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1.  18β-glycyrrhetinic acid inhibits rotavirus replication in culture 
Virology Journal  2012;9:96.
Background
Glycyrrhizin (GA) and primary metabolite 18β-glycyrrhetinic acid (GRA) are pharmacologically active components of the medicinal licorice root, and both have been shown to have antiviral and immunomodulatory properties. Although these properties are well established, the mechanisms of action are not completely understood. In this study, GA and GRA were tested for the ability to inhibit rotavirus replication in cell culture, toward a long term goal of discovering natural compounds that may complement existing vaccines.
Methods
Epithelial cells were treated with GA or GRA various times pre- or post-infection and virus yields were measured by immunofluorescent focus assay. Levels of viral proteins VP2, VP6, and NSP2 in GRA treated cells were measured by immunoblot to determine if there was an effect of GRA treatment on the accumulation of viral protein.
Results
GRA treatment reduced rotavirus yields by 99% when added to infected cultures post-- virus adsorption, whereas virus yields in GA treated cultures were similar to mock treated controls. Time of addition experiments indicated that GRA-mediated replication inhibition likely occurs at a step or steps subsequent to virus entry. The amounts of VP2, VP6 and NSP2 were substantially reduced when GRA was added to cultures up to two hours post-entry.
Conclusions
GRA, but not GA, has significant antiviral activity against rotavirus replication in vitro, and studies to determine whether GRA attenuates rotavirus replication in vivo are underway.
doi:10.1186/1743-422X-9-96
PMCID: PMC3478227  PMID: 22616823
Rotavirus; Licorice; 18beta-glycyrrhetinic acid; Antiviral
2.  Mitogen-Activated Protein Kinase Phosphatase 2 Regulates the Inflammatory Response in Sepsis▿  
Infection and Immunity  2010;78(6):2868-2876.
Sepsis results from a dysregulation of the regulatory mechanisms of the pro- and anti-inflammatory response to invading pathogens. The mitogen-activated protein (MAP) kinase cascades are key signal transduction pathways involved in the cellular production of cytokines. The dual-specific phosphatase 1 (DUSP 1), mitogen-activated protein kinase phosphatase-1 (MKP-1), has been shown to be an important negative regulator of the inflammatory response by regulating the p38 and Jun N-terminal protein kinase (JNK) MAP kinase pathways to influence pro- and anti-inflammatory cytokine production. MKP-2, also a dual-specific phosphatase (DUSP 4), is a phosphatase highly homologous with MKP-1 and is known to regulate MAP kinase signaling; however, its role in regulating the inflammatory response is not known. We hypothesized a regulatory role for MKP-2 in the setting of sepsis. Mice lacking the MKP-2 gene had a survival advantage over wild-type mice when challenged with intraperitoneal lipopolysaccharide (LPS) or a polymicrobial infection via cecal ligation and puncture. The MKP-2−/− mice also exhibited decreased serum levels of both pro-inflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-1β [IL-1β], IL-6) and anti-inflammatory cytokines (IL-10) following endotoxin challenge. Isolated bone marrow-derived macrophages (BMDMs) from MKP-2−/− mice showed increased phosphorylation of the extracellular signal-regulated kinase (ERK), decreased phosphorylation of JNK and p38, and increased induction of MKP-1 following LPS stimulation. The capacity for cytokine production increased in MKP-2−/− BMDMs following MKP-1 knockdown. These data support a mechanism by which MKP-2 targets ERK deactivation, thereby decreasing MKP-1 and thus removing the negative inhibition of MKP-1 on cytokine production.
doi:10.1128/IAI.00018-10
PMCID: PMC2876557  PMID: 20351138
3.  18β-Glycyrrhetinic Acid Delivered Orally Induces Isolated Lymphoid Follicle Maturation at the Intestinal Mucosa and Attenuates Rotavirus Shedding 
PLoS ONE  2012;7(11):e49491.
Glycyrrhizin, an abundant bioactive component of the medicinal licorice root is rapidly metabolized by gut commensal bacteria into 18β-glycyrrhetinic acid (GRA). Either or both of these compounds have been shown to have antiviral, anti-hepatotoxic, anti-ulcerative, anti-tumor, anti-allergenic and anti-inflammatory activity in vitro or in vivo. In this study, the ability of GRA to modulate immune responses at the small intestinal mucosa when delivered orally was investigated. Analysis of cytokine transcription in duodenal and ileal tissue in response to GRA treatment revealed a pattern of chemokine and chemokine receptor gene expression predictive of B cell recruitment to the gut. Consistent with this finding, GRA induced increases in CD19+ B cells in the lamina propria and B220+ B cell aggregates framed by CD11c+ dendritic cells in structures resembling isolated lymphoid follicles (ILF). Using a mouse model of rotavirus infection, GRA reduced the duration of viral antigen shedding, and endpoint serum antibody titers were higher in GRA-treated animals. Together the data suggest GRA delivered orally augments lymphocyte recruitment to the intestinal mucosa and induces maturation of B cell-rich ILF independently of ectopic antigenic stimulus. These results provide further support a role for dietary ligands in modulation of dynamic intestinal lymphoid tissue.
doi:10.1371/journal.pone.0049491
PMCID: PMC3496704  PMID: 23152913
4.  Phosphorylation of the Kinase Interaction Motif in Mitogen-activated Protein (MAP) Kinase Phosphatase-4 Mediates Cross-talk between Protein Kinase A and MAP Kinase Signaling Pathways* 
The Journal of Biological Chemistry  2011;286(44):38018-38026.
Background: DUSP9/MKP-4 dephosphorylates and inactivates MAP kinases.
Results: The kinase interaction motif of DUSP9/MKP-4 is phosphorylated by protein kinase A, and this reduces its ability to interact with and inactivate MAP kinase substrates.
Conclusion: DUSP9/MKP-4 represents a point of cross-talk between PKA and MAP kinase signaling.
Significance: Cross-talk between distinct signal transduction pathways may be important in determining physiological response.
MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site 55RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo.
doi:10.1074/jbc.M111.255844
PMCID: PMC3207433  PMID: 21908610
MAP Kinases (MAPKs); p38 MAPK; Protein Kinase A (PKA); Protein Phosphatase; Protein Phosphorylation; Signal Transduction
5.  Diversity of two-component systems: insights into the signal transduction mechanism by the  Staphylococcus aureus two-component system GraSR 
F1000Research  2014;3:252.
The response to cationic antimicrobial peptides (CAMPs) in Staphylococcus aureus relies on a two-component system (TCS), GraSR, an auxiliary protein GraX and an ATP-binding cassette (ABC) transporter, VraF/G. To understand the signal transduction mechanism by GraSR, we investigated the kinase activity of the cytoplasmic domain of histidine kinase GraS and the interaction with its cognate response regulator GraR. We also investigated interactions among the auxiliary protein GraX, GraS/R and the ATPase protein of the ABC transporter, VraF. We found that GraS lacks autophosphorylation activity, unlike a similar histidine kinase, BceS, of Bacillus subtilis. In addition, the interaction between GraS and GraR is very weak in comparison to the stronger interaction observed between BceS and its conjugated response regulator, BceR, suggesting that CAMP signaling may not flow directly from GraS to GraR. We found that the auxiliary protein GraX interacts with VraF and GraR, and requires the histidine phosphotransfer and dimerization domain of GraS to interact with this protein. Further, VraF requires the GraS region that connects the membrane-bound domain with the cytoplasmic domain of this protein for interaction with GraS. The interactions of GraX with GraS/R and VraF indicate that GraX may serve as a scaffold to bring these proteins in close proximity to GraS, plausibly to facilitate activation of GraS to ultimately transduce the signal to GraR.
doi:10.12688/f1000research.5512.1
PMCID: PMC4314665
Two-component systems; histidine kinases; response regulators; GraSR; Staphylococcus aureus; cationic antimicrobial peptides
6.  Diversity of two-component systems: insights into the signal transduction mechanism by the  Staphylococcus aureus two-component system GraSR 
F1000Research  2014;3:252.
The response to cationic antimicrobial peptides (CAMPs) in Staphylococcus aureus relies on a two-component system (TCS), GraSR, an auxiliary protein GraX and an ATP-binding cassette (ABC) transporter, VraF/G. To understand the signal transduction mechanism by GraSR, we investigated the kinase activity of the cytoplasmic domain of histidine kinase GraS and the interaction with its cognate response regulator GraR. We also investigated interactions among the auxiliary protein GraX, GraS/R and the ATPase protein of the ABC transporter, VraF. We found that GraS lacks autophosphorylation activity, unlike a similar histidine kinase, BceS, of Bacillus subtilis. In addition, the interaction between GraS and GraR is very weak in comparison to the stronger interaction observed between BceS and its conjugated response regulator, BceR, suggesting that CAMP signaling may not flow directly from GraS to GraR. We found that the auxiliary protein GraX interacts with VraF and GraR, and requires the histidine phosphotransfer and dimerization domain of GraS to interact with this protein. Further, VraF requires the GraS region that connects the membrane-bound domain with the cytoplasmic domain of this protein for interaction with GraS. The interactions of GraX with GraS/R and VraF indicate that GraX may serve as a scaffold to bring these proteins in close proximity to GraS, plausibly to facilitate activation of GraS to ultimately transduce the signal to GraR.
doi:10.12688/f1000research.5512.2
PMCID: PMC4314665
Two-component systems; histidine kinases; response regulators; GraSR; Staphylococcus aureus; cationic antimicrobial peptides
7.  Protective effect of DNA-mediated immunization with liposome-encapsulated GRA4 against infection of Toxoplasma gondii *  
The dense granule protein 4 (GRA4) is a granular protein from Toxoplasma gondii, and is a candidate for vaccination against this parasite. In this study, the plasmid pcDNA3.1-GRA4 (pGRA4), encoding for the GRA4 antigen, was incorporated by the dehydration-rehydration method into liposomes composed of 16 mmol/L egg phosphatidylcholine (PC), 8 mmol/L dioleoyl phosphatidylethanolamine (DOPE), and 4 mmol/L 1,2-diodeoyl-3-(trimethylammonium) propane (DOTAP). C57BL/6 mice and BALB/c mice were immunized intramuscularly three times with liposome-encapsulated pGRA4 to determine whether DNA immunization could elicit a protective immune response to T. gondii. Enzyme-linked immunosorbent assay (ELISA) of sera from immunized mice showed that liposome-encapsulated pGRA4 generated high levels of IgG antibodies to GRA4. Production of primary interferon (IFN)-γ and interleukin (IL)-2 in GRA4-stimulated splenocytes from vaccinated mice suggested a modulated Th1-type response. 72.7% of C57BL/6 mice immunized with liposome-encapsulated pGRA4 survived the challenge with 80 tissue cysts of ME49 strain, whereas C57BL/6 mice immunized with pGRA4 had only a survival rate of 54.5%. When immunized BALB/c mice were intraperitoneally challenged with 103 tachyzoites of the highly virulent RH strain, the survival time of mice immunized with liposome-encapsulated pGRA4 was markedly longer than that of other groups. Our observations show that liposome-encapsulated pGRA4 enhanced the protective effect against infection of T. gondii.
doi:10.1631/jzus.B0820300
PMCID: PMC2704969  PMID: 19585669
DNA vaccine; Granule protein 4 (GRA4); Liposome; Toxoplasma gondii
8.  18β-Glycyrrhetinic Acid Inhibits Methicillin-Resistant Staphylococcus aureus Survival and Attenuates Virulence Gene Expression 
Methicillin-resistant Staphylococcus aureus (MRSA) has become a major source of infection in hospitals and in the community. Increasing antibiotic resistance in S. aureus strains has created a need for alternative therapies to treat disease. A component of the licorice root Glycyrrhiza spp., 18β-glycyrrhetinic acid (GRA), has been shown to have antiviral, antitumor, and antibacterial activity. This investigation explores the in vitro and in vivo effects of GRA on MRSA pulsed-field gel electrophoresis (PFGE) type USA300. GRA exhibited bactericidal activity at concentrations exceeding 0.223 μM. Upon exposure of S. aureus to sublytic concentrations of GRA, we observed a reduction in expression of key virulence genes, including saeR and hla. In murine models of skin and soft tissue infection, topical GRA treatment significantly reduced skin lesion size and decreased the expression of saeR and hla genes. Our investigation demonstrates that at high concentrations GRA is bactericidal to MRSA and at sublethal doses it reduces virulence gene expression in S. aureus both in vitro and in vivo.
doi:10.1128/AAC.01023-12
PMCID: PMC3535912  PMID: 23114775
9.  MAP Kinase Phosphatase-2 Plays a Critical Role in Response to Infection by Leishmania mexicana 
PLoS Pathogens  2010;6(11):e1001192.
