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• W2015705955 abstract "Pattern recognition receptors contain a binding domain for pathogen-associated molecular patterns coupled to a signaling domain that regulates transcription of host immune response genes. Here, a novel mechanism that links pathogen recognition to channel activation and downstream signaling is proposed. We demonstrate that an intracellular sodium channel variant, human macrophage SCN5A, initiates signaling and transcription through a calcium-dependent isoform of adenylate cyclase, ADCY8, and the transcription factor, ATF2. Pharmacological stimulation with a channel agonist or treatment with cytoplasmic poly(I:C), a mimic of viral dsRNA, activates this pathway to regulate expression of SP100-related genes and interferon β. Electrophysiological analysis reveals that the SCN5A variant mediates nonselective outward currents and a small, but detectable, inward current. Intracellular poly(I:C) markedly augments an inward voltage-sensitive sodium current and inhibits the outward nonselective current. These results suggest human macrophage SCN5A initiates signaling in an innate immune pathway relevant to antiviral host defense. It is postulated that SCN5A is a novel pathogen sensor and that this pathway represents a channel activation-dependent mechanism of transcriptional regulation. Pattern recognition receptors contain a binding domain for pathogen-associated molecular patterns coupled to a signaling domain that regulates transcription of host immune response genes. Here, a novel mechanism that links pathogen recognition to channel activation and downstream signaling is proposed. We demonstrate that an intracellular sodium channel variant, human macrophage SCN5A, initiates signaling and transcription through a calcium-dependent isoform of adenylate cyclase, ADCY8, and the transcription factor, ATF2. Pharmacological stimulation with a channel agonist or treatment with cytoplasmic poly(I:C), a mimic of viral dsRNA, activates this pathway to regulate expression of SP100-related genes and interferon β. Electrophysiological analysis reveals that the SCN5A variant mediates nonselective outward currents and a small, but detectable, inward current. Intracellular poly(I:C) markedly augments an inward voltage-sensitive sodium current and inhibits the outward nonselective current. These results suggest human macrophage SCN5A initiates signaling in an innate immune pathway relevant to antiviral host defense. It is postulated that SCN5A is a novel pathogen sensor and that this pathway represents a channel activation-dependent mechanism of transcriptional regulation. Characterization of innate immune signal transduction has focused primarily on ligand-receptor interactions between pathogen-associated molecular patterns and pattern recognition receptors (PRR) 2The abbreviations used are: PRRpattern recognition receptorAdcyadenylate cyclaseATF2activating transcription factor 2BMDMbone marrow-derived macrophageMDMmonocyte-derived macrophageNMDGN-methyl-d-glucaminepoly(I:C)polyinosinic:polycytidylic acidRYRryanodine receptorSCNsodium channelANOVAanalysis of varianceAUCarea under the curveHBSSHanks' buffered saline solutionQPCRquantitative PCRIFNBinterferon βLMWlow molecular weightDiSBAC2(3)bis-(1,3-diethylthiobarbituric acid) trimethine oxonol. (1.Janeway Jr., C.A. How the immune system works to protect the host from infection: a personal view.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 7461-7468Crossref PubMed Scopus (247) Google Scholar, 2.Takeuchi O. Akira S. Pattern recognition receptors and inflammation.Cell. 2010; 140: 805-820Abstract Full Text Full Text PDF PubMed Scopus (5704) Google Scholar). PRR contain a binding domain for pathogen-associated molecular patterns and a signaling domain that initiates downstream activation of transcription factors relevant to host defense. However, alternative biological mechanisms also may initiate signaling in innate immune cells and include those pathways utilized by excitable tissues such as neurons and muscle. These excitable cells express voltage-gated channels that mediate not only ionic flux but also regulate activity-dependent regulation of gene transcription and some long term forms of memory (3.Lynch M.A. Long-term