FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1490290744> ?p ?o ?g. }

• W1490290744 endingPage "828" @default.
• W1490290744 startingPage "820" @default.
• W1490290744 abstract "The ability of neonatal and adult cardiomyocytes to activate the nuclear factor (NF)-κB pathway in response to lipopolysaccharide and interleukin-1β challenge has been investigated and compared with that of peritoneal macrophages. The activation of the IκB kinase and the phosphorylation and degradation of IκBα and IκBβ was much lower in adult cardiomyocytes than in the neonatal counterparts and macrophages. This restricted activation of the NF-κB pathway resulted in a significant reduction in the time of nuclear activation of NF-κB, as deduced by electrophoretic mobility shift assays and in the transcription of target genes, such as IκBα, cyclooxygenase-2 (COX-2) and nitric-oxide synthase-2 (NOS-2). Studies on chromatin immunoprecipitation showed binding of NF-κB proteins to the regulatory κB sites identified in the promoters of the IκBα, COX-2, and NOS-2 genes in macrophages and, to a lower extent, in neonatal cardiomyocytes. The binding to these κB sites in adult cardiomyocytes was observed only in the IκBα promoter and was minimal or absent in the COX-2 and NOS-2 promoters, respectively, suggesting a restricted activation of NF-κB-regulated genes in these cells. These data indicate that the function of the NF-κB pathway in adult cardiomyocytes is limited in time, which results in the expression of a reduced number of genes and provides a functional explanation for the absence of NOS-2 inducibility in these cells under proinflammatory conditions. The ability of neonatal and adult cardiomyocytes to activate the nuclear factor (NF)-κB pathway in response to lipopolysaccharide and interleukin-1β challenge has been investigated and compared with that of peritoneal macrophages. The activation of the IκB kinase and the phosphorylation and degradation of IκBα and IκBβ was much lower in adult cardiomyocytes than in the neonatal counterparts and macrophages. This restricted activation of the NF-κB pathway resulted in a significant reduction in the time of nuclear activation of NF-κB, as deduced by electrophoretic mobility shift assays and in the transcription of target genes, such as IκBα, cyclooxygenase-2 (COX-2) and nitric-oxide synthase-2 (NOS-2). Studies on chromatin immunoprecipitation showed binding of NF-κB proteins to the regulatory κB sites identified in the promoters of the IκBα, COX-2, and NOS-2 genes in macrophages and, to a lower extent, in neonatal cardiomyocytes. The binding to these κB sites in adult cardiomyocytes was observed only in the IκBα promoter and was minimal or absent in the COX-2 and NOS-2 promoters, respectively, suggesting a restricted activation of NF-κB-regulated genes in these cells. These data indicate that the function of the NF-κB pathway in adult cardiomyocytes is limited in time, which results in the expression of a reduced number of genes and provides a functional explanation for the absence of NOS-2 inducibility in these cells under proinflammatory conditions. The ability of the heart to respond to proinflammatory cytokines and pathogen-associated molecular patterns has been reported by several authors.1Koyanagi M Egashira K Kitamoto S Ni W Shimokawa H Takeya M Yoshimura T Takeshita A Role of monocyte chemoattractant protein-1 in cardiovascular remodeling induced by chronic blockade of nitric oxide synthesis.Circulation. 2000; 102: 2243-2248Crossref PubMed Scopus (107) Google Scholar, 2Knuefermann P Nemoto S Baumgarten G Misra A Sivasubramanian N Carabello BA Vallejo JG Cardiac inflammation and innate immunity in septic shock: is there a role for toll-like receptors?.Chest. 2002; 121: 1329-1336Crossref PubMed Scopus (63) Google Scholar, 3Frishman WH Ismail AA Role of infection in atherosclerosis and coronary artery disease: a new therapeutic target?.Cardiol Rev. 2002; 10: 199-210Crossref PubMed Scopus (29) Google Scholar However, in isolated cardiac cells from neonatal or adult animals there are discrepancies regarding the conditions required to observe the expression of genes involved in the inflammatory response such as nitric-oxide synthase-2 (NOS-2), cyclooxygenase-2 (COX-2), and matrix metalloproteinases.4Bradford Sanders D Hunter K Wu Y Jablonowski C Bahl JJ Larson DF Modulation of the inflammatory response in the cardiomyocyte and macrophage.J Extra Corpor Technol. 