In this study we generated a novel dual specific phosphatase 4 (DUSP4) deletion mouse using a targeted deletion strategy in order to examine the role of MAP kinase phosphatase-2 (MKP-2) in immune responses. Lipopolysaccharide (LPS) induced a rapid, time and concentration-dependent increase in MKP-2 protein expression in bone marrow-derived macrophages from MKP-2+/+ but not from MKP-2−/− mice. LPS-induced JNK and p38 MAP kinase phosphorylation was significantly increased and prolonged in MKP-2−/− macrophages whilst ERK phosphorylation was unaffected. MKP-2 deletion also potentiated LPS-stimulated induction of the inflammatory cytokines, IL-6, IL-12p40, TNF-α, and also COX-2 derived PGE2 production. However surprisingly, in MKP-2−/− macrophages, there was a marked reduction in LPS or IFNγ-induced iNOS and nitric oxide release and enhanced basal expression of arginase-1, suggesting that MKP-2 may have an additional regulatory function significant in pathogen-mediated immunity. Indeed, following infection with the intracellular parasite Leishmania mexicana, MKP-2−/− mice displayed increased lesion size and parasite burden, and a significantly modified Th1/Th2 bias compared with wild-type counterparts. However, there was no intrinsic defect in MKP-2−/− T cell function as measured by anti-CD3 induced IFN-γ production. Rather, MKP-2−/− bone marrow-derived macrophages were found to be inherently more susceptible to infection with Leishmania mexicana, an effect reversed following treatment with the arginase inhibitor nor-NOHA. These findings show for the first time a role for MKP-2 in vivo and demonstrate that MKP-2 may be essential in orchestrating protection against intracellular infection at the level of the macrophage.
Author Summary
In cells of the immune system are switch-on enzymes called kinases which regulate responses to infectious agents such as Leishmania. However, in the same cells there are switch-off enzymes known as phosphatases which function to turn off the kinases once they have done their work. A lot of studies have focussed on the role of kinases but not phosphatases in response to infection; we therefore generated a novel mouse in which the gene for one of these phosphatases, called MKP-2, has been deleted. We found that in the absence of this phosphatase unexpected things happened. The profile of inflammatory proteins, produced by a special cell of the immune system, called a macrophage, that functions to regulate infection by Leishmania, changed in ways which meant the macrophage could either fight infection very effectively or very weakly. In actual fact, we found that the macrophages with no MKP-2 fought off Leishmania poorly and mice deficient in MKP-2 had a modified immune response favouring the growth of the parasite. This is the first study to give critical insight into the role of MKP-2 in fighting Leishmania infection and demonstrates very well the importance of this class of enzyme in pathogen biology.
doi:10.1371/journal.ppat.1001192
PMCID: PMC2978729  PMID: 21085614
10.  GraXSR Proteins Interact with the VraFG ABC Transporter To Form a Five-Component System Required for Cationic Antimicrobial Peptide Sensing and Resistance in Staphylococcus aureus 
The GraSR two-component system (TCS) controls cationic antimicrobial peptide (CAMP) resistance in Staphylococcus aureus through the synthesis of enzymes that increase bacterial cell surface positive charges, by d-alanylation of teichoic acids and lysylination of phosphatidylglycerol, leading to electrostatic repulsion of CAMPs. The GraS histidine kinase belongs to the “intramembrane-sensing kinases” subfamily, with a structure featuring a short amino-terminal sensing domain, and two transmembrane helices separated only by a short loop, thought to be buried in the cytoplasmic membrane. The GraSR TCS is in fact a multicomponent system, requiring at least one accessory protein, GraX, in order to function, which, as we show here, acts by signaling through the GraS kinase. The graXRS genes are located immediately upstream from genes encoding an ABC transporter, vraFG, whose expression is controlled by GraSR. We demonstrated that the VraFG transporter does not act as a detoxification module, as it cannot confer resistance when produced on its own, but instead plays an essential role by sensing the presence of CAMPs and signaling through GraS to activate GraR-dependent transcription. A bacterial two-hybrid approach, designed to identify interactions between the GraXSR and VraFG proteins, was carried out in order to understand how they act in detecting and signaling the presence of CAMPs. We identified many interactions between these protein pairs, notably between the GraS kinase and both GraX and the VraG permease, indicating the existence of an original five-component system involved in CAMP sensing and signal transduction to promote S. aureus resistance.
doi:10.1128/AAC.05054-11
PMCID: PMC3264281  PMID: 22123691
11.  Human Innate Immunity to Toxoplasma gondii Is Mediated by Host Caspase-1 and ASC and Parasite GRA15 
mBio  2013;4(4):e00255-13.
ABSTRACT  
Interleukin-1β (IL-1β) functions as a key regulator of inflammation and innate immunity. The protozoan parasite Toxoplasma gondii actively infects human blood monocytes and induces the production of IL-1β; however, the host and parasite factors that mediate IL-1β production during T. gondii infection are poorly understood. We report that T. gondii induces IL-1β transcript, processing/cleavage, and release from infected primary human monocytes and THP-1 cells. Treating monocytes with the caspase-1 inhibitor Ac-YVAD-CMK reduced IL-1β release, suggesting a role for the inflammasome in T. gondii-induced IL-1β production. This was confirmed by performing short hairpin RNA (shRNA) knockdown of caspase-1 and of the inflammasome adaptor protein ASC. IL-1β induction required active parasite invasion of monocytes, since heat-killed or mycalolide B-treated parasites did not induce IL-1β. Among the type I, II, and III strains of T. gondii, the type II strain induced substantially more IL-1β mRNA and protein release than did the type I and III strains. Since IL-1β transcript is known to be induced downstream of NF-κB signaling, we investigated a role for the GRA15 protein, which induces sustained NF-κB signaling in a parasite strain-specific manner. By infecting human monocytes with a GRA15-knockout type II strain and a type I strain stably expressing type II GRA15, we determined that GRA15 is responsible for IL-1β induction during T. gondii infection of human monocytes. This research defines a pathway driving human innate immunity by describing a role for the classical inflammasome components caspase-1 and ASC and the parasite GRA15 protein in T. gondii-induced IL-1β production.
