTP observed during late-stage infection were insufficient to halt runaway cytokine levels. While antisense inhibitors of the post-transcriptional targeters of TTP identified here could conceivably be used further to augment TTP regulation of cytokines, it is possible that high levels of TTP are undesirable. Additional research is needed to characterize members of the miRNA/TTP/cytokine regulatory network and identify nodes that may be best targeted therapeutically to ameliorate the effects of chronic inflammation in retrovirus-associated CNS disease.
PMCID: PMC3766027  PMID: 24103357
Cytokine; RNA-binding protein; Tristetraprolin; microRNA; Human immunodeficiency virus; HIV-associated neurocognitive disorder
18.  miR-29a inhibition normalizes HuR over-expression and aberrant AU-rich mRNA stability in invasive cancer 
The Journal of Pathology  2013;230(1):28-38.
The activities of RNA-binding proteins are perturbed in several pathological conditions, including cancer. These proteins include tristetraprolin (TTP, ZFP36) and HuR (ELAVL1), which respectively promote the decay or stability of adenylate-uridylate-rich (AU-rich) mRNAs. Here, we demonstrated that increased stabilization and subsequent over-expression of HuR mRNA were coupled to TTP deficiency. These findings were observed in breast cancer cell lines with an invasive phenotype and were further confirmed in ZFP36-knockout mouse fibroblasts. We show that TTP–HuR imbalance correlated with increased expression of AU-rich element (ARE) mRNAs that code for cancer invasion genes. The microRNA miR-29a was abundant in invasive breast cancer cells when compared to non-tumourigenic cell types. When normal breast cells were treated with miR-29a, HuR mRNA and protein expression were up-regulated. MiR-29a recognized a seed target in the TTP 3′ UTR and a cell-permeable miR-29a inhibitor increased TTP activity towards HuR 3′ UTR. This led to HuR mRNA destabilization and restoration of the aberrant TTP–HuR axis. Subsequently, the cancer invasion factors uPA, MMP-1 and MMP-13, and cell invasiveness, were decreased. The TTP:HuR mRNA ratios were also perturbed in samples from invasive breast cancer patients when compared with normal tissues, and were associated with invasion gene expression. This study demonstrates that an aberrant ARE-mediated pathway in invasive cancer can be normalized by targeting the aberrant and functionally coupled TTP–HuR axis, indicating a potential therapeutic approach. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
PMCID: PMC3732382  PMID: 23401122
cancer invasion; breast cancer; post-transcriptional control; miRNA; mRNA stability; AU-rich elements
19.  Tristetraprolin-driven regulatory circuit controls quality and timing of mRNA decay in inflammation 
Inflammatory gene activation must be rigorously controlled to ensure a rapid, but transient, response. In this work, a regulatory circuit is revealed that governs the destabilization of inflammatory mRNAs and plays an essential role in re-establishing immune homeostasis after inflammatory stimulus.
We describe a regulatory circuit that governs the sequential destabilization of inflammatory mRNAs. This circuit limits potentially deleterious inflammatory mRNA accumulation, yet it prevents premature removal of those mRNAs that are still needed.We show that the sequential destabilization of inflammatory mRNAs is driven by the continuous inverse coupling of p38 MAPK activity profile with the mRNA-destabilizing function of tristetraprolin (TTP) during the entire inflammatory response. This control mechanism ensures that with time, the TTP-dependent mRNA decay gradually spreads resulting in cumulative elimination of 30% of inflammation-induced unstable mRNAs in macrophages.We generated mice with myeloid cell-specific TTP deletion to provide evidence for the function of this regulatory circuit in vivo. These animals are hypersensitive to LPS and display a dysbalanced cytokine production whose pattern is agreement with our model of sequential destabilization the individual mRNAs by TTP.We propose that myeloid TTP is critically involved in the re-installment of immune homeostasis after inflammatory stimulus rather than in the maintenance of steady-state immune homeostasis.
