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1.  Dual-Specificity Phosphatase 1 and Tristetraprolin Cooperate To Regulate Macrophage Responses to Lipopolysaccharide 
Dual-specificity phosphatase (DUSP) 1 dephosphorylates and inactivates members of the MAPK superfamily, in particular, JNKs, p38α, and p38β MAPKs. It functions as an essential negative regulator of innate immune responses, hence disruption of the Dusp1 gene renders mice extremely sensitive to a wide variety of experimental inflammatory challenges. The principal mechanisms behind the overexpression of inflammatory mediators by Dusp1−/− cells are not known. In this study, we use a genetic approach to identify an important mechanism of action of DUSP1, involving the modulation of the activity of the mRNA-destabilizing protein tristetraprolin. This mechanism is key to the control of essential early mediators of inflammation, TNF, CXCL1, and CXCL2, as well as the anti-inflammatory cytokine IL-10. The same mechanism also contributes to the regulation of a large number of transcripts induced by treatment of macrophages with LPS. These findings demonstrate that modulation of the phosphorylation status of tristetraprolin is an important physiological mechanism by which innate immune responses can be controlled.
PMCID: PMC4472943  PMID: 26019272
2.  Dominant Suppression of Inflammation via Targeted Mutation of the mRNA Destabilizing Protein Tristetraprolin 
In myeloid cells, the mRNA-destabilizing protein tristetraprolin (TTP) is induced and extensively phosphorylated in response to LPS. To investigate the role of two specific phosphorylations, at serines 52 and 178, we created a mouse strain in which those residues were replaced by nonphosphorylatable alanine residues. The mutant form of TTP was constitutively degraded by the proteasome and therefore expressed at low levels, yet it functioned as a potent mRNA destabilizing factor and inhibitor of the expression of many inflammatory mediators. Mice expressing only the mutant form of TTP were healthy and fertile, and their systemic inflammatory responses to LPS were strongly attenuated. Adaptive immune responses and protection against infection by Salmonella typhimurium were spared. A single allele encoding the mutant form of TTP was sufficient for enhanced mRNA degradation and underexpression of inflammatory mediators. Therefore, the equilibrium between unphosphorylated and phosphorylated TTP is a critical determinant of the inflammatory response, and manipulation of this equilibrium may be a means of treating inflammatory pathologies.
PMCID: PMC4472942  PMID: 26002976
3.  Heat Shock Protein B1-Deficient Mice Display Impaired Wound Healing 
PLoS ONE  2013;8(10):e77383.
There is large literature describing in vitro experiments on heat shock protein (hsp)B1 but understanding of its function in vivo is limited to studies in mice overexpressing human hspB1 protein. Experiments in cells have shown that hspB1 has chaperone activity, a cytoprotective role, regulates inflammatory gene expression, and drives cell proliferation. To investigate the function of the protein in vivo we generated hspB1-deficient mice. HspB1-deficient fibroblasts display increased expression of the pro-inflammatory cytokine, interleukin-6, compared to wild-type cells, but reduced proliferation. HspB1-deficient fibroblasts exhibit reduced entry into S phase and increased expression of cyclin-dependent kinase inhibitors p27kip1 and p21waf1. The expression of hspB1 protein and mRNA is also controlled by the cell cycle. To investigate the physiological function of hspB1 in regulating inflammation and cell proliferation we used an excisional cutaneous wound healing model. There was a significant impairment in the rate of healing of wounds in hspB1-deficient mice, characterised by reduced re-epithelialisation and collagen deposition but also increased inflammation. HspB1 deficiency augments neutrophil infiltration in wounds, driven by increased chemokine (C-X-C motif) ligand 1 expression. This appears to be a general mechanism as similar results were obtained in the air-pouch and peritonitis models of acute inflammation.
PMCID: PMC3797036  PMID: 24143227
4.  Anti-inflammatory effects of selective glucocorticoid receptor modulators are partially dependent on up-regulation of dual specificity phosphatase 1 
British Journal of Pharmacology  2012;165(4b):1124-1136.
It is thought that the anti-inflammatory effects of glucocorticoids (GCs) are largely due to GC receptor (GR)-mediated transrepression of NF-κB and other transcription factors, whereas side effects are caused by activation of gene expression (transactivation). Selective GR modulators (SGRMs) that preferentially promote transrepression should retain anti-inflammatory properties whilst causing fewer side effects. Contradicting this model, we found that anti-inflammatory effects of the classical GC dexamethasone were partly dependent on transactivation of the dual specificity phosphatase 1 (DUSP1) gene. We wished to determine whether anti-inflammatory effects of SGRMs are also mediated by DUSP1.
Dissociated properties of two SGRMs were confirmed using GR- and NF-κB-dependent reporters, and capacity to activate GC-responsive elements of the DUSP1 gene was tested. Effects of SGRMs on the expression of DUSP1 and pro-inflammatory gene products were assessed in various cell lines and in primary murine Dusp1+/+ and Dusp1−/−macrophages.
