The RNA-binding Protein Tristetraprolin Research Articles

The RNA Binding Protein Tristetraprolin Contributes to CFTR mRNA Stability in Cystic Fibrosis.

Cystic Fibrosis (CF) is the most common inherited disorder and is characterized by an inflammatory phenotype. Here, we found that in bronchial epithelium reconstituted form lung tissue biopsies from patients with CF, the RNA-binding protein tristetraprolin (TTP), a key regulator of inflammation, is dysregulated in cells that strongly express cytokines and interleukins. TTP activity is regulated by extensive post-translational modifications, particularly phosphorylation. We found that in addition to mRNA downregulation, phosphorylated TTP (which cannot bind to mRNA) accumulated in CF cultures, suggesting that the imbalance in TTP phosphorylation status could contribute to the inflammatory phenotype in CF. We confirmed TTP destabilizing role on IL8 mRNA through its 3'UTR sequence in CF cells. We next demonstrated that TTP phosphorylation is mainly regulated by MK2 through activation of ERK, which also was hyperphosphorylated. TTP is considered a mRNA decay factor with some exception, and we present a new positive role of TTP in CF cultures. We determined that TTP binds to specific ARE motifs on the 3'UTR of mRNA sequences and also, for the first time, to the 3'UTR of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) where TTP binding stabilizes the mRNA level. This study identified new partners that can be targeted in CF and proposes a new way to control CFTR gene expression.

Targeting Tristetraprolin Expression or Functional Activity Regulates Inflammatory Response Induced by MSU Crystals.

The RNA-binding protein tristetraprolin (TTP) is an anti-inflammatory factor that prompts the mRNA decay of target mRNAs and is involved in inflammatory diseases such as rheumatoid arthritis (RA). TTP is regulated by phosphorylation, and protein phosphatase 2A (PP2A) can dephosphorylate TTP to activate its mRNA-degrading function. Some small molecules can enhance PP2A activation. Short interfering RNA (siRNA) targeting TTP expression or PP2A agonist (Arctigenin) was administered to monosodium urate (MSU) crystal-induced J774A.1 cells, and the expression of inflammatory related genes was detected by RT-PCR and Western blot assays. The effects of Arctigenin in mouse models of acute inflammation induced by MSU crystals, including peritonitis and arthritis, were evaluated. The data indicated that TTP expression levels and endogenous PP2A activity were increased in MSU-crystal treated J774A.1 cells. TTP knockdown exacerbated inflammation-related genes expression and NLRP3 inflammasome activation. However, PP2A agonist treatment (Arctigenin) suppressed MSU crystal-induced inflammation in J774A.1 cells. Arctigenin also relieved mitochondrial reactive oxygen species (mtROS) production and improved lysosomal membrane permeability in MSU crystal-treated J774A.1 cells. Moreover, TTP knockdown reversed the anti-inflammatory and antioxidant effects of Arctigenin. Oral administration of Arctigenin significantly alleviated foot pad swelling, the number of inflammatory cells in peritoneal lavage fluids and the production of IL-1β in the mouse model of inflammation induced by MSU crystals. Collectively, these data imply that targeting TTP expression or functional activity may provide a potential therapeutic strategy for inflammation caused by MSU crystals.

Role of Tristetraprolin in the Resolution of Inflammation.

Simple SummaryChronic inflammatory diseases account for up to 60% of deaths worldwide and, thus, are considered a great threat for human health by the World Health Organization. Nevertheless, acute inflammatory reactions are an integral part of the host defense against invading pathogens or injuries. To avoid excessive damage due to the persistence of a highly reactive environment, inflammations need to resolve in a coordinate and timely manner, ensuring for the immunological normalization of the affected tissues. Since post-transcriptional regulatory mechanisms are essential for effective resolution, the present review discusses the key role of the RNA-binding and post-transcriptional regulatory protein tristetraprolin in establishing resolution of inflammation.Inflammation is a crucial part of immune responses towards invading pathogens or tissue damage. While inflammatory reactions are aimed at removing the triggering stimulus, it is important that these processes are terminated in a coordinate manner to prevent excessive tissue damage due to the highly reactive inflammatory environment. Initiation of inflammatory responses was proposed to be regulated predominantly at a transcriptional level, whereas post-transcriptional modes of regulation appear to be crucial for resolution of inflammation. The RNA-binding protein tristetraprolin (TTP) interacts with AU-rich elements in the 3′ untranslated region of mRNAs, recruits deadenylase complexes and thereby facilitates degradation of its targets. As TTP regulates the mRNA stability of numerous inflammatory mediators, it was put forward as a crucial post-transcriptional regulator of inflammation. Here, we summarize the current understanding of the function of TTP with a specific focus on its role in adding to resolution of inflammation.