potentiation and memory.Physiol. Rev. 2004; 84: 87-136Crossref PubMed Scopus (1434) Google Scholar). pattern recognition receptor adenylate cyclase activating transcription factor 2 bone marrow-derived macrophage monocyte-derived macrophage N-methyl-d-glucamine polyinosinic:polycytidylic acid ryanodine receptor sodium channel analysis of variance area under the curve Hanks' buffered saline solution quantitative PCR interferon β low molecular weight bis-(1,3-diethylthiobarbituric acid) trimethine oxonol. One candidate molecule is a unique splice variant of the sodium channel gene SCN5A (4.Carrithers M.D. Dib-Hajj S. Carrithers L.M. Tokmoulina G. Pypaert M. Jonas E.A. Waxman S.G. Expression of the voltage-gated sodium channel NaV1.5 in the macrophage late endosome regulates endosomal acidification.J. Immunol. 2007; 178: 7822-7832Crossref PubMed Scopus (98) Google Scholar, 5.Carrithers L.M. Hulseberg P. Sandor M. Carrithers M.D. The human macrophage sodium channel NaV1.5 regulates mycobacteria processing through organelle polarization and localized calcium oscillations.FEMS Immunol. Med. Microbiol. 2011; 63: 319-327Crossref PubMed Scopus (41) Google Scholar, 6.Carrithers M.D. Chatterjee G. Carrithers L.M. Offoha R. Iheagwara U. Rahner C. Graham M. Waxman S.G. Regulation of podosome formation in macrophages by a splice variant of the sodium channel SCN8A.J. Biol. Chem. 2009; 284: 8114-8126Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 7.Rahgozar K. Wright E. Carrithers L.M. Carrithers M.D. Mediation of protection and recovery from experimental autoimmune encephalomyelitis by macrophages expressing the human voltage-gated sodium channel NaV1.5.J. Neuropathol. Exp. Neurol. 2013; 72: 489-504Crossref PubMed Scopus (12) Google Scholar). Prior studies demonstrated that the SCN5A variant encodes a channel that regulates phagocytosis, endosomal acidification, calcium signaling, and phenotypic differentiation in human macrophages. Unlike voltage-gated sodium channels in excitable tissues, this macrophage variant is expressed on endosomes intracellularly and not at the plasma membrane. Because of an exon deletion in the extracellular selectivity filter, channel activation does not elicit typical action potentials as in excitable tissues but does mediate ionic flux in response to pharmacological stimulation. Because this channel is not expressed in murine macrophages, in vivo characterization has been performed in a knock-in transgenic model (7.Rahgozar K. Wright E. Carrithers L.M. Carrithers M.D. Mediation of protection and recovery from experimental autoimmune encephalomyelitis by macrophages expressing the human voltage-gated sodium channel NaV1.5.J. Neuropathol. Exp. Neurol. 2013; 72: 489-504Crossref PubMed Scopus (12) Google Scholar). The central questions of this study were to assess how an intracellular voltage-gated channel could mediate innate immune signaling, regulate transcription, and function as a pathogen sensor. In mouse macrophages that express the human SCN5A variant and primary human monocyte-derived macrophages, we demonstrate that channel expression and activation are associated with a signaling pathway that links activation of a calcium-dependent adenylate cyclase isoform, ADCY8, to the transcription factor ATF2. In an ATF2-dependent manner, SCN5A expression and activation in macrophages regulates expression of host antiviral response genes, Sp100 and β-interferon (IFNB), and an innate immune signaling pathway initiated by cytosolic poly(I:C). Channel activation by poly(I:C) markedly increases an inward voltage-sensitive sodium current and decreases a nonspecific outward current. Experimental mouse strains (C57BL6cfms-hSCN5A) were bred at our on-campus breeding facility (Biotron Facility), where our transgenic colony is maintained. Experimental mice were transferred to an approved University of Wisconsin animal facility for the performance and monitoring of all in vivo experiments. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee of the University of Wisconsin, Madison (protocol numbers M024031 and M02544). Genotyping was performed at Mouse Genotype (Escondido, CA). Primary mouse bone marrow cells were obtained from femurs and tibias of transgenic and wild type littermate control mice and were differentiated to macrophages in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), sodium pyruvate, nonessential amino acids, and mCSF (macrophage colony stimulating factor, 20 ng/ml) for 7–10 days. Human CD14+ peripheral blood monocytes were obtained from Lonza and differentiated to macrophages in the same media. HEK-293F cells were obtained from Invitrogen and maintained as a suspension culture in 293 Freestyle media. Plasmid transfections of empty vector (pcDNA3.1 hygro) or a vector-containing construct (human macrophage SCN5A, accession number KC858891 (7.Rahgozar K. Wright E. Carrithers L.M. Carrithers M.D. Mediation of protection and recovery from experimental autoimmune encephalomyelitis by macrophages expressing the human voltage-gated sodium channel NaV1.5.J. Neuropathol. Exp. Neurol. 2013; 72: 489-504Crossref PubMed Scopus (12) Google Scholar)) were performed using TransIT X2 transfection reagent (Mirus). Mouse bone marrow-derived macrophages from transgenic and littermate controls were lysed by passage through a 27-gauge needle in PBS containing 0.1% Triton X-100, protease, and phosphatase inhibitors (Thermo Scientific Pierce). The lysate was cleared by centrifugation and incubated with rabbit anti-NaV1.5 (SCN5A, Alomone Labs) at a 1:100 dilution and anti-rabbit magnetic microbeads (Miltenyi) for 15 min. Beads were then applied to a micro-column, washed, and collected by elution and subsequent centrifugation. Nano LC-MS/MS was performed at Applied Biomics (Hayward, CA). For sample preparation, proteins were exchanged into 50 mm ammonium bicarbonate buffer. DTT was added to a final concentration of 10 mm and incubated at 60 °C for 30 min, followed by cooling down to room temperature. Iodoacetamide was then added to a final concentration of 10 mm and incubated in the dark for 30 min at room temperature. The proteins were then digested by trypsin (Promega) overnight at 37 °C. Nano-LC was carried out using a Dionex Ultimate 3000 (Milford, MA). Mobile phase solvents A and B were 0.1% TFA (v/v) in water and 0.1% TFA (v/v) in 80% acetonitrile, respectively. Tryptic peptides were loaded into a μ-Precolumn Cartridge and separated on an acetonitrile gradient (ranging from 5 to 60%) on a Nano LC column. Fractions were collected at 20-s intervals followed by mass spectrometry analysis on AB SCIEX TOF/TOFTM 5800 system (AB SCIEX). Mass spectra were acquired in reflectron positive ion mode. TOF/TOF tandem MS fragmentation spectra were acquired for each ion, averaging 4000 laser shots per fragmentation spectrum (excluding trypsin autolytic peptides and other known background ions). Both of the resulting peptide mass and the associated fragmentation spectra were submitted to GPS Explorer workstation equipped with MASCOT search engine (Matrix Science, London, UK) to search the database of Swiss-Prot. Searches were performed without constraining protein molecular weight or isoelectric point, with variable carbamidomethylation of cysteine and oxidation of methionine residues, and with one missed cleavage also allowed in the search parameters. One-dimensional Western blot was performed by Applied Biomics (Hayward, CA). For sample preparation, lysate or immunoprecipitation pellets were exchanged into cell lysis buffer (30 mm Tris-HCl, pH 8.8, containing 7 m urea, 2 m thiourea, and 4% CHAPS). Protein concentration was measured using Bio-Rad protein assay method. For each sample, 30 μg of protein was mixed with 1.0 μl of diluted CyDye and kept in the dark on ice for 10 min. The labeling reaction was stopped by adding 1.0 μl of 10 mm lysine to each sample and incubating in dark on ice for an additional 15 min. The labeled samples were then mixed with 120 μg of unlabeled material and exchanged into SDS gel loading buffer (100 mm Tris-Cl, pH 6.8, 4% (w/v) SDS, 0.2% (w/v) bromphenol blue, 20% (v/v) glycerol); DTT was added to the final concentration of 200 mm. The sample was heated at 95 °C for 5 min before loading into the wells. The SDS gels were run at 15 °C. Gel images were scanned immediately following SDS-PAGE using Typhoon TRIO (GE Healthcare). The gels were transferred to Immobilon-FL PVDF membranes (EMD Millipore, Billerica, MA), and the resulting images were immediately scanned using Typhoon TRIO. Membranes were blocked in 2.5% fish gelatin for 2 