Stuart Kellie

Expression analysis of G Protein-Coupled Receptors in mouse macrophages

BackgroundMonocytes and macrophages express an extensive repertoire of G Protein-Coupled Receptors (GPCRs) that regulate inflammation and immunity. In this study we performed a systematic micro-array analysis of GPCR expression in primary mouse macrophages to identify family members that are either enriched in macrophages compared to a panel of other cell types, or are regulated by an inflammatory stimulus, the bacterial product lipopolysaccharide (LPS).ResultsSeveral members of the P2RY family had striking expression patterns in macrophages; P2ry6 mRNA was essentially expressed in a macrophage-specific fashion, whilst P2ry1 and P2ry5 mRNA levels were strongly down-regulated by LPS. Expression of several other GPCRs was either restricted to macrophages (e.g. Gpr84) or to both macrophages and neural tissues (e.g. P2ry12, Gpr85). The GPCR repertoire expressed by bone marrow-derived macrophages and thioglycollate-elicited peritoneal macrophages had some commonality, but there were also several GPCRs preferentially expressed by either cell population.ConclusionThe constitutive or regulated expression in macrophages of several GPCRs identified in this study has not previously been described. Future studies on such GPCRs and their agonists are likely to provide important insights into macrophage biology, as well as novel inflammatory pathways that could be future targets for drug discovery.

Phosphorylation regulates tau interactions with Src homology 3 domains of phosphatidylinositol 3-kinase, phospholipase C gamma 1, Grb2, and Src family kinases

The microtubule-associated protein tau can associate with various other proteins in addition to tubulin, including the SH3 domains of Src family tyrosine kinases. Tau is well known to aggregate to form hyperphosphorylated filamentous deposits in several neurodegenerative diseases (tauopathies) including Alzheimer disease. We now report that tau can bind to SH3 domains derived from the p85α subunit of phosphatidylinositol 3-kinase, phospholipase Cγ1, and the N-terminal (but not the C-terminal) SH3 of Grb2 as well as to the kinases Fyn, cSrc, and Fgr. However, the short inserts found in neuron-specific isoforms of Src prevented the binding of tau. The experimentally determined binding of tau peptides is well accounted for when modeled into the peptide binding cleft in the SH3 domain of Fyn. After phosphorylation in vitro or in transfected cells, tau showed reduced binding to SH3 domains; no binding was detected with hyperphosphorylated tau isolated from Alzheimer brain, but SH3 binding was restored by phosphatase treatment. Tau mutants with serines and threonines replaced by glutamate, to mimic phosphorylation, showed reduced SH3 binding. These results strongly suggest that tau has a potential role in cell signaling in addition to its accepted role in cytoskeletal assembly, with regulation by phosphorylation that may be disrupted in the tauopathies including Alzheimer disease.

Tyrosine 394 Is Phosphorylated in Alzheimer's Paired Helical Filament Tau and in Fetal Tau with c-Abl as the Candidate Tyrosine Kinase

Tau is a major microtubule-associated protein of axons and is also the principal component of the paired helical filaments (PHFs) that comprise the neurofibrillary tangles found in Alzheimers disease and other tauopathies. Besides phosphorylation of tau on serine and threonine residues in both normal tau and tau from neurofibrillary tangles, Tyr-18 was reported to be a site of phosphorylation by the Src-family kinase Fyn. We examined whether tyrosine residues other than Tyr-18 are phosphorylated in tau and whether other tyrosine kinases might phosphorylate tau. Using mass spectrometry, we positively identified phosphorylated Tyr-394 in PHF-tau from an Alzheimer brain and in human fetal brain tau. When wild-type human tau was transfected into fibroblasts or neuroblastoma cells, treatment with pervanadate caused tau to become phosphorylated on tyrosine by endogenous kinases. By replacing each of the five tyrosines in tau with phenylalanine, we identified Tyr-394 as the major site of tyrosine phosphorylation in tau. Tyrosine phosphorylation of tau was inhibited by PP2 (4-amino-5-(4-chlorophenyl-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), which is known to inhibit Src-family kinases and c-Abl. Cotransfection of tau and kinases showed that Tyr-18 was the major site for Fyn phosphorylation, but Tyr-394 was the main residue for Abl. In vitro, Abl phosphorylated tau directly. Abl could be coprecipitated with tau and was present in pretangle neurons in brain sections from Alzheimer cases. These results show that phosphorylation of tau on Tyr-394 is a physiological event that is potentially part of a signal relay and suggest that Abl could have a pathogenic role in Alzheimers disease.