The SGRMs were able to up-regulate DUSP1 in several cell types, and this response correlated with the ability of the compounds to suppress COX-2 expression. Several anti-inflammatory effects of SGRMs were ablated or significantly impaired in Dusp1−/− macrophages.
Like dexamethasone, SGRMs appear to exert anti-inflammatory effects partly via the up-regulation of DUSP1. This finding has implications for how potentially therapeutic novel GR ligands are identified and assessed.
PMCID: PMC3346253  PMID: 21718312
immunopharmacology; nuclear receptors; steroids/neurosteroids; anti-inflammatory drugs; gene transcription; inflammation
5.  Cyclin-Dependent Kinase 6 Phosphorylates NF-κB P65 at Serine 536 and Contributes to the Regulation of Inflammatory Gene Expression 
PLoS ONE  2012;7(12):e51847.
Nuclear factor kappa-B (NF-κB) activates multiple genes with overlapping roles in cell proliferation, inflammation and cancer. Using an unbiased approach we identified human CDK6 as a novel kinase phosphorylating NF-κB p65 at serine 536. Purified and reconstituted CDK6/cyclin complexes phosphorylated p65 in vitro and in transfected cells. The physiological role of CDK6 for basal as well as cytokine-induced p65 phosphorylation or NF-κB activation was revealed upon RNAi-mediated suppression of CDK6. Inhibition of CDK6 catalytic activity by PD332991 suppressed activation of NF-κB and TNF-induced gene expression. In complex with a constitutively active viral cyclin CDK6 stimulated NF-κB p65-mediated transcription in a target gene specific manner and this effect was partially dependent on its ability to phosphorylate p65 at serine 536. Tumor formation in thymi and spleens of v-cyclin transgenic mice correlated with increased levels of p65 Ser536 phosphorylation, increased expression of CDK6 and upregulaton of the NF-κB target cyclin D3. These results suggest that aberrant CDK6 expression or activation that is frequently observed in human tumors can contribute through NF-κB to chronic inflammation and neoplasia.
PMCID: PMC3530474  PMID: 23300567
7.  Antiinflammatory effects of dexamethasone are partly dependent on induction of dual specificity phosphatase 1 
The Journal of Experimental Medicine  2006;203(8):1883-1889.
Glucocorticoids (GCs), which are used in the treatment of immune-mediated inflammatory diseases, inhibit the expression of many inflammatory mediators. They can also induce the expression of dual specificity phosphatase 1 (DUSP1; otherwise known as mitogen-activated protein kinase [MAPK] phosphatase 1), which dephosphorylates and inactivates MAPKs. We investigated the role of DUSP1 in the antiinflammatory action of the GC dexamethasone (Dex). Dex-mediated inhibition of c-Jun N-terminal kinase and p38 MAPK was abrogated in DUSP1−/− mouse macrophages. Dex-mediated suppression of several proinflammatory genes (including tumor necrosis factor, cyclooxygenase 2, and interleukin 1α and 1β) was impaired in DUSP1−/− mouse macrophages, whereas other proinflammatory genes were inhibited by Dex in a DUSP1-independent manner. In vivo antiinflammatory effects of Dex on zymosan-induced inflammation were impaired in DUSP1−/− mice. Therefore, the expression of DUSP1 is required for the inhibition of proinflammatory signaling pathways by Dex in mouse macrophages. Furthermore, DUSP1 contributes to the antiinflammatory effects of Dex in vitro and in vivo.
PMCID: PMC2118371  PMID: 16880258
8.  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
11.  Dexamethasone Causes Sustained Expression of Mitogen-Activated Protein Kinase (MAPK) Phosphatase 1 and Phosphatase-Mediated Inhibition of MAPK p38 
Molecular and Cellular Biology  2002;22(22):7802-7811.
The stress-activated protein kinase p38 stabilizes a number of mRNAs encoding inflammatory mediators, such as cyclooxygenase 2 (Cox-2). In HeLa cells the anti-inflammatory glucocorticoid dexamethasone destabilizes Cox-2 mRNA by inhibiting p38 function. Here we demonstrate that this effect is phosphatase dependent. Furthermore, in HeLa cells dexamethasone induced the sustained expression of mitogen-activated protein kinase phosphatase 1 (MKP-1), a potent inhibitor of p38 function. The inhibition of p38 and the induction of MKP-1 by dexamethasone occurred with similar dose dependence and kinetics. No other known p38 phosphatases were induced by dexamethasone, and other cell types which failed to express MKP-1 also failed to inhibit p38 in response to dexamethasone. The proinflammatory cytokine interleukin 1 (IL-1) induced MKP-1 expression in a p38-dependent manner and acted synergistically with dexamethasone to induce MKP-1 expression. In HeLa cells treated with IL-1 or IL-1 and dexamethasone, the dynamics of p38 activation mirrored the expression of MKP-1. These observations suggest that MKP-1 participates in a negative-feedback loop which regulates p38 function and that dexamethasone may inhibit proinflammatory gene expression in part by inducing MKP-1 expression.
