Each week we showcase a hot new cell signaling article from a Nature Publishing Group journal. Free full text access to the paper will be maintained for three months, after which the paper will be accessible via the Research Library.
Mast cells release inflammatory substances to trigger and maintain the allergic response. Their differentiation and activation are regulated by the stem-cell factor (SCF) and immunoglobulin E (IgE) signaling pathways, respectively. Activation of SCF receptors and engagement of IgE by allergen result in downstream phosphatidylinositol-3-OH kinase (PI(3)K) signaling. Ali et al. now report that specific inactivation of the PI(3)K p110δ isoform protects mice against anaphylactic allergic responses.
Leukocytes primarily express the p110δ catalytic subunit isoform of PI(3)K, as opposed to the ubiquitously expressed p110α and p110β isoforms. To investigate the role of p110δ in leukocytes, the authors isolated bone-marrow mast cells (BMMCs) from mice harboring a p110δ loss-of-function mutation (p110δD910A/D910A). p110δ-mutant BMMCs exhibited defects in proliferation and had reduced levels of interleukin-3 (IL-3; a mast cell proliferation and differentiation factor) and/or SCF-induced DNA synthesis. Moreover, their ability to adhere to fibronectin or migrate towards SCF was severely impaired when compared with wild-type cells.
By using a series of in vitro studies, the authors showed that, in p110δ-mutant BMMCs, SCF- or IL-3 -induced phosphorylation of PKB was severely reduced, or even absent, whereas phosphorylation of Erk remained unaffected. In addition, they demonstrated that total class IA PI(3)K activity was reduced by up to 90%. Thus, they concluded that the p110δ isoform was a major contributor to PI(3)K signaling downstream of both SCF and IL-3.
To strengthen the physiological significance of their findings, Ali et al. examined the capacity of mutant BMMCs to release inflammatory mediators in response to antigen in complex with IgE. They showed that p110δD910A/D910A BMMCs exhibited a 50% reduction in FcεRI-induced degranulation, compared with wild-type cells. Consistently, pre-incubation of wild-type BMMCs or human mast cells with a specific p110δ inhibitor reduced degranulation to similar levels and also blocked the capacity of SCF to potentiate degranulation by low doses of antigen, while leaving mutant BMMCs unaffected. p110δD910A/D910A BMMCs were also defective in the release of inflammatory cytokines such as interleukin-6 (IL-6) and tumor-necrosis factor α (TNF - α).
Finally, the authors examined the ability of p110δD910A/D910A mice to respond to immune challenge in a passive cutaneous anaphylaxis (PCA) model. They observed that PCA was markedly reduced in both the ear and back dermis of mutant mice, and that this result could be mimicked by treating mice with a p110δ-specific inhibitor. Collectively, the work by Ali et al. demonstrates an important role for the p110δ isoform of PI(3)K in immune homeostasis and mast-cell allergies, and has wider implications in designing specific PI(3)K compounds for therapeutic intervention.
Myrto Raftopoulou Signaling Gateway
article
Khaled Ali, Antonio Bilancio, Matthew Thomas, Wayne Pearce, Alasdair M. Gilfillan, Christine Tkaczyk, Nicolas Kuehn, Alexander Gray, June Giddings, Emma Peskett, Roy Fox, Ian Bruce, Christoph Walker, Carol Sawyer, Klaus Okkenhaug, Peter Finan & Bart Vanhaesebroeck Essential role for the p110δ phosphoinositide 3-kinase in the allergic response Nature, 431, 1007 – 1011 (21 October 2004); doi:10.1038/nature02991
| Full Text (HTML)
| PDF
| Subscribe to Nature
The morbidity of carcinomas is exacerbated by local tissue invasion and metastization, a process which is reminiscent of the epithelial-mesenchymal transition (EMT) that occurs during embryogenesis. A key step in EMT is the downregulation of E-cadherin levels, and E-cadherin loss has been associated with many metastatic tumors. Snail is a transcriptional repressor that regulates E-cadherin levels during EMT. Zhou et al. now propose a new mechanism by which GSK-3β regulates the stability of Snail through phosphorylation, implicating both Snail and GSK-3β in EMT and tumour invasion.
