Deficiency of Tristetraprolin Triggers Hyperthermia through Enhancing Hypothalamic Inflammation.

Tristetraprolin (TTP), an RNA-binding protein, controls the stability of RNA by capturing AU-rich elements on their target genes. It has recently been identified that TTP serves as an anti-inflammatory protein by guiding the unstable mRNAs of pro-inflammatory proteins in multiple cells. However, it has not yet been investigated whether TTP affects the inflammatory responses in the hypothalamus. Since hypothalamic inflammation is tightly coupled to the disturbance of energy homeostasis, we designed the current study to investigate whether TTP regulates hypothalamic inflammation and thereby affects energy metabolism by utilizing TTP-deficient mice. We observed that deficiency of TTP led to enhanced hypothalamic inflammation via stimulation of a variety of pro-inflammatory genes. In addition, microglial activation occurred in the hypothalamus, which was accompanied by an enhanced inflammatory response. In line with these molecular and cellular observations, we finally confirmed that deficiency of TTP results in elevated core body temperature and energy expenditure. Taken together, our findings unmask novel roles of hypothalamic TTP on energy metabolism, which is linked to inflammatory responses in hypothalamic microglial cells.

Tristetraprolin Overexpression in Non-hematopoietic Cells Protects Against Acute Lung Injury in Mice.

Tristetraprolin (TTP) is a mRNA binding protein that binds to adenylate-uridylate-rich elements within the 3' untranslated regions of certain transcripts, such as tumor necrosis factor (Tnf) mRNA, and increases their rate of decay. Modulation of TTP expression is implicated in inflammation; however, its role in acute lung inflammation remains unknown. Accordingly, we tested the role of TTP in lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. LPS-challenged TTP-knockout (TTPKO) mice, as well as myeloid cell-specific TTP-deficient (TTPmyeKO) mice, exhibited significant increases in lung injury, although these responses were more robust in the TTPKO. Mice with systemic overexpression of TTP (TTPΔARE) were protected from ALI, as indicated by significantly reduced neutrophilic infiltration, reduced levels of neutrophil chemoattractants, and histological parameters of ALI. Interestingly, while irradiated wild-type (WT) mice reconstituted with TTPKO hematopoietic progenitor cells (HPCs) showed exaggerated ALI, their reconstitution with the TTPΔARE HPCs mitigated ALI. The reconstitution of irradiated TTPΔARE mice with HPCs from either WT or TTPΔARE donors conferred significant protection against ALI. In contrast, irradiated TTPΔARE mice reconstituted with TTPKO HPCs had exaggerated ALI, but the response was milder as compared to WT recipients that received TTPKO HPCs. Finally, the reconstitution of irradiated TTPKO recipient mice with TTPΔARE HPCs did not confer any protection to the TTPKO mice. These data together suggest that non-HPCs-specific overexpression of TTP within the lungs protects against ALI via downregulation of neutrophil chemoattractants and reduction in neutrophilic infiltration.

Tristetraprolin Regulates TH17 Cell Function and Ameliorates DSS-Induced Colitis in Mice.

TH17 cells have been extensively investigated in inflammation, autoimmune diseases, and cancer. The precise molecular mechanisms for TH17 cell regulation, however, remain elusive, especially regulation at the post-transcriptional level. Tristetraprolin (TTP) is an RNA-binding protein important for degradation of the mRNAs encoding several proinflammatory cytokines. With newly generated T cell-specific TTP conditional knockout mice (CD4CreTTPf/f), we found that aging CD4CreTTPf/f mice displayed an increase of IL-17A in serum and spontaneously developed chronic skin inflammation along with increased effector TH17 cells in the affected skin. TTP inhibited TH17 cell development and function by promoting IL-17A mRNA degradation. In a DSS-induced colitis model, CD4CreTTPf/f mice displayed severe colitis and had more TH17 cells and serum IL-17A compared with wild-type mice. Furthermore, neutralization of IL-17A reduced the severity of colitis. Our results reveal a new mechanism for regulating TH17 function and TH17-mediated inflammation post-transcriptionally by TTP, suggests that TTP might be a novel therapeutic target for the treatment of TH17-mediated diseases.

Tristetraprolin regulates necroptosis during tonic Toll-like receptor 4 (TLR4) signaling in murine macrophages.