IMPORTANCE
Monocytes are immune cells that protect against infection by increasing inflammation and antimicrobial activities in the body. Upon infection with the parasitic pathogen Toxoplasma gondii, human monocytes release interleukin-1β (IL-1β), a “master regulator” of inflammation, which amplifies immune responses. Although inflammatory responses are critical for host defense against infection, excessive inflammation can result in tissue damage and pathology. This delicate balance underscores the importance of understanding the mechanisms that regulate IL-1β during infection. We have investigated the molecular pathway by which T. gondii induces the synthesis and release of IL-1β in human monocytes. We found that specific proteins in the parasite and the host cell coordinate to induce IL-1β production. This research is significant because it contributes to a greater understanding of human innate immunity to infection and IL-1β regulation, thereby enhancing our potential to modulate inflammation in the body.
doi:10.1128/mBio.00255-13
PMCID: PMC3705447  PMID: 23839215
12.  Strain-specific activation of the NF-κB pathway by GRA15, a novel Toxoplasma gondii dense granule protein 
The Toxoplasma gondii granule protein GRA15 activates the NF-κB pathway.
NF-κB is an integral component of the immune response to Toxoplasma gondii. Although evidence exists that T. gondii can directly modulate the NF-κB pathway, the parasite-derived effectors involved are unknown. We determined that type II strains of T. gondii activate more NF-κB than type I or type III strains, and using forward genetics we found that this difference is a result of the polymorphic protein GRA15, a novel dense granule protein which T. gondii secretes into the host cell upon invasion. A GRA15-deficient type II strain has a severe defect in both NF-κB nuclear translocation and NF-κB–mediated transcription. Furthermore, human cells expressing type II GRA15 also activate NF-κB, demonstrating that GRA15 alone is sufficient for NF-κB activation. Along with the rhoptry protein ROP16, GRA15 is responsible for a large part of the strain differences in the induction of IL-12 secretion by infected mouse macrophages. In vivo bioluminescent imaging showed that a GRA15-deficient type II strain grows faster compared with wild-type, most likely through its reduced induction of IFN-γ. These results show for the first time that a dense granule protein can modulate host signaling pathways, and dense granule proteins can therefore join rhoptry proteins in T. gondii’s host cell–modifying arsenal.
doi:10.1084/jem.20100717
PMCID: PMC3023140  PMID: 21199955
13.  Extracellular Signal-Regulated Kinases Phosphorylate Mitogen-Activated Protein Kinase Phosphatase 3/DUSP6 at Serines 159 and 197, Two Sites Critical for Its Proteasomal Degradation 
Molecular and Cellular Biology  2005;25(2):854-864.
Mitogen-activated protein (MAP) kinase phosphatases (MKPs) are dual-specificity phosphatases that dephosphorylate phosphothreonine and phosphotyrosine residues within MAP kinases. Here, we describe a novel posttranslational mechanism for regulating MKP-3/Pyst1/DUSP6, a member of the MKP family that is highly specific for extracellular signal-regulated kinase 1 and 2 (ERK1/2) inactivation. Using a fibroblast model in which the expression of either MKP-3 or a more stable MKP-3-green fluorescent protein (GFP) chimera was induced by tetracycline, we found that serum induces the phosphorylation of MKP-3 and its subsequent degradation by the proteasome in a MEK1 and MEK2 (MEK1/2)-ERK1/2-dependent manner. In vitro phosphorylation assays using glutathione S-transferase (GST)-MKP-3 fusion proteins indicated that ERK2 could phosphorylate MKP-3 on serines 159 and 197. Tetracycline-inducible cell clones expressing either single or double serine mutants of MKP-3 or MKP-3-GFP confirmed that these two sites are targeted by the MEK1/2-ERK1/2 module in vivo. Double serine mutants of MKP-3 or MKP-3-GFP were more efficiently protected from degradation than single mutants or wild-type MKP-3, indicating that phosphorylation of either serine by ERK1/2 enhances proteasomal degradation of MKP-3. Hence, double mutation caused a threefold increase in the half-life of MKP-3. Finally, we show that the phosphorylation of MKP-3 has no effect on its catalytic activity. Thus, ERK1/2 exert a positive feedback loop on their own activity by promoting the degradation of MKP-3, one of their major inactivators in the cytosol, a situation opposite to that described for the nuclear phosphatase MKP-1.
doi:10.1128/MCB.25.2.854-864.2005
PMCID: PMC543408  PMID: 15632084
14.  T Cell Hypo-Responsiveness against Leishmania major in MAP Kinase Phosphatase (MKP) 2 Deficient C57BL/6 Mice Does Not Alter the Healer Disease Phenotype 
We have recently demonstrated that MAP kinase phosphatase 2 (MKP-2) deficient C57BL/6 mice, unlike their wild-type counterparts, are unable to control infection with the protozoan parasite Leishmania mexicana. Increased susceptibility was associated with elevated Arginase-1 levels and reduced iNOS activity in macrophages as well as a diminished TH1 response. By contrast, in the present study footpad infection of MKP-2−/− mice with L. major resulted in a healing response as measured by lesion size and parasite numbers similar to infected MKP-2+/+ mice. Analysis of immune responses following infection demonstrated a reduced TH1 response in MKP-2−/− mice with lower parasite specific serum IgG2b levels, a lower frequency of IFN-γ and TNF-α producing CD4+ and CD8+ T cells and lower antigen stimulated spleen cell IFN-γ production than their wild-type counterparts. However, infected MKP-2−/− mice also had similarly reduced levels of antigen induced spleen and lymph node cell IL-4 production compared with MKP-2+/+ mice as well as reduced levels of parasite-specific IgG1 in the serum, indicating a general T cell hypo-responsiveness. Consequently the overall TH1/TH2 balance was unaltered in MKP-2−/− compared with wild-type mice. Although non-stimulated MKP-2−/− macrophages were more permissive to L. major growth than macrophages from MKP-2+/+ mice, reflecting their reduced iNOS and increased Arginase-1 expression, LPS/IFN-γ activation was equally effective at controlling parasite growth in MKP-2−/− and MKP-2+/+ macrophages. Consequently, in the absence of any switch in the TH1/TH2 balance in MKP-2−/− mice, no significant change in disease phenotype was observed.