Hepatic tristetraprolin promotes insulin resistance through RNA destabilization of FGF21.

The role of posttranscriptional metabolic gene regulatory programs in diabetes is not well understood. Here, we show that the RNA-binding protein tristetraprolin (TTP) is reduced in the livers of diabetic mice and humans and is transcriptionally induced in response to insulin treatment in murine livers in vitro and in vivo. Liver-specific Ttp-KO (lsTtp-KO) mice challenged with high-fat diet (HFD) have improved glucose tolerance and peripheral insulin sensitivity compared with littermate controls. Analysis of secreted hepatic factors demonstrated that fibroblast growth factor 21 (FGF21) is posttranscriptionally repressed by TTP. Consistent with increased FGF21, lsTtp-KO mice fed HFD have increased brown fat activation, peripheral tissue glucose uptake, and adiponectin production compared with littermate controls. Downregulation of hepatic Fgf21 via an adeno-associated virus-driven shRNA in mice fed HFD reverses the insulin-sensitizing effects of hepatic Ttp deletion. Thus, hepatic TTP posttranscriptionally regulates systemic insulin sensitivity in diabetes through liver-derived FGF21.

Tristetraprolin Overexpression in Gastric Cancer Cells Suppresses PD-L1 Expression and Inhibits Tumor Progression by Enhancing Antitumor Immunity.

The RNA-binding protein tristetraprolin (TTP) binds to adenosine-uridine AU-rich elements in the 3′-untranslated region of messenger RNAs and facilitates rapid degradation of the target mRNAs. Therefore, it regulates the expression of multiple cancer and immunity-associated transcripts. Furthermore, a lack of TTP in cancer cells influences cancer progression and predicts poor survival. Although the functions of TTP on cancer cells have previously been researched, the mechanism of TTP on the interaction between cancer cells with their microenvironment remains undiscovered. In this study, we admed to determine the role of cancer cell TTP during the interaction between tumor and immune cells, specifically regulatory T cells (Tregs). We evaluate the capability of TTP to modulate the antitumor immunity of GC and explored the underlying mechanism. The overexpression of TTP in GC cells dramatically increased peripheral blood mononuclear lymphocyte (PBML) -mediated cytotoxicity against GC cells. Increased cytotoxicity against TTP-overexpressed GC cells by PBMLs was determined by Treg development and infiltration. Surprisingly, we found the stabilization of programmed death-ligand 1 (PD-L1) mRNA was declining while TTP was elevated. The PD-L1 protein level was reduced in TTP-abundant GC cells. PD-L1 gas been found to play a pivotal role in Treg development and functional maintenance in immune system. Taken together, our results suggest the overexpression of TTP in GC cells not only affects cell survival and apoptosis but also increases PBMLs -mediated cytotoxicity against GC cells to decelerate tumor progression. Moreover, we identified PD-L1 as a critical TTP-regulated factor that contributes to inhibiting antitumor immunity.

The RNA-binding protein Tristetraprolin (TTP) is a critical negative regulator of the NLRP3 inflammasome

The NLRP3 inflammasome is a central regulator of inflammation in many common diseases, including atherosclerosis and type 2 diabetes, driving the production of pro-inflammatory mediators such as IL-1β and IL-18. Due to its function as an inflammatory gatekeeper, expression and activation of NLRP3 need to be tightly regulated. In this study, we highlight novel post-transcriptional mechanisms that can modulate NLRP3 expression. We have identified the RNA-binding protein Tristetraprolin (TTP) as a negative regulator of NLRP3 in human macrophages. TTP targets AU-rich elements in the NLRP3 3'-untranslated region (UTR) and represses NLRP3 expression. Knocking down TTP in primary macrophages leads to an increased induction of NLRP3 by LPS, which is also accompanied by increased Caspase-1 and IL-1β cleavage upon NLRP3, but not AIM2 or NLRC4 inflammasome activation. Furthermore, we found that human NLRP3 can be alternatively polyadenylated, producing a short 3'-UTR isoform that excludes regulatory elements, including the TTP- and miRNA-223-binding sites. Because TTP also represses IL-1β expression, it is a dual inhibitor of the IL-1β system, regulating expression of the cytokine and the upstream controller NLRP3.

