Phosphatase MKP-1 Research Articles

The reduced SCFA-producing gut microbes are involved in the inflammatory activation in Kawasaki disease.

Kawasaki disease (KD), an acute febrile systemic vasculitis in children, has become the leading cause of acquired heart disease in developed countries. Recently, the altered gut microbiota was found in KD patients during the acute phase. However, little is known about its characteristics and role in the pathogenesis of KD. In our study, an altered gut microbiota composition featured by the reduction in SCFAs-producing bacteria was demonstrated in the KD mouse model. Next, probiotic Clostridium butyricum (C. butyricum) and antibiotic cocktails were respectively employed to modulate gut microbiota. The use of C. butyricum significantly increased the abundance of SCFAs-producing bacteria and attenuated the coronary lesions with reduced inflammatory markers IL-1β and IL-6, but antibiotics depleting gut bacteria oppositely deteriorated the inflammation response. The gut leakage induced by dysbiosis to deteriorate the host's inflammation was confirmed by the decreased intestinal barrier proteins Claudin-1, Jam-1, Occludin, and ZO-1, and increased plasma D-lactate level in KD mice. Mechanistically, SCFAs, the major beneficial metabolites of gut microbes to maintain the intestinal barrier integrity and inhibit inflammation, was also found decreased, especially butyrate, acetate and propionate, in KD mice by gas chromatography-mass spectrometry (GC-MS). Moreover, the reduced expression of SCFAs transporters, monocarboxylate transporter 1 (MCT-1) and sodium-dependent monocarboxylate transporter 1 (SMCT-1), was also shown in KD mice by western blot and RT-qPCR analyses. As expected, the decrease of fecal SCFAs production and barrier dysfunction were improved by oral C. butyricum treatment but was deteriorated by antibiotics. In vitro, butyrate, not acetate or propionate, increased the expression of phosphatase MKP-1 to dephosphorylate activated JNK, ERK1/2 and p38 MAPK against excessive inflammation in RAW264.7 macrophages. It suggests a new insight into probiotics and their metabolites supplements to treat KD.

MKP3 Overexpression and ERK1/2 Phosphorylation in Dopaminergic Neurons Regulate DAT Trafficking and Function

Dopamine is a neurotransmitter that plays a critical role in movement, motivation, and reward. Dopamine neurotransmission is regulated by the dopamine transporter (DAT), which translocates dopamine from the extracellular space to the presynaptic neuron. DAT is a target for psychostimulants such as cocaine, giving DAT a key role in psychostimulant‐use disorders. DAT cell‐surface expression regulated by trafficking to and from the plasma membrane is an important mechanism for controlling DAT function. Two signaling pathways that contribute to DAT trafficking are the protein kinase C (PKC) and mitogen activated protein kinases (MAPKs), including the extracellular signal‐regulated kinase (ERK1/2) and p38 MAPK. ERK1/2 is inactivated by the MAP kinase phosphatase MKP3, which dephosphorylates and inactivates ERK1/2. Previous data from the lab demonstrated that activation of PKC by administration of phorbol 12‐myristate 13‐acetate (PMA) in an in vitro system results in decreased DAT cell‐surface expression, and overexpression of MKP3 attenuates this effect. Furthermore, other studies have shown that ERK1/2 activation, downstream of PKC activation, may lead to phosphorylation of DAT on residue threonine 53 (Thr53), however the significance of this phosphorylation is not completely understood. In order to characterize the contributions of MKP3 and ERK1/2 to DAT phosphorylation and trafficking in vivo, we have developed two viral constructs based on adeno‐associated viral (AAV) vectors that enable cre recombinase (cre)‐dependent expression. One vector cre‐dependently expresses MKP3. The other vector is based on a genetic tool named OptoSOS. This vector enables cre‐dependent, blue‐light induced activation of ERK1/2, and as a result, can spatiotemporally activate intracellular ERK1/2 signaling. Both viral vectors are employed with transgenic rats that express cre in tyrosine hydroxylase‐positive cells (TH‐Cre rats) to drive expression in dopaminergic neurons. From these studies, we find that MKP3 overexpression and resulting ERK1/2 inactivation increases DAT surface expression by blocking the internalization and degradation of the transporter leading to an accumulation of DAT on the cell‐surface. Interestingly, MKP3 overexpression also decreases phosphorylation at Thr53. While we have shown that ERK inactivation results in increased DAT cell‐surface expression, we have employed the OptoSOS system to activate ERK1/2 and assess resulting changes in DAT trafficking. We have confirmed that ERK1/2 is activated by blue‐light in a cre‐dependent manner, and similar to what we observe with MKP3 overexpression, ERK1/2 activation results in changes in DAT cell‐surface expression and phosphorylation. Taken together, our results indicate that MKP3 and ERK1/2 serve critical physiological roles in DAT regulation and can be utilized to investigate the mechanistic relationship between DAT trafficking, phosphorylation, and transporter activity.