The necrosome is a protein complex required for signaling in cells that results in necroptosis, which is also dependent on tumor necrosis factor receptor (TNF-R) signaling. TNFα promotes necroptosis, and its expression is facilitated by mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2) but is inhibited by the RNA-binding protein tristetraprolin (TTP, encoded by the Zfp36 gene). We have stimulated murine macrophages from WT, MyD88-/-, Trif-/-, MyD88-/-Trif-/-, MK2-/-, and Zfp36-/- mice with graded doses of lipopolysaccharide (LPS) and various inhibitors to evaluate the role of various genes in Toll-like receptor 4 (TLR4)-induced necroptosis. Necrosome signaling, cytokine production, and cell death were evaluated by immunoblotting, ELISA, and cell death assays, respectively. We observed that during TLR4 signaling, necrosome activation is mediated through the adaptor proteins MyD88 and TRIF, and this is inhibited by MK2. In the absence of MK2-mediated necrosome activation, lipopolysaccharide-induced TNFα expression was drastically reduced, but MK2-deficient cells became highly sensitive to necroptosis even at low TNFα levels. In contrast, during tonic TLR4 signaling, WT cells did not undergo necroptosis, even when MK2 was disabled. Of note, necroptosis occurred only in the absence of TTP and was mediated by the expression of TNFα and activation of JUN N-terminal kinase (JNK). These results reveal that TTP plays an important role in inhibiting TNFα/JNK-induced necrosome signaling and resultant cytotoxicity.

Tristetraprolin Is Required for Alveolar Bone Homeostasis.

Tristetraprolin (TTP) is an RNA-binding protein that targets numerous immunomodulatory mRNA transcripts for degradation. Many TTP targets are key players in the pathogenesis of periodontal bone loss, including tumor necrosis factor-α. To better understand the extent that host immune factors play during periodontal bone loss, we assessed alveolar bone levels, inflammation and osteoclast activity in periodontal tissues, and immune response in draining cervical lymph nodes in TTP-deficient and wild-type (WT) mice in an aging study. WT and TTP-deficient (knockout [KO]) mice were used for all studies under specific pathogen-free conditions. Data were collected on mice aged 3, 6, and 9 mo. Microcomputed tomography (µCT) was performed on maxillae where 3-dimensional images were generated and bone loss was assessed. Decalcified sections of specimens were scored for inflammation and stained with tartrate-resistant acid phosphate (TRAP) to visualize osteoclasts. Immunophenotyping was performed on single-cell suspensions isolated from primary and peripheral lymphoid tissues using flow cytometry. Results presented indicate that TTP KO mice had significantly more alveolar bone loss over time compared with WT controls. Bone loss was associated with significant increases in inflammatory cell infiltration and an increased percentage of alveolar bone surfaces apposed with TRAP+ cells. Furthermore, it was found that the draining cervical lymph nodes were significantly enlarged in TTP-deficient animals and contained a distinct pathological immune profile compared with WT controls. Finally, the oral microbiome in the TTP KO mice was significantly different with age from WT cohoused mice. The severe bone loss, inflammation, and increased osteoclast activity observed in these mice support the concept that TTP plays a critical role in the maintenance of alveolar bone homeostasis in the presence of oral commensal flora. This study suggests that TTP is required to inhibit excessive inflammatory host responses that contribute to periodontal bone loss, even in the absence of specific periodontal pathogens.

Impaired insulin signaling upon loss of ovarian function is associated with a reduction of tristetraprolin and an increased stabilization of chemokine in adipose tissue.

Loss of ovarian function can activate inflammation and lead to insulin resistance (IR). IR is also a core feature of obesity and obesity-associated metabolic dysfunction. Tristetraprolin/zinc finger protein 36 (TTP) interferes with TNF-α production by destabilizing TNF-α mRNA, and mice deficient in TTP develop a complex syndrome of inflammatory disease (Carballo et al., 1998; Taylor et al., 1999). We hypothesized that ovariectomy (OVX) might also prime inflammation by reducing tristetraprolin/zinc finger protein 36 (TTP) levels. We used a mouse OVX model to study impaired insulin signaling due to loss of ovarian function by evaluating Akt activity upon insulin stimulus. Impaired insulin signaling was initially detected in adipose tissue (AT) at 4 weeks after OVX, and then spread to liver and muscle, finally resulting in systemic IR at 12 weeks after OVX. OVX decreased TTP protein levels and increased adipocyte size, oxidative stress, chemokine expression and fat mass in AT by 4 weeks after surgery. TTP deficiency due to TTP gene deletion induced aberrant insulin signaling and increased chemokine expression and macrophage numbers in AT but did not increase adipocyte size, oxidative stress, or fat mass, suggesting that it promotes insulin signaling by decreasing AT inflammation independent of oxidative stress and adiposity. OVX, like TTP deficiency, increased the stability of chemokine transcripts as assessed from their half-lives. Our data indicate that the impaired insulin signaling resulting from OVX is due to an OVX-induced reduction of TTP and the resulting stabilization of inflammatory chemokines.