Author Summary
Leishmania species are parasites that are of extensive public health importance in the tropics and subtropics. Within humans the parasites are intracellular and reside particularly within macrophages. Classical activation of macrophages by Interferon-γ (IFN-γ) induces the enzyme nitric oxide synthase (iNOS) and parasites are killed via the production of nitric oxide (NO) from the substrate L-arginine. Alternative activation by Interleukin-4 (IL-4) results in Arginase-1 expression, which depletes L-arginine and facilitates parasite growth. We have recently shown that MAP Kinase Phosphatase-2 (MKP-2) suppresses macrophage Arginase-1 and that C57BL/6 mice with a deletion of this gene are subsequently extremely susceptible to New World cutaneous leishmaniasis caused by Leishmania mexicana. Surprisingly, MKP-2 deficient mice have been shown here to be resistant to Old World cutaneous leishmaniasis caused by L. major. We show that during infection with L. major, enhanced Arginase-1 in MKP-2 deficient mice serves to induce a generalized T cell hypo-responsiveness so that IFN-γ and IL-4 levels are equally suppressed compared with intact mice. In addition, unlike L. mexicana, classically activated MKP-2 deficient macrophages were able to control L. major growth equally as well as MKP-2 intact macrophages, highlighting a fundamental difference in the control of these two species.
doi:10.1371/journal.pntd.0002064
PMCID: PMC3578781  PMID: 23437409
15.  Mapping MKP-3/FOXO1 Interaction and Evaluating the Effect on Gluconeogenesis 
PLoS ONE  2012;7(7):e41168.
Background
MAP kinase phosphatase 3 (MKP-3) is known to attenuate the ERK signaling pathway. It has been recently demonstrated that MKP-3 is also a player in promoting hepatic glucose output in obese state by interacting and activating FOXO1. Reduction of hepatic MKP-3 expression is sufficient to reduce blood glucose levels in both diet-induced and genetically obese mice.
Methodology/Principal Findings
In current study, the mechanism of MKP-3/FOXO1 interaction and the effects on transcription of gluconeogenic gene and glucose output was investigated in Fao hepatoma cells by using mutated MKP-3 and FOXO1 adenoviral constructs. The results indicate that MKP-3 phosphatase activity is not required for MKP-3/FOXO1 interaction but is essential for FOXO1 nuclear translocation and MKP-3 promoted gluconeogenesis. Compared to GFP control (1±0.38), MKP-3 increased G6Pase gene expression by 242% (3.42±0.62) while inactive MKP-3 does not change G6Pase expression (0.98±0.17). The residues 200–260 of MKP-3 and the residues 360–456 of FOXO1 are essential for mediating MKP-3/FOXO1 interaction. Interestingly, ERK phosphorylation deficient but not Akt phosphorylation deficient FOXO1 mutant lost interaction with MKP-3. Furthermore, in vivo experiments showed that Akt phosphorylation resistant FOXO1 3A mutant is sufficient to rescue the hypoglycemia caused by MKP-3 knock down in the liver of lean mice (from 141±6.78 to 209±14.64 mg/dL).
Conclusions/Significance
1) Critical residues mediating MKP-3/FOXO1 interaction have been identified; 2) ERK phosphorylation deficient FOXO1 mutant is as potent as Akt phosphorylation deficient FOXO1 mutant in activating transcription of gluconeogenic genes; 3) Constitutively active FOXO1 can rescue the hypoglycemic effect caused by reduced hepatic MKP-3 expression in vivo.
doi:10.1371/journal.pone.0041168
PMCID: PMC3405053  PMID: 22848439
16.  DNA prime and peptide boost immunization protocol encoding the Toxoplasma gondii GRA4 induces strong protective immunity in BALB/c mice 
BMC Infectious Diseases  2013;13:494.
Background
Toxoplasma gondii is a widespread intracellular parasite, which infects most vertebrate animal hosts and causes zoonotic infection in humans. Vaccine strategy remains a promising method for the prevention and control of toxoplasmosis. T. gondii GRA4 protein has been identified as a potential candidate for vaccine development. In our study, we evaluated the immune response induced by four different immunization vaccination strategies encoding TgGRA4.
Methods
BALB/c mice were intramuscularly (i.m.) immunized four times according to specific immunization schedules. Generally, mice in experimental groups were immunized with polypeptide, pGRA4, peptide/DNA, or DNA/peptide, and mice in the control groups were injected with PBS or pEGFP. After immunization, the levels of IgG antibodies and cytokine productions were determined by enzyme-linked immunosorbent assays (ELISA). The survival time of mice was also evaluated after challenge infection with the highly virulent T. gondii RH strain.
Results
The results showed that mice vaccinated with different immunization regimens (polypeptide, pGRA4, peptide/DNA, or DNA/peptide) elicited specific humoral and cellular responses, with high levels of total IgG, IgG2a isotype and gamma interferon (IFN-γ), which suggested a specific Th1 immunity was activated. After lethal challenge, an increased survival time was observed in immunized mice (11.8 ± 4.8 days) compared to the control groups injected with PBS or pEGFP (P < 0.05). Mice injected with PBS or pEGFP died within 8 days, and there was no significant difference in the protection level in two groups (P > 0.05).
Conclusions
These results demonstrated that this DNA prime and peptide boost immunization protocol encoding the TgGRA4 can elicit the highest level of humoral and cellular immune responses compared to other immunized groups, which is a promising approach to increase the efficacy of DNA immunization.
doi:10.1186/1471-2334-13-494
PMCID: PMC3871000  PMID: 24148219
17.  Molecular Basis of Resistance to Muramidase and Cationic Antimicrobial Peptide Activity of Lysozyme in Staphylococci 
PLoS Pathogens  2007;3(7):e102.