Dual control of MAPK activities by AP2C1 and MKP1 MAPK phosphatases regulates defence responses in Arabidopsis.

Mitogen-activated protein kinase (MAPK) cascades transmit environmental signals and induce stress and defence responses in plants. These signalling cascades are negatively controlled by specific Ser/Thr protein phosphatases of the type 2C (PP2C) and dual-specificity phosphatase (DSP) families that inactivate stress-induced MAPKs; however, the interplay between phosphatases of these different types has remained unknown. This work reveals that different Arabidopsis MAPK phosphatases, the PP2C-type AP2C1 and the DSP-type MKP1, exhibit both specific and overlapping functions in plant stress responses. Each single mutant, ap2c1 and mkp1, and the ap2c1 mkp1 double mutant displayed enhanced stress-induced activation of the MAPKs MPK3, MPK4, and MPK6, as well as induction of a set of transcription factors. Moreover, ap2c1 mkp1 double mutants showed an autoimmune-like response, associated with increased levels of the stress hormones salicylic acid and ethylene, and of the phytoalexin camalexin. This phenotype was reduced in the ap2c1 mkp1 mpk3 and ap2c1 mkp1 mpk6 triple mutants, suggesting that the autoimmune-like response is due to MAPK misregulation. We conclude that the evolutionarily distant MAPK phosphatases AP2C1 and MKP1 contribute crucially to the tight control of MAPK activities, ensuring appropriately balanced stress signalling and suppression of autoimmune-like responses during plant growth and development.

Early p38 Activation Regulated by MKP-1 Is Determinant for High Levels of IL-10 Expression Through TLR2 Activation.

Toll-like receptors (TLRs) play a crucial role in the recognition of pathogen-derived components as a first line of defense against infections. It has been suggested that depending on the nature of the pathogens, TLRs activation induce a distinct cytokine profile that may contribute to the polarization of the acquired immune response. Here, we investigated the early MAPK signaling activation via TLR4 and TLR2 receptors and its impact in differential cytokine profile by macrophages. We found that TLR2 ligands activated MAPKs p38 and ERK earlier compared to the TLR4 ligand LPS in macrophages. Higher IL-10/IL-12 and IL-10/TNF-α ratios were also observed at later time points in response to TLR2 ligands compared to LPS. The results also indicate an earlier activation of the phosphatase MKP-1 and that MKP-1 KO macrophages show a prolongation in p38 phosphorylation in response to TLR2 stimulation. Furthermore, p38 is critical for IL-10 expression in response to TLR2 ligands, which triggers the macrophage change to a M2 and regulatory phenotype in contrast to the M1 phenotype induced by TLR4 activation. Therefore, the early TLR2-mediated p38 induction contributes for the high IL-10 production, likely as a virulence strategy to suppress host Th1 response against certain types of pathogens.

Increased hepatic JNK activation by ethanol is mediated by transcriptional suppression of mitogen‐activated protein kinase phosphatase 1 (Mkp1): role of cAMP‐specific protein kinase A

