A moderate increase in ambient temperature modulates the Atlantic cod (Gadus morhua) spleen transcriptome response to intraperitoneal viral mimic injection.

A moderate increase in ambient temperature modulates the Atlantic cod (Gadus morhua) spleen transcriptome response to intraperitoneal viral mimic injection.

BMC Genomics. 2012 Aug 28;13(1):431

Authors: Hori TS, Gamperl AK, Booman M, Nash GW, Rise ML

Abstract
ABSTRACT: BACKGROUND: Atlantic cod (Gadus morhua) reared in sea-cages can experience large variations in temperature, and these have been shown to affect their immune function. We used the new 20 K Atlantic cod microarray to investigate how a water temperature change which simulates that seen in Newfoundland during the spring-summer (i.e. from 10[DEGREE SIGN]C to 16[DEGREE SIGN]C, 1[DEGREE SIGN]C increase every 5 days) impacted the cod spleen transcriptome response to the intraperitoneal injection of a viral mimic (polyriboinosinic polyribocytidylic acid, pIC). RESULTS: The temperature regime alone did not cause any significant increases in plasma cortisol levels and only minor changes in spleen gene transcription. However, it had a considerable impact on the fish spleen transcriptome response to pIC [290 and 339 significantly differentially expressed genes between 16[DEGREE SIGN]C and 10[DEGREE SIGN]C at 6 and 24 hours post-injection (HPI), respectively]. Seventeen microarray-identified transcripts were selected for QPCR validation based on immune-relevant functional annotations. Fifteen of these transcripts (i.e. 88%), including DHX58, STAT1, IRF7, ISG15, RSAD2 and IkappaBalpha, were shown by QPCR to be significantly induced by pIC. CONCLUSIONS: The temperature increase appeared to accelerate the spleen immune transcriptome response to pIC. We found 41 and 999 genes differentially expressed between fish injected with PBS vs. pIC at 10[DEGREE SIGN]C and sampled at 6HPI and 24HPI, respectively. In contrast, there were 656 and 246 genes differentially expressed between fish injected with PBS vs. pIC at 16[DEGREE SIGN]C and sampled at 6HPI and 24HPI, respectively. Our results indicate that the modulation of mRNA expression of genes belonging to the NF-kappaB and type I interferon signal transduction pathways may play a role in controlling temperature-induced changes in the spleen's transcript expression response to pIC. Moreover, interferon effector genes such as ISG15 and RSAD2 were differentially expressed between fish injected with pIC at 10[DEGREE SIGN]C vs. 16[DEGREE SIGN]C at 6HPI. These results substantially increase our understanding of the genes and molecular pathways involved in the negative impacts of elevated ambient temperature on fish health, and may also be valuable to our understanding of how accelerated global climate change could impact cold-water marine finfish species.

PMID: 22928584 [PubMed - as supplied by publisher]

NF-kB signaling pathway NF-kB pathway NF-kB signaling

Directed molecular evolution of DREADDs: a generic approach to creating next-generation RASSLs.

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Directed molecular evolution of DREADDs: a generic approach to creating next-generation RASSLs.

Nat Protoc. 2010 Mar;5(3):561-73

Authors: Dong S, Rogan SC, Roth BL

Abstract
G protein-coupled receptors (GPCRs) and their downstream signaling cascades contribute to most physiological processes and a variety of human diseases. Isolating the effects of GPCR activation in an in vivo experimental setting is challenging as exogenous ligands have off-target effects and endogenous ligands constantly modulate the activity of native receptors. Highly specific designer drug-designer receptor complexes are a valuable tool for elucidating the effects of activating particular receptors and signaling pathways within selected cell types in vivo. In this study, we describe a generic protocol for the directed molecular evolution of designer receptors exclusively activated by designer drugs (DREADDs). First, the yeast system is validated with the template receptor. Second, a mutant library is generated by error-prone PCR. Third, the library is screened by drug-dependent yeast growth assays. Mutants exhibiting the desired properties are selected for further rounds of mutagenesis or for characterization in mammalian systems. In total, these steps should take 6-8 weeks of experimentation and should result in the evolution of a receptor to be activated by the chosen ligand. This protocol should help improve the experimental targeting of select cell populations.

PMID: 20203671 [PubMed - indexed for MEDLINE]

GPCR Signaling G-protein Receptors gpcr pathway

Lipid raft-mediated regulation of G-protein coupled receptor signaling by ligands which influence receptor dimerization: a computational study.