2001; 33: 167-174PubMed Google Scholar, 5Kinugawa K Shimizu T Yao A Kohmoto O Serizawa T Takahashi T Transcriptional regulation of inducible nitric oxide synthase in cultured neonatal rat cardiac myocytes.Circ Res. 1997; 81: 911-921Crossref PubMed Scopus (103) Google Scholar, 6Li YY Feng YQ Kadokami T McTiernan CF Draviam R Watkins SC Feldman AM Myocardial extracellular matrix remodeling in transgenic mice overexpressing tumor necrosis factor α can be modulated by anti-tumor necrosis factor α therapy.Proc Natl Acad Sci USA. 2000; 97: 12746-12751Crossref PubMed Scopus (286) Google Scholar The nuclear factor (NF)-κB family of transcription factors plays an essential role in regulating the induction of genes involved in inflammation, and it has been shown that they are important mediators of the inflammatory response in the heart. The presence of p65, p50, IκBα, and IκBβ in the myocardium has been described in postnatal and adult heart,7Frantz S Fraccarollo D Wagner H Behr TM Jung P Angermann CE Ertl G Bauersachs J Sustained activation of nuclear factor κB and activator protein 1 in chronic heart failure.Cardiovasc Res. 2003; 57: 749-756Crossref PubMed Scopus (142) Google Scholar, 8Norman DA Yacoub MH Barton PJ Nuclear factor NF-κB in myocardium: developmental expression of subunits and activation by interleukin-1 β in cardiac myocytes in vitro.Cardiovasc Res. 1998; 39: 434-441Crossref PubMed Scopus (26) Google Scholar but less is known regarding the role of c-Rel that mediates late events in the transcriptional activity of the NF-κB/c-Rel complex, preferentially in immune cells.9Jankowska EA von Haehling S Czarny A Zaczynska E Kus A Anker SD Banasiak W Ponikowski P Activation of the NF-κB system in peripheral blood leukocytes from patients with chronic heart failure.Eur J Heart Fail. 2005; 7: 984-990Crossref PubMed Scopus (27) Google Scholar The p65 protein is a key transcriptionally active component of NF-κB, but the activation of this factor mainly depends on the phosphorylation of inhibitory molecules including IκBα. However, optimal induction of NF-κB target genes also requires phosphorylation of NF-κB proteins such as p65.10Viatour P Merville MP Bours V Chariot A Phosphorylation of NF-κB and IκB proteins: implications in cancer and inflammation.Trends Biochem Sci. 2005; 30: 43-52Abstract Full Text Full Text PDF PubMed Scopus (1179) Google Scholar We have shown that recently isolated neonatal and adult cardiomyocytes are unable to express most of the NF-κB-regulated enzymes related to inflammation, except after a prolonged period of culture in the case of neonatal cells.11Goren N Cuenca J Martin-Sanz P Bosca L Attenuation of NF-κB signalling in rat cardiomyocytes at birth restricts the induction of inflammatory genes.Cardiovasc Res. 2004; 64: 289-297Crossref PubMed Scopus (31) Google Scholar This effect is attributable, at least in part, to a reduced activation of the IκB kinase/NF-κB pathway observed in cardiomyocytes11Goren N Cuenca J Martin-Sanz P Bosca L Attenuation of NF-κB signalling in rat cardiomyocytes at birth restricts the induction of inflammatory genes.Cardiovasc Res. 2004; 64: 289-297Crossref PubMed Scopus (31) Google Scholar but exerts differential responses depending on the genes considered; in particular, the expression of NOS-2 is significantly impaired. To gain insight on the mechanisms that regulate the expression of NF-κB-dependent genes in the cardiomyocyte, we used the in situ method of chromatin immunoprecipitation (ChIP) for the study of the transcription of IκBα, COX-2, and NOS-2, as three genes related to NF-κB activation. Our data show that after lipopolysaccharide (LPS) and interleukin (IL)-1β challenge, the activation of the IκB kinase (IKK) exhibits marked differences among cells, with adult cardiomyocytes being the less activated. Neonatal and adult cardiomyocytes expressed IκB proteins and COX-2. However, adult cardiomyocytes failed to express NOS-2 indicating an inefficient capacity of the NF-κB complex to accomplish the transcription of this gene. This behavior was compared with that of macrophages, which exhibited a robust activation of the NF-κB pathway, resulting