Thioredoxin-interacting protein gene expression via MondoA is rapidly and transiently suppressed during inflammatory responses.

Whereas accumulating evidence indicates that a number of inflammatory genes are induced by activation of nuclear factor-κB and other transcription factors, less is known about genes that are suppressed by proinflammatory stimuli. Here we show that expression of thioredoxin-interacting protein (Txnip) is dramatically suppressed both in mRNA and protein levels upon stimulation with lipopolysaccharide in mouse and human macrophages. In addition to lipopolysaccharide, a Toll-like receptor 4 ligand, stimulation with other Toll-like receptor ligands such as CpG DNA also suppressed Txnip expression. Not only the Toll-like receptor ligands, but also other proinflammatory stimulators, such as interleukin-1β and tumor necrosis factor-α elicited the similar response in fibroblasts. Suppression of Txnip by lipopolysaccharide is accompanied by a decrease of the glucose sensing transcription factor MondoA in the nuclei and dissociation of the MondoA:Mlx complex that bound to the carbohydrate-response elements in the Txnip promoter in unstimulated cells. Lipopolysaccharide-mediated decrease of nuclear MondoA was inhibited in the presence of 2-deoxyglucose. Furthermore, blockage of glyceraldehyde-3-phosphate dehydrogenase by iodoacetate alleviated the suppression of Txnip mRNA by lipopolysaccharide, suggesting the involvement of glucose-metabolites in the regulation. Since Txnip is implicated in the regulation of glucose metabolism, this observation links between inflammatory responses and metabolic regulation

Targeted disruption of Np95 gene renders murine embryonic stem cells hypersensitive to DNA damaging agents and DNA replication blocks

NP95, which contains a ubiquitin-like domain, a cyclin A/E-Cdk2 phosphorylation site, a retinoblastoma (Rb) binding motif, and a ring finger domain, has been shown to be colocalized as foci with proliferating cell nuclear antigen in early and mid-S phase nuclei. We established Np95 nulligous embryonic stem cells by replacing the exons 2-7 of the Np95 gene with a neo cassette and by selecting out a spontaneously occurring homologous chromosome crossing over with a higher concentration of neomycin. Np95-null cells were more sensitive to x-rays, UV light, N-methyl-N"-nitro-N-nitrosoguanidine (MNNG), and hydroxyurea than embryonic stem wild type (Np95(+/+)) or heterozygously inactivated (Np95(+/-)) cells. Expression of transfected Np95 cDNA in Np95-null cells restored the resistance to x-rays, UV, MNNG, or hydroxyurea concurrently to a level similar to that of Np95(+/-) cells, although slightly below that of wild type (Np95(+/+)) cells. These findings suggest that NP95 plays a role in the repair of DNA damage incurred by these agents. The frequency of spontaneous sister chromatid exchange was significantly higher for Np95-null cells than for Np95(+/+) cells or Np95(+/-) cells (p < 0.001). We conclude that NP95 functions as a common component in the multiple response pathways against DNA damage and replication arrest and thereby contributes to genomic stability

Effects of various inhibitors on <i>Txnip</i> suppression in response to LPS.

<p>(A and B) RAW264.7 cells were treated with Vehicle (dimethyl sulfoxide in (A) or diethyl ether in (B)), 10 µM compound C (Comp. C), or 50 nM okadaic acid for 1 h, followed by stimulation with 100 ng/ml LPS for 90 min. (C) RAW264.7 cells were treated with 0.5 mM iodoacetate or sodium acetate for 1 h, followed by treatment with 100 ng/ml LPS, 5 mM sodium azide, or 5 µM rotenone for 90 min. <i>Txnip</i> mRNA copy numbers normalized to that of β-actin are shown. Data shown are mean ± S.E. of triplicate samples of a representative of at least three independent experiments.</p

Suppression of <i>Txnip</i> expression by proinflammatory stimulation.

<p>BMDM (A), NIH3T3 (B), or RAW264.7 (C) cells were stimulated with 2 µM CpG DNA 100 ng/ml LPS, 10 ng/ml IL-1β, 10 ng/ml TNF-α, or 10 ng/ml IL-10. Cells were lysed at the indicated time after stimulation. Txnip, β-actin, phosphorylated STAT3 (pSTAT3), or STAT3 proteins were detected by western blotting. Data shown are a representative of at least three independent experiments.</p

Suppression of <i>Txnip</i> expression by LPS.

<p>RAW264.7 cells (A and B), BMDM (C and D), or differentiated THP-1 cells (E) were stimulated with 100 ng/ml LPS. Cells were lysed at the indicated time after stimulation. <i>Txnip</i> mRNA copy numbers normalized to that of <i>β-actin</i> are shown (A and C). Data shown are mean ± S.E. of at least 4 independent experiments. Txnip and β-actin proteins were detected by western blotting (B, D, and E). Data shown are a representative of at least three independent experiments.</p

Dissociation of MondoA:Mlx from the <i>Txnip</i> promoter on LPS stimulation.

<p>(A) Chromatin immunoprecipitation assay was performed to examine occupation of Mlx, MondoA, and NF-YA in <i>Txnip</i> promoter region with RAW264.7 cells stimulated with or without 100 ng/ml LPS for 45 min. Enriched DNAs was eluted and <i>Txnip</i> promoter region was quantified by quantitative PCR. Data shown are mean ± S.E. of duplicate samples. (B) RAW264.7 cells stimulated with or without 100 ng/ml LPS for 45 min. Cells were fixed, permeabilized, and stained with anti-MondoA antibody and Alexa488-conjugated polyclonal anti-rabbit IgG antibody. Fluorescence microscopic images were obtained and analyzed by three-dimentional deconvolution. (C) Nuclear and cytoplasmic fractions of RAW264.7 cells stimulated with or without 100 ng/ml LPS for 45 min were analyzed by western blotting with anti-<i>MondoA</i>, histone deacetylase 1 (<i>Hdac1</i>) or ribosomal S6 ribosomal protein (<i>Rps6</i>). Data shown are a representative of at least three independent experiments.</p