Regulation of anoxic death in Caenorhabditis elegans by mammalian apoptosis signal-regulating kinase (ASK) family proteins

Cells and organisms face anoxia in a wide variety of contexts, including ischemia and hibernation. Cells respond to anoxic conditions through multiple signaling pathways. We report that NSY-1, the Caenorhabditis elegans ortholog of mammalian apoptosis signal-regulating kinase (ASK) family of MAP kinase (MAPK) kinase kinases (MAP3Ks), regulates viability of animals in anoxia. Loss-of-function mutations of nsy-1 increased survival under anoxic conditions, and increased survival was also observed in animals with mutations in tir-1 and the MAPK kinase (MAP2K) sek-1, which are upstream and downstream factors of NSY-1, respectively. Consistent with these findings, anoxia was found to activate the p38 MAPK ortholog PMK-1, and this was suppressed in nsy-1 and tir-1 mutant animals. Furthermore, double-mutant analysis showed that the insulin-signaling pathway, which also regulates viability in anoxia, functioned in parallel to NSY-1. These results suggest that the TIR-1-NSY-1-SEK-1-PMK-1 pathway plays important roles in the reponse to anoxia in C. elegans.     

The Evolutionarily Conserved Mediator Subunit MDT-15/MED15 Links Protective Innate Immune Responses and Xenobiotic Detoxification

Metazoans protect themselves from environmental toxins and virulent pathogens through detoxification and immune responses. We previously identified a small molecule xenobiotic toxin that extends survival of Caenorhabditis elegans infected with human bacterial pathogens by activating the conserved p38 MAP kinase PMK-1 host defense pathway. Here we investigate the cellular mechanisms that couple activation of a detoxification response to innate immunity. From an RNAi screen of 1,420 genes expressed in the C. elegans intestine, we identified the conserved Mediator subunit MDT-15/MED15 and 28 other gene inactivations that abrogate the induction of PMK-1-dependent immune effectors by this small molecule. We demonstrate that MDT-15/MED15 is required for the xenobiotic-induced expression of p38 MAP kinase PMK-1-dependent immune genes and protection from Pseudomonas aeruginosa infection. We also show that MDT-15 controls the induction of detoxification genes and functions to protect the host from bacteria-derived phenazine toxins. These data define a central role for MDT-15/MED15 in the coordination of xenobiotic detoxification and innate immune responses.     

Caenorhabditis elegans innate immune response triggered by Salmonella enterica requires intact LPS and is mediated by a MAPK signaling pathway

Compared to mammals, insects, and plants, relatively little is known about innate immune responses in the nematode Caenorhabditis elegans. Previous work showed that Salmonella enterica serovars cause a persistent infection in the C. elegans intestine that triggers gonadal programmed cell death (PCD) and that C. elegans cell death (ced) mutants are more susceptible to Salmonella-mediated killing. To further dissect the role of PCD in C. elegans innate immunity, we identified both C. elegans and S. enterica factors that affect the elicitation of Salmonella-induced PCD. Salmonella-elicited PCD was shown to require the C. elegans homolog of the mammalian p38 mitogen-activated protein kinase (MAPK) encoded by the pmk-1 gene. Inactivation of pmk-1 by RNAi blocked Salmonella-elicited PCD, and epistasis analysis showed that CED-9 lies downstream of PMK-1. Wild-type Salmonella lipopolysaccharide (LPS) was also shown to be required for the elicitation of PCD, as well as for persistence of Salmonella in the C. elegans intestine. However, a presumptive C. elegans TOLL signaling pathway did not appear to be required for the PCD response to Salmonella. These results establish a PMK-1-dependant PCD pathway as a C. elegans innate immune response to Salmonella.     

Staphylococcal biofilm exopolysaccharide protects against Caenorhabditis elegans immune defenses

Staphylococcus epidermidis and Staphylococcus aureus are leading causes of hospital-acquired infections that have become increasingly difficult to treat due to the prevalence of antibiotic resistance in these organisms. The ability of staphylococci to produce biofilm is an important virulence mechanism that allows bacteria both to adhere to living and artificial surfaces and to resist host immune factors and antibiotics. Here, we show that the icaADBC locus, which synthesizes the biofilm-associated polysaccharide intercellular adhesin (PIA) in staphylococci, is required for the formation of a lethal S. epidermidis infection in the intestine of the model nematode Caenorhabditis elegans. Susceptibility to S. epidermidis infection is influenced by mutation of the C. elegans PMK-1 p38 mitogen-activated protein (MAP) kinase or DAF-2 insulin-signaling pathways. Loss of PIA production abrogates nematocidal activity and leads to reduced bacterial accumulation in the C. elegans intestine, while overexpression of the icaADBC locus in S. aureus augments virulence towards nematodes. PIA-producing S. epidermidis has a significant survival advantage over ica-deficient S. epidermidis within the intestinal tract of wild-type C. elegans, but not in immunocompromised nematodes harboring a loss-of-function mutation in the p38 MAP kinase pathway gene sek-1. Moreover, sek-1 and pmk-1 mutants are equally sensitive to wild-type and icaADBC-deficient S. epidermidis. These results suggest that biofilm exopolysaccharide enhances virulence by playing an immunoprotective role during colonization of the C. elegans intestine. These studies demonstrate that C. elegans can serve as a simple animal model for studying host-pathogen interactions involving staphylococcal biofilm exopolysaccharide and suggest that the protective activity of biofilm matrix represents an ancient conserved function for resisting predation.     