in the expression of higher levels of COX-2 and NOS-2. LPS from Salmonella typhimurium and all other reagents not specified were from Sigma Chemical Co. (St. Louis, MO). Cytokines were from Roche (Mannheim, Germany). Collagenase type II was from Worthington (Lakewood, NJ). Antibodies were obtained from Santa Cruz Laboratories (Santa Cruz, CA) or Calbiochem (San Jose, CA). Tissue culture dishes were from Falcon (Lincoln Park, NJ), and culture media were from BioWhittaker (Walkersville, MD). Neonatal cardiomyocytes (NeoC) were isolated from 1-day-old BALB/c mice as follows. The hearts were pooled, minced, disaggregated mechanically, and digested for 15 minutes at 37°C with 0.8% collagenase (type II, Worthington Biochemical Corp.) in phosphate-buffered saline (PBS), according to a previous protocol of successive digestions.11Goren N Cuenca J Martin-Sanz P Bosca L Attenuation of NF-κB signalling in rat cardiomyocytes at birth restricts the induction of inflammatory genes.Cardiovasc Res. 2004; 64: 289-297Crossref PubMed Scopus (31) Google Scholar The cell pellets were resuspended in Dulbecco's modified Eagle's medium/F-12/M-199 medium [4:1 (v/v)], supplemented with 10% fetal bovine serum (FBS) and antibiotics (penicillin, 100 U/ml; streptomycin, 100 μg/ml). The isolated cells were incubated for 1 hour in an uncoated flask, allowing the nonmyocytic population to differentially attach to the flask. Then, the cell suspension was sedimented by centrifugation at low speed (80 × g), and cells were distributed in plates precoated with a 2% solution of gelatin. After overnight incubation, the dishes were washed with PBS, and the medium was replaced. Usually, cells were plated at 4 × 104 cells/cm2 and used after 3 days in culture.11Goren N Cuenca J Martin-Sanz P Bosca L Attenuation of NF-κB signalling in rat cardiomyocytes at birth restricts the induction of inflammatory genes.Cardiovasc Res. 2004; 64: 289-297Crossref PubMed Scopus (31) Google Scholar Ventricular myocytes (AdtC) were isolated as previously described.12Poon BY Ward CA Giles WR Kubes P Emigrated neutrophils regulate ventricular contractility via α4 integrin.Circ Res. 1999; 84: 1245-1251Crossref PubMed Scopus (31) Google Scholar, 13Fernández-Velasco M Goren N Benito G Blanco-Rivero J Bosca L Delgado C Regional distribution of hyperpolarization-activated current (If) and hyperpolarization-activated cyclic nucleotide-gated channel mRNA expression in ventricular cells from control and hypertrophied rat hearts.J Physiol. 2003; 553: 395-405Crossref PubMed Scopus (99) Google Scholar In brief, 8-week-old male mice were anesthetized (92:7 mg/kg ketamine/xylazine) and sacrificed by cervical dislocation. Hearts were rapidly removed and placed into Tyrode buffer (140 mmol/L NaCl, 5.4 mmol/L KCl, 1 mmol/L Na2HPO4, 5 mmol/L HEPES, 10 mmol/L glucose, and 1 mmol/L MgCl2, pH 7.4), containing 1 mmol/L CaCl2 at 4°C and then cannulated via the aorta for retrograde perfusion of the coronary arteries. Hearts were perfused with Tyrode buffer containing 1 mmol/L CaCl2 at 2 ml/minute for 5 minutes at 37°C and then Tyrode buffer without CaCl2 at 2 ml/minute for 5 minutes. Final perfusion continued at 2 ml/minute for 8 to 10 minutes with Tyrode buffer containing 40 μmol/L CaCl2, 20 μg/ml collagenase (Yakult Pharmaceuticals Co., Seoul, Korea), and 4 μg/ml protease (Sigma). Ventricles were then minced in Tyrode buffer containing 1 mmol/L CaCl2, 500 μg/ml collagenase, 100 μg/ml protease, and 2.5% bovine serum albumin (Sigma). Ventricular tissue segments were put into a shaking water bath for 10 to 20 minutes at 37°C to complete dispersion of myocytes. Myocytes were cultured at a density of 1 to 2 × 104 cells/cm2 on laminin-coated tissue cultureware. The cardiomyocytes were cultured in Dulbecco's modified Eagle's medium with 10% FBS, 10 μg of transferrin, 10 μg/ml insulin, minimal essential medium with vitamin mix and nonessential amino acids, and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin), and used within the next 24 hours. For both NeoC and AdtC cultures, the viability of the cells was determined by propidium iodide exclusion (>90%), and the purity by immunocytochemistry for α-actin and cardiac actinin (>85% positive). HL-1 cells are cardiomyocytes derived from a mouse atrial tumor and were kindly provided by