Background Increased and sustained JNK activation has been shown to be detrimental for hepatocyte survival and liver injury in various diseases, including alcohol-associated liver disease (ALD). Among three JNK isoforms, JNK1 and 2 are expressed in hepatocytes and both play a role in mouse models of ALD. Mkp1 phosphatase has been identified as a regulator of JNK activation. A previous study showed that Mkp1 degradation plays a critical role in sustained JNK activation and hepatocyte death from acute ethanol exposure. However, the effect of chronic ethanol exposure on hepatocyte Mkp1 expression is not known. Methods Male C57B/6 mice were subjected to intragastric (iG) feeding of ethanol for 8 weeks with weekly ethanol binge (5g/kg) starting in the second week of iG feeding. Pair-fed control mice were fed an isocaloric high-fat liquid diet. Plasma ALT, AST and histologic grading were performed to evaluate steatosis, liver inflammation and injury. Liver immunohistochemistry was performed to evaluate apoptosis (TUNEL staining). Myeloperoxidase (MPO) activity assay and chloroacetate esterase (CAE) staining for neutrophil infiltration were also performed. Liver RNA sequencing was performed on an Illumina NextSeq 500. Data analysis was done using the Tuxedo suite pipeline. Validation studies were carried out by performing real time qPCR and Western blot. Primary mouse hepatocytes were exposed to 50 mM ethanol for 48 hours followed by TNF stimulation. Statistical analysis was performed using ANOVA. Results Histological examination of liver sections confirmed the development of severe alcohol-associated steatohepatitis in ethanol fed mice via significant micro- and macro-vesicular steatosis, polymorphonuclear leukocyte (PMN) infiltration, CAE and TUNEL-positive cells. ALT and AST levels were significantly elevated in ethanol-fed mice. JNK activation was evident by Western blot analysis of phosphorylated JNK levels. RNA Seq analysis showed significant changes in expression of several Mkps; however, only Mkp1 levels were significantly downregulated, which was further validated by real time qPCR. Importantly, ethanol exposure of primary mouse hepatocytes led to a significant decrease in Mkp1 mRNA expression which further declined following TNF stimulation. These changes in Mkp1 were accompanied by sustained JNK activation and cell death. Moreover, ethanol exposure and TNF stimulation markedly increased cAMP specific PDE4 expression resulting in reduction in hepatocyte cAMP levels. Significantly, PDE4 inhibition and PKA specific agonists prevented the suppressive effect of ethanol and TNF on Mkp1 and resultant activation of JNK and cell death. Conclusion Our results strongly implicate ethanol-induced decline in hepatic Mkp1 as a significant mechanism regulating JNK activation and hepatocyte death in ALD. Further, these data strongly suggest that the ethanol induced decrease in cAMP/PKA signaling plays a causal role in regulating the Mkp1/JNK axis in hepatocytes and development of liver injury.

The anti-fibrotic actions of relaxin are mediated through AT2 R-associated protein phosphatases via RXFP1-AT2 R functional crosstalk in human cardiac myofibroblasts.

Fibrosis is a hallmark of several cardiovascular diseases. The relaxin family peptide receptor 1 (RXFP1) agonist, relaxin, has rapidly occurring anti-fibrotic actions which are mediated through RXFP1 and angiotensin II receptor crosstalk on renal and cardiac myofibroblasts. Here, we investigated whether this would allow relaxin to indirectly activate angiotensin II type 2 receptor (AT2 R)-specific signal transduction in primary human cardiac myofibroblasts (HCMFs). The anti-fibrotic effects of recombinant human relaxin (RLX; 16.8nM) or the AT2 R-agonist, Compound 21 (C21; 1μM), were evaluated in TGF-β1-stimulated HCMFs, in the absence or presence of an RXFP1 antagonist (1μM) or AT2 R antagonist (0.1μM) to confirm RXFP1-AT2 R crosstalk. Competition binding for RXFP1 was determined. Western blotting was performed to determine which AT2 R-specific protein phosphatases were expressed by HCMFs; then, the anti-fibrotic effects of RLX and/or C21 were evaluated in the absence or presence of pharmacological inhibition (NSC95397 (1μM) for MKP-1; okadaic acid (10nM) for PP2A) or siRNA-knockdown of these phosphatasesafter 72hours. The RLX- or C21-induced increase in ERK1/2 and nNOS phosphorylation, and decrease in α-SMA (myofibroblast differentiation) and collagen-I expression by HCMFs was abrogated by pharmacological blockade of RXFP1 or the AT2 R, confirming RXFP1-AT2 R crosstalk in these cells. HCMFs were found to express AT2 R-dependent MKP-1 and PP2A phosphatases, while pharmacological blockade or siRNA-knockdown of either phosphatase also abolished RLX and/or C21 signal transduction in HCMFs (all P<.05 vs RLX or C21 alone). These findings demonstrated that RLX can indirectly activate AT2 R-dependent phosphatase activity in HCMFs by signaling through RXFP1-AT2 R crosstalk, which have important therapeutic implications for its anti-fibrotic actions.

