Background/Aims: Although the cytotoxicity of aspirin against the intestinal epithelium is a major clinical problem, little is known about its pathogenesis. We assessed the involvement of Multi Drug ...Resistance (MDR) 1 in intestinal epithelial cell injury caused by aspirin using MDR1 gene-transfected Caco2 cells. Methods: Caco2 cells were treated with various concentrations of aspirin for 24 h. After treatment of Caco2 cells with verapamil, a specific inhibitor of MDR1, we assessed the extent of cell injury using a WST-8 assay at 24 h after aspirin-stimulation. We performed the same procedure in MDR1 gene-transfected Caco2 cells. To determine the function of MDR1 in the metabolism of aspirin, flux study was performed using 14C-labeled aspirin. Results: The level of aspirin-induced cell injury was higher in verapamil-treated Caco2 cells than in control cells and was less serious in MDR1-transfected Caco2 cells than in control vector-transfected cells. The efflux of 14C-labeled aspirin was higher in verapamil-treated Caco2 cells than in control cells. Conclusion: These data suggest that aspirin effux occurs through the MDR1 transporter and that the MDR1 transporter is involved in the pathogenesis of aspirin-induced cell injury.
Immunoglobulin A (IgA)-mediated mucosal immunity is important for the host because it contributes to reducing infection risk and to establishing host-microbe symbiosis. BTB and CNC homology 1 (Bach1) ...is a transcriptional repressor with physiological and pathophysiological functions that are of particular interest for their relation to gastrointestinal diseases. However, Bach1 effects on IgA-mediated mucosal immunity remain unknown. For this study using Bach1-deficient (Bach1 -/- ) mice, we investigated the function of Bach1 in IgA-mediated mucosal immunity. Intestinal mucosa, feces, and plasma IgA were examined using immunosorbent assay. After cell suspensions were prepared from Peyer's patches and colonic lamina propria, they were examined using flow cytometry. The expression level of polymeric immunoglobulin receptor (pIgR), which plays an important role in the transepithelial transport of IgA, was evaluated using Western blotting, quantitative real-time PCR, and immunohistochemistry. Although no changes in the proportions of IgA-producing cells were observed, the amounts of IgA in the intestinal mucosa were increased in Bach1 -/- mice. Furthermore, plasma IgA was increased in Bach1 -/- mice, but fecal IgA was decreased, indicating that Bach1 -/- mice have abnormal secretion of IgA into the intestinal lumen. In fact, Bach1 deficiency reduced pIgR expression in colonic mucosa at both the protein and mRNA levels. In the human intestinal epithelial cell line LS174T, suppression of Bach1 reduced pIgR mRNA stability. In contrast, overexpression of Bach1 increased pIgR mRNA stability. These results demonstrate that Bach1 deficiency causes abnormal secretion of IgA into the intestinal lumen via suppression of pIgR expression.
The androgen receptor (AR) possesses a polymorphic polyglutamine tract (polyQ), whose length is inversely correlated with its transcriptional activity. Here, we investigated whether 6 and 5 ...repetitive glutamine tracts (Q6 and Q5, respectively) in the N-terminal domain of AR also have effects on AR transactivation. In a reporter gene assay using two-tandem repeats of an androgen response element, deletion of glutamine tracts significantly increased AR transactivation in the following order: wild-type
<
a single deletion of polyQ or Q5
<
double deletion of polyQ and Q6
<
double deletion of polyQ and Q5
<
triple deletion. Deletion of polyQ alone or combined deletion of polyQ and Q5 from an AR mutant lacking the ligand-binding domain