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Lipid raft-mediated regulation of G-protein coupled receptor signaling by ligands which influence receptor dimerization: a computational study.

PLoS One. 2009;4(8):e6604

Authors: Fallahi-Sichani M, Linderman JJ

Abstract
G-protein coupled receptors (GPCRs) are the largest family of cell surface receptors; they activate heterotrimeric G-proteins in response to ligand stimulation. Although many GPCRs have been shown to form homo- and/or heterodimers on the cell membrane, the purpose of this dimerization is not known. Recent research has shown that receptor dimerization may have a role in organization of receptors on the cell surface. In addition, microdomains on the cell membrane termed lipid rafts have been shown to play a role in GPCR localization. Using a combination of stochastic (Monte Carlo) and deterministic modeling, we propose a novel mechanism for lipid raft partitioning of GPCRs based on reversible dimerization of receptors and then demonstrate that such localization can affect GPCR signaling. Modeling results are consistent with a variety of experimental data indicating that lipid rafts have a role in amplification or attenuation of G-protein signaling. Thus our work suggests a new mechanism by which dimerization-inducing or inhibiting characteristics of ligands can influence GPCR signaling by controlling receptor organization on the cell membrane.

PMID: 19668374 [PubMed - indexed for MEDLINE]

NF-kB pathway NF-kB signaling NF-kappaB signaling pathway

G-protein-coupled receptors in drug discovery: nanosizing using cell-free technologies and molecular biology approaches.

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G-protein-coupled receptors in drug discovery: nanosizing using cell-free technologies and molecular biology approaches.

J Biomol Screen. 2005 Dec;10(8):765-79

Authors: Leifert WR, Aloia AL, Bucco O, Glatz RV, McMurchie EJ

Abstract
Signal transduction by G-protein-coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. Signal transduction has been studied extensively with both cell-based systems and assays comprising isolated signaling components. Interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high throughput screening, biosensors, and so on will focus greater attention on assay development to allow for miniaturization, ultra-high throughput and, eventually, microarray/biochip assay formats. Although cell-based assays are adequate for many GPCRs, it is likely that these formats will limit the development of higher density GPCR assay platforms mandatory for other applications. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR assay platforms adaptable for such applications as microarrays. The authors review current cell-free GPCR assay technologies and molecular biological approaches for construction of novel, functional GPCR assays.

PMID: 16234342 [PubMed - indexed for MEDLINE]

gpcr pathway NF-κB NF-kB signaling pathway

Statins suppress apolipoprotein CIII-induced vascular endothelial cell activation and monocyte adhesion.

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Statins suppress apolipoprotein CIII-induced vascular endothelial cell activation and monocyte adhesion.

Eur Heart J. 2012 Aug 26;

Authors: Zheng C, Azcutia V, Aikawa E, Figueiredo JL, Croce K, Sonoki H, Sacks FM, Luscinskas FW, Aikawa M

Abstract
AimsActivation of vascular endothelial cells (ECs) contributes importantly to inflammation and atherogenesis. We previously reported that apolipoprotein CIII (apoCIII), found abundantly on circulating triglyceride-rich lipoproteins, enhances adhesion of human monocytes to ECs in vitro. Statins may exert lipid-independent anti-inflammatory effects. The present study examined whether statins suppress apoCIII-induced EC activation in vitro and in vivo.Methods and resultsPhysiologically relevant concentrations of purified human apoCIII enhanced attachment of the monocyte-like cell line THP-1 to human saphenous vein ECs (HSVECs) or human coronary artery ECs (HCAECs) under both static and laminar shear stress conditions. This process mainly depends on vascular cell adhesion molecule-1 (VCAM-1), as a blocking VCAM-1 antibody abolished apoCIII-induced monocyte adhesion. ApoCIII significantly increased VCAM-1 expression in HSVECs and HCAECs. Pre-treatment with statins suppressed apoCIII-induced VCAM-1 expression and monocyte adhesion, with two lipophilic statins (pitavastatin and atorvastatin) exhibiting inhibitory effects at lower concentration than those of hydrophilic pravastatin. Nuclear factor ?B (NF-?B) mediated apoCIII-induced VCAM-1 expression, as demonstrated via loss-of-function experiments, and pitavastatin treatment suppressed NF-?B activation. Furthermore, in the aorta of hypercholesterolaemic Ldlr(-/-) mice, pitavastatin administration in vivo suppressed VCAM-1 mRNA and protein, induced by apoCIII bolus injection. Similarly, in a subcutaneous dorsal air pouch mouse model of leucocyte recruitment, apoCIII injection induced F4/80+ monocyte and macrophage accumulation, whereas pitavastatin administration reduced this effect.ConclusionsThese findings further establish the direct role of apoCIII in atherogenesis and suggest that anti-inflammatory effects of statins could improve vascular disease in the population with elevated plasma apoCIII.