Regulation of Drosophila p38 activation by specific MAP2 kinase and MAP3 kinase in response to different stimuli

The p38 mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in cellular responses to inflammatory stimuli and environmental stress. Activation of p38 is mediated through phosphorylation by upstream MAPKK, which in turn is activated by MAPKKK. However, the mechanism of how different upstream MAP2Ks and MAP3Ks specifically contribute to p38 activation in response to different stimuli is still not clearly understood. By using double-stranded RNA-mediated interference (RNAi) in Drosophila cells, we demonstrate that D-MKK3 is a major MAP2K responsible for D-p38 activation by UV, heat shock, NaCl or peptiodglycan (PGN). Stimulation of UV and PGN activates D-p38 through D-MEKK1, heat shock-induced activation of D-p38 signals through both D-MEKK1 and D-ASK1. On the other hand, maximal activation of D-p38 by NaCl requires the expression of four MAP3Ks.     

A decline in p38 MAPK signaling underlies immunosenescence in Caenorhabditis elegans

The decline in immune function with aging, known as immunosenescence, has been implicated in evolutionarily diverse species, but the underlying molecular mechanisms are not understood. During aging in Caenorhabditis elegans, intestinal tissue deterioration and the increased intestinal proliferation of bacteria are observed, but how innate immunity changes during C. elegans aging has not been defined. Here we show that C. elegans exhibits increased susceptibility to bacterial infection with age, and we establish that aging is associated with a decline in the activity of the conserved PMK-1 p38 mitogen-activated protein kinase pathway, which regulates innate immunity in C. elegans. Our data define the phenomenon of innate immunosenescence in C. elegans in terms of the age-dependent dynamics of the PMK-1 innate immune signaling pathway, and they suggest that a cycle of intestinal tissue aging, immunosenescence, and bacterial proliferation leads to death in aging C. elegans.     

Human pathogenic fungus Trichophytonschoenleinii activates the NLRP3 inflammasome

The fungus Trichophyton schoenleinii (T. schoenleinii) is the causative agent of Trichophytosis and Tinea favosa of the scalp in certain regions of Eurasia and Africa. Human innate immune system plays an important role in combating with various pathogens including fungi. The inflammasome is one of the most critical arms of host innate immunity, which is a protein complex controlling maturation of IL-1β. To clarify whether T. schoenleinii is able to activate the inflammasome, we analyzed human monocytic cell line THP-1 for IL-1β production upon infection with T. schoenleinii strain isolated from Tinea favosa patients, and rapid IL-1β secretion from THP-1 cells was observed. Moreover, applying competitive inhibitors and gene specific silencing with shRNA, we found that T. schoenleinii induced IL-1β secretion, ASC pyroptosome formation as well as caspase-1 activation were all dependent on NLRP3. Cathepsin B activity, ROS production and K⁺ efflux were required for the inflammasome activation by T. schoenleinii. Our data thus reveal that the NLRP3 inflammasome plays an important role in host defense against T. schoenleinii, and suggest that manipulating NLRP3 signaling can be a novel approach for control of diseases caused by T. schoenleinii infection.     

Regulation of a DLK-1 and p38 MAP kinase pathway by the ubiquitin ligase RPM-1 is required for presynaptic development

Synapses display a stereotyped ultrastructural organization, commonly containing a single electron-dense presynaptic density surrounded by a cluster of synaptic vesicles. The mechanism controlling subsynaptic proportion is not understood. Loss of function in the C. elegans rpm-1 gene, a putative RING finger/E3 ubiquitin ligase, causes disorganized presynaptic cytoarchitecture. RPM-1 is localized to the presynaptic periactive zone. We report that RPM-1 negatively regulates a p38 MAP kinase pathway composed of the dual leucine zipper-bearing MAPKKK DLK-1, the MAPKK MKK-4, and the p38 MAP kinase PMK-3. Inactivation of this pathway suppresses rpm-1 loss of function phenotypes, whereas overexpression or constitutive activation of this pathway causes synaptic defects resembling rpm-1(lf) mutants. DLK-1, like RPM-1, is localized to the periactive zone. DLK-1 protein levels are elevated in rpm-1 mutants. The RPM-1 RING finger can stimulate ubiquitination of DLK-1. Our data reveal a presynaptic role of a previously unknown p38 MAP kinase cascade.     