3’UTR AU-Rich Elements (AREs) and the RNA-Binding Protein Tristetraprolin (TTP) Are Not Required for the LPS-Mediated Destabilization of Phospholipase-Cβ-2 mRNA in Murine Macrophages

We have shown previously that bacterial lipopolysaccharide (LPS)-mediated suppression of phospholipase-Cβ-2 (PLCβ-2) expression is involved in M1 (inflammatory) to M2-like (wound healing) phenotypic switching of macrophages triggered by adenosine. This suppression is mediated post-transcriptionally by destabilization of PLCβ-2 mRNA (messenger ribonucleic acid). To investigate the mechanism of this LPS-mediated destabilization, we examined the roles of RNA-binding agents including microRNAs and RNA-binding proteins that are involved in regulating stability of mRNAs encoding growth factors, inflammatory mediators, and proto-oncogenes. Adenylate and uridylate (AU)-rich elements (AREs) in 3'UTRs are specific recognition sites for RNA-binding proteins including tristetraprolin (TTP), HuR, and AUF1 and for microRNAs that are involved in regulating mRNA stability. In this study, we investigated the role of TTP and AREs in regulating PLCβ-2 mRNA stability. The 3'UTR of the PLCβ-2 gene was inserted into the pLightswitch luciferase reporter plasmid and transfected into RAW264.7 cells. LPS suppressed luciferase expression from this reporter. Luciferase expression from mutant 3'UTR constructs lacking AREs was similarly downregulated, suggesting that these regions are not required for LPS-mediated suppression of PLCβ-2. TTP was rapidly upregulated in both primary murine macrophages and RAW264.7 cells in response to LPS. Suppression of PLCβ-2 by LPS was examined using macrophages from mice lacking TTP (TTP-/-). LPS suppressed PLCβ-2 expression to the same extent in wild type (WT) and TTP-/- macrophages. Also, the rate of decay of PLCβ-2 mRNA in LPS-treated macrophages following transcriptional blockade was similar in WT and TTP-/- macrophages, clearly indicating that TTP is not involved in LPS-mediated destabilization of PLCβ-2 mRNA in macrophages.

Tristetraprolin exerts tumor suppressive functions on the tumorigenesis of glioma by targeting IL-13

The RNA-binding protein tristetraprolin (TTP) is an adenine/uridine (AU)-rich elements (AREs)-binding protein that can induce the decay of AREs containing mRNAs. In this study, we demonstrated that TTP is significantly down-regulated in human glioma tissue samples and cell lines. It is also associated with diminished survival in glioma patients. Gain- and loss-of-function studies demonstrated that TTP inhibited the growth, migration and invasion of glioma cells through regulation of interleukin (IL)-13. Furthermore, mechanistic investigations showed that TTP attenuated activation of PI3K/Akt/mTOR pathway by IL-13, and the ectopic expression of IL-13 markedly abrogated the anti-invasive effect of TTP. Additionally, TTP were found inversely correlated with IL-13 in glioma specimens. In conclusion, our results suggested that the low expression of TTP is significantly associated with the growth and metastasis of human glioma cells by targeting IL-13, while TTP may be a potential therapeutic target for glioma treatment.

Destabilization of the ornithine decarboxylase mRNA transcript by the RNA-binding protein tristetraprolin.

Ornithine decarboxylase (ODC) is the first and usually rate-limiting enzyme in the polyamine biosynthetic pathway. In a normal physiological state, ODC is tightly regulated. However, during neoplastic transformation, ODC expression becomes upregulated. The studies described here show that the ODC mRNA transcript is destabilized by the RNA-binding protein tristetraprolin (TTP). We show that TTP is able to bind to the ODC mRNA transcript in both non-transformed RIE-1 cells and transformed Ras12V cells. Moreover, using mouse embryonic fibroblast cell lines that are devoid of a functional TTP protein, we demonstrate that in the absence of TTP both ODC mRNA stability and ODC enzyme activity increase when compared to wild-type cells. Finally, we show that the ODC 3' untranslated region contains cis acting destabilizing elements that are affected by, but not solely dependent on, TTP expression. Together, these data support the hypothesis that TTP plays a role in the post-transcriptional regulation of the ODC mRNA transcript.