Mechanisms for Temporal Information Coding in Inflammatory JNK Signaling

JNK (c‐Jun‐N‐terminal kinase) is a stress‐activated protein kinase that plays key roles in mediating stress and inflammatory response such as cell death, proliferation, and cytokine production. Several studies have uncovered that the duration of JNK signaling determines the specificity of downstream cellular events that lead to either cell‐death or survival. However, the question of how JNK is dynamically regulated in cells remains elusive. In this study, we aimed to reveal the physiological importance of the dynamics of JNK signaling in downstream gene expression by elucidating how inflammatory cytokine IL‐1β regulates a temporal pattern of JNK signaling in living HeLa cells. Here, using fluorescence imaging and our novel JNK activity reporter based on FRET, we succeeded in the quantitative evaluation of JNK dynamics at single cell resolution upon IL‐1β stimulation. Periodic pulsatile IL‐1β‐stimuli to cells were employed to analyze the regulatory mechanism of JNK activity. Our analysis revealed that JNK can be activated either transiently or repeatedly depending on the interval of stimulation pulses, while the initial peak amplitude of JNK activity was almost the same. We found that a simple negative‐feedback loop from a MAPK phosphatase MKP1 to its upstream kinase p38 MAPK accounts for the observed temporal dynamics of JNK activity, which was further verified by biochemical analysis and mathematical model simulation. The temporal aspects of IL‐1β stimulation were reflected in the gene expression profile obtained from HeLa cells after stimulation, in which the specific set of JNK‐dependent genes were more upregulated by repetitive IL‐1β stimulation pulses compared to that by continuous IL‐1β exposure. Our results indicate that the dynamic behavior of JNK in response to IL‐1β stimulation is regulated through dephosphorylation of JNK by MKP1 whose expression is positively regulated by p38 MAPK and down‐regulated by its phosphatase activity on p38 MAPK. In addition, JNK activity as well as downstream gene expression specificity could be modulated just by altering the temporal pattern of input IL‐1β stimuli. In conclusion, we demonstrated that the information of temporally fluctuating cytokine stimulation can be converted into the activation dynamics of JNK signaling, and the downstream gene expression specificity can be encoded by the temporal dynamics of JNK activity. Such a temporal information coding mechanism may have physiological relevance for inducing an adequate cellular response to a wide range of fluctuating cytokine levels through precise control of gene expression.Support or Funding InformationThis work was supported in part by JSPS KAKENHI [Grant numbers JP19K06548, JP17H06017 to T.T., JP18K06859 to S.A‐A.].

JNK-Dependent cJun Phosphorylation Mitigates TGFβ- and EGF-Induced Pre-Malignant Breast Cancer Cell Invasion by Suppressing AP-1-Mediated Transcriptional Responses.

Transforming growth factor-β (TGFβ) has both tumor-suppressive and tumor-promoting effects in breast cancer. These functions are partly mediated through Smads, intracellular transcriptional effectors of TGFβ. Smads form complexes with other DNA-binding transcription factors to elicit cell-type-dependent responses. Previously, we found that the collagen invasion and migration of pre-malignant breast cancer cells in response to TGFβ and epidermal growth factor (EGF) critically depend on multiple Jun and Fos components of the activator protein (AP)-1 transcription factor complex. Here we report that the same process is negatively regulated by Jun N-terminal kinase (JNK)-dependent cJun phosphorylation. This was demonstrated by analysis of phospho-deficient, phospho-mimicking, and dimer-specific cJun mutants, and experiments employing a mutant version of the phosphatase MKP1 that specifically inhibits JNK. Hyper-phosphorylation of cJun by JNK strongly inhibited its ability to induce several Jun/Fos-regulated genes and to promote migration and invasion. These results show that MEK-AP-1 and JNK-phospho-cJun exhibit distinct pro- and anti-invasive functions, respectively, through differential regulation of Smad- and AP-1-dependent TGFβ target genes. Our findings are of importance for personalized cancer therapy, such as for patients suffering from specific types of breast tumors with activated EGF receptor-Ras or inactivated JNK pathways.

Regulation of apoptosis in the ischemic penumbra in the first day post-stroke.

Regulation of apoptosis in the ischemic penumbra in the first day post-stroke.

Cdk5 Deletion Enhances the Anti-inflammatory Potential of GC-Mediated GR Activation During Inflammation.