PMCID: PMC134716  PMID: 12391149
12.  Preface 
Arthritis Research  2002;4(Suppl 3):I.
PMCID: PMC3273048
13.  Glucocorticoids: do we know how they work? 
Arthritis Research  2002;4(3):146-150.
It is not known to what extent glucocorticoid hormones cause their anti-inflammatory actions and their undesirable side effects by the same or different molecular mechanisms. Glucocorticoids combine with a cytoplasmic receptor that alters gene expression in two ways. One way is dependent on the receptor's binding directly to DNA and acting (positively or negatively) as a transcription factor. The other is dependent on its binding to and interfering with other transcription factors. Both mechanisms could underlie suppression of inflammation. The liganded receptor binds and inhibits the inflammatory transcription factors activator protein-1 and NF-κB. It also directly induces anti-inflammatory genes such as that encoding the protein inhibitor of NF-κB. Recent work has shown that glucocorticoids inhibit signalling in the mitogen-activated protein kinase pathways that mediate the expression of inflammatory genes. This inhibition is dependent on de novo gene expression. It is important to establish the significance of these different mechanisms for the various physiological effects of glucocorticoids, because it may be possible to produce steroid-related drugs that selectively target the inflammatory process.
PMCID: PMC128923  PMID: 12010562
glucocorticoid; inflammation; JNK; MAP kinase; NF-κB
14.  Mitogen-Activated Protein Kinase p38 Controls the Expression and Posttranslational Modification of Tristetraprolin, a Regulator of Tumor Necrosis Factor Alpha mRNA Stability 
Molecular and Cellular Biology  2001;21(19):6461-6469.
Signal transduction pathways regulate gene expression in part by modulating the stability of specific mRNAs. For example, the mitogen-activated protein kinase (MAPK) p38 pathway mediates stabilization of tumor necrosis factor alpha (TNF-α) mRNA in myeloid cells stimulated with bacterial lipopolysaccharide (LPS). The zinc finger protein tristetraprolin (TTP) is expressed in response to LPS and regulates the stability of TNF-α mRNA. We show that stimulation of RAW264.7 mouse macrophages with LPS induces the binding of TTP to the TNF-α 3′ untranslated region. The p38 pathway is required for the induction of TNF-α RNA-binding activity and for the expression of TTP protein and mRNA. Following stimulation with LPS, TTP is expressed in multiple, differentially phosphorylated forms. We present evidence that phosphorylation of TTP is mediated by the p38-regulated kinase MAPKAPK2 (MAPK-activated protein kinase 2). Our findings demonstrate a direct link between a specific signal transduction pathway and a specific RNA-binding protein, both of which are known to regulate TNF-α gene expression at a posttranscriptional level.
PMCID: PMC99793  PMID: 11533235
15.  Dexamethasone Destabilizes Cyclooxygenase 2 mRNA by Inhibiting Mitogen-Activated Protein Kinase p38 
Molecular and Cellular Biology  2001;21(3):771-780.
The stability of cyclooxygenase 2 (Cox-2) mRNA is regulated positively by proinflammatory stimuli acting through mitogen-activated protein kinase (MAPK) p38 and negatively by anti-inflammatory glucocorticoids such as dexamethasone. A tetracycline-regulated reporter system was used to investigate mechanisms of regulation of Cox-2 mRNA stability. Dexamethasone was found to destabilize β-globin–Cox-2 reporter mRNAs by inhibiting p38. This inhibition occurred at the level of p38 itself: stabilization of reporter mRNA by a kinase upstream of p38 was blocked by dexamethasone, while stabilization by a kinase downstream of p38 was insensitive to dexamethasone. Inhibition of p38 activity by dexamethasone was observed in a variety of cell types treated with different activating stimuli. Furthermore, inhibition of p38 was antagonized by the anti-glucocorticoid RU486 and was delayed and actinomycin D sensitive, suggesting that ongoing glucocorticoid receptor-dependent transcription is required.
PMCID: PMC86669  PMID: 11154265
16.  The 3′ Untranslated Region of Tumor Necrosis Factor Alpha mRNA Is a Target of the mRNA-Stabilizing Factor HuR 
Molecular and Cellular Biology  2001;21(3):721-730.