Dr. W.C. Claycomb, Louisiana State University Health Science Center, New Orleans, LA, and cultured in Claycomb medium (JRH Biosciences, Lenexa, KS) containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.1 mmol/L norepinephrine, and 2 mmol/L l-glutamine. Cells were cultivated at 37°C under 5% CO2 on fibronectin-coated flasks. HL-1 is a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte.14Claycomb WC Lanson Jr, NA Stallworth BS Egeland DB Delcarpio JB Bahinski A Izzo Jr, NJ HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte.Proc Natl Acad Sci USA. 1998; 95: 2979-2984Crossref PubMed Scopus (1240) Google Scholar Elicited peritoneal macrophages were prepared from male mice 4 days after intraperitoneal administration of 1 ml of 10% thioglycollate broth.15Terenzi F Diaz-Guerra MJ Casado M Hortelano S Leoni S Bosca L Bacterial lipopeptides induce nitric oxide synthase and promote apoptosis through nitric oxide-independent pathways in rat macrophages.J Biol Chem. 1995; 270: 6017-6021Crossref PubMed Scopus (90) Google Scholar Cells were seeded at 2 × 106 in 6-cm plates or 5 × 105 in 24-multiwell plates and cultured with RPMI 1640 medium supplemented with 10% heat-inactivated FBS and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) at 37°C in an humidified atmosphere with 5% CO2. After incubation for 4 hours, nonadherent cells were removed, and remnant cells were cultured for the indicated times.16Callejas NA Fernandez-Martinez A Castrillo A Bosca L Martin-Sanz P Selective inhibitors of cyclooxygenase-2 delay the activation of nuclear factor κ B and attenuate the expression of inflammatory genes in murine macrophages treated with lipopolysaccharide.Mol Pharmacol. 2003; 63: 671-677Crossref PubMed Scopus (17) Google Scholar Cells were transfected per triplicate using JetPeI transfection reagent (PolyPlus Transfection, San Marcos, CA) with a JetPeI/DNA ratio of 3:1 and in the presence of 2% FBS. Twenty-four hours after transfection, the culture medium was changed, and cells were activated with the indicated stimuli. The vectors used were p-Luc, (κB)3-Luc, p-p65, c-Rel, and the respective dominant-negative forms p-DN-p65 and p-DN-c-Rel, respectively. These vectors were provided generously by Dr. M. Fresno, Universidad Autónoma de Madrid, Madrid, Spain, and have been described previously.17Martin AG San Antonio B Fresno M Regulation of nuclear factor κB transactivation. Implication of phosphatidylinositol 3-kinase and protein kinase C zeta in c-Rel activation by tumor necrosis factor α.J Biol Chem. 2001; 276: 15840-15849Crossref PubMed Scopus (90) Google Scholar, 18Martin AG Fresno M Tumor necrosis factor-α activation of NF-κB requires the phosphorylation of Ser-471 in the transactivation domain of c-Rel.J Biol Chem. 2000; 275: 24383-24391Crossref PubMed Scopus (63) Google Scholar Reporter activities were assayed using the dual luciferase assay system per the manufacturer's instructions (Promega, Madison, WI). For normalization of transfection efficiency, cells were co-transfected with the reporter plasmid pTK Renilla (Promega), and luminescence data were expressed as the relative light ratio between both enzymes. A vector encoding the GFP gene was used for the evaluation of the transfection efficiency. The amounts of nitrite and nitrate that accumulate in the culture medium were determined after reduction of nitrates to nitrites followed by the determination of NO2− levels with Griess reagent.11Goren N Cuenca J Martin-Sanz P Bosca L Attenuation of NF-κB signalling in rat cardiomyocytes at birth restricts the induction of inflammatory genes.Cardiovasc Res. 2004; 64: 289-297Crossref PubMed Scopus (31) Google Scholar The levels of PGE2 were determined using a Biotrak kit from Amersham, Arlington Heights, IL, following the instructions of the supplier. Cells were resuspended in PBS 1× and incubated 15 minutes with a fluorescein isothiocyanate/phycoerythrin-conjugated antibody (Ab) against the indicated TLR (eBioscience, San Diego, CA). The fluorescence intensity was measured using a Quantum MESF kit (Bangs Laboratories, Inc., Fishers, IN). Cultured cells were washed with PBS, scraped off the dishes in ice-cold PBS, and centrifuged. Cell pellets were homogenized in 200 μl of buffer A (10 mmol/L HEPES, pH 7.9, 1 mmol/L ethylenediamine tetraacetic