Crystal structure of Toll-like receptor adaptor MAL/TIRAP reveals the molecular basis for signal transduction and disease protection

Initiation of the innate immune response requires agonist recognition by pathogen-recognition receptors such as the Toll-like receptors (TLRs). Toll/interleukin-1 receptor (TIR) domain-containing adaptors are critical in orchestrating the signal transduction pathways after TLR and interleukin-1 receptor activation. Myeloid differentiation primary response gene 88 (MyD88) adaptor-like (MAL)/TIR domain-containing adaptor protein (TIRAP) is involved in bridging MyD88 to TLR2 and TLR4 in response to bacterial infection. Genetic studies have associated a number of unique single-nucleotide polymorphisms in MAL with protection against invasive microbial infection, but a molecular understanding has been hampered by a lack of structural information. The present study describes the crystal structure of MAL TIR domain. Significant structural differences exist in the overall fold of MAL compared with other TIR domain structures: A sequence motif comprising a β-strand in other TIR domains instead corresponds to a long loop, placing the functionally important “BB loop” proline motif in a unique surface position in MAL. The structure suggests possible dimerization and MyD88-interacting interfaces, and we confirm the key interface residues by coimmunoprecipitation using site-directed mutants. Jointly, our results provide a molecular and structural basis for the role of MAL in TLR signaling and disease protection.

Functional and structural properties of mammalian acyl-coenzyme A thioesterases.

Acyl-coenzyme A thioesterases (Acots) play important cellular roles in mammalian fatty acid metabolism through modulation of cellular concentrations of activated fatty acyl-CoAs. Acots catalyse the hydrolysis of the thioester bond present within acyl-CoA ester molecules to yield coenzyme A (CoASH) and the corresponding non-esterified fatty acid. Acyl-CoA thioesterases are expressed ubiquitously in both prokaryotes and eukaryotes and, in higher order organisms, the enzymes are expressed and localised in a tissue-dependent manner within the cytosol, mitochondria, peroxisomes and endoplasmic reticulum. Recent studies have led to advances in the functional and structural characterization of many mammalian Acot family members. These include the structure determination of both type-I and type-II Acot family members, structural elucidation of the START domain of ACOT11, identification of roles in arachidonic acid and inflammatory prostaglandin production by Acot7, and inclusion of a 13th Acot family member. Here, we review and analyse the current literature on mammalian Acots with respect to their characterization and summarize the current knowledge on the structure, function and regulation of this enzyme family.

Translocation of pp60c-src to the cytoskeleton during platelet aggregation.

The high amount of pp60c‐src in platelets has led to speculation that this kinase is responsible for tyrosine‐specific phosphorylation of cellular proteins during platelet activation by different agonists, and is, therefore, implicated in signal transduction of these cells. Unlike pp60v‐src, the association of which with the cytoskeleton appears to be a prerequisite for transformation, pp60c‐src is detergent‐soluble in fibroblasts overexpressing the c‐src gene, and its role in normal cellular function remains elusive. To gain a better understanding of the function of pp60c‐src we have investigated the subcellular distribution of pp60c‐src and its relationship to the cytoskeleton during platelet activation. Quantitative immunoblotting and immunoprecipitation have revealed that pp60c‐src is detergent‐soluble in resting platelets, while 40% of total platelet pp60c‐src becomes associated with the cytoskeletal fraction upon platelet activation. We have also shown that a small pool of pp60c‐src is associated with the membrane skeletal fraction which remains unchanged during the activation process. The interaction of pp60c‐src with cytoskeletal proteins strongly correlates with aggregation and is mediated by GPIIb/IIIa receptor‐fibrinogen binding. We suggest that the translocation of pp60c‐src to the cytoskeleton and its association with cytoskeletal proteins may regulate tyrosine phosphorylation in platelets.

G‐protein‐coupled receptor expression, function, and signaling in macrophages

G‐protein‐coupled receptors (GPCRs) are widely targeted in drug discovery. As macrophages are key cellular mediators of acute and chronic inflammation, we review here the role of GPCRs in regulating macrophage function, with a focus on contribution to disease pathology and potential therapeutic applications. Within this analysis, we highlight novel GPCRs with a macrophage‐restricted expression profile, which provide avenues for further exploration. We also review an emerging literature, which documents novel roles for GPCR signaling components in GPCR‐independent signaling in macrophages. In particular, we examine the crosstalk between GPCR and TLR signaling pathways and highlight GPCR signaling molecules which are likely to have uncharacterized functions in this cell lineage.

Clusterin regulates β-amyloid toxicity via Dickkopf-1-driven induction of the wnt–PCP–JNK pathway