It has been shown recently that modification of peptidoglycan by O-acetylation renders pathogenic staphylococci resistant to the muramidase activity of lysozyme. Here, we show that a Staphylococcus aureus double mutant defective in O-acetyltransferase A (OatA), and the glycopeptide resistance-associated two-component system, GraRS, is much more sensitive to lysozyme than S. aureus with the oatA mutation alone. The graRS single mutant was resistant to the muramidase activity of lysozyme, but was sensitive to cationic antimicrobial peptides (CAMPs) such as the human lysozyme-derived peptide 107R-A-W-V-A-W-R-N-R115 (LP9), polymyxin B, or gallidermin. A comparative transcriptome analysis of wild type and the graRS mutant revealed that GraRS controls 248 genes. It up-regulates global regulators (rot, sarS, or mgrA), various colonization factors, and exotoxin-encoding genes, as well as the ica and dlt operons. A pronounced decrease in the expression of the latter two operons explains why the graRS mutant is also biofilm-negative. The decrease of the dlt transcript in the graRS mutant correlates with a 46.7% decrease in the content of esterified d-alanyl groups in teichoic acids. The oatA/dltA double mutant showed the highest sensitivity to lysozyme; this mutant completely lacks teichoic acid–bound d-alanine esters, which are responsible for the increased susceptibility to CAMPs and peptidoglycan O-acetylation. Our results demonstrate that resistance to lysozyme can be dissected into genes mediating resistance to its muramidase activity (oatA) and genes mediating resistance to CAMPs (graRS and dlt). The two lysozyme activities act synergistically, as the oatA/dltA or oatA/graRS double mutants are much more susceptible to lysozyme than each of the single mutants.
Author Summary
In humans, lysozyme plays an important role in the suppression of bacterial infections. However, some bacterial pathogens, such as Staphylococcus aureus, are completely resistant to lysozyme. Here we demonstrate that lysozyme acts on S. aureus in two ways: as a muramidase (cell wall lytic enzyme) and as a cationic antimicrobial peptide (CAMP). S. aureus has developed resistance mechanisms against both activities by modifying distinct cell wall structures. Modification of the peptidoglycan by O-acetylation (OatA) renders the cells resistant to the muramidase activity. Modification of teichoic acids by d-alanine esterification (Dlt) renders the cells resistant to lysozyme's CAMPs and other CAMPs. Transcriptome analysis of the glycopeptide resistance-associated (GraRS) two-component system revealed that this global regulator controls 248 genes such as other global regulators, colonization factors, or exotoxin-encoding genes. Since GraRS also upregulates the dlt operon, it was not surprising that in the graRS mutant teichoic acid d-alanylation is markedly decreased, which explains its increased sensitivity to CAMPs. By comparative analysis of mutants we were able to dissect genes that were responsive to the dual activities of lysozyme. Here we show how efficiently S. aureus is protected from the human defense system, which enables this pathogen to cause persistent infections.
doi:10.1371/journal.ppat.0030102
PMCID: PMC1933452  PMID: 17676995
18.  MAP Kinase Phosphatase 1 (MKP-1/DUSP1) is Neuroprotective in Huntington’s Disease Via Additive Effects of JNK and p38 Inhibition 
We previously demonstrated that sodium butyrate is neuroprotective in Huntington’s disease (HD) mice and that this therapeutic effect is associated with increased expression of mitogen-activated protein kinase/dual-specificity phosphatase 1 (MKP-1/DUSP1). Here we show that enhancing MKP-1 expression is sufficient to achieve neuroprotection in lentiviral models of HD. Wild-type MKP-1 overexpression inhibited apoptosis in primary striatal neurons exposed to an N-terminal fragment of polyglutamine-expanded huntingtin (Htt171-82Q), blocking caspase-3 activation and significantly reducing neuronal cell death. This neuroprotective effect of MKP-1 was demonstrated to be dependent on its enzymatic activity, being ablated by mutation of its phosphatase domain and being attributed to inhibition of specific MAP kinases (MAPKs). Overexpression of MKP-1 prevented the polyglutamine-expanded huntingtin-induced activation of c-Jun N-terminal kinases (JNKs) and p38 MAPKs, whereas extracellular signal-regulated kinase 1/2 (ERK1/2) activation was not altered by either polyglutamine-expanded Htt or MKP-1. Moreover, mutants of MKP-1 that selectively prevented p38 or JNK binding confirmed the important dual contributions of p38 and JNK regulation to MKP-1-mediated neuroprotection. These results demonstrate additive effects of p38 and JNK MAPK inhibition by MKP-1 without consequence to ERK activation in this striatal neuron-based paradigm. MKP-1 also provided neuroprotection in vivo in a lentiviral model of HD neuropathology in rat striatum. Taken together, these data extend previous evidence that JNK- and p38-mediated pathways contribute to HD pathogenesis and, importantly, show that therapies simultaneously inhibiting both JNK and p38 signalling pathways may lead to improved neuroprotective outcomes.
doi:10.1523/JNEUROSCI.4965-11.2013
PMCID: PMC3711389  PMID: 23392662
19.  Transcriptional Induction of MKP-1 in Response to Stress Is Associated with Histone H3 Phosphorylation-Acetylation 
Molecular and Cellular Biology  2001;21(23):8213-8224.
Mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1) has been shown to play a critical role in mediating the feedback control of MAP kinase cascades in a variety of cellular processes, including proliferation and stress responsiveness. Although MKP-1 expression is induced by a broad array of extracellular stimuli, the mechanisms mediating its induction remain poorly understood. Here we show that MKP-1 mRNA was potently induced by arsenite and ultraviolet light and modestly increased by heat shock and hydrogen peroxide. Interestingly, arsenite also dramatically induces phosphorylation-acetylation of histone H3 at a global level which precedes the induction of MKP-1 mRNA. The transcriptional induction of MKP-1, histone H3 modification, and elevation in MKP-1 mRNA in response to arsenite are all partially prevented by the p38 MAP kinase inhibitor SB203580, suggesting that the p38 pathway is involved in these processes. Finally, analysis of the DNA brought down by chromatin immunoprecipitation (ChIP) reveals that arsenite induces phosphorylation-acetylation of histone H3 associated with the MKP-1 gene and enhances binding of RNA polymerase II to MKP-1 chromatin. ChIP assays following exposure to other stress agents reveal various degrees of histone H3 modification at the MKP-1 chromatin. The differential contribution of p38 and ERK MAP kinases in mediating MKP-1 induction by different stress agents further illustrates the complexity and versatility of stress-induced MKP-1 expression. Our results strongly suggest that chromatin remodeling after stress contributes to the transcriptional induction of MKP-1.
doi:10.1128/MCB.21.23.8213-8224.2001
PMCID: PMC99986  PMID: 11689710
20.  Toxoplasma gondii Rhoptry 16 Kinase Promotes Host Resistance to Oral Infection and Intestinal Inflammation Only in the Context of the Dense Granule Protein GRA15 
Infection and Immunity  2013;81(6):2156-2167.
Toxoplasma gondii transmission between intermediate hosts is dependent on the ingestion of walled cysts formed during the chronic phase of infection. Immediately following consumption, the parasite must ensure survival of the host by preventing adverse inflammatory responses and/or by limiting its own replication. Since the Toxoplasma secreted effectors rhoptry 16 kinase (ROP16) and dense granule 15 (GRA15) activate the JAK-STAT3/6 and NF-κB signaling pathways, respectively, we explored whether a particular combination of these effectors impacted intestinal inflammation and parasite survival in vivo. Here we report that expression of the STAT-activating version of ROP16 in the type II strain (strain II+ROP16I) promotes host resistance to oral infection only in the context of endogenous GRA15 expression. Protection was characterized by a lower intestinal parasite burden and dampened inflammation. Host resistance to the II+ROP16I strain occurred independently of STAT6 and the T cell coinhibitory receptors B7-DC and B7-H1, two receptors that are upregulated by ROP16. In addition, coexpression of ROP16 and GRA15 enhanced parasite susceptibility within tumor necrosis factor alpha/gamma interferon-stimulated macrophages in a STAT3/6-independent manner. Transcriptional profiling of infected STAT3- and STAT6-deficient macrophages and parasitized Peyer's patches from mice orally challenged with strain II+ROP16I suggested that ROP16 activated STAT5 to modulate host gene expression. Consistent with this supposition, the ROP16 kinase induced the sustained phosphorylation and nuclear localization of STAT5 in Toxoplasma-infected cells. In summary, only the combined expression of both GRA15 and ROP16 promoted host resistance to acute oral infection, and Toxoplasma may possibly target the STAT5 signaling pathway to generate protective immunity in the gut.
doi:10.1128/IAI.01185-12
PMCID: PMC3676013  PMID: 23545295
21.  Reciprocal Regulation of Extracellular Signal Regulated Kinase 1/2 and Mitogen Activated Protein Kinase Phosphatase-3 
Toxicology and applied pharmacology  2008;232(3):408-417.
Mitogen activated protein kinase phosphatase-3 (MKP-3) is a putative tumor suppressor. When transiently overexpressed, MKP-3 dephosphorylates and inactivates extracellular signal regulated kinase (ERK) 1/2. Little is known about the roles of endogenous MKP-3, however. We previously showed that MKP-3 is upregulated in cell lines that express oncogenic Ras. Here we tested the roles of endogenous MKP-3 in modulating ERK1/2 under conditions of chronic stimulation of the Ras/Raf/MEK1/2/ERK1/2 pathway by expression of oncogenic Ras. We used two cell lines: H-ras MCF10A, breast epithelial cells engineered to express H-Ras, and DLD-1, colon cancer cells that express endogenous Ki-Ras. First, we found that MKP-3 acts in a negative feedback loop to suppress basal ERK1/2 when oncogenic Ras stimulates the Ras/Raf/MEK1/2/ERK1/2 cascade. ERK1/2 was required to maintain elevated MKP-3, indicative of a negative feedback loop. Accordingly, knockdown of MKP-3, via siRNA, increased ERK1/2 phosphorylation. Second, by using siRNA, we found that MKP-3 helps establish the sensitivity of ERK1/2 to extracellular activators by limiting the duration of ERK1/2 phosphorylation. Third, we found that the regulation of ERK1/2 by MKP-3 is countered by the complex regulation of MKP-3 by ERK1/2. Potent ERK1/2 activators stimulated the loss of MKP-3 within 30 minutes due to an ERK1/2-dependent decrease in MKP-3 protein stability. MKP-3 levels recovered within 120 minutes due to ERK1/2-dependent resynthesis. Preventing MKP-3 resynthesis, via siRNA, prolonged ERK1/2 phosphorylation. Altogether, these results suggest that under the pressure of oncogenic Ras expression, MKP-3 reins in ERK1/2 by serving in ERK1/2-dependent negative feedback pathways.
doi:10.1016/j.taap.2008.08.007
PMCID: PMC2581931  PMID: 18771677
Mitogen activated protein kinase phosphatase-3; extracellular signal regulated kinase; Ras
22.  Differential Induction of Isolated Lymphoid Follicles in the Gut by 18β-Glycyrrhetinic Acid 
PLoS ONE  2014;9(7):e100878.