The RNA-Binding Proteins Zfp36l1 and Zfp36l2 Enforce the Thymic ß-Selection Checkpoint by Limiting DNA Damage Response Signaling and Cell Cycle Progression.

The RNA-binding proteins Zfp36l1 and Zfp36l2 act redundantly to enforce the ß-selection checkpoint during thymopoiesis, yet their molecular targets remain largely unknown. In this study, we identify these targets on a genome-wide scale in primary mouse thymocytes and show that Zfp36l1/l2 regulate DNA damage response and cell cycle transcripts to ensure proper ß-selection. Double-negative 3 thymocytes lacking Zfp36l1/l2 share a gene expression profile with postselected double-negative 3b cells despite the absence of intracellular TCRß and reduced IL-7 signaling. Our findings show that in addition to controlling the timing of proliferation at ß-selection, posttranscriptional control by Zfp36l1/l2 limits DNA damage responses, which are known to promote thymocyte differentiation. Zfp36l1/l2 therefore act as posttranscriptional safeguards against chromosomal instability and replication stress by integrating pre-TCR and IL-7 signaling with DNA damage and cell cycle control.

Deficiency of the placenta- and yolk sac-specific tristetraprolin family member ZFP36L3 identifies likely mRNA targets and an unexpected link to placental iron metabolism.

The ZFP36L3 protein is a rodent-specific, placenta- and yolk sac-specific member of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins. These proteins bind to AU-rich elements in target mRNAs, and promote their deadenylation and decay. We addressed the hypotheses that the absence of ZFP36L3 would result in the accumulation of target transcripts in placenta and/or yolk sac, and that some of these would be important for female reproductive physiology and overall fecundity. Mice deficient in ZFP36L3 exhibited decreased neonatal survival rates, but no apparent morphological changes in the placenta or surviving offspring. We found Zfp36l3 to be paternally imprinted, with profound parent-of-origin effects on gene expression. The protein was highly expressed in the syncytiotrophoblast cells of the labyrinth layer of the placenta, and the epithelial cells of the yolk sac. RNA-Seq of placental mRNA from Zfp36l3 knockout (KO) mice revealed many significantly upregulated transcripts, whereas there were few changes in KO yolk sacs. Many of the upregulated placental transcripts exhibited decreased decay rates in differentiated trophoblast stem cells derived from KO blastocysts. Several dozen transcripts were deemed high probability targets of ZFP36L3; these include proteins known to be involved in trophoblast and placenta physiology. Type 1 transferrin receptor mRNA was unexpectedly decreased in KO placentas, despite an increase in its stability in KO stem cells. This receptor is crucial for placental iron uptake, and its decrease was accompanied by decreased iron stores in the KO fetus, suggesting that this intrauterine deficiency might have deleterious consequences in later life.

Tristetraprolin (TTP) coordinately regulates primary and secondary cellular responses to proinflammatory stimuli.

TTP is an anti-inflammatory protein that acts by binding to AREs in its target mRNAs, such as Tnf mRNA, and promoting their deadenylation and decay. TNF released from inflammatory cells can then stimulate gene expression in tissue cells, such as fibroblasts. To determine whether TTP could affect the decay of TNF-induced transcripts in fibroblasts, we exposed primary embryonic fibroblasts and stable fibroblast cell lines, derived from WT and TTP KO mice, to TNF. The decay rates of transcripts encoded by several early-response genes, including Cxcl1, Cxcl2, Ier3, Ptgs2, and Lif, were significantly slowed in TTP-deficient fibroblasts after TNF stimulation. These changes were associated with TTP-dependent increases in CXCL1, CXCL2, and IER3 protein levels. The TTP-susceptible transcripts contained multiple, conserved, closely spaced, potential TTP binding sites in their 3'-UTRs. WT TTP, but not a nonbinding TTP zinc finger mutant, bound to RNA probes that were based on the mRNA sequences of Cxcl1, Cxcl2, Ptgs2, and Lif. TTP-promoted decay of transcripts encoding chemokines and other proinflammatory mediators is thus a critical post-transcriptional regulatory mechanism in the response of secondary cells, such as fibroblasts, to TNF released from primary immune cells.