h with shaking. The membranes were then incubated in primary antibodies (mouse anti-ATF2 and rabbit anti-phospho-ATF2, Santa Cruz Biotechnologies) in 1× TBST buffer with shaking overnight. The membranes were washed four times, 10 min each, with shaking in 1× TBST buffer. The membranes were then incubated using CyDye-conjugated secondary antibodies at a dilution of 1:2000 in 1× TBST buffer with shaking for 2 h. The membranes were then washed four times, 10 min each, with shaking in 1× TBST buffer. The membranes were dried, and the images were scanned using Typhoon TRIO. The scanned images were then analyzed by ImageJ. For immunofluorescence staining, cell monolayers were fixed in 4% paraformaldehyde, washed with PBS, and blocked in PBS containing 5% serum, 0.1% Triton X-100, and 1% BSA. Primary and secondary antibodies were diluted in blocking solution. For primary antibody staining, anti-ATF2 antibodies were from Santa Cruz Biotechnology (goat antiphospho-ATF2-Thr-71; rabbit anti-ATF2 C terminus; 1:50 dilution), and isotype controls were from eBioscience. Alexa dye-labeled secondary antibodies (donkey anti-goat and anti-rabbit) were from Invitrogen. Rhodamine-labeled poly(I:C) was obtained from Invivogen, and staining was performed on live cells at 200 ng/ml for 20 min. Treated cells were washed extensively with PBS prior to fixation and imaging. Fluorescent images were acquired and analyzed using a Zeiss Axiovert 200 fluorescent microscope equipped with an Axiocam-MRm CCD camera (Zeiss) and AxioVision version 4.8 software. Primary cells were transfected in serum-free OptiMEM media that contained 100 nm retinoic acid using the TransIT X2 transfection reagent (Mirus) and pooled ON-TARGETplus siRNA for each target (Dharmacon). Cells were maintained in media with transfection complexes for 48 h prior to experiments. Knockdown was confirmed by QPCR and was greater than 75%. For QPCR experiments, cells were treated with veratridine (100 μm; Tocris) or DMSO vehicle in OptiMEM media. For immunoprecipitation and live cell imaging experiments, treatment was performed in HBSS supplemented with 10 mm Hepes. Poly(I:C) (LMW; Invivogen) complexes for acute treatments (2 h) were generated in OptiMEM using 10 μg/ml poly(I:C) and 30 μl of TransIT X2 transfection reagent/ml media (30 min at 25 °C). For transfected and naked poly(I:C), the final concentration during cell treatments was 200 ng/ml. RNA purification, reverse transcription, QPCR, and data analysis were performed as described previously (5.Carrithers L.M. Hulseberg P. Sandor M. Carrithers M.D. The human macrophage sodium channel NaV1.5 regulates mycobacteria processing through organelle polarization and localized calcium oscillations.FEMS Immunol. Med. Microbiol. 2011; 63: 319-327Crossref PubMed Scopus (41) Google Scholar). Data were acquired on a Cepheid SmartCycler and analyzed by the ΔCt method with normalization to either mouse GAPDH or human hydroxymethylbilane synthase. The following TaqMan primers were obtained from Applied Biosystems/Invitrogen: Hs00165693_m1 (human SCN5A), Mm99999915_g1 (mouse Gapdh), Mm00449735_m1 (mouse Sp100), Mm04204797_m1 (mouse Gm7609), Hs00162109_m1 (human SP100), Hs01077958_s1 (human IFNB1), AIGJQ71 (HSV thymidine kinase), and AIHSPDQ (HSV glycoprotein B (8.Mott K.R. Underhill D. Wechsler S.L. Town T. Ghiasi H. A role for the JAK-STAT1 pathway in blocking replication of HSV-1 in dendritic cells and macrophages.Virol. J. 2009; 6: 56Crossref PubMed Scopus (34) Google Scholar)). Total RNA was prepared from mouse bone marrow-derived macrophages using column purification (Qiagen RNeasy). Labeling, hybridization, and scanning were performed at the Gene Expression Center at the University of Wisconsin Biotechnology Center (Madison, WI). Affymetrix mouse 2.0ST whole transcriptome arrays were utilized. Data were analyzed using NetAffx software (Affymetrix). Fold change (hSCN5A-transgenic macrophages versus wild type cells) and p values were based on three separate RNA preparations for each condition. Primary mouse bone marrow-derived macrophages and human monocyte-derived macrophages were grown on collagen-coated glass coverslips (Mat-Tek). siRNA treatment was performed 48 h prior to use in experiments. For imaging, cells were maintained with HBSS supplemented