acid (EDTA), 1 mmol/L ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 10 mmol/L KCl, 1 mmol/L dithiothreitol, 0.5 mmol/L phenylmethyl sulfonyl fluoride, 40 μg/ml leupeptin, 2 μg/ml tosyl-lysyl-chloromethane, 5 mmol/L NaF, 1 mmol/L NaVO4, and 10 mmol/L Na2MoO4), and Nonidet P-40 was added to reach 0.5% (v/v). After 15 minutes at 4°C, the tubes were gently vortexed for 15 seconds, and nuclei were collected by centrifugation at 8000 × g for 15 minutes. The supernatants were stored at −80°C (cytosolic extracts), and the pellets were resuspended in 50 μl of buffer A supplemented with 20% (v/v) glycerol and 0.4 mol/L KCl, and mixed for 30 minutes at 4°C. Nuclear proteins were obtained by centrifugation at 13,000 × g for 15 minutes, and aliquots of the supernatant (nuclear extracts) were stored at −80°C. For Western blot analysis, cytosolic and nuclear proteins were boiled in Laemmli sample buffer, and equal amounts of protein (30 to 15 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Macrophages and NeoC and AdtC cells (5 to 6 × 106) were stimulated, and at the indicated times the cells were homogenized in 1 ml of buffer A and centrifuged for 10 minutes in a microcentrifuge. The IKK from equal amounts of protein from these supernatants was immunoprecipitated with 1 μg of anti-IKK2 Ab. After washing with 4 ml of buffer A, the pellet was resuspended in kinase buffer (20 mmol/L HEPES, pH 7.4, 0.1 mmol/L EDTA, 100 mmol/L NaCl, 1 mmol/L dithiothreitol, 0.5 mmol/L phenylmethyl sulfonyl fluoride, 2 μg/ml aprotinin, 10 μg/ml leupeptin, 2 μg/ml TLCK, 5 mmol/L NaF, 1 mmol/L NaVO4, 10 mmol/L Na2MoO4, and 10 nmol/L okadaic acid). Kinase activity was assayed in 100 μl of kinase buffer containing 50 ng of precipitated protein. Myelin basic protein was used as substrate in the presence of 50 μmol/L [γ-32P]ATP (0.5 μCi). Reactions were stopped with 1 ml of ice-cold buffer A supplemented with 5 mmol/L EDTA, and after retention onto a P81 Whatman filter and counted. The oligonucleotide sequence corresponding to the NF-κB site of the rat COX-2 promoter (5′-GGCAAGGGGATTCCCTTAGTT-3′)11Goren N Cuenca J Martin-Sanz P Bosca L Attenuation of NF-κB signalling in rat cardiomyocytes at birth restricts the induction of inflammatory genes.Cardiovasc Res. 2004; 64: 289-297Crossref PubMed Scopus (31) Google Scholar was annealed with the complementary sequence by incubation for 5 minutes at 85°C in 10 mmol/L Tris-HCl, pH 8.0, 50 mmol/L NaCl, 10 mmol/L MgCl2, 1 mmol/L dithiothreitol. Aliquots (100 ng) were end-labeled with Klenow enzyme fragment in the presence of 50 μCi of [α-32P]dCTP and the other unlabeled dNTPs in a final volume of 50 μl. DNA probe (5 × 104 dpm) was used for each binding assay: 5 μg of nuclear protein were incubated for 15 minutes at 4°C with the probe and with 1 μg of poly (dI-dC), 5% glycerol, 1 mmol/L EDTA, 10 mmol/L KCl, 5 mmol/L MgCl2, 1 mmol/L dithiothreitol, and 10 mmol/L Tris-HCl (pH 7.8) in a final volume of 20 μl. The DNA-protein complexes were separated on native 6% polyacrylamide gels in 0.5% Tris-borate-EDTA buffer. Supershift assays were performed by incubating the nuclear extracts (3 μg of protein) with the indicated antibodies against NF-κB proteins (p50, p65, and c-Rel) before the addition of the probe. The protein levels of IκBα, IκBβ, p65, c-Rel, and α/β-actin were determined in cytosolic or nuclear extracts as indicated. Equal amounts of protein (15 to 30 μg) were size-fractionated in a 10% acrylamide gel, transferred to a Hybond P membrane (Amersham), and after blocking with 5% nonfat dry milk, were incubated with the corresponding antibodies and visualized by enhanced chemiluminescence. Different exposure times were performed with each blot to ensure the linearity of the band intensities. Band intensities were measured on a densitometric scanner (Amersham) and expressed in arbitrary units. Total RNA was extracted from frozen cells by using TRIzol reagent (Life Technologies, Inc., Grand Island, NY). Total RNA (1 μg per sample) was reverse-transcribed with oligo(dT) as primer using Expand Reverse Transcriptase (Roche, Basel, Switzerland) according to the manufacturer's protocol. Q-RT-PCR was performed using SYBR Green PCR kit (PE Applied Biosystems, Foster City, CA) in an Applied Biosystems 7700 sequence detector. Primer sequences