For a successful yet controlled immune response, cells need to specifically destabilize inflammatory mRNAs but prevent premature removal of those still used. The regulatory circuits controlling quality and timing in the global inflammatory mRNA decay are not understood. Here, we show that the mRNA-destabilizing function of the AU-rich element-binding protein tristetraprolin (TTP) is inversely regulated by the p38 MAPK activity profile such that after inflammatory stimulus the TTP-dependent decay is initially limited to few mRNAs. With time, the TTP-dependent decay gradually spreads resulting in cumulative elimination of one third of inflammation-induced unstable mRNAs in macrophages in vitro. We confirmed this sequential decay model in vivo since LPS-treated mice with myeloid TTP ablation exhibited similar cytokine dysregulation profile as macrophages. The mice were hypersensitive to LPS but otherwise healthy with no signs of hyperinflammation seen in conventional TTP knockout mice demonstrating the requirement for myeloid TTP in re-installment but not maintenance of immune homeostasis. These findings reveal a TTP- and p38 MAPK-dominated regulatory mechanism that is vital for balancing acute inflammation by a temporally and qualitatively controlled mRNA decay.
PMCID: PMC3737733  PMID: 22186734
immune homeostasis; inflammation; mRNA stability; p38 MAPK; tristetraprolin
20.  Tristetraprolin Regulates Interleukin-6 Expression Through p38 MAPK-Dependent Affinity Changes with mRNA 3′ Untranslated Region 
Tristetraprolin (TTP) is a well-characterized, zinc finger-containing, RNA-binding protein. TTP targets tumor necrosis factor α for degradation via the 3′ untranslated region (3′UTR). Although AU-rich elements (AREs) in the 3′UTR of interleukin-6 (IL-6) mRNA dictate mRNA degradation, the role of TTP in the post-transcriptional regulation of IL-6 gene expression is unclear. Here we used TTP-deficient mice to test the hypothesis that IL-6 expression is influenced by TTP. Genetic and siRNA-mediated knockdown of TTP resulted in increased IL-6 production and overexpression of TTP had the reverse effect. IL-6 and tumor necrosis factor α production were elevated after injection of IL-1β in TTP-deficient mice. Further, embryonic fibroblasts from these mice (mouse embryonic fibroblasts) exhibited greater IL-6 mRNA expression and longer half-life than wild-type mouse embryonic fibroblasts. Overexpression of TTP reduced IL-6 3′UTR luciferase reporter activity in an ARE-dependent manner. Proximal and distal regions of the 3′UTR acted synergistically to produce the full repression of TTP. Mutation-based luciferase assays show that ARE2, ARE3, and ARE4 are required for TTP-mediated repression. The constitutively activated p38-MK2 pathway abrogated TTP-mediated repression of IL-6 3′UTR reporter activity. RNA immunoprecipitation assay indicated that the deficiency of p38α resulted in the increased affinity of TTP to IL-6 mRNA. Taken together, we propose that TTP downregulates IL-6 gene expression at the post-transcriptional level by targeting ARE elements in the 3′UTR region.
PMCID: PMC3151618  PMID: 21457063
21.  Phosphorylation of Human Tristetraprolin in Response to Its Interaction with the Cbl Interacting Protein CIN85 
PLoS ONE  2010;5(3):e9588.
Tristetraprolin (TTP) is the prototype member of a family of CCCH tandem zinc finger proteins and is considered to be an anti-inflammatory protein in mammals. TTP plays a critical role in the decay of tumor necrosis factor alpha (TNF) mRNA, among others, by binding AU-rich RNA elements in the 3′-untranslated regions of this transcript and promoting its deadenylation and degradation.