Tristetraprolin induces cell cycle arrest in breast tumor cells through targeting AP-1/c-Jun and NF-κB pathway.

The main characteristic of cancers, including breast cancer, is the ability of cancer cells to proliferate uncontrollably. However, the underlying mechanisms of cancer cell proliferation, especially those regulated by the RNA binding protein tristetraprolin (TTP), are not completely understood. In this study, we found that TTP inhibits cell proliferation in vitro and suppresses tumor growth in vivo through inducing cell cycle arrest at the S phase. Our studies demonstrate that TTP inhibits c-Jun expression through the C-terminal Zn finger and therefore increases Wee1 expression, a regulatory molecule which controls cell cycle transition from the S to the G2 phase. In contrast to the well-known function of TTP in regulating mRNA stability, TTP inhibits c-Jun expression at the level of transcription by selectively blocking NF-κB p65 nuclear translocation. Reconstitution of NF-κB p65 completely abolishes the inhibition of c-Jun transcription by TTP. Moreover, reconstitution of c-Jun in TTP-expressing breast tumor cells diminishes Wee1 overexpression and promotes cell proliferation. Our results indicate that TTP suppresses c-Jun expression that results in Wee1 induction which causes cell cycle arrest at the S phase and inhibition of cell proliferation. Our study provides a new pathway for TTP function as a tumor suppressor which could be targeted in tumor treatment.

Histone Deacetylase Inhibitors Activate Tristetraprolin Expression through Induction of Early Growth Response Protein 1 (EGR1) in Colorectal Cancer Cells.

The RNA-binding protein tristetraprolin (TTP) promotes rapid decay of mRNAs bearing 3' UTR AU-rich elements (ARE). In many cancer types, loss of TTP expression is observed allowing for stabilization of ARE-mRNAs and their pathologic overexpression. Here we demonstrate that histone deacetylase (HDAC) inhibitors (Trichostatin A, SAHA and sodium butyrate) promote TTP expression in colorectal cancer cells (HCA-7, HCT-116, Moser and SW480 cells) and cervix carcinoma cells (HeLa). We found that HDAC inhibitors-induced TTP expression, promote the decay of COX-2 mRNA, and inhibit cancer cell proliferation. HDAC inhibitors were found to promote TTP transcription through activation of the transcription factor Early Growth Response protein 1 (EGR1). Altogether, our findings indicate that loss of TTP in tumors occurs through silencing of EGR1 and suggests a therapeutic approach to rescue TTP expression in colorectal cancer.

Abstract 3197: RNase-L modulates transcriptional and post-transcriptional responses to mitogenic stimuli through serum response factor and tristetraprolin.