Tristetraprolin mediates anti-inflammatory effect of carbon monoxide against DSS-induced colitis.

Endogenous carbon monoxide (CO) exerts anti-inflammatory effects. Tristetraprolin (TTP) is known to destabilize pro-inflammatory transcripts. Here we found that exogenous CO enhanced the decay of TNF-α mRNA and suppressed TNF-α expression in LPS-activated macrophages from wild-type (WT) mice. However, TTP deficiency abrogated the effects of exogenous CO. While CO treatment prior to DSS administration in WT mice significantly reduced inflammatory cytokine levels and colitis, it failed to reduce the pro-inflammatory cytokine levels and colitis in TTP knockout (KO) mice. Our results demonstrate that TTP is a key factor mediating the anti-inflammatory action of CO in DSS-induced colitis.

Differential post-transcriptional regulation of IL-10 by TLR2 and TLR4-activated macrophages.

The activation of TLRs by microbial molecules triggers intracellular-signaling cascades and the expression of cytokines such as IL-10. Il10 expression is tightly controlled to ensure effective immune responses, while preventing pathology. Maximal TLR-induction of Il10 transcription in macrophages requires signaling through the MAPKs, ERK, and p38. Signals via p38 downstream of TLR4 activation also regulate IL-10 at the post-transcriptional level, but whether this mechanism operates downstream of other TLRs is not clear. We compared the regulation of IL-10 production in TLR2 and TLR4-stimulated BM-derived macrophages and found different stability profiles for the Il10 mRNA. TLR2 signals promoted a rapid induction and degradation of Il10 mRNA, whereas TLR4 signals protected Il10 mRNA from rapid degradation, due to the activation of Toll/IL-1 receptor domain-containing adaptor inducing IFN-ß (TRIF) and enhanced p38 signaling. This differential post-transcriptional mechanism contributes to a stronger induction of IL-10 secretion via TLR4. Our study provides a molecular mechanism for the differential IL-10 production by TLR2- or TLR4-stimulated BMMs, showing that p38-induced stability is not common to all TLR-signaling pathways. This mechanism is also observed upon bacterial activation of TLR2 or TLR4 in BMMs, contributing to IL-10 modulation in these cells in an infection setting.

Suppression of IL-12 production by tristetraprolin through blocking NF-kcyB nuclear translocation.

Tristetraprolin (TTP), an mRNA-binding protein, plays a significant role in regulating the expression of adenylate-uridylate-rich elements containing mRNAs. Mice deficient of TTP (TTP(-/-)) develop a systemic autoimmune inflammatory syndrome characterized by cachexia, conjunctivitis, and dermatitis. IL-12 plays a crucial role in immune defense against infectious and malignant diseases. In this study, we found increased production of IL-12 during endotoxic shock and enhanced Th1 cells in TTP knockout mice. The levels of IL-12 p70 and p40 protein as well as p40 and p35 mRNA were also increased in activated macrophages deficient of TTP. In line with these findings, overexpression of TTP suppressed IL-12 p35 and p40 expression at the mRNA and promoter level, whereas it surprisingly had little effects on their mRNA stability. Our data showed that the inhibitory effects of TTP on p35 gene transcription were completely rescued by overexpression of NF-кB p65 and c-Rel but not by the p50 in activated macrophages. Our data further indicated that TTP acquired its inhibition on IL-12 expression through blocking nuclear translocation of NF-кB p65 and c-Rel while enhancing p50 upon stimulation. In summary, our study reveals a novel pathway through which TTP suppresses IL-12 production in macrophages, resulting in suppression of Th1 cell differentiation. This study may provide us with therapeutic targets for treatment of inflammatory and autoimmune disorders.

Tristetraprolin regulation of interleukin 23 mRNA stability prevents a spontaneous inflammatory disease.