were: NOS-2 (mouse) forward, 5′-CAGCTGGGCTGTACAAACCTT-3′, reverse 5′-CATTGGAAGTGAAGCGTTTCG-3′; COX-2 (mouse) forward, 5′-GCTGTACAAGCAGTGGC AAAG-3′, reverse 5′-GCGTTTGCGGTACTCATTGAGA-3′; IκBα (mouse) forward, 5′-GCTCCGAGACTTTCGAGGAA-3′, reverse 5′-TTGTAGTTGGTGGCCTGCA G-3′. All samples were analyzed in the same run for 18S expression for normalization: forward, 5′-AACACGGGAAACCTCACCC-3′ and reverse, 5′-CCACCAACTAAGAACGGCCA-3′. PCR parameters were 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The quantitative expression values were extrapolated from standard curves for 18S mRNA. The replicates were then averaged, and fold induction was determined, considering the value at zero time as 1. ChIP assay was performed according to Saccani and colleagues.19Saccani S Pantano S Natoli G p38-Dependent marking of inflammatory genes for increased NF-κB recruitment.Nat Immunol. 2002; 3: 69-75Crossref PubMed Scopus (627) Google Scholar, 20Saccani S Pantano S Natoli G Two waves of nuclear factor κB recruitment to target promoters.J Exp Med. 2001; 193: 1351-1359Crossref PubMed Scopus (337) Google Scholar In brief, after stimulation, cells were fixed by adding directly to the medium formaldehyde solution to reach a final concentration of 1%. After incubation for 15 minutes, the cells were washed extensively with PBS, scraped off the dishes, and centrifuged. Cells were resuspended in ice-cold buffer I (10 mmol/L HEPES, pH 7.9, 10 mmol/L EDTA, 0.5 mmol/L ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and 0.25% Triton X-100), centrifuged, and the pellet resuspended in buffer II (10 mmol/L HEPES, pH 7.9, 1 mmol/L EDTA, 0.5 mmol/L ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and 200 mmol/L NaCl). After centrifugation, nuclei were lysed with ice-cold lysis buffer (50 mmol/L Tris-HCl, pH 8.0, 10 mmol/L EDTA, 1% SDS, and protease inhibitors). Chromatin was sheared by sonication (5 × 18 seconds each at 10% power), centrifuged, and diluted five times with the IP dilution buffer (20 mmol/L Tris-HCl, pH 8.0,; 2 mmol/L EDTA, 1% Triton X-100, 150 mmol/L NaCl, and protease inhibitors). The extracts were precleared for 30 minutes with a 50% suspension of salmon sperm-saturated protein A/G agarose. Immunoprecipitations with anti-p65 or anti-c-Rel Abs (Santa Cruz Biotechnologies) or with no Ab, were performed overnight at 4°C. Immune complexes were collected with protein A/G agarose and washed sequentially for 5 minutes each in buffer W150 (20 mmol/L Tris-HCl, pH 8.0, 2 mmol/L EDTA, 1% Triton X-100, 0.1% SDS, and 150 mmol/L NaCl), buffer W500 (20 mmol/L Tris-HCl, pH 8.0, 2 mmol/L EDTA, 1% Triton X-100, 0.1% SDS, and 500 mmol/L NaCl), and buffer WIII (10 mmol/L Tris-HCl, pH 8.0, 1 mmol/L EDTA, 0.25 mol/L LiCl, 1% Nonidet P-40, and 1% deoxycholate). Precipitates were then washed twice with TE (10 mmol/L Tris-HCl, pH 8.0, and 1 mmol/L EDTA), extracted with TES (25 mmol/L Tris-HCl, pH 7.5, 10 mmol/L EDTA, and 0.5% SDS) and heated at 65°C to elute the chromatin. After proteinase K digestion, chromatin DNA was extracted with phenol-chloroform followed by ethanol precipitation. PCR reactions were performed with promoter-specific primers and normalized with the input controls. Primer sequences are shown in Table 1. After amplification, the PCR products were electrophoresed in a 2% agarose gel and visualized by ethidium bromide staining.Table 1Primers for ChIP AssaysGenePrimersCOX-2Forward 5′-TTAACCGGTAGCTGTGTGCGT-3′Reverse 5′-CCCGGGATCTAAGGTCCTAACT-3′NOS-2Forward 5′-GACCATGCGAAGATGAGTGGA-3′Reverse 5′-TGAAAGTGAAATATTGACAGTGTT AGGG-3′IκBα (1)Forward 5′-GACTTTCCAGCCACTCAGGG-3′Reverse 5′-CCTAAACCCAGGGCCGG-3′IκBα (2)Forward 5′-TACTGAGTGCAGGCTGCAGG-3′Reverse 5′-GGTCATGCACAGGGAACTTTTT-3′IκBα (3)Forward 5′-ACCCCAGGGAAAGAAGGGTT-3′Reverse 5′-CTGGAAAGTCCTCCCGACC-3′ Open table in a new tab Unless otherwise stated, data are expressed as mean ± SD. To compare means between two independent experiments the Mann-Whitney rank sum test was used. Statistical analysis of mRNA expression data was performed by using the two-tailed homoscedastic t-test. The results were considered significant at P < 0.05. Data were analyzed by SPSS for Windows statistical package version 9.0.1 (SPSS Inc., Chicago, IL). The response of primary cultures of neonatal (NeoC) and adult (AdtC) cardiomyocytes to tumor