Abstract RNase-L is an endoribonuclease that functions in a range of important physiologic activities including the innate immune response and control of cell proliferation and is dysregulated in pathologic conditions including cancer. RNase-L is tightly regulated and requires the binding of 2’,5’-linked oligoadenylates (2-5A: px5′A(2′p5′A)n; x=1-2;n>2) to induce oligomerization and enzymatic activity. Activated RNase-L cleaves single-stranded regions of cellular and pathogen RNAs, and distinct profiles of RNA are targeted by RNase-L to mediate its biologic functions in different cellular contexts. Therefore an understanding of the mechanism(s) by which specific RNAs are targeted for RNase-L cleavage is essential to modulate its substrate selection and biologic activity for therapeutic applications. Towards this goal, we identified the RNA binding protein tristetraprolin (TTP) as a novel RNase-L interacting partner. TTP binds to A-U-rich elements (AREs) in the 3’-untranslated region (3’UTR) of target mRNAs, including its own mRNA, to promote their degradation. We hypothesize that TTP functions to direct RNase-L cleavage of a subset of ARE mRNAs. Consistent with this prediction, RNase-L interacted with TTP mRNA, and decreased steady-state levels and increased half-life of TTP mRNA in RNase-L-/- fibroblasts. In addition to this direct effect on basal TTP mRNA stability, RNase-L inhibited the transcriptional induction of TTP primary transcripts following serum stimulation. In this context, we determined that RNase-L targeted the mRNA encoding serum response factor (SRF), a transcription factor that induces the expression of proliferation and tumorigenesis-associated genes. The TTP promoter contains SRF binding sites suggesting that the RNase-L-mediated downregulation of SRF indirectly reduced TTP transcription; experimental validation of this regulation is in progress. Together these data support a model in which following mitogen stimulation, RNase-L constrains the transcriptional induction of proliferation-associated genes via its regulation of SRF and interacts with TTP to post-transcriptionally downregulate an overlapping set of mRNAs. The RNase-L-dependent regulation of TTP, which itself functions to limit the mitogenic response, suggests that RNase-L contributes to proliferative homeostasis through the regulation of both positive and negative effectors. These findings have implications for the therapeutic targeting of RNase-L-mediated cleavage in regenerative repair and tumorigenesis. Citation Format: Sarah E. Brennan-Laun, X-L Li, Heather J. Ezelle, Gerald M. Wilson, Bret A. Hassel. RNase-L modulates transcriptional and post-transcriptional responses to mitogenic stimuli through serum response factor and tristetraprolin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3197. doi:10.1158/1538-7445.AM2013-3197

Depletion of tristetraprolin in breast cancer cells increases interleukin-16 expression and promotes tumor infiltration with monocytes/macrophages

The RNA-binding protein tristetraprolin (TTP) destabilizes target messenger RNAs (mRNAs) containing AU-rich elements within their 3' untranslated region. Thereby, it controls the expression of multiple inflammatory and tumor-associated transcripts. Moreover, a loss of TTP in tumors predicts disease-associated survival. Although tumor intrinsic functions of TTP have previously been studied, the impact of TTP on the interaction of tumors with their microenvironment remains elusive. As immune cell infiltration into tumors is a critical determinant for tumor progression, this study aimed at determining the influence of tumor cell TTP on the interaction between tumor and immune cells, specifically monocytes (MO)/macrophages (MΦ). Knockdown (k/d) of TTP in T47D breast cancer cells enhanced tumor growth both in vitro and in vivo and increased infiltration of MO into 3D tumor spheroids in vitro and of MΦ into tumor xenografts in vivo. Enhanced migration of MO toward supernatants of TTP-deficient tumor spheroids was determined as the underlying principle. Interestingly, we noticed interleukin-16 (IL-16) mRNA stabilization when TTP was depleted. In line, IL-16 protein levels were elevated in TTP-deficient spheroids and their supernatants as well as in TTP k/d tumor xenografts and critically contributed to the enhanced chemotactic behavior. In summary, we show that the loss of TTP in tumors not only affects tumor cell proliferation and survival but also enhances infiltration of MO/MΦ into the tumors, which is typically associated with poor prognosis. Moreover, we identified IL-16 as a critical TTP-regulated chemotactic factor that contributes to MO/MΦ migration.

Cutting Edge: IL-10–Mediated Tristetraprolin Induction Is Part of a Feedback Loop That Controls Macrophage STAT3 Activation and Cytokine Production

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 posttranscriptionally 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.

A Combination of Hypoxia and Lipopolysaccharide Activates Tristetraprolin to Destabilize Proinflammatory mRNAs such as Tumor Necrosis Factor-α

Inflammation is often accompanied by hypoxia because of the high oxygen consumption of invading bacteria and immune cells. During resolution of inflammation, the formation of inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha), which is produced by macrophages, needs to be terminated. We show in RAW264.7 macrophages that TNF-alpha mRNA as well as intracellular and secreted TNF-alpha protein levels are reduced after prolonged incubations with lipopolysaccharide (LPS) under hypoxic conditions. The decrease in TNF-alpha was mediated by destabilization of TNF-alpha mRNA via a 3'-untranslated region-dependent mechanism. Specifically, the RNA-binding protein tristetraprolin (TTP) increased at mRNA and protein levels after 16-hour incubations with LPS under hypoxia. Interestingly, TTP accumulated in a dephosphorylated and active form, and this accumulation was attributable to reduced p38 mitogen-activated protein kinase activity under these conditions. Knockdown of TTP by small interfering RNA abolished destabilization of TNF-alpha mRNA. Prolonged incubations with LPS under hypoxia also reduced mRNA amounts and stability of other proinflammatory mediators such as macrophage inflammatory protein-2, interleukin-6, and granulocyte macrophage colony-stimulating factor. Therefore, we propose that hypoxia plays a key role during resolution of inflammation by activating posttranscriptional, TTP-dependent regulatory mechanisms.