Interleukin (IL) 12 and IL23 are two related heterodimeric cytokines produced by antigen-presenting cells. The balance between these two cytokines plays a crucial role in the control of Th1/Th17 responses and autoimmune inflammation. Most studies focused on their transcriptional regulation. Herein, we explored the role of the adenine and uridine-rich element (ARE)-binding protein tristetraprolin (TTP) in influencing mRNA stability of IL12p35, IL12/23p40, and IL23p19 subunits. LPS-stimulated bone marrow-derived dendritic cells (BMDCs) from TTP(-/-) mice produced normal levels of IL12/23p40. Production of IL12p70 was modestly increased in these conditions. In contrast, we observed a strong impact of TTP on IL23 production and IL23p19 mRNA stability through several AREs in the 3' untranslated region. TTP(-/-) mice spontaneously develop an inflammatory syndrome characterized by cachexia, myeloid hyperplasia, dermatitis, and erosive arthritis. We observed IL23p19 expression within skin lesions associated with exacerbated IL17A and IL22 production by infiltrating γδ T cells and draining lymph node CD4 T cells. We demonstrate that the clinical and immunological parameters associated with TTP deficiency were completely dependent on the IL23-IL17A axis. We conclude that tight control of IL23 mRNA stability by TTP is critical to avoid severe inflammation.

Life without TTP: apparent absence of an important anti-inflammatory protein in birds.

Both innate and adaptive immunity in birds are different from their mammalian counterparts. Understanding bird immunity is important because of the enormous potential impact of avian infectious diseases, both in their role as food animals and as potential carriers of zoonotic diseases in man. The anti-inflammatory protein tristetraprolin (TTP) is an important component of the mammalian innate immune response, in that it binds to and destabilizes key cytokine mRNAs. TTP knockout mice exhibit a severe systemic inflammatory syndrome, and they are abnormally sensitive to innate immune stimuli such as LPS. TTP orthologs have been found in most vertebrates studied, including frogs. Here, we attempted to identify TTP orthologs in chicken and other birds, using database searches and deep mRNA sequencing. Although sequences encoding the two other widely expressed TTP family members, ZFP36L1 and ZFP36L2, were identified, we did not find sequences corresponding to TTP in any bird species. Sequences corresponding to TTP were identified in both lizards and alligators, close evolutionary relatives of birds. The induction kinetics of Zfp36l1 and Zfp36l2 mRNAs in LPS-stimulated chicken macrophages or serum-stimulated chick embryo fibroblasts did not resemble the normal mammalian TTP response to these stimuli, suggesting that the other two family members might not compensate for the TTP deficiency in regulating rapidly induced mRNA targets. Several mammalian TTP target transcripts have chicken counterparts that contain one or more potential TTP binding sites, raising the possibility that birds express other proteins that subsume TTP's function as a rapidly inducible regulator of AU-rich element (ARE)-dependent mRNA turnover.

ZFP36L2 is required for self-renewal of early burst-forming unit erythroid progenitors.

Stem cells and progenitors in many lineages undergo self-renewing divisions, but the extracellular and intracellular proteins that regulate this process are largely unknown. Glucocorticoids stimulate red blood cell formation by promoting self-renewal of early burst-forming unit-erythroid (BFU-E) progenitors. Here we show that the RNA-binding protein ZFP36L2 is a transcriptional target of the glucocorticoid receptor (GR) in BFU-Es and is required for BFU-E self-renewal. ZFP36L2 is normally downregulated during erythroid differentiation from the BFU-E stage, but its expression is maintained by all tested GR agonists that stimulate BFU-E self-renewal, and the GR binds to several potential enhancer regions of ZFP36L2. Knockdown of ZFP36L2 in cultured BFU-E cells did not affect the rate of cell division but disrupted glucocorticoid-induced BFU-E self-renewal, and knockdown of ZFP36L2 in transplanted erythroid progenitors prevented expansion of erythroid lineage progenitors normally seen following induction of anaemia by phenylhydrazine treatment. ZFP36L2 preferentially binds to messenger RNAs that are induced or maintained at high expression levels during terminal erythroid differentiation and negatively regulates their expression levels. ZFP36L2 therefore functions as part of a molecular switch promoting BFU-E self-renewal and a subsequent increase in the total numbers of colony-forming unit-erythroid (CFU-E) progenitors and erythroid cells that are generated.

LPS-induced production of TNF-α and IL-6 in mast cells is dependent on p38 but independent of TTP.