necrosis factor (TNF)-α, IL-1β, and LPS in terms of NOS-2 and COX-2 expression (two well-characterized NF-κB-dependent genes) and nitrite and PGE2 synthesis was analyzed and compared with that of peritoneal macrophages treated under identical conditions. Figure 1A shows the response of these cells to combinations of these proinflammatory stimuli, and maximal accumulation of NOx and/or PGE2 in the culture medium was obtained when LPS and IL-1β were combined. TNF-α promoted a significant apoptosis in cardiomyocytes after 24 hours of culture and for this reason it was omitted in the proinflammatory mixture. As Figure 1, A and B, shows, AdtC failed to express NOS-2, and only moderate levels of COX-2 were detected, whereas NeoC and macrophages exhibited a robust expression of both NOS-2 and COX-2. Agreement was observed between the levels of NOx and PGE2 and the corresponding protein levels determined by Western blot. In addition to these measurements, the relative levels of TLR4 and TLR2 in the cell membrane were determined in intact cultured cells. Macrophages exhibited approximately twofold and fourfold higher levels than those measured in NeoC and AdtC, respectively (Figure 1C); however, the levels of IKK2, p65, and IκBα were quite similar in the three cell types, and only a minor content of IκBβ was observed in AdtC (Figure 1D). In view of these data, we compared the ability of these cells to activate the NF-κB pathway after proinflammatory challenge. As Figure 2A shows, on normalization to the basal levels, macrophages and NeoC exhibited a rapid degradation of IκBα and IκBβ, as a result of the activation of IKK and phosphorylation of IκBα in S32 in response to LPS and IL-1β (Figure 2B). However, AdtC exhibited a moderate phosphorylation of IκBα in S32 and a decrease of both IκB proteins, minimal in the case of IκBβ, followed by a rapid resynthesis of IκBα. When the activation of IKK was determined in these cells, the activation in AdtC represented only 21% and 32% of the activity in macrophages and NeoC, respectively, after 15 minutes of stimulation (Figure 2C). These differences in IKK activity and IκB degradation and recovery resulted in different responses in the mRNA levels of the genes studied: IκBα, NOS-2, and COX-2, with a significantly attenuated response in the case of the NOS-2 gene in AdtC (Figure 3; to note the difference in scales).Figure 2Time course of IκB degradation and IKK activity in macrophages and cardiomyocytes challenged with LPS/IL-1β. Primary cultures of macrophages (Mϕ), neonatal cardiomyocytes (NeoC), and adult cardiomyocytes (AdtC) were treated as described in Figure 1 and the time course of the levels of IκBα, IκBβ, and β- and α-actin were determined by Western blot. A: The means of the levels of IκB proteins after normalization for the β- and α-actin content in macrophages and cardiomyocytes, respectively, were plotted as percentage versus 0 minutes (n = 4 experiments) (a representative blot is shown). B: To evaluate the phosphorylation in S32 of IκBα, cells were stimulated with LPS/IL-1β for 15 minutes in the presence of 20 μmol/L MG132 to prevent proteasomal degradation, and the levels of phospho-S32-IκBα were determined by Western blot, using GAPDH for the normalization of the blots, and expressed as the percentage of activation measured in macrophages. C: IKK activity was determined after immunoprecipitation of cell extracts and in vitro assay of the kinase activity using myelin basic protein as substrate. Results show the mean ± SD of at least three experiments. *P < 0.01 and #P < 0.01 versus the corresponding condition in macrophages and NeoC, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Time c" @default.
• W1490290744 created "2016-06-24" @default.
• W1490290744 creator A5017459306 @default.
• W1490290744 creator A5028322178 @default.
• W1490290744 creator A5031573079 @default.
• W1490290744 creator A5031878146 @default.
• W1490290744 creator A5081185662 @default.
• W1490290744 date "2007-09-01" @default.
• W1490290744 modified "2023-10-18" @default.
• W1490290744 title "Selective Impairment of Nuclear Factor-κB-Dependent Gene Transcription in Adult Cardiomyocytes" @default.
• W1490290744 cites W1585358533 @default.
• W1490290744 cites W1635131150 @default.
• W1490290744 cites W1874207496 @default.
