In vivo pattern of lipopolysaccharide and anti-CD3-induced NF-kappa B activation using a novel gene-targeted enhanced GFP reporter gene mouse.
NF-kappa B is a family of transcription factors involved in regulating cell death/survival, differentiation, and inflammation. Although the transactivation ability of NF-kappa B has been extensively studied in vitro, limited information is available on the spatial and temporal transactivation pattern in vivo. To investigate the kinetics and cellular localization of NF-kappa B-induced transcription, we created a transgenic mouse expressing the enhanced GFP (EGFP) under the transcriptional control of NF-kappa B cis elements (cis-NF-kappa B(EGFP)). A gene-targeting approach was used to insert a single copy of a NF-kappa B-dependent EGFP reporter gene 5' of the X-linked hypoxanthine phosphoribosyltransferase locus in mouse embryonic stem cells. Embryonic fibroblasts, hepatic stellate cells, splenocytes, and dendritic cells isolated from cis-NF-kappa B(EGFP) mice demonstrated a strong induction of EGFP in response to LPS, anti-CD3, or TNF-alpha that was blocked by the NF-kappa B inhibitors BAY 11-7082 and NEMO-binding peptide. Chromatin immunoprecipitation analysis demonstrated RelA binding to the cis-NF-kappa B(EGFP) promoter. Adenoviral delivery of NF-kappa B-inducing kinase strongly induced EGFP expression in the liver of cis-NF-kappa B(EGFP) mice. Similarly, mice injected with anti-CD3 or LPS showed increased EGFP expression in mononuclear cells, lymph node, spleen, and liver as measured by flow cytometry and/or fluorescence microscopy. Using whole organ imaging, LPS selectively induced EGFP expression in the duodenum and proximal jejunum, but not in the ileum and colon. Confocal analysis indicated EGFP expression was primarily found in lamina propria mononuclear cells. In summary, the cis-NF-kappa B(EGFP) mouse will serve as a valuable tool to address multiple questions regarding the cell-specific and real-time activation of NF-kappa B during normal and diseased states.
Magness
Scott T
ST
Department of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA.
Jijon
Humberto
H
Van Houten Fisher
Nancy
N
Sharpless
Ned E
NE
Brenner
David A
DA
Jobin
Christian
C
D000818:Animals; D005786:Gene Expression Regulation / Q000187:drug effects*; D018390:Gene Targeting; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007421:Intestine, Small / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008099:Liver / Q000378:metabolism; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D008198:Lymph Nodes / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D016853:Muromonab-CD3 / Q000494:pharmacology; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D009928:Organ Specificity; D010455:Peptides / Q000494:pharmacology; D013154:Spleen / Q000378:metabolism; D013450:Sulfones / Q000494:pharmacology; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D019076:Transgenes; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
C434003:3-(4-methylphenylsulfonyl)-2-propenenitrile; D008070:Lipopolysaccharides; D008164:Luminescent Proteins; D016853:Muromonab-CD3; C467063:NBD peptide, mouse; D016328:NF-kappa B; D009570:Nitriles; D010455:Peptides; D013450:Sulfones; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; D007041:Hypoxanthine Phosphoribosyltransferase