The suppression of activated pro-inflammatory macrophages during immune response has a major impact on the outcome of many inflammatory diseases including sepsis and rheumatoid arthritis. The pro- and anti-inflammatory functions of macrophages have been widely studied, whereas their regulation under immunosuppressive treatments such as glucocorticoid (GC) therapy is less well-understood. GC-mediated glucocorticoid receptor (GR) activation is crucial to mediate anti-inflammatory effects. In addition, the anti-cancer drug roscovitine, that is currently being tested in clinical trials, was recently described to regulate inflammatory processes by inhibiting different Cdks such as cyclin-dependent kinase 5 (Cdk5). Cdk5 was identified as a modulator of inflammatory processes in different immune cells and furthermore described to influence GR gene expression in the brain. Whether roscovitine can enhance the immunosuppressive effects of GCs and if the inhibition of Cdk5 affects GR gene regulatory function in innate immune cells, such as macrophages, has not yet been investigated. Here, we report that roscovitine enhances the immunosuppressive Dexamethasone (Dex) effect on the inducible nitric oxide synthase (iNos) expression, which is essential for immune regulation. Cdk5 deletion in macrophages prevented iNos protein and nitric oxide (NO) generation after a combinatory treatment with inflammatory stimuli and Dex. Cdk5 deletion in macrophages attenuated the GR phosphorylation on serine 211 after Dex treatment alone and in combination with inflammatory stimuli, but interestingly increased the GR-dependent anti-inflammatory target gene dual-specificity phosphatase 1 (Dusp1, Mkp1). Mkp1 phosphatase activity decreases the activation of its direct target p38Mapk, reduced iNos expression and NO production upon inflammatory stimuli and Dex treatment in the absence of Cdk5. Taken together, we identified Cdk5 as a potential novel regulator of NO generation in inflammatory macrophages under GC treatment. Our data suggest that GC treatment in combination with specific Cdk5 inhibtior(s) provides a stronger suppression of inflammation and could thus replace high-dose GC therapy which has severe side effects in the treatment of inflammatory diseases.

Apoptosis regulation in the penumbra after ischemic stroke: expression of pro- and antiapoptotic proteins.

Ischemic stroke is the leading cause of human disability and mortality in the world. The main problem in stroke therapy is the search of efficient neuroprotector capable to rescue neurons in the potentially salvageable transition zone (penumbra), which is expanding after brain damage. The data on molecular mechanisms of penumbra formation and expression of diverse signaling proteins in the penumbra during first 24h after ischemic stroke are discussed. Two basic features of cell death regulation in the ischemic penumbra were observed: (1) both apoptotic and anti-apoptotic proteins are simultaneously over-expressed in the penumbra, so that the fate of individual cells is determined by the balance between these opposite tendencies. (2) Similtaneous and concerted up-regulation in the ischemic penumbra of proteins that execute apoptosis (caspases 3, 6, 7; Bcl-10, SMAC/DIABLO, AIF, PSR), signaling proteins that regulate different apoptosis pathways (p38, JNK, DYRK1A, neurotrophin receptor p75); transcription factors that control expression of various apoptosis regulation proteins (E2F1, p53, c-Myc, GADD153); and proteins, which are normally involved in diverse cellular functions, but stimulate apoptosis in specific situations (NMDAR2a, Par4, GAD65/67, caspase 11). Hence, diverse apoptosis initiation and regulation pathways are induced simultaneously in penumbra from very different initial positions. Similarly, various anti-apoptotic proteins (Bcl-x, p21/WAF-1, MDM2, p63, PKBα, ERK1, RAF1, ERK5, MAKAPK2, protein phosphatases 1α and MKP-1, estrogen and EGF receptors, calmodulin, CaMKII, CaMKIV) are upregulated. These data provide an integral view of neurodegeneration and neuroprotection in penumbra. Some discussed proteins may serve as potential targets for anti-stroke therapy.

Chronic adolescent stress alters learning and cognition in adult female rats

Corticosteroids are effective anti-inflammatory therapies widely utilized in chronic respiratory diseases. But these medicines can lose their efficacy during respiratory infection resulting in disease exacerbation. Further in vitro research is required to understand how infection worsens lung function control in order to advance therapeutic options to treat infectious exacerbation in the future. In this study, we utilize a cellular model of bacterial exacerbation where we pretreat A549 lung epithelial cells with the synthetic bacterial lipoprotein Pam3CSK4 (a TLR2 ligand) to mimic bacterial infection and tumor necrosis factor α (TNFα) to simulate inflammation. Under these conditions, Pam3CSK4 induces corticosteroid insensitivity; demonstrated by substantially reduced ability of the corticosteroid dexamethasone to repress TNFα-induced interleukin 6 secretion. We then explored the molecular mechanism responsible and found that corticosteroid insensitivity induced by bacterial mimics was not due to altered translocation of the glucocorticoid receptor into the nucleus, nor an impact on the NF-κB pathway. Moreover, Pam3CSK4 did not affect corticosteroid-induced upregulation of anti-inflammatory MAPK deactivating phosphatase—MKP-1. However, Pam3CSK4 can induce oxidative stress and we show that a proportion of the MKP-1 produced in response to corticosteroid in the context of TLR2 ligation was rendered inactive by oxidation. Thus to combat inflammation in the context of bacterial exacerbation we sought to discover effective strategies that bypassed this road-block. We show for the first time that known (FTY720) and novel (theophylline) activators of the phosphatase PP2A can serve as non-steroidal anti-inflammatory alternatives and/or corticosteroid-sparing approaches in respiratory inflammation where corticosteroid insensitivity exists.