with 10 mm Hepes and treated with either veratridine (100 μm) or DMSO vehicle. Live cell imaging was performed on a Zeiss Axiovert 200 fluorescent microscope with a ×40/0.75 objective (Neofluar) in an environmental chamber, and time lapse images were acquired with a QuantEM:512SC EMCCD camera. Time-lapse sequences were acquired using Zeiss AxioVision 4.6.3 software and analyzed using the Time Series Analyzer plugin (Balaji) for ImageJ software (rsb.info.nih.gov). Subsequent statistical analysis was performed using KaleidaGraph (Synergy) and MATLAB (MathWorks). For single cell detection of cAMP, mouse bone marrow-derived macrophages were transfected with a plasmid encoding human GFP-labeled CNGA2 (cyclic nucleotide-gated channel). Two days later, cells were labeled with the red fluorescent dye DiSBAC2(3) (9.Willoughby D. Cooper D.M. Live-cell imaging of cAMP dynamics.Nat. Methods. 2008; 5: 29-36Crossref PubMed Scopus (95) Google Scholar). Cells were observed in the presence and absence of veratridine, a sodium channel agonist (100 μm at 100 s). Background fluorescent responses over time in DiSBAC2(3) labeled cells were measured in GFP-negative cells. Responses in GFP-positive cells were acquired in the time-lapse mode (every 10 s for 720 s) by AxioVision software, and background responses were subtracted. Further analysis was performed using ImageJ and MATLAB (MathWorks). Normalized fluorescent responses (fluorescent ratio, Ft/F0) versus time were plotted. For calcium measurements, human monocyte derived macrophages were labeled with 1 μm Fluo 4 NW (Invitrogen) for 30 min. Images were acquired every 10 s for 600 s and then analyzed using ImageJ and MATLAB. cAMP concentration in lysates from monocyte-derived macrophages was determined by a commercially available ELISA kit (colorimetric; Cell Biolabs, San Diego). Plates were scanned on a Victor3 plate reader (PerkinElmer Life Sciences), and data were analyzed using KaleidaGraph. Monocyte-derived macrophages were infected with HSV-1 (KOS strain, ATCC VR-1493) for 2 h at a multiplicity of infection of 1:1 in HBSS supplemented with 10 mm Hepes. Cells were washed and incubated for an additional 22 h in HBSS/Hepes prior to assays. Whole cell patch clamp analysis of transfected HEK-293F cells that were grown as suspension cultures was performed on a planar chip system (Nanion, Munich, Germany). Microchips had an equivalent resistance of 2–5 megohms. Cells were placed on the microchip, and giga-ohm seals (1–4 giga-ohms) were obtained with an automated suction device (port-a-patch). Mean cell capacitance was 6.7 ± 0.3 picofarads (n = 9). Whole cell currents were recorded for 20 millisiemens in 20-mV increments from −120 to +60 mV utilizing pClamp 10.3.1 software. Data were acquired at a sampling frequency of 20 kHz and a low pass Bessel filter setting at 5 kHz. Capacitative transients and series resistance were compensated (∼80%) with amplifier settings (Axopatch 200B) in conjunction with pClamp software. P/4 leak subtraction was performed for all recordings used for analysis. All recordings were performed at room temperature. Intracellular solutions (pH 7.2, 285 mosm) contained either 50 mm CsCl, 10 mm NaCl, 60 mm cesium fluoride, 20 mm EGTA, and 10 mm Hepes (cesium internal solution) or 50 mm KCl, 10 mm NaCl, 60 mm potassium fluoride, 20 mm EGTA, and 10 mm Hepes (potassium internal solution). For experiments with poly(I:C) treatment, 200 ng/ml LMW poly(I:C) was added directly to the internal solution of the microchip. Extracellular solutions (pH 7.4, 298 mosm) contained either 140 mm NaCl, 4 mm KCl, 1 mm MgCl2, 2 mm CaCl2, 5 mm d-glucose, and 10 mm Hepes (sodium external solution) or 100 mm NMDG, 30 mm CaCl2, 10 mm d-glucose, and 20 mm Hepes (NMDG-calcium external solution). As indicated, ambroxol (50 μm final concentration), an inhibitor of tetrodotoxin-resistant sodium channels, was added to the external bath solution. Data were analyzed as indicated using ImageJ, AxioVision, KaleidaGraph (Student's t test and ANOVA), MATLAB, and pClamp. Although prior work suggested that SCN5A plays a central role in human macrophage regulation, its intracellular signaling mechanisms remained unknown. Our initial goal in this study was to identify potentially novel signaling mechanisms for SCN5A in macrophages by using an unbiased, discovery-based approach. To identify potential signaling mechanisms, immunoprecipitation of SCN5A from bone marrow-derived macrophages (BMDM) from C57BL6cfms-hSCN5A mice was performed to analyze its interactions by nano-LC-MS/MS. These transgenic mice express the human macrophage splice variant of SCN5A under the control of a macrophage-specific promoter (7.Rahgozar K. Wright E. Carrithers L.M. Carrithers M.D. Mediation of protection and recovery from experimental autoimmune encephalomyelitis by macrophages expressing the human voltage-gated sodium channel NaV1.5.J. Neuropathol. Exp. Neurol. 