The production of the proinflammatory cytokines TNF-α and IL-6 is regulated by various mRNA-binding proteins, influencing stability and translation of the respective transcripts. Research in macrophages has shown the importance of the p38-MK2-tristetraprolin (TTP) axis for regulation of TNF-α mRNA stability and translation. In the current study we examined a possible involvement of p38 and TTP in LPS-induced cytokine production in bone marrow-derived mast cells (BMMCs). Using pharmacological inhibitors we initially found a strong dependence of LPS-induced TNF-α and IL-6 production on p38 activation, whereas activation of the Erk pathway appeared dispensable. LPS treatment also induced p38-dependent expression of the TTP gene. This prompted us to analyze the proinflammatory cytokine response in BMMCs generated from TTP-deficient mice. Unexpectedly, there were no significant differences in cytokine production between TTP-deficient and WT BMMCs in response to LPS. Gene expression and cytokine production of TNF-α and IL-6 as well as stability of the TNF-α transcript were comparable between TTP-deficient and WT BMMCs. In contrast to TTP mRNA expression, TTP protein expression could not be detected in BMMCs. While we successfully precipitated and detected TTP from lysates of LPS-stimulated RAW 264.7 macrophages, this was not accomplished from BMMC lysates. In contrast, we found mRNA and protein expressions of the other TIS11 family members connected to regulation of mRNA stability, BRF1 and BRF2, and detected their interaction with 14-3-3 proteins. These data suggest that control of cytokine mRNA stability and translation in MCs is exerted by proteins different from TTP.

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.

Myeloid-specific tristetraprolin deficiency in mice results in extreme lipopolysaccharide sensitivity in an otherwise minimal phenotype.

Tristetraprolin (TTP) is a mRNA-destabilizing protein that binds to AU-rich elements in labile transcripts, such as the mRNA encoding TNF, and promotes their deadenylation and degradation. TTP-deficient (knockout [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 Abs, 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 LPS-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 with 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 LPS 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 posttranscriptional regulation of TNF mRNA stability.

Deletion of tristetraprolin caused spontaneous reactive granulopoiesis by a non-cell-autonomous mechanism without disturbing long-term hematopoietic stem cell quiescence.

Tristetraprolin (TTP, Zfp36, Nup475, Tis11) dramatically reduces the stability of target mRNAs by binding to AU-rich elements in their 3' untranslated regions. Through this mechanism, TTP functions as a rheostatic, temporal regulator of gene expression. TTP knockout (KO) mice exhibit completely penetrant granulocytic hyperplasia. We have shown that the hematopoietic stem-progenitor cell compartment in TTP KO mice is also altered. Although no change was detected in long-term hematopoietic stem cell (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, multipotent progenitors, and granulocyte-monocyte progenitors. 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 wild-type bone marrow into TTP KO mice and found the "reactive granulopoiesis" phenocopied, indicating a non-hematopoietic stem-progenitor cell-autonomous mechanism. Correspondingly, we found elevated levels of the granulopoietic TTP targets IL-1ß, TNF-α, 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-1ß, TNF-α, and IL-6 transcripts in the livers of TTP KO mice and no detectable difference in the bone marrows. 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.

MAPK signaling pathways regulate IL-8 mRNA stability and IL-8 protein expression in cystic fibrosis lung epithelial cell lines.

Cystic fibrosis (CF) is characterized by a massive proinflammatory phenotype in the lung, caused by mutations in the CFTR gene. IL-8 and other proinflammatory mediators are elevated in the CF airway, and the immediate mechanism may depend on disease-specific stabilization of IL-8 mRNA in CF lung epithelial cells. MAPK signaling pathways impact directly on IL-8 protein expression in CF cells, and we have hypothesized that the mechanism may also involve stabilization of the IL-8 mRNA. To test this hypothesis, we have examined the effects of pharmacological and molecular inhibitors of p38, and downstream MK2, ERK1/2, and JNK, on stability of IL-8 mRNA in CF lung epithelial cells. We previously showed that tristetraprolin (TTP) was constitutively low in CF and that raising TTP destabilized the IL-8 mRNA. We therefore also tested these effects on CF lung epithelial cells stably expressing TTP. TTP binds to AU-rich elements in the 3'-UTR of the IL-8 mRNA. We find that inhibition of p38 and ERK1/2 reduces the stability of IL-8 mRNA in parental CF cells. However, neither intervention further lowers TTP-dependent destabilization of IL-8 mRNA. By contrast, inhibition of the JNK-2 pathway has no effect on IL-8 mRNA stability in parental CF cell, but rather increases the stability of the message in cells expressing high levels of TTP. However, we find that inhibition of ERK1/2 or p38 leads to suppression of the effect of JNK-2 inhibition on IL-8 mRNA stability. These data thus lend support to our hypothesis that constitutive MAPK signaling and proteasomal activity might also contribute, along with aberrantly lower TTP, to the proinflammatory phenotype in CF lung epithelial cells by increasing IL-8 mRNA stability and IL-8 protein expression.