IFN-γ stimulates CpG-induced IL-10 production in Bcells via p38 and JNK signalling pathways.

The production of IL-10, a potent immunosuppressive cytokine, must be strictly regulated to ensure a balanced immune response. IFN-γ, a key cytokine in multiple immune processes and pathologies, is known as an inhibitor of IL-10 production by monocytes and macrophages, but also has some regulatory functions. In the present study, we explored the role of IFN-γ on Toll-like receptor (TLR)-induced IL-10 production in murine peritoneal and spleen cells and in human peripheral blood mononuclear cells. IFN-γ inhibited IL-10 production induced by TLR2, TLR3, TLR4 and TLR7/8 agonists, but stimulated IL-10 production when cells were triggered with CpG oligodeoxynucleotides, a specific TLR9 agonist. The stimulatory effect of IFN-γ on TLR9-induced IL-10 was restricted to Bcells. In line with the increased IL-10, Bcells stimulated with CpG and IFN-γ profoundly inhibited CD4 T cell proliferation. Further research into the mechanisms involved, revealed that the mitogen-activated protein kinases p38 and JNK are essential players in this stimulatory effect, and that the phosphatase MKP1 - an inhibitor of p38 and JNK activity - is downregulated after combined stimulation with IFN-γ and CpG. Our data may represent a novel immunoregulatory role of IFN-γ in Bcells after triggering of TLR9, by stimulating IL-10 production.

IL-36 and IL-1/IL-17 Drive Immunity to Oral Candidiasis via Parallel Mechanisms.

Protection against microbial infection by the induction of inflammation is a key function of the IL-1 superfamily, including both classical IL-1 and the new IL-36 cytokine families. Candida albicans is a frequent human fungal pathogen causing mucosal infections. Although the initiators and effectors important in protective host responses to C. albicans are well described, the key players in driving these responses remain poorly defined. Recent work has identified a central role played by IL-1 in inducing innate Type-17 immune responses to clear C. albicans infections. Despite this, lack of IL-1 signaling does not result in complete loss of immunity, indicating that there are other factors involved in mediating protection to this fungus. In this study, we identify IL-36 cytokines as a new player in these responses. We show that C. albicans infection of the oral mucosa induces the production of IL-36. As with IL-1α/β, induction of epithelial IL-36 depends on the hypha-associated peptide toxin Candidalysin. Epithelial IL-36 gene expression requires p38-MAPK/c-Fos, NF-κB, and PI3K signaling and is regulated by the MAPK phosphatase MKP1. Oral candidiasis in IL-36R-/- mice shows increased fungal burdens and reduced IL-23 gene expression, indicating a key role played by IL-36 and IL-23 in innate protective responses to this fungus. Strikingly, we observed no impact on gene expression of IL-17 or IL-17-dependent genes, indicating that this protection occurs via an alternative pathway to IL-1-driven immunity. Thus, IL-1 and IL-36 represent parallel epithelial cell-driven protective pathways in immunity to oral C. albicans infection.

PO-476 Increased ERK phosphorylation as a candidate driver of resistance to the experimental cancer drug AT13148

IntroductionThe AGC family of serine/threonine protein kinases comprises a number of drug targets with therapeutic potential for the treatment of cancer. AT13148 is an ATP competitive AGC kinase inhibitor, currently in phase 1 clinical trials, with activity against several members of the AGC kinase family, including ROCK 1/2, AKT, and p70S6K. The development of acquired drug resistance in the clinic is an issue with many kinase inhibitors. Elucidating acquired mechanisms of resistance early in the development of an experimental cancer drug may therefore provide opportunities to circumvent or overcome resistance and offer opportunities for new target drug discovery. The aim of this project was therefore to develop preclinical models of AT13148 resistance and identify potential driver mechanisms prior to clinical emergence.Material and methodsDose-escalation of AT13148 was used to generate resistance in the A2780 human ovarian cancer cell line, with isogenic cloning subsequently used to isolate resistant sub-clones. Combination assays and cross-profiling with a panel of compounds were performed using a 96 hour sulforhodamine B (SRB) cell viability assay. Investigations of changes in cell signalling pathways were conducted using a range of techniques including western blot analysis.Results and discussionsIsogenic AT13148 resistant sub-clones exhibited between 8 and 21-fold resistance to AT13148. No changes in ROCK, AKT and p70S6K signalling were detected in these sub-clones versus parental cells. However, there were stark differences in MAPK (Mitogen Activated Protein Kinase) signalling; increased ERK 1/2 phosphorylation concurrent with a decrease in MEK 1/2 phosphorylation. In addition, two of the resistant sub-clones were sensitised to AT13148 on exposure to the ERK inhibitor GDC-0994, implicating ERK as a driver of resistance. These sub-clones also exhibited cross-resistance to MEK inhibitors (PD-0325901 and selumetinib), with ERK phosphorylation also shown to be refractory to PD-0325901. Loss of MKP-3 expression, an ERK 1/2 phosphatase, was subsequently detected in these AT13148 resistant sub-clones and is hypothesised as a potential mechanism of increased ERK phosphorylation and therefore resistance.ConclusionThe loss of the phosphatase MKP-3 and subsequent increase in ERK phosphorylation is a candidate mechanism of resistance to AT13148 and warrants further investigation.