Methodology/Principal Findings
We used yeast two-hybrid analysis to identify potential protein binding partners for human TTP (hTTP). Various regions of hTTP recovered 31 proteins that fell into 12 categories based on sequence similarities. Among these, the interactions between hTTP and CIN85, cytoplasmic poly (A) binding protein (PABP), nucleolin and heat shock protein 70 were confirmed by co-immunoprecipitation experiments. CIN85 and hTTP co-localized in the cytoplasm of cells as determined by confocal microscopy. CIN85 contains three SH3 domains that specifically bind a unique proline-arginine motif (PXXXPR) found in several CIN85 effectors. We found that the SH3 domains of CIN85 bound to a PXXXPR motif located near the C-terminus of hTTP. Co-expression of CIN85 with hTTP resulted in the increased phosphorylation of hTTP at serine residues in positions 66 and 93, possibly due in part to the demonstrated association of mitogen-activated protein kinase kinase kinase 4 (MEKK4) to both proteins. The presence of CIN85 did not appear to alter hTTP's binding to RNA probes or its stimulated breakdown of TNF mRNA.
These studies describe interactions between hTTP and nucleolin, cytoplasmic PABP, heat shock protein 70 and CIN85; these interactions were initially discovered by two-hybrid analysis, and confirmed by co-immunoprecipitation. We found that CIN85 binding to a C-terminal motif within hTTP led to the increased phosphorylation of hTTP, possibly through enhanced association with MEKK4. The functional consequences to each of the members of this putative complex remain to be determined.
PMCID: PMC2833206  PMID: 20221403
22.  Chorioallantoic Fusion Defects and Embryonic Lethality Resulting from Disruption of Zfp36L1, a Gene Encoding a CCCH Tandem Zinc Finger Protein of the Tristetraprolin Family 
Molecular and Cellular Biology  2004;24(14):6445-6455.
The mouse gene Zfp36L1 encodes zinc finger protein 36-like 1 (Zfp36L1), a member of the tristetraprolin (TTP) family of tandem CCCH finger proteins. TTP can bind to AU-rich elements within the 3′-untranslated regions of the mRNAs encoding tumor necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), leading to accelerated mRNA degradation. TTP knockout mice exhibit an inflammatory phenotype that is largely due to increased TNF secretion. Zfp36L1 has activities similar to those of TTP in cellular RNA destabilization assays and in cell-free RNA binding and deadenylation assays, suggesting that it may play roles similar to those of TTP in mammalian physiology. To address this question we disrupted Zfp36L1 in mice. All knockout embryos died in utero, most by approximately embryonic day 11 (E11). Failure of chorioallantoic fusion occurred in about two-thirds of cases. Even when fusion occurred, by E10.5 the affected placentas exhibited decreased cell division and relative atrophy of the trophoblast layers. Although knockout embryos exhibited neural tube abnormalities and increased apoptosis within the neural tube and also generalized runting, these and other findings may have been due to deficient placental function. Embryonic expression of Zfp36L1 at E8.0 was greatest in the allantois, consistent with a potential role in chorioallantoic fusion. Fibroblasts derived from knockout embryos had apparently normal levels of fully polyadenylated compared to deadenylated GM-CSF mRNA and normal rates of turnover of this mRNA species, both sensitive markers of TTP deficiency in cells. We postulate that lack of Zfp36L1 expression during mid-gestation results in the abnormal stabilization of one or more mRNAs whose encoded proteins lead directly or indirectly to abnormal placentation and fetal death.
PMCID: PMC434251  PMID: 15226444
23.  Tristetraprolin is required for full anti-inflammatory response of murine macrophages to IL-10 1 
IL-10 is essential for inhibiting chronic and acute inflammation by decreasing the amounts of proinflammatory cytokines made by activated macrophages. IL-10 controls pro-inflammatory cytokine and chemokine production indirectly via the transcription factor Stat3. One of the most physiologically significant IL-10 targets is tumor necrosis factor-alpha (TNFalpha), a potent pro-inflammatory mediator that is the target for multiple anti-TNFalpha clinical strategies in Crohn’s Disease and rheumatoid arthritis. The anti-inflammatory effects of IL-10 seem to be mediated by several incompletely understood transcriptional and posttranscriptional mechanisms. Here we show that in LPS-activated bone marrow-derived murine macrophages, IL-10 reduces the mRNA and protein levels of TNFalpha and IL-1alpha in part through the RNA destabilizing factor tristetraprolin (TTP). TTP is known for its central role in destabilizing mRNA molecules containing class II AU-rich elements in 3′ untranslated regions. We found that IL-10 initiates a Stat3-dependent increase of TTP expression accompanied by a delayed decrease of p38MAPK activity. The reduction of p38MAPK activity releases TTP from the p38MAPK-mediated inhibition thereby resulting in diminished mRNA and protein levels of proinflammatory cytokines. These findings establish that TTP is required for full responses of bone marrow-derived murine macrophages to IL-10.
