Pathogenic infections and tissue injuries trigger the assembly of inflammasomes, cytosolic protein complexes that activate caspase-1, leading to cleavage of pro-IL-1β and pro-IL-18 and to pyroptosis, ...a proinflammatory cell death program. Although microbial recognition by Toll-like receptors (TLRs) is known to induce the synthesis of the major caspase-1 substrate pro-IL-1β, the role of TLRs has been considered limited to up-regulation of the inflammasome components. During infection with a virulent microbe, TLRs and nucleotide-binding oligomerization domain-like receptors (NLRs) are likely activated simultaneously. To examine the requirements and outcomes of combined activation, we stimulated TLRs and a specific NLR, nucleotide binding and oligomerization, leucine-rich repeat, pyrin domain-containing 3 (NLRP3), simultaneously and discovered that such activation triggers rapid caspase-1 cleavage, leading to secretion of presynthesized inflammatory molecules and pyroptosis. This acute caspase-1 activation is independent of new protein synthesis and depends on the TLR-signaling molecule IL-1 receptor-associated kinase (IRAK-1) and its kinase activity. Importantly, Listeria monocytogene s induces NLRP3-dependent rapid caspase-1 activation and pyroptosis, both of which are compromised in IRAK-1–deficient macrophages. Our results reveal that simultaneous sensing of microbial ligands and virulence factors by TLRs and NLRP3, respectively, leads to a rapid TLR- and IRAK-1–dependent assembly of the NLRP3 inflammasome complex, and that such activation is important for release of alarmins, pyroptosis, and early IFN-γ production by memory CD8 T cells, all of which could be critical for early host defense.
Regulation of intracellular cyclic adenosine 3 ′,5 ′-monophosphate (cAMP) is integral in mediating cell growth, cell differentiation, and immune responses in hematopoietic cells. To facilitate ...studies of cAMP regulation we developed a BRET (bioluminescence resonance energy transfer) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in vivo. This sensor was used to characterize three distinct pathways for modulation of cAMP synthesis stimulated by presumed Gs-dependent receptors for isoproterenol and prostaglandin E2. Whereas two ligands, uridine 5 ′-diphosphate and complement C5a, appear to use known mechanisms for augmentation of cAMP via Gq/calcium and Gi, the action of sphingosine 1-phosphate (S1P) is novel. In these cells, S1P, a biologically active lysophospholipid, greatly enhances increases in intracellular cAMP triggered by the ligands for Gs-coupled receptors while having only a minimal effect by itself. The enhancement of cAMP by S1P is resistant to pertussis toxin and independent of intracellular calcium. Studies with RNAi and chemical perturbations demonstrate that the effect of S1P is mediated by the S1P2 receptor and the heterotrimeric G13 protein. Thus in these macrophage cells, all four major classes of G proteins can regulate intracellular cAMP.
Cellular information processing requires the coordinated activity of a large network of intracellular signalling pathways. Cross-talk between pathways provides for complex non-linear responses to ...combinations of stimuli, but little is known about the density of these interactions in any specific cell. Here, we have analysed a large-scale survey of pathway interactions carried out by the Alliance for Cellular Signalling (AfCS) in RAW 264.7 macrophages. Twenty-two receptor-specific ligands were studied, both alone and in all pairwise combinations, for Ca2+ mobilization, cAMP synthesis, phosphorylation of many signalling proteins and for cytokine production. A large number of non-additive interactions are evident that are consistent with known mechanisms of cross-talk between pathways, but many novel interactions are also revealed. A global analysis of cross-talk suggests that many external stimuli converge on a relatively small number of interaction mechanisms to provide for context-dependent signalling.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
4.
Sulindac Inhibits Activation of the NF-κB Pathway Yamamoto, Yumi; Yin, Min-Jean; Lin, Keng-Mean ...