Processing of Candida albicans Ece1p Is Critical for Candidalysin Maturation and Fungal Virulence.

ABSTRACTCandida albicans is an opportunistic fungal pathogen responsible for superficial and life-threatening infections in humans. During mucosal infection, C. albicans undergoes a morphological transition from yeast to invasive filamentous hyphae that secrete candidalysin, a 31-amino-acid peptide toxin required for virulence. Candidalysin damages epithelial cell plasma membranes and stimulates the activating protein 1 (AP-1) transcription factor c-Fos (via p38–mitogen-activated protein kinase [MAPK]), and the MAPK phosphatase MKP1 (via extracellular signal-regulated kinases 1 and 2 [ERK1/2]–MAPK), which trigger and regulate proinflammatory cytokine responses, respectively. The candidalysin toxin resides as a discrete cryptic sequence within a larger 271-amino-acid parental preproprotein, Ece1p. Here, we demonstrate that kexin-like proteinases, but not secreted aspartyl proteinases, initiate a two-step posttranslational processing of Ece1p to produce candidalysin. Kex2p-mediated proteolysis of Ece1p after Arg61 and Arg93, but not after other processing sites within Ece1p, is required to generate immature candidalysin from Ece1p, followed by Kex1p-mediated removal of a carboxyl arginine residue to generate mature candidalysin. C. albicans strains harboring mutations of Arg61 and/or Arg93 did not secrete candidalysin, were unable to induce epithelial damage and inflammatory responses in vitro, and showed attenuated virulence in vivo in a murine model of oropharyngeal candidiasis. These observations identify enzymatic processing of C. albicans Ece1p by kexin-like proteinases as crucial steps required for candidalysin production and fungal pathogenicity.

Mitogen activated protein kinase 6 and MAP kinase phosphatase 1 are involved in the response of Arabidopsis roots to L-glutamate.

The function and components of L-glutamate signaling pathways in plants have just begun to be elucidated. Here, using a combination of genetic and biochemical strategies, we demonstrated that a MAPK module is involved in the control of root developmental responses to this amino acid. Root system architecture plays an essential role in plant adaptation to biotic and abiotic factors via adjusting signal transduction and gene expression. L-Glutamate (L-Glu), an amino acid with neurotransmitter functions in animals, inhibits root growth, but the underlying genetic mechanisms are poorly understood. Through a combination of genetic analysis, in-gel kinase assays, detailed cell elongation and division measurements and confocal analysis of expression of auxin, quiescent center and stem cell niche related genes, the critical roles of L-Glu in primary root growth acting through the mitogen-activated protein kinase 6 (MPK6) and the dual specificity serine-threonine-tyrosine phosphatase MKP1 could be revealed. In-gel phosphorylation assays revealed a rapid and dose-dependent induction of MPK6 and MPK3 activities in wild-type Arabidopsis seedlings in response to L-Glu. Mutations in MPK6 or MKP1 reduced or increased root cell division and elongation in response to L-Glu, possibly modulating auxin transport and/or response, but in a PLETHORA1 and 2 independent manner. Our data highlight MPK6 and MKP1 as components of an L-Glu pathway linking the auxin response, and cell division for primary root growth.