2013; 72: 489-504Crossref PubMed Scopus (12) Google Scholar). Nano-LC/MS analysis demonstrated known and novel protein-protein interactions with human SCN5A (C.I. >95%). Consistent with prior studies of the cardiac channel variant, the macrophage variant binds to PDZ (PSD95/Dlg/ZO-1 domain) (10.Ou Y. Strege P. Miller S.M. Makielski J. Ackerman M. Gibbons S.J. Farrugia G. Syntrophin γ2 regulates SCN5A gating by a PDZ domain-mediated interaction.J. Biol. Chem. 2003; 278: 1915-1923Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) and ankyrin-domain containing proteins (11.Mohler P.J. Rivolta I. Napolitano C. LeMaillet G. Lambert S. Priori S.G. Bennett V. Nav1.5 E1053K mutation causing Brugada syndrome blocks binding to ankyrin-G and expression of Nav1.5 on the surface of cardiomyocytes.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 17533-17538Crossref PubMed Scopus (309) Google Scholar); these interactions mediate connections to the cytoskeleton (data not shown). In addition, consistent with prior cellular analysis of SCN5A subcellular localization in macrophages, the channel interacts with proteins found in acidic organelles such as late endosomes (4.Carrithers M.D. Dib-Hajj S. Carrithers L.M. Tokmoulina G. Pypaert M. Jonas E.A. Waxman S.G. Expression of the voltage-gated sodium channel NaV1.5 in the macrophage late endosome regulates endosomal acidification.J. Immunol. 2007; 178: 7822-7832Crossref PubMed Scopus (98) Google Scholar) and lysosomes. These included lysosomal acid phosphatase and a proton transporter. These findings provided an initial validation measure for the specificity of the immunoprecipitation and nano-LC/MS analysis. In addition to known interactions, a novel interaction was identified (Table 1) between SCN5A and the cAMP-dependent transcription factor ATF2 that is relevant to cellular signaling and transcriptional regulation. To confirm the protein-protein interaction with ATF2, Western blot analysis following immunoprecipitation for macrophage SCN5A was performed (Fig. 1). Co-immunoprecipitation of ATF2 was observed in transgenic BMDM but not in the wild type condition. Although co-immunoprecipitation does not demonstrate direct binding, the results suggested a possible association within a subcellular signaling complex. Based on these results, it was reasoned that macrophage SCN5A could regulate ATF2-dependent transcription.TABLE 1Mass spectroscopy analysis of SCN5A-ATF2 protein-protein interaction Open table in a new tab When ATF2 is activated in the cytosol through phosphorylation, it translocates from the cytosol to the nucleus. Treatment of SCN5A+ macrophages with veratridine (100 μm for 15 min), a voltage-gated sodium channel agonist, resulted in a significant increase in phosphorylated ATF2 (Thr-71 phosphorylation site) in the nucleus (Fig. 2A). This translocation and increase in phosphorylated ATF2 was significantly greater than that observed in wild type cells. To confirm these results, Western blot analysis was performed and demonstrated increased levels of phospho-ATF2 in SCN5A+ macrophages that were treated with veratridine but no related increase in wild type cells (Fig. 2B). Because cAMP can activate ATF2 (12.Liao H. Hyman M.C. Baek A.E. Fukase K. Pinsky D.J. cAMP/CREB-mediated transcriptional regulation of ectonucleoside triphosphate diphosphohydrolase 1 (CD39) expression.J. Biol. Chem. 2010; 285: 14791-14805Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), this interaction provides a potential link between channel activation, cAMP signaling, and transcriptional pathways. Based on these findings, it was hypothesized that SCN5A regulates cellular phenotype and