The TGFβ-induced phosphorylation and activation of p38 mitogen-activated protein kinase is mediated by MAP3K4 and MAP3K10 but not TAK1

The signalling pathways downstream of the transforming growth factor beta (TGFβ) family of cytokines play critical roles in all aspects of cellular homeostasis. The phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) has been implicated in TGFβ-induced epithelial-to-mesenchymal transition and apoptosis. The precise molecular mechanisms by which TGFβ cytokines induce the phosphorylation and activation of p38 MAPK are unclear. In this study, I demonstrate that TGFβ-activated kinase 1 (TAK1/MAP3K7) does not play a role in the TGFβ-induced phosphorylation and activation of p38 MAPK in MEFs and HaCaT keratinocytes. Instead, RNAi-mediated depletion of MAP3K4 and MAP3K10 results in the inhibition of the TGFβ-induced p38 MAPK phosphorylation. Furthermore, the depletion of MAP3K10 from cells homozygously knocked-in with a catalytically inactive mutant of MAP3K4 completely abolishes the TGFβ-induced phosphorylation of p38 MAPK, implying that among MAP3Ks, MAP3K4 and MAP3K10 are sufficient for mediating the TGFβ-induced activation of p38 MAPK.     

MEKK3 regulates IFN-gamma production in T cells through the Rac1/2-dependent MAPK cascades

MEKK3 is a conserved Ser/Thr protein kinase belonging to the MAPK kinase kinase (MAP3K) family. MEKK3 is constitutively expressed in T cells, but its function in T cell immunity has not been fully elucidated. Using Mekk3 T cell conditional knockout (T-cKO) mice, we show that MEKK3 is required for T cell immunity in vivo. Mekk3 T-cKO mice had reduced T cell response to bacterial infection and were defective in clearing bacterial infections. The Ag-induced cytokine production, especially IFN-γ production, was impaired in Mekk3-deficient CD4 T cells. The TCR-induced ERK1/2, JNK, and p38 MAPKs activation was also defective in Mekk3-deficient CD4 T cells. In vitro, MEKK3 is not required for Th1 and Th2 cell differentiation. Notably, under a nonpolarizing condition (Th0), Mekk3 deficiency led to a significant reduction of IFN-γ production in CD4 T cells. Furthermore, the IL-12/IL-18-driven IFN-γ production and MAPK activation in Mekk3-deficient T cells was not affected suggesting that MEKK3 may selectively mediate the TCR-induced MAPK signals for IFN-γ production. Finally, we found that MEKK3 activation by TCR stimulation requires Rac1/2. Taken together, our study reveals a specific role of MEKK3 in mediating the TCR signals for IFN-γ production.     

p38 MAPK autophosphorylation drives macrophage IL-12 production during intracellular infection

The intracellular protozoan Toxoplasma gondii triggers rapid MAPK activation in mouse macrophages (Mphi). We used synthetic inhibitors and dominant-negative Mphi mutants to demonstrate that T. gondii triggers IL-12 production in dependence upon p38 MAPK. Chemical inhibition of stress-activated protein kinase/JNK showed that this MAPK was also required for parasite-triggered IL-12 production. Examination of upstream MAPK kinases (MKK) 3, 4, and 6 that function as p38 MAPK activating kinases revealed that parasite infection activates only MKK3. Nevertheless, in MKK3(-/-) Mphi, p38 MAPK activation was near normal and IL-12 production was unaffected. Recently, MKK-independent p38alpha MAPK activation via autophosphorylation was described. Autophosphorylation depends upon p38alpha MAPK association with adaptor protein, TGF-beta-activated protein kinase 1-binding protein-1. We observed TGF-beta-activated protein kinase 1-binding protein-1-p38alpha MAPK association that closely paralleled p38 MAPK phosphorylation during Toxoplasma infection of Mphi. Furthermore, a synthetic p38 catalytic-site inhibitor blocked tachyzoite-induced p38alpha MAPK phosphorylation. These data are the first to demonstrate p38 MAPK autophosphorylation triggered by intracellular infection.     

Role of C/EBP-β, p38 MAPK, and MKK6 in IL-1β-mediated C3 gene regulation in astrocytes

Complement component C3, the central player in the complement cascade and the pro-inflammatory cytokine IL-1β is expressed by activated glial cells and may contribute to neurodegeneration. This study examines the regulation of the expression of C3 by IL-1β in astroglial cells focusing on the role of the upstream kinase MKK6, p38-α MAPK, and C/EBP-β isoforms (LAP1, LAP2, or LIP) in astroglial cells. Activation of human astroglial cell line, U373 with IL-1β, led to the induction of C3 mRNA and protein expression as determined by real-time RT-PCR and Western blot analysis, respectively. This induction was suppressed by the pharmacological inhibitor of p38 MAPK (i.e., SB202190-HCl), suggesting the involvement of p38 MAPK in C3 gene expression. IL-1β also induced C3 promoter activity in U373 cells in a MAP kinase- and C/EBP-β-dependent manner. Cotransfection of C3 luciferase reporter construct with constitutively active form of the upstream kinase in the MAP kinase cascade, that is, MKK6 (the immediate upstream activator of p38 kinase) resulted in marked stimulation of the promoter activity, whereas overexpression of a dominant negative forms of MKK6 and p38α MAPK inhibited C3 promoter activity. Furthermore, a mutant form of C/EBP-β, LAP(T235A) showed reduction in IL-1β-mediated C3 promoter activation. These results suggest that the p38α, MAPK, and MKK6 play prominent roles in IL-1β and C/EBP-β-mediated C3 gene expression in astrocytes.