PMID: 22927557 [PubMed - as supplied by publisher]

NF-κB NF-kB signaling pathway NF-kB pathway

The role of Raf kinase inhibitor protein in rheumatoid fibroblast-like synoviocytes invasiveness and cytokine and matrix metalloproteinase expression.

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The role of Raf kinase inhibitor protein in rheumatoid fibroblast-like synoviocytes invasiveness and cytokine and matrix metalloproteinase expression.

Inflammation. 2012 Apr;35(2):474-83

Authors: Ahn JK, Hwang JW, Bae EK, Lee J, Jeon CH, Koh EM, Cha HS

Abstract
Fibroblast-like synoviocytes (FLS) play an important role in the pathogenesis of rheumatoid arthritis. Raf kinase inhibitor protein (RKIP) negatively regulates the Raf/MEK/ERK and NF-?B pathway. The role of RKIP in rheumatoid FLS is unknown. The purpose of the present study was to investigate the function of RKIP in rheumatoid FLS. Rheumatoid FLS were transfected with either RKIP-expressing plasmids or RKIP small interfering RNA (siRNA). RKIP protein was detected in rheumatoid synovial tissue (ST) and FLS. RKIP overexpression significantly decreased IL-6 mRNA expression in TNF-?-stimulated rheumatoid FLS. RKIP overexpression also showed a decreased trend in IL-8, MMP-1, and MMP-3 mRNA expression in TNF-?-stimulated rheumatoid FLS. RKIP silencing resulted in significantly increased MMP-1 and MMP-3 mRNA expression in TNF-?-stimulated rheumatoid FLS. RKIP silencing also increased IL-6 and IL-8 mRNA expression in TNF-?-stimulated rheumatoid FLS, but this increase did not reach statistical significance. TNF-?-induced ERK and NF-?B activation was suppressed in FLS with RKIP overexpression. RKIP silencing resulted in a significantly higher invasion index in TNF-?-stimulated rheumatoid FLS compared to controls. These results suggest that RKIP might be a potential therapeutic target for rheumatoid arthritis.

PMID: 21556737 [PubMed - indexed for MEDLINE]

gpcr pathway NF-κB NF-kB signaling pathway

Chemokines, selectins and intracellular calcium flux: temporal and spatial cues for leukocyte arrest.

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Chemokines, selectins and intracellular calcium flux: temporal and spatial cues for leukocyte arrest.

Front Immunol. 2012;3:188

Authors: Dixit N, Simon SI

Abstract
Leukocyte trafficking to acute sites of injury or infection requires spatial and temporal cues that fine tune precise sites of firm adhesion and guide migration to endothelial junctions where they undergo diapedesis to sites of insult. Many detailed studies on the location and gradient of chemokines such as IL-8 and other CXCR ligands reveal that their recognition shortly after selectin-mediated capture and rolling exerts acute effects on integrin activation and subsequent binding to their ligands on the endothelium, which directs firm adhesion, adhesion strengthening, and downstream migration. In this process, G-protein coupled receptor (GPCR) signaling has been found to play an integral role in activating and mobilizing intracellular stores of calcium, GTPases such as Rap-1 and Rho and cytokeletal proteins such as Talin and F-actin to facilitate cell polarity and directional pseudopod formation. A critical question remaining is how intracellular Ca(2+) flux from CRAC channels such as Orai1 synergizes with cytosolic stores to mediate a rapid flux which is critical to the onset of PMN arrest and polarization. Our review will highlight a specific role for calcium as a signaling messenger in activating focal clusters of integrins bound to the cytoskeleton which allows the cell to attain a migratory phenotype. The precise interplay between chemokines, selectins, and integrins binding under the ubiquitous presence of shear stress from blood flow provides an essential cooperative signaling mechanism for effective leukocyte recruitment.

PMID: 22787461 [PubMed - in process]

GPCR Signaling G-protein Receptors gpcr pathway