Circadian clock regulates hepatic polyploidy by modulating Mkp1-Erk1/2 signaling pathway

Liver metabolism undergoes robust circadian oscillations in gene expression and enzymatic activity essential for liver homeostasis, but whether the circadian clock controls homeostatic self-renewal of hepatocytes is unknown. Here we show that hepatocyte polyploidization is markedly accelerated around the central vein, the site of permanent cell self-renewal, in mice deficient in circadian Period genes. In these mice, a massive accumulation of hyperpolyploid mononuclear and binuclear hepatocytes occurs due to impaired mitogen-activated protein kinase phosphatase 1 (Mkp1)-mediated circadian modulation of the extracellular signal-regulated kinase (Erk1/2) activity. Time-lapse imaging of hepatocytes suggests that the reduced activity of Erk1/2 in the midbody during cytokinesis results in abscission failure, leading to polyploidization. Manipulation of Mkp1 phosphatase activity is sufficient to change the ploidy level of hepatocytes. These data provide clear evidence that the Period genes not only orchestrate dynamic changes in metabolic activity, but also regulate homeostatic self-renewal of hepatocytes through Mkp1-Erk1/2 signaling pathway.

Suppression of inflammatory and infection responses in lung macrophages by eucalyptus oil and its constituent 1,8-cineole: Role of pattern recognition receptors TREM-1 and NLRP3, the MAP kinase regulator MKP-1, and NFκB.

Eucalyptus oil (EO) used in traditional medicine continues to prove useful for aroma therapy in respiratory ailments; however, there is a paucity of information on its mechanism of action and active components. In this direction, we investigated EO and its dominant constituent 1,8–cineole (eucalyptol) using the murine lung alveolar macrophage (AM) cell line MH-S. In an LPS-induced AM inflammation model, pre-treatment with EO significantly reduced (P ≤0.01or 0.05) the pro-inflammatory mediators TNF-α, IL-1 (α and β), and NO, albeit at a variable rate and extent; 1,8-cineole diminished IL-1 and IL-6. In a mycobacterial-infection AM model, EO pre-treatment or post-treatment significantly enhanced (P ≤0.01) the phagocytic activity and pathogen clearance. 1,8-cineole also significantly enhanced the pathogen clearance though the phagocytic activity was not significantly altered. EO or 1,8-cineole pre-treatment attenuated LPS-induced inflammatory signaling pathways at various levels accompanied by diminished inflammatory response. Among the pattern recognition receptors (PRRs) involved in LPS signaling, the TREM pathway surface receptor (TREM-1) was significantly downregulated. Importantly, the pre-treatments significantly downregulated (P ≤0.01) the intracellular PRR receptor NLRP3 of the inflammasome, which is consistent with the decrease in IL-1β secretion. Of the shared downstream signaling cascade for these PRR pathways, there was significant attenuation of phosphorylation of the transcription factor NF-κB and p38 (but increased phosphorylation of the other two MAP kinases, ERK1/2 and JNK1/2). 1,8-cineole showed a similar general trend except for an opposite effect on NF-κB and JNK1/2. In this context, either pre-treatment caused a significant downregulation of MKP-1 phosphatase, a negative regulator of MAPKs. Collectively, our results demonstrate that the anti-inflammatory activity of EO and 1,8-cineole is modulated via selective downregulation of the PRR pathways, including PRR receptors (TREM-1 and NLRP3) and common downstream signaling cascade partners (NF-κB, MAPKs, MKP-1). To our knowledge, this is the first report on the modulatory role of TREM-1 and NLRP3 inflammasome pathways and the MAPK negative regulator MKP-1 in context of the anti-inflammatory potential of EO and its constituent 1,8-cineole.

Microsomal Prostaglandin E Synthase-1 Expression in Inflammatory Conditions Is Downregulated by Dexamethasone: Seminal Role of the Regulatory Phosphatase MKP-1.

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible enzyme situated downstream of cyclo-oxygenase-2, promoting the excessive PGE2 production in inflammation. Dexamethasone is known to suppress mPGES-1 but the mechanisms regulating mPGES-1 expression remain poorly known. MKP-1 is a phosphatase controlling the proinflammatory MAP kinase pathways p38 and JNK, thus limiting the inflammatory responses. We have now investigated the role of MKP-1 and MAP kinases p38 and JNK in the regulation of mPGES-1 expression by dexamethasone. Dexamethasone increased MKP-1 and decreased mPGES-1 expression in J774 macrophages and in peritoneal macrophages from wild-type but not from MKP-1 deficient mice. Dexamethasone also reduced p38 and JNK phosphorylation along with enhancement of MKP-1, while inhibition of JNK reduced mPGES-1 expression. These findings were also translated to in vivo conditions as dexamethasone downregulated mPGES-1 expression in paw inflammation in wild-type but not in MKP-1 deficient mice. In conclusion, dexamethasone was found to downregulate mPGES-1 expression through enhanced MKP-1 expression and reduced JNK phosphorylation in inflammatory conditions. The results extend the understanding on the regulation of mPGES-1 expression and highlight the potential of MKP-1 as an anti-inflammatory drug target.