function through regulation of downstream transcriptional mechanisms. To assess this possible mechanism, global gene expression analysis was performed on BMDM from transgenic and wild type littermate mice (Fig. 3A). In the absence of any exogenous treatment, there was a statistically significant increase in the expression of genes (Gm7609 and Sp100rs) that are duplications or fusion variants of mouse Sp100 (13.Guldner H.H. Szostecki C. Schröder P. Matschl U. Jensen K. Lüders C. Will H. Sternsdorf T. Splice variants of the nuclear dot-associated Sp100 protein contain homologies to HMG-1 and a human nuclear phosphoprotein-box motif.J. Cell Sci. 1999; 112: 733-747PubMed Google Scholar). Most variants of Sp100 localize to a specialized nuclear structure called the nuclear body; it can function as a transcriptional repressor, mediate antiviral host defense, and regulate mRNA splicing and translation. Many of these variants are splice variants of the Sp100 gene. However, the mouse Sp100rs and Gm7609 variants are gene duplications with distinct promoter regions. Gm7609 is a fusion gene that contains Csprs (component of Sp100rs), the 5′ portion of Sp100, and a predicted G-protein domain. To validate the microarray analysis, QPCR analysis was performed to analyze mRNA expression of mouse Gm7609 and the 3′ region of Sp100. As described above, Gm7609 contains unique domains and the 5′ region of Sp100. Consistent with the microarray data, significantly increased expression of Gm7609 mRNA was observed in SCN5A+ macrophages as compared with wild type cells but not in transcripts that contain the 3′ region of Sp100 (Fig. 3B and Table 2). These results suggested that expression of human macrophage SCN5A in mouse BMDM increases expression of an ATF2-regulated gene.TABLE 2QPCR analysis of hSCN5A+ bone marrow-derived macrophages demonstrates increased expression of mouse Gm7609 mRNA but not Sp100 transcripts that contain the 3′ gene sequencesConditionSp100-related Gm7609 mRNA copies/Gapdh × 10−5Sp100 3′ region copies/Gapdh × 10−2hSCN5A-untreated8.0 ± 0.8ap < 0.01 (Student t test, n = 5).1.5 ± 0.1Wild type-untreated2.7 ± 0.7ap < 0.01 (Student t test, n = 5).1.8 ± 0.2a p < 0.01 (Student t test, n = 5). Open table in a new tab The next goal was to determine whether channel activation regulates this pathway. Activity-dependent regulation of gene transcription occurs in neurons to regulate cellular plasticity and memory. Based on this mechanism, it was reasoned that channel activation in macrophages also could be associated with transcriptional regulation. To test this hypothesis, expression of Gm7609 transcripts was examined in the presence and absence of pharmacological activation. Treatment with the channel agonist veratridine (100 μm for 2 h) induced an early transcriptional response in transgenic cells but not wild type (Fig. 3C and Table 3). This increase in Gm7609 transcripts was ∼3–4-fold greater than that observed in the untreated condition. There was no effect on the expression of transcripts that contain the 3′ region of Sp100.TABLE 3Veratridine treatment increases Gm7609 transcription in hSCN5A+ BMDMConditionSp100-related Gm7609 mRNA copies/Gapdh × 10−5hSCN5A-veratridine29.5 ± 4.5ap < 0.01 for hSCN5A-veratridine treated versus all conditions (ANOVA, n = 5).hSCN5A-DMSO vehicle3.6 ± 0.1ap < 0.01 for hSCN5A-veratridine treated versus all conditions (ANOVA, n = 5).Wild type-veratridine1.6 ± 0.1ap < 0.01 for hSCN5A-veratridine treated versus all conditions (ANOVA, n = 5).Wild type-DMSO vehicle1.5 ± 0.1ap < 0.01 for hSCN5A-veratridine treated versus all conditions (ANOVA, n = 5).a p < 0.01 for hSCN5A-veratridine treated versus all conditions (ANOVA, n = 5). Open table in a new tab To analyze the relevance of ATF2 to transcriptional regulation by SCN5A, the promoter sequence of the mouse Gm7609" @default.
• W2015705955 created "2016-06-24" @default.
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• W2015705955 date "2014-12-01" @default.
• W2015705955 modified "2023-09-29" @default.
• W2015705955 title "Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense" @default.
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• W2015705955 doi "https://doi.org/10.1074/jbc.m114.611962" @default.
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