Although several human cancers exhibit increased Snail mRNA levels, Snail protein is hardly detectable. Through a series of in vitro and in vivo approaches, Zhou et al. have offered an explanation for this observation. They show that the highly labile Snail protein is stabilized by inhibition of the proteasome or GSK-3β. Sequence analysis predicted two consensus motifs in Snail for GSK-3β phosphorylation, and this kinase associates with Snail to phosphorylate two serines in motif I and four serines in motif II. Phosphorylation at the first motif regulates the degradation of Snail, whereas phosphorylation at the second motif regulates its subcellular localization. These findings led to a model whereby GSK-3β phosphorylates Snail in the nucleus at motif II, leading to nuclear export, whereas additional phosphorylation by GSK-3β at motif I results in association of Snail with the ubiquitin ligase component β-Trcp, and its subsequent degradation by the proteasome.
To strengthen their hypothesis, Zhou et al. examined the correlation between GSK-3β activity and the cellular levels of Snail in response to upstream signaling. They found that activation of the PI3K and MAPK pathways by IGF or EGF, respectively, led to suppression of GSK3β activity and stabilization of Snail protein. Both the PI3K and MAPK pathways have been implicated in EMT and tumor invasion. The authors show that expression of a mutant of Snail (Snail-6SA) refractory to phosphorylation was sufficient to induce EMT-like morphological changes including increased motility, a phenotype rescued by expression of β-catenin.
The work by Zhou et al. provides new insights into the regulation of EMT and offers a better understanding of tumor invasion and metastasis. Snail is not, however, the only transcription factor to be implicated in EMT, and loss of E-cadherin is not sufficient to induce metastization, as discussed in the accompanying News and Views article. It will be interesting, therefore, to see how future research will add to our understanding of the complex interplay between transcription factors and upstream signaling pathways during EMT and tumor progression.
Myrto Raftopoulou Signaling Gateway
article
Binhua P. Zhou, Jiong Deng, Weiya Xia, Jihong Xu, Yan M. Li, Mehmet Gunduz & Mien-Chie Hung Dual regulation of Snail by GSK-3β-mediated phosphorylation in control of epithelial-mesenchymal transition Nature Cell Biology, 6, 931 – 940 (2004); doi:10.1038/ncb1173
| Full Text (HTML)
| PDF
| Subscribe to Nature Cell Biology
In the nematode worm Caenorhabditis elegans, dopamine controls locomotion in response to food. Chase et al. now report that dopamine functions extrasynaptically through two different classes of G-protein coupled receptors: D1- and D2-like. These receptors are co-expressed in cholinergic motor neurons, resulting in activation of antagonistic signaling pathways.
In a process controlled by dopamine signaling, C. elegans slow down when they encounter food. Here, the authors show that mutants lacking the D2-like dopamine receptor dop-3 do not exhibit this response; however, the slowing down phenotype was rescued by the additional deletion of the D1-like dop-1 gene.
The addition of high levels of exogenous dopamine causes paralysis in C. elegans. Unlike wild-type worms, dop-3 mutants continued to move in the presence of high dopamine levels. Again, the dop-1 dop-3 double mutant restored wild-type behavior; thus, the authors conclude that activation of DOP-3 is responsible for controlling locomotion in response to both endogenous and exogenous dopamine. Furthermore, this activation can be antagonized by signaling through DOP-1.
Ventral cord motor neurons, which are composed of cholinergic and GABAergic neurons, directly control C. elegans locomotion. By using fluorescent reporters, Chase et al. found that DOP-1 was expressed strongly in cholinergic motor neurons, whereas DOP-3 was expressed predominantly in GABAergic neurons; however, weak expression was also observed in cholinergic neurons. The specific expression of DOP-3 in cholinergic neurons rescued the dop-3-mutant phenotype, and, similarly, the specific expression of DOP-1 in cholinergic neurons rescued the dop-1 dop-3 mutant phenotype. Thus, DOP-1 and DOP-3 both function in cholinergic neurons to mediate dopamine signaling and consequently locomotive responses.
To identify candidates that were mediating the effects of DOP-1 and DOP-3, the authors screened for mutants resistant to the paralyzing effects of dopamine, and isolated components of the Gα0 and Gαq pathways. Consistent with this, mutants with reduced Gα0 (GOA-1 in C. elegans) or increased Gαq (EGL-30 in C. elegans) signaling exhibited resistance to exogenous dopamine. They thus propose a mechanism whereby extrasynaptic dopamine binds to co-localized D1- and D2-like receptors, but results in two antagonistic signaling events through the actions of Gαq and Gαo, respectively.
As discussed in the accompanying News and Views article, the signaling pathways identified in this study differ from the canonical view of mammalian dopamine signaling. However, the high degree of conservation of proteins between C. elegans and humans suggests that the nematode may be a good model system to further study the mechanistic details of the D1 and D2 antagonism. Ultimately, this may open up new possibilities for therapeutic intervention in schizophrenia and Parkinson's disease.
Joanne Kotz, Assistant Editor Nature Chemical Biology
article
Daniel L Chase, Judy S Pepper & Michael R Koelle Mechanism of extrasynaptic dopamine signaling in Caenorhabditis elegans Nature Neuroscience, 7, 1096 – 1103 (2004); doi:10.1038/nn1316
| Full Text (HTML)
| PDF
| Subscribe to Nature Neuroscience
Innate and adaptive immune responses can be activated by pathogen-associated molecules, such as lipopolysaccharide (LPS), binding to Toll-like receptors (TLRs) on macrophages and dendritic cells. Negative regulation of TLR signaling may be an important mechanism for preventing excessive immune responses to pathogens, which can lead to toxic shock. Boone et al. now report that A20 mediates deubiquitination of the signaling molecule TRAF6, which is required to terminate TLR-induced NF-κB activity.
A20 was known to inhibit tumor necrosis factor (TNF)-induced NF-κB activity. However, mice deficient in both A20 and TNF receptors showed symptoms characteristic of an immune homeostasis failure, demonstrating that A20 also played a role in limiting TNF-independent signals. In comparison with wild-type mice, injection of a sub-lethal dose of LPS into mice with A20-deficient splenic macrophages resulted in increased TNF production and symptoms of toxic shock. Thus, A20 restricts LPS-mediated signaling through TLRs.
Bone marrow-derived macrophages (BMDMs) were found to constitutively express A20, with increased A20 levels observed in response to LPS. Compared to wild-type cells, A20 deficient BMDMs produced more TNF, interleukin 6 (IL-6) and nitric oxide (NO) in response to LPS. This increase in pro-inflammatory molecule production was also seen when macrophages were stimulated through different TLRs, supporting a role for A20 in regulating downstream signaling components that are shared by all TLRs. Increased levels of IKK kinase activity were seen with A20-TNF double-deficient macrophages compared with TNF-deficient cells, suggesting that A20 is involved in terminating NF-κB activity by regulating IKK activation.
The N-terminal domain of A20 has sequence similarity to proteins involved in cleaving ubiquitin chains, and contains the conserved DXXC motif implicated in catalysis. Mutation of the conserved cysteine prevented A20-mediated antagonism of LPS-stimulated NF-κB activity. TRAF6 is subject to K63-linked ubiquitination, and this post-translational modification is involved in activating TLR signaling to NF-κB. A20, but not the cysteine mutant, cleaved ubiquitin dimers in vitro and reduced levels of TRAF6 ubiquitination in cell lysates.
The deubiquitinating activity of A20 has now been implicated in regulating both TLR and TNF-mediated NF-κB activity. The cylindromatosis gene product, CylD, can also deubiquitinate multiple proteins involved in the immune response including TRAF6 and IKKγ. Future experiments will be directed toward defining the function of CylD and other ubiquitin-modifying enzymes. The role of ubiquitination in TLR signaling has wide implications for the molecular understanding of immune homeostasis and septic shock.
Joanne Kotz, Assistant Editor Nature Chemical Biology
article
David L Boone, Emre E Turer, Eric G Lee, Regina-Celeste Ahmad, Matthew T Wheeler, Colleen Tsui, Paula Hurley, Marcia Chien, Sophia Chai, Osamu Hitotsumatsu, Elizabeth McNally, Cecile Pickart & Averil Ma The ubiquitin-modifying enzyme A20 is required for termination of Toll-like receptor responses Nature Immunology, 5, 1052 – 1060 (2004); doi:10.1038/ni1110
| Full Text (HTML)
| PDF
| Subscribe to Nature Immunology
news and views
Neal Silverman & Katherine Fitzgerald DUBbing down innate immunity Nature Immunology, 5, 1010 – 1012 (2004); doi:10.1038/ni1004-1010
| Full Text (HTML)
| PDF
| Subscribe to Nature Immunology
Immunological synapses: Directing cell fate
At a key branch-point of the immune system, naive T-helper lymphocytes (Thp) are directed toward either the Th1 or Th2 pathway. Two cytokines, interferon-γ (IFNγ) and interleukin-4 (IL-4), are critical early effectors of T-helper commitment that favor the Th1 and Th2 pathways, respectively. How these signals are coordinated with T-cell receptor (TCR) activation is not known. Immunological synapses are clusters of signaling molecules at the point of contact between T cells and antigen presenting cells. Maldonado et al. now demonstrate co-polarization of TCR and IFNγ receptors (IFNGR) at the synapses of activated Thp, thus suggesting a role for immunological synapses in the direction of T cell development.
At the point of initial conjugate formation between Thp and mature splenic dendritic cells (DC), the TCR and IFNGR were randomly distributed on the cell surface. Following 30 minutes of cell–cell contact, TCR and IFNGR congregated at the cellular interface. Consistent with this, time-lapse microscopy of cells showed low Ca2+ levels and uniform receptor distribution prior to Thp–DC contact, whereas, following Ca2+ influx, TCR and IFNGR were observed to progressively migrate towards the point of cell–cell contact. In Thp activated by antibody-mediated TCR crosslinking, the authors again observed co-localization specific to IFNGR and co-polarization of TCR and INFGR. Thp activation induced significant receptor co-recruitment in cells from a Th1-prone mouse strain, B6. In contrast, cells from a Th2-prone strain, BALB/c, showed 200-fold less TCR and IFNGR co-polarization. These results suggest that genetic differences in Thp developmental tendencies may be mediated by changes in receptor trafficking.
Presence of Th2-promoting IL-4 completely prevented TCR crosslinking-induced receptor migration. This inhibition was observed whether IL-4 was present prior to Thp activation or added after activation. In Stat6 null Thp cells deficient in IL-4 receptor-mediated signaling, the inhibitory effect of IL-4 was abolished. These results suggest a mechanism in which a Th1-promoting complex is assembled at the immunological synapse in the absence of an IL-4 inhibitory signal.
One role of receptor co-localization at the immunological synapse could be to increase receptor concentration at the point of cytokine production. The authors speculate that receptor co-polarization may also control cell fate in other cells where lipid rafts control effector function.
Joanne Kotz, Assistant Editor Nature Chemical Biology
article
Roberto A. Maldonado, Darrell J. Irvine, Robert Schreiber & Laurie H. Glimcher A role for the immunological synapse in lineage commitment of CD4 lymphocytes Nature, 431, 527 – 532 (30 September 2004); doi:10.1038/nature02916
| Full Text (HTML)
| PDF
| Subscribe to Nature