• W1490290744 cites W1965269412 @default.
• W1490290744 cites W1974162725 @default.
• W1490290744 cites W1975185970 @default.
• W1490290744 cites W1988036810 @default.
• W1490290744 cites W1991970275 @default.
• W1490290744 cites W1994446230 @default.
• W1490290744 cites W2001013005 @default.
• W1490290744 cites W2005327503 @default.
• W1490290744 cites W2027178254 @default.
• W1490290744 cites W2028959692 @default.
• W1490290744 cites W2028989473 @default.
• W1490290744 cites W2029062125 @default.
• W1490290744 cites W2036888095 @default.
• W1490290744 cites W2039104446 @default.
• W1490290744 cites W2058520191 @default.
• W1490290744 cites W2058526434 @default.
• W1490290744 cites W2065198767 @default.
• W1490290744 cites W2074994532 @default.
• W1490290744 cites W2077468629 @default.
• W1490290744 cites W2084182872 @default.
• W1490290744 cites W2085926398 @default.
• W1490290744 cites W2087298963 @default.
• W1490290744 cites W2088068709 @default.
• W1490290744 cites W2090919432 @default.
• W1490290744 cites W2106877484 @default.
• W1490290744 cites W2124734965 @default.
• W1490290744 cites W2130994795 @default.
• W1490290744 cites W2132103002 @default.
• W1490290744 cites W2134633678 @default.
• W1490290744 cites W2136853725 @default.
• W1490290744 cites W2148027060 @default.
• W1490290744 cites W2152098493 @default.
• W1490290744 cites W2153992771 @default.
• W1490290744 cites W2154171168 @default.
• W1490290744 cites W2157942810 @default.
• W1490290744 cites W2160117562 @default.
• W1490290744 cites W2162019549 @default.
• W1490290744 cites W2162460978 @default.
• W1490290744 cites W2167636398 @default.
• W1490290744 cites W2398032313 @default.
• W1490290744 doi "https://doi.org/10.2353/ajpath.2007.061076" @default.
• W1490290744 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1959507" @default.
• W1490290744 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17675583" @default.
• W1490290744 hasPublicationYear "2007" @default.
• W1490290744 type Work @default.
• W1490290744 sameAs 1490290744 @default.
• W1490290744 citedByCount "9" @default.
• W1490290744 countsByYear W14902907442012 @default.
• W1490290744 countsByYear W14902907442013 @default.
• W1490290744 countsByYear W14902907442014 @default.
• W1490290744 countsByYear W14902907442016 @default.
• W1490290744 countsByYear W14902907442023 @default.
• W1490290744 crossrefType "journal-article" @default.
• W1490290744 hasAuthorship W1490290744A5017459306 @default.
• W1490290744 hasAuthorship W1490290744A5028322178 @default.
• W1490290744 hasAuthorship W1490290744A5031573079 @default.
• W1490290744 hasAuthorship W1490290744A5031878146 @default.
• W1490290744 hasAuthorship W1490290744A5081185662 @default.
• W1490290744 hasBestOaLocation W14902907441 @default.
• W1490290744 hasConcept C104317684 @default.
• W1490290744 hasConcept C138885662 @default.
• W1490290744 hasConcept C179926584 @default.
• W1490290744 hasConcept C2780114586 @default.
• W1490290744 hasConcept C41895202 @default.
• W1490290744 hasConcept C54355233 @default.
• W1490290744 hasConcept C86339819 @default.
• W1490290744 hasConcept C86803240 @default.
• W1490290744 hasConcept C95444343 @default.
• W1490290744 hasConceptScore W1490290744C104317684 @default.
• W1490290744 hasConceptScore W1490290744C138885662 @default.
• W1490290744 hasConceptScore W1490290744C179926584 @default.
• W1490290744 hasConceptScore W1490290744C2780114586 @default.
• W1490290744 hasConceptScore W1490290744C41895202 @default.
• W1490290744 hasConceptScore W1490290744C54355233 @default.
• W1490290744 hasConceptScore W1490290744C86339819 @default.
• W1490290744 hasConceptScore W1490290744C86803240 @default.
• W1490290744 hasConceptScore W1490290744C95444343 @default.
• W1490290744 hasIssue "3" @default.
• W1490290744 hasLocation W14902907441 @default.
• W1490290744 hasLocation W14902907442 @default.
• W1490290744 hasLocation W14902907443 @default.
• W1490290744 hasLocation W14902907444 @default.
• W1490290744 hasOpenAccess W1490290744 @default.
• W1490290744 hasPrimaryLocation W14902907441 @default.
• W1490290744 hasRelatedWork W1818003313 @default.

• next