PMCID: PMC2755621  PMID: 19542371
Monocytes/Macrophages; Cytokines; Inflammation
24.  The p38/MK2-Driven Exchange between Tristetraprolin and HuR Regulates AU–Rich Element–Dependent Translation 
PLoS Genetics  2012;8(9):e1002977.
TNF expression of macrophages is under stringent translational control that depends on the p38 MAPK/MK2 pathway and the AU–rich element (ARE) in the TNF mRNA. Here, we elucidate the molecular mechanism of phosphorylation-regulated translation of TNF. We demonstrate that translation of the TNF-precursor at the ER requires expression of the ARE–binding and -stabilizing factor human antigen R (HuR) together with either activity of the p38 MAPK/MK2 pathway or the absence of the ARE-binding and -destabilizing factor tristetraprolin (TTP). We show that phosphorylation of TTP by MK2 decreases its affinity to the ARE, inhibits its ability to replace HuR, and permits HuR-mediated initiation of translation of TNF mRNA. Since translation of TTP's own mRNA is also regulated by this mechanism, an intrinsic feedback control of the inflammatory response is ensured. The phosphorylation-regulated TTP/HuR exchange at target mRNAs provides a reversible switch between unstable/non-translatable and stable/efficiently translated mRNAs.
Author Summary
For immediate response and better control of gene expression, eukaryotic cells have developed means to specifically regulate the stability and translation of pre-formed mRNA transcripts. This post-transcriptional regulation of gene expression is realized by a variety of mRNA-binding proteins, which target specific mRNA sequence elements in a signal-dependent manner. Here we describe a molecular switch mechanism where the exchange of two mRNA-binding proteins is regulated by stress and inflammatory signals. This switch operates between stabilization and efficient translation of the target mRNA, when the activator protein of translational initiation binds instead of the phosphorylated destabilizing protein, and translational arrest and degradation of the target, when the non-phosphorylated destabilizing protein replaces the activator. This mechanism is specific to the mRNA of the inflammatory cytokine tumor necrosis factor (TNF)-α and the mRNA of its regulator protein TTP and, hence, enables fast inflammatory response and its stringent feedback control.
PMCID: PMC3459988  PMID: 23028373
25.  Interferons limit inflammatory responses by induction of tristetraprolin 
Blood  2006;107(12):4790-4797.
Interferons (IFNs) are cytokines with pronounced proinflammatory properties. Here we provide evidence that IFNs play a key role also in decline of inflammation by inducing expression of tristetraprolin (TTP). TTP is an RNA-binding protein that destabilizes several AU-rich element-containing mRNAs including TNFα. By promoting mRNA decay TTP significantly contributes to cytokine homeostasis. Now we report that IFNs strongly stimulate expression of TTP if a co-stimulatory stress signal is provided. IFN-induced expression of TTP depends on the IFN-activated transcription factor STAT1, and the co-stimulatory stress signal requires p38 MAPK. Within the TTP promoter we have identified a functional gamma interferon-activated sequence that recruits STAT1. Consistently, STAT1 is required for full expression of TTP in response to LPS that stimulates both p38 MAPK and, indirectly, interferon signaling. We demonstrate that in macrophages IFN-induced TTP protein limits LPS-stimulated expression of several proinflammatory genes such as TNFα, IL-6, Ccl2 and Ccl3. Thus, our findings establish a link between interferon responses and TTP-mediated mRNA decay during inflammation, and propose a novel immunomodulatory role of IFNs.
PMCID: PMC3963709  PMID: 16514065
Immunobiology; innate immunity; monocyte and macrophage biology

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