Although the mechanism of Aβ action in the pathogenesis of Alzheimer’s disease (AD) has remained elusive, it is known to increase the expression of the antagonist of canonical wnt signalling, Dickkopf-1 (Dkk1), whereas the silencing of Dkk1 blocks Aβ neurotoxicity. We asked if clusterin, known to be regulated by wnt, is part of an Aβ/Dkk1 neurotoxic pathway. Knockdown of clusterin in primary neurons reduced Aβ toxicity and DKK1 upregulation and, conversely, Aβ increased intracellular clusterin and decreased clusterin protein secretion, resulting in the p53-dependent induction of DKK1. To further elucidate how the clusterin-dependent induction of Dkk1 by Aβ mediates neurotoxicity, we measured the effects of Aβ and Dkk1 protein on whole-genome expression in primary neurons, finding a common pathway suggestive of activation of wnt–planar cell polarity (PCP)–c-Jun N-terminal kinase (JNK) signalling leading to the induction of genes including EGR1 (early growth response-1), NAB2 (Ngfi-A-binding protein-2) and KLF10 (Krüppel-like factor-10) that, when individually silenced, protected against Aβ neurotoxicity and/or tau phosphorylation. Neuronal overexpression of Dkk1 in transgenic mice mimicked this Aβ-induced pathway and resulted in age-dependent increases in tau phosphorylation in hippocampus and cognitive impairment. Furthermore, we show that this Dkk1/wnt–PCP–JNK pathway is active in an Aβ-based mouse model of AD and in AD brain, but not in a tau-based mouse model or in frontotemporal dementia brain. Thus, we have identified a pathway whereby Aβ induces a clusterin/p53/Dkk1/wnt–PCP–JNK pathway, which drives the upregulation of several genes that mediate the development of AD-like neuropathologies, thereby providing new mechanistic insights into the action of Aβ in neurodegenerative diseases.

Macrophage Activation and Differentiation Signals Regulate Schlafen-4 Gene Expression: Evidence for Schlafen-4 as a Modulator of Myelopoiesis

Background The ten mouse and six human members of the Schlafen (Slfn) gene family all contain an AAA domain. Little is known of their function, but previous studies suggest roles in immune cell development. In this report, we assessed Slfn regulation and function in macrophages, which are key cellular regulators of innate immunity. Methodology/Principal Findings Multiple members of the Slfn family were up-regulated in mouse bone marrow-derived macrophages (BMM) by the Toll-like Receptor (TLR)4 agonist lipopolysaccharide (LPS), the TLR3 agonist Poly(I∶C), and in disease-affected joints in the collagen-induced model of rheumatoid arthritis. Of these, the most inducible was Slfn4. TLR agonists that signal exclusively through the MyD88 adaptor protein had more modest effects on Slfn4 mRNA levels, thus implicating MyD88-independent signalling and autocrine interferon (IFN)-β in inducible expression. This was supported by the substantial reduction in basal and LPS-induced Slfn4 mRNA expression in IFNAR-1−/− BMM. LPS causes growth arrest in macrophages, and other Slfn family genes have been implicated in growth control. Slfn4 mRNA levels were repressed during macrophage colony-stimulating factor (CSF-1)-mediated differentiation of bone marrow progenitors into BMM. To determine the role of Slfn4 in vivo, we over-expressed the gene specifically in macrophages in mice using a csf1r promoter-driven binary expression system. Transgenic over-expression of Slfn4 in myeloid cells did not alter macrophage colony formation or proliferation in vitro. Monocyte numbers, as well as inflammatory macrophages recruited to the peritoneal cavity, were reduced in transgenic mice that specifically over-expressed Slfn4, while macrophage numbers and hematopoietic activity were increased in the livers and spleens. Conclusions Slfn4 mRNA levels were up-regulated during macrophage activation but down-regulated during differentiation. Constitutive Slfn4 expression in the myeloid lineage in vivo perturbs myelopoiesis. We hypothesise that the down-regulation of Slfn4 gene expression during macrophage differentiation is a necessary step in development of this lineage.

Gangliosides as receptors for fibronectin? Comparison of cell spreading on a ganglioside-specific ligand with that on fibronectin.

Abstract We have examined the possibility that gangliosides act as the cell surface receptor for fibronectin, as previously suggested by the data of Kleinman et al. (Proc natl acad sci US 76 (1979) 3367), using three different approaches. 1. 1. Gangliosides inhibited the spreading of both CHO and BHK cells on fibronectin-coated substrates. 50% inhibition of cell spreading was produced by 1.0 and 3.5 × 10 −5 M di- and trisialogangliosides respectively, although monosialogangliosides were less effective inhibitors. The inhibition was apparently due to an interaction of gangliosides with fibronectin and not due to a direct effect of gangliosides on the cells. 2. 2. Using anti-fibronectin antibodies, 125 I-labelled protein A, and gangliosides adsorbed to polystyrene tubes, we have provided direct evidence that fibronectin will bind to gangliosides. However, the interaction is apparently of low affinity compared with binding of cholera toxin to gangliosides. 3. 3. We have compared the ability of BALB/c 3T3 cells to spread on fibronectin with that on substrates coated with a ganglioside-specific ligand, cholera toxin B-subunit. Cells plated onto fibronectin-coated substrates rapidly (within 60 min) adopted a well spread morphology, whereas spreading on substrates coated with the toxin B-subunit was less extensive even after 2 h. In addition, the organization of F-actin within cells spread on the two types of substrate was also quite different. We conclude that the interaction of cells with fibronectin may well be influenced by membrane-bound gangliosides. It is unlikely, however, that binding of fibronectin to such gangliosides can lead to the cytoskeletal reorganization which is characteristic of cells spread on fibronectin.