18β-glycyrrhetinic acid (GRA) is a pharmacologically active component of licorice root with documented immunomodulatory properties. We reported that GRA administered orally to mice induces B cell recruitment to isolated lymphoid follicles (ILF) in the small intestine and shortens the duration of rotavirus antigen shedding. ILF are dynamic lymphoid tissues in the gut acquired post-natally upon colonization with commensal bacteria and mature through B cell recruitment to the follicles, resulting in up-regulation of IgA synthesis in response to changes in the composition of microbiota. In this study, we investigated potential mechanisms by which GRA induces ILF maturation in the ileum and the colon using mice depleted of enteric bacteria and a select group of mice genetically deficient in pattern recognition receptors. The data show GRA was unable to induce ILF maturation in ileums of mice devoid of commensal bacteria, MyD88−/− or NOD2−/− mice, but differentially induced ILF in colons. Increased expression of chemokine and chemokine receptor genes that modulate B and T cell recruitment to the mucosa were in part dependent on NOD2, TLR, and signaling adaptor protein MyD88. Together the results suggest GRA induces ILF through cooperative signals provided by bacterial ligands under normal conditions to induce B cell recruitment to ILF to the gut, but that the relative contribution of these signals differ between ileum and colon.
doi:10.1371/journal.pone.0100878
PMCID: PMC4081046  PMID: 24992099
23.  A Toxoplasma dense granule protein, GRA24, modulates the early immune response to infection by promoting a direct and sustained host p38 MAPK activation 
The Journal of Experimental Medicine  2013;210(10):2071-2086.
Toxoplasma gondii secretes a novel dense granule protein, GRA24, that traffics from the vacuole to the host cell nucleus where it prolongs p38a activation and correlates with proinflammatory cytokine production.
Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan parasite that resides inside a parasitophorous vacuole. During infection, Toxoplasma actively remodels the transcriptome of its hosting cells with profound and coupled impact on the host immune response. We report that Toxoplasma secretes GRA24, a novel dense granule protein which traffics from the vacuole to the host cell nucleus. Once released into the host cell, GRA24 has the unique ability to trigger prolonged autophosphorylation and nuclear translocation of the host cell p38α MAP kinase. This noncanonical kinetics of p38α activation correlates with the up-regulation of the transcription factors Egr-1 and c-Fos and the correlated synthesis of key proinflammatory cytokines, including interleukin-12 and the chemokine MCP-1, both known to control early parasite replication in vivo. Remarkably, the GRA24–p38α complex is defined by peculiar structural features and uncovers a new regulatory signaling path distinct from the MAPK signaling cascade and otherwise commonly activated by stress-related stimuli or various intracellular microbes.
doi:10.1084/jem.20130103
PMCID: PMC3782045  PMID: 24043761
24.  Tay Bridge Is a Negative Regulator of EGFR Signalling and Interacts with Erk and Mkp3 in the Drosophila melanogaster Wing 
PLoS Genetics  2013;9(12):e1003982.
The regulation of Extracellular regulated kinase (Erk) activity is a key aspect of signalling by pathways activated by extracellular ligands acting through tyrosine kinase transmembrane receptors. In this process, participate proteins with kinase activity that phosphorylate and activate Erk, as well as different phosphatases that inactivate Erk by de-phosphorylation. The state of Erk phosphorylation affects not only its activity, but also its subcellular localization, defining the repertoire of Erk target proteins, and consequently, the cellular response to Erk. In this work, we characterise Tay bridge as a novel component of the EGFR/Erk signalling pathway. Tay bridge is a large nuclear protein with a domain of homology with human AUTS2, and was previously identified due to the neuronal phenotypes displayed by loss-of-function mutations. We show that Tay bridge antagonizes EGFR signalling in the Drosophila melanogaster wing disc and other tissues, and that the protein interacts with both Erk and Mkp3. We suggest that Tay bridge constitutes a novel element involved in the regulation of Erk activity, acting as a nuclear docking for Erk that retains this protein in an inactive form in the nucleus.
Author Summary
Extracellular regulated kinases (Erk) mediate signalling by pathways activated by tyrosine kinase transmembrane receptors. The level of activated Erk depends on a highly regulated balance between cytoplasmic kinases and nuclear/cytoplasmic phosphatases, which determine the state of Erk phosphorylation. This affects Erk activity and its subcellular localization, defining the repertoire of Erk targets, and consequently, the cellular response to Erk. In this work, we use a genetic approach to characterise the gene tay bridge as a novel component of the EGFR/Erk signalling pathway. Tay bridge has a domain of homology with human AUTS2, and was previously identified due to the neuronal phenotypes displayed by loss-of-function mutations. We show that Tay bridge antagonizes EGFR signalling in the Drosophila melanogaster wing disc and other tissues, and that the protein interacts with both Erk and Mkp3. We suggest that Tay bridge constitutes a novel element involved in the regulation of Erk activity, acting as a nuclear docking for Erk that retains this protein in an inactive form in the nucleus. These results could provide important insights into the clinical consequences of AUTS2 mutations in humans, which are related to behavioural perturbations including autism, mental retardation, Attention Deficit Hyperactivity Disorder and alcohol drinking behaviour.
doi:10.1371/journal.pgen.1003982
PMCID: PMC3861119  PMID: 24348264
25.  Cannabinoid receptor type 2 activation induces a microglial anti-inflammatory phenotype and reduces migration via MKP induction and ERK dephosphorylation 
Molecular Pain  2009;5:25.
Background
Cannabinoid receptor type 2 (CBR2) inhibits microglial reactivity through a molecular mechanism yet to be elucidated. We hypothesized that CBR2 activation induces an anti-inflammatory phenotype in microglia by inhibiting extracellular signal-regulated kinase (ERK) pathway, via mitogen-activated protein kinase-phosphatase (MKP) induction. MKPs regulate mitogen activated protein kinases, but their role in the modulation of microglial phenotype is not fully understood.
Results
JWH015 (a CBR2 agonist) increased MKP-1 and MKP-3 expression, which in turn reduced p-ERK1/2 in LPS-stimulated primary microglia. These effects resulted in a significant reduction of tumor necrosis factor-α (TNF) expression and microglial migration. We confirmed the causative link of these findings by using MKP inhibitors. We found that the selective inhibition of MKP-1 by Ro-31-8220 and PSI2106, did not affect p-ERK expression in LPS+JWH015-treated microglia. However, the inhibition of both MKP-1 and MKP-3 by triptolide induced an increase in p-ERK expression and in microglial migration using LPS+JWH015-treated microglia.
Conclusion
Our results uncover a cellular microglial pathway triggered by CBR2 activation. These data suggest that the reduction of pro-inflammatory factors and microglial migration via MKP-3 induction is part of the mechanism of action of CBR2 agonists. These findings may have clinical implications for further drug development.
doi:10.1186/1744-8069-5-25
PMCID: PMC2704199  PMID: 19476641

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