Posttranscriptional regulation is important for tumor necrosis factor alpha (TNF-α) expression in monocytes and macrophages, and an AU-rich element (ARE) in the 3′ untranslated region (UTR) of TNF-α mRNA is implicated in control of its translation and mRNA stability. Regulation of mRNA turnover is thought to be mediated by trans-acting proteins, which bind the ARE and stabilize or destabilize the transcript. However, with the exception of the destabilizing factor tristetraprolin, the identity and function of the proteins binding the TNF-α mRNA ARE have not been established. To identify other proteins involved in the posttranscriptional control of TNF-α, the subcellular location of TNF-α mRNA was determined in the macrophage-like cell line RAW 264.7. TNF-α mRNA was located in the pellet following centrifugation of cytoplasm at 100,000 × g (P100 fraction). This fraction also contained proteins which formed two distinct ARE-specific complexes with the TNF-α mRNA 3′ UTR in electrophoretic mobility shift assays (EMSAs). A protein present in these two complexes was purified and identified by peptide mass mapping and tandem mass spectrometry as HuR. In EMSAs both complexes were supershifted by an anti-HuR antibody, while Western blotting also demonstrated the presence of HuR in the P100 extract. A HeLa cell tetracycline-regulated reporter system was used to determine the effect of HuR on mRNA stability. In this system, overexpression of HuR resulted in stabilization of an otherwise unstable reporter-mRNA containing the TNF-α ARE. These results demonstrate that the TNF-α ARE is a target of the mRNA-stabilizing factor HuR.
PMCID: PMC86664  PMID: 11154260
17.  Regulation of Cyclooxygenase 2 mRNA Stability by the Mitogen-Activated Protein Kinase p38 Signaling Cascade 
Molecular and Cellular Biology  2000;20(12):4265-4274.
A tetracycline-regulated reporter system was used to investigate the regulation of cyclooxygenase 2 (Cox-2) mRNA stability by the mitogen-activated protein kinase (MAPK) p38 signaling cascade. The stable β-globin mRNA was rendered unstable by insertion of the 2,500-nucleotide Cox-2 3′ untranslated region (3′ UTR). The chimeric transcript was stabilized by a constitutively active form of MAPK kinase 6, an activator of p38. This stabilization was blocked by SB203580, an inhibitor of p38, and by two different dominant negative forms of MAPK-activated protein kinase 2 (MAPKAPK-2), a kinase lying downstream of p38. Constitutively active MAPKAPK-2 was also able to stabilize chimeric β-globin–Cox-2 transcripts. The MAPKAPK-2 substrate hsp27 may be involved in stabilization, as β-globin–Cox-2 transcripts were partially stabilized by phosphomimetic mutant forms of hsp27. A short (123-nucleotide) fragment of the Cox-2 3′ UTR was necessary and sufficient for the regulation of mRNA stability by the p38 cascade and interacted with a HeLa protein immunologically related to AU-rich element/poly(U) binding factor 1.
PMCID: PMC85794  PMID: 10825190
18.  Fibroblast Growth Factor 2 Drives Changes in Gene Expression Following Injury to Murine Cartilage In Vitro and In Vivo 
Arthritis and Rheumatism  2013;65(9):2346-2355.
The articular cartilage is known to be highly mechanosensitive, and a number of mechanosensing mechanisms have been proposed as mediators of the cellular responses to altered mechanical load. These pathways are likely to be important in tissue homeostasis as well as in the pathogenesis of osteoarthritis. One important injury-activated pathway involves the release of pericellular fibroblast growth factor 2 (FGF-2) from the articular cartilage. Using a novel model of murine cartilage injury and surgically destabilized joints in mice, we examined the extent to which FGF-2 contributes to the cellular gene response to injury.
Femoral epiphyses from 5-week-old wild-type mice were avulsed and cultured in serum-free medium. Explant lysates were Western blotted for phospho-JNK, phospho-p38, and phospho-ERK or were fixed for immunohistochemical analysis of the nuclear translocation of p65 (indicative of NF-κB activation). RNA was extracted from injured explants, rested explants that had been stimulated with recombinant FGF-2 or FGF-18, or whole joints from either wild-type mice or FGF-2−/− mice. Reverse transcription–polymerase chain reaction was performed to examine a number of inflammatory response genes that had previously been identified in a microarray analysis.
Murine cartilage avulsion injury resulted in rapid activation of the 3 MAP kinase pathways as well as NF-κB. Almost all genes identified in murine joints following surgical destabilization were also regulated in cartilage explants upon injury. Many of these genes, including those for activin A (Inhba), tumor necrosis factor–stimulated gene 6 (Tnfaip6), matrix metalloproteinase 19 (Mmp19), tissue inhibitor of metalloproteinases 1 (Timp1), and podoplanin (Pdpn), were significantly FGF-2 dependent following injury to cartilage in vitro and to joint tissues in vivo.
FGF-2–dependent gene expression occurs in vitro and in vivo in response to cartilage/joint injury in mice.
PMCID: PMC3992838  PMID: 23740825

Results 1-18 (18)