Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling by array of murine cardiomyocytes with and without expression of constitutively active IKK2

ABSTRACT: Inflammation is a major factor in heart disease. IÎºB kinase (IKK) and its downstream target NF-ÎºB are regulators of inflammation and are activated in cardiac disorders, but their precise contributions and targets are unclear. We analyzed IKK/NF-ÎºB function in the heart by a gain-of-function approach, generating an inducible transgenic mouse model with cardiomyocyte-specific expression of constitutively active IKK2 (IKK2-CA). In adult animals, IKK2 activation led to inflammatory dilated cardiomyopathy and heart failure. Transgenic hearts showed infiltration with CD11b+ cells, fibrosis, fetal reprogramming, and atrophy of myocytes with strong constitutively active IKK2 expression. To gain insight into proximal events after activation of IKK2/NF-ÎºB, we isolated cardiomyocytes and activated the transgene in vitro, thus avoiding interference of other cell types. Gene expression analysis revealed up-regulation of transcripts of chemotactic cytokines, adhesion molecules, and a striking number of IFN-regulated genes in cardiomyocytes expressing IKK2-CA. In addition, apoptotic and anti-apoptotic genes were up-regulated.

ORGANISM(S): Mus musculus

SUBMITTER: Harald Jakob Maier

PROVIDER: E-MTAB-3975 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Abstract. Deregulated c-MYC is found in a variety of cancers where it promotes proliferation as well as apoptosis. In many hematological malignancies enhanced NF-kB exerts prosurvival functions. Here we investigated the role of NF-kB in mouse and human c-MYC-transformed lymphomas. The NF-kB-pathway is extinguished in murine lymphoma cells and extrinsic stimuli typically inducing NF-kB activity fail to activate this pathway. Genetic activation of the NF-kB pathway induces apoptosis in these cells, while inhibition of NF-kB by an IkBa superrepressor provides a selective advantage in vivo. Furthermore, in human BurkittÂ´s lymphoma cells we find that NF-kB activation induces apoptosis. NF-kB upregulates Fas and predisposes to Fas-induced cell death, which is caspase 8 mediated and can be prevented by CFLAR overexpression. We conclude that c-MYC overexpression sensitizes cells to NF-kB-induced apoptosis and persistent inacvtivity of NF-kB signaling is a prerequisite for MYC-mediated tumorigenesis. We could also show that low immunogenicity and Fas insensitivity of MYC driven lymphoma cells is reversed by activation of NF-kB. Our observations provide a molecular explanation for the described absence of the NF-kB signaling in BurkittÂ´s lymphoma and question the applicability of NF-kB inhibitors as candidates for treatment of this cancer. Experiment Overall Design: Ramos cells were transfected in triplicates with pRTS-GFP (A+,C+,D+) or pRTS-CA-IKK2 (E+,F+,H+), selected with hygromycin and transgene expression was induced with doxycycline (0,5mg/ml) for 48hrs. RNA was isolated with RNeasy mini kit (Qiagen, Venlo, Netherlands) and Gene expression profiling (GEP) was performed using Affymetrix Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA, USA). 2 Âµg of total RNA were labeled using the GeneChipÂ® One-Cycle Target Labeling assay kit (Affymetrix). After hybridization arrays were stained and washed in a FS 450 Fluidics station (Affymetrix) before imaging on an Affymetrix GeneChip (3000) scanner. Raw data were generated using the GCOS 1.4 software (Affymetrix). Probe level data were obtained using the Robust Multichip Average (RMA) normalization algorithm and CEL files were loaded into Genesifter (GeneSifter.Net, VizX Laboratories, Seattle, WA, USA). Genes were identified as differentially expressed among the two classes if a two-sample T-test revealed a nominal significance level of 0.05 and the ratio between the two classes was at least 2 fold. Calculation of false discovery rate was done according to the method of Benjamini and Hochberg. Biological significance was determined using Gene Ontolgy reports.

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Transcription profiling by array of murine cardiomyocytes with and without expression of constitutively active IKK2

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