Journal of biological chemistry/The Journal of biological chemistry,
09/1999, Letnik:
274, Številka:
38
Journal Article
Recenzirano
Odprti dostop
Sulindac is a non-steroidal anti-inflammatory agent that is related both structurally and pharmacologically to indomethacin. In addition to its anti-inflammatory properties, sulindac has been ...demonstrated to have a role in the prevention of colon cancer. Both its growth inhibitory and anti-inflammatory properties are due at least in part to its ability to decrease prostaglandin synthesis by inhibiting the activity of cyclooxygenases. Recently, we demonstrated that both aspirin and sodium salicylate, but not indomethacin, inhibited the activity of an IκB kinase β (IKKβ) that is required to activate the nuclear factor-κB (NF-κB) pathway. In this study, we show that sulindac and its metabolites sulindac sulfide and sulindac sulfone can also inhibit the NF-κB pathway in both colon cancer and other cell lines. Similar to our previous results with aspirin, this inhibition is due to sulindac-mediated decreases in IKKβ kinase activity. Concentrations of sulindac that inhibit IKKβ activity also reduce the proliferation of colon cancer cells. These results suggest that the growth inhibitory and anti-inflammatory properties of sulindac may be regulated in part by inhibition of kinases that regulate the NF-κB pathway.
We previously reported that expression of the transcription factor interferon regulatory factor 1 (IRF1) is an early, critical maladaptive signal expressed by renal tubules during murine ischemic ...acute kidney injury (AKI). We now show that IRF1 mediates signals from reactive oxygen species (ROS) generated during ischemic AKI and that these signals ultimately result in production of α-subtypes of type I interferons (IFNαs). We found that genetic knockout of the common type I IFN receptor (IFNARI-/-) improved kidney function and histology during AKI. There are major differences in the spatial-temporal production of the two major IFN subtypes, IFNβ and IFNαs: IFNβ expression peaks at 4 h, earlier than IFNαs, and continues at the same level at 24 h; expression of IFNαs also increases at 4 h but continues to increase through 24 h. The magnitude of the increase in IFNαs relative to baseline is much greater than that of IFNβ. We show by immunohistology and study of isolated cells that IFNβ is produced by renal leukocytes and IFNαs are produced by renal tubules. IRF1, IFNαs, and IFNARI were found on the same renal tubules during ischemic AKI. Furthermore, we found that ROS induced IFNα expression by renal tubules in vitro. This expression was inhibited by small interfering RNA knockdown of IRF1. Overexpression of IRF1 resulted in the production of IFNαs. Furthermore, we found that IFNα stimulated production of maladaptive proinflammatory CXCL2 by renal tubular cells. Altogether our data support the following autocrine pathway in renal tubular cells: ROS > IRF1 > IFNα > IFNARI > CXCL2.
Studies in fibroblasts, neurons, and platelets have demonstrated the integration of signals from different G protein-coupled receptors (GPCRs) in raising intracellular free Ca2+. To study signal ...integration in macrophages, we screened RAW264.7 cells and bone marrow-derived macrophages (BMDM) for their Ca2+ response to GPCR ligands. We found a synergistic response to complement component 5a (C5a) in combination with uridine 5′-diphosphate (UDP), platelet activating factor (PAF), or lysophosphatidic acid (LPA). The C5a response was Gαi-dependent, whereas the UDP, PAF, and LPA responses were Gαq-dependent. Synergy between C5a and UDP, mediated by the C5a and P2Y6 receptors, required dual receptor occupancy, and affected the initial release of Ca2+ from intracellular stores as well as sustained Ca2+ levels. C5a and UDP synergized in generating inositol 1,4,5-trisphosphate, suggesting synergy in activating phospholipase C (PLC) β. Macrophages expressed transcripts for three PLCβ isoforms (PLCβ2, PLCβ3, and PLCβ4), but GPCR ligands selectively used these isoforms in Ca2+ signaling. C5a predominantly used PLCβ3, whereas UDP used PLCβ3 but also PLCβ4. Neither ligand required PLCβ2. Synergy between C5a and UDP likewise depended primarily on PLCβ3. Importantly, the Ca2+ signaling deficiency observed in PLCβ3-deficient BMDM was reversed by re-constitution with PLCβ3. Neither phosphatidylinositol (PI) 3-kinase nor protein kinase C was required for synergy. In contrast to Ca2+, PI 3-kinase activation by C5a was inhibited by UDP, as was macropinocytosis, which depends on PI 3-kinase. PLCβ3 may thus provide a selective target for inhibiting Ca2+ responses to mediators of inflammation, including C5a, UDP, PAF, and LPA.
Regulation of β-Catenin Function by the IκB Kinases Lamberti, Carmela; Lin, Keng-Mean; Yamamoto, Yumi ...
Journal of biological chemistry/The Journal of biological chemistry,
11/2001, Letnik:
276, Številka:
45
Journal Article
Recenzirano
Odprti dostop
Both the β-catenin and the nuclear factor κB (NF-κB) proteins are important regulators of gene expression and cellular proliferation. Two kinases, IKKα and IKKβ, are critical activators of the NF-κB ...pathway. Here we present evidence that these kinases are also important in the regulation of β-catenin function. IKKα- and IKKβ-deficient mouse embryo fibroblasts exhibited different patterns of β-catenin cellular localization. IKKβ decreases β-catenin-dependent transcriptional activation, while IKKα increases β-catenin-dependent transcriptional activity. IKKα and IKKβ interact with and phosphorylate β-catenin using both in vitro and in vivo assays. Our results suggest that differential interactions of β-catenin with IKKα and IKKβ may in part be responsible for regulating β-catenin protein levels and cellular localization and integrating signaling events between the NF-κB and Wingless pathways.
Distinct activities of toxin B up‐regulate PLCβ3 or down‐regulate PLCβ4‐dependent IP3‐Ca2+ coupling.
Clostridium difficile toxins cause acute colitis by disrupting the enterocyte barrier and ...promoting inflammation. ToxB from C. difficile inactivates Rho family GTPases and causes release of cytokines and eicosanoids by macrophages. We studied the effects of ToxB on GPCR signaling in murine RAW264.7 macrophages and found that ToxB elevated Ca2+ responses to Gαi‐linked receptors, including the C5aR, but reduced responses to Gαq‐linked receptors, including the UDP receptors. Other Rho inhibitors also reduced UDP Ca2+ responses, but they did not affect C5a responses, suggesting that ToxB inhibited UDP responses by inhibiting Rho but enhanced C5a responses by other mechanisms. By using PLCβ isoform‐deficient BMDM, we found that ToxB inhibited Ca2+ signaling through PLCβ4 but enhanced signaling through PLCβ3. Effects of ToxB on GPCR Ca2+ responses correlated with GPCR use of PLCβ3 versus PLCβ4. ToxB inhibited UDP Ca2+ signaling without reducing InsP3 production or the sensitivity of cellular Ca2+ stores to exogenous InsP3, suggesting that ToxB impairs UDP signaling at the level of InsP3/Ca2+coupling. In contrast, ToxB elevated InsP3 production by C5a, and the enhancement of Ca2+ signaling by C5a was prevented by inhibition of PLA2 or 5‐LOX but not COX, implicating LTs but not prostanoids in the mechanism. In sum, ToxB has opposing, independently regulated effects on Ca2+ signaling by different GPCR‐linked PLCβ isoforms in macrophages.
Hydrolysis of GTP by dynamin is essential for budding clathrin-coated vesicles from the plasma membrane. Two distinct domains
of dynamin are implicated in the interactions with dynamin GTPase ...activators. Microtubules and Grb2 bind to the carboxyl-terminal
proline/arginine-rich domain (PRD), whereas phosphoinositides bind to the pleckstrin homology (PH) domain. In this study we
tested the effect of different phosphoinositides on dynamin GTPase activity and found that the best activator is phosphatidylinositol
4,5-bisphosphate followed by 1- O -(1,2-di- O -palmitoyl- sn -glycerol-3-benzyloxyphosphoryl)- d - myo -inositol 3,4,5-triphosphate. Phosphatidylinositol 4-phosphate was a weak activator and phosphatidylinositol 3,4-bisphosphate
did not activate GTPase at all. We then addressed the question of whether both domains of dynamin, PRD and PH, can be engaged
simultaneously, and determined the effects of dual occupancy on dynamin GTPase activity. We found that Grb2 and phosphatidylinositol
4,5-bisphosphate together increased the dynamin GTPase activity up to 4-fold higher than that obtained by these activators
tested separately, and also reduced the dynamin concentration required for half-maximal activities by 3-fold. These results
indicate that both stimulators can bind to dynamin simultaneously resulting in superactivation of dynamin GTPase activity.
We propose that SH3-containing proteins such as Grb2 bind to the dynamin PRD to target it to clathrin-coated pits and prime
it for superactivation by phosphoinositides.
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