IL-17 Regulates CXCL1 mRNA Stability via an AUUUA/Tristetraprolin-Independent Sequence

IL-17 contributes to inflammatory response in part by promoting enhanced expression of chemokines, such as CXCL1, by prolonging the t(1/2) of this constitutively unstable mRNA. Although IL-17 is a weak stimulus for transcription of the CXCL1 gene, it strongly potentiates message accumulation via stabilization when the mRNA is transcribed in cells stimulated with TNF. In myeloid cells, LPS-induced CXCL1 mRNA stabilization is dependent on AUUUA-containing sequence motifs that are recognized by the RNA binding protein tristetraprolin (TTP). Using deletion and site-specific mutagenesis, we report that IL-17-mediated stabilization of CXCL1 mRNA in nonmyeloid cells depends on a sequence that does not contain the AUUUA motif. Furthermore, a specific two-nucleotide mutation within this region markedly abrogates sensitivity for IL-17-mediated stabilization. Consistent with this finding, the IL-17-sensitive sequence does not exhibit increased instability in the presence of TTP, and CXCL1 mRNA remains unstable and can be stabilized in response to treatment with IL-17 in embryo fibroblasts from mice in which the TTP gene has been deleted. Whereas the RNA binding protein KSRP has been shown to participate in regulating the instability of human CXCL8 mRNA, inhibitory RNA-based reduction in KSRP does not effect the instability mediated by the IL-17-sensitive sequence motif. These findings suggest that IL-17-mediated chemokine mRNA stabilization in nonmyeloid cells uses a mechanism that is distinct from that operating to control AU-rich mRNA stability in myeloid cells.

Role of the RNA-binding protein tristetraprolin in glucocorticoid-mediated gene regulation.

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.

Role of the RNA-binding Protein Tristetraprolin (TTP) in Glucocorticoid (GC)-mediated Gene Regulation

Role of the RNA-binding Protein Tristetraprolin (TTP) in Glucocorticoid (GC)-mediated Gene Regulation

Tristetraprolin regulates Cyclin D1 and c-Myc mRNA stability in response to rapamycin in an Akt-dependent manner via p38 MAPK signaling

The differential expression of the critical cell cycle control proteins cyclin D1 and c-myc has been shown to result in Akt-dependent hypersensitivity of tumor cells to mTOR inhibitors. We have previously demonstrated that the differential utilization of internal ribosome entry sites within the mRNAs of these transcripts allows maintenance of protein synthesis in the face of rapamycin (rapa) exposure in an Akt-dependent manner. Here, we demonstrate that in addition to this mechanism, cyclin D1 and c-myc mRNA stability is also coordinately regulated following rapa treatment depending on Akt activity status. We identify A/U-rich response elements within the 3' untranslated regions (UTRs) of these transcripts, which confer the observed differential stabilities and show that the RNA-binding protein, tristetraprolin (TTP), interacts with these elements. We also present evidence that TTP accumulates in response to rapa exposure, binds to the cis-acting elements within the cyclin D1 and c-myc 3' UTRs and is differentially serine phosphorylated in an Akt-dependent manner. Furthermore, the differential phosphorylation status of TTP results in its sequestration by 14-3-3 proteins in quiescent Akt-containing cells. Finally, siRNA-mediated knockdown of TTP expression or inhibiting a known regulator of TTP phosphorylation, p38 MAP kinase, abolishes the effects on cyclin D1 and c-myc mRNA stability. We assume that the differential control of cyclin D1 and c-myc mRNA stability and translational efficiency constitutes a coordinate response to rapa contributing to the maintenance of expression of these determinants in rapa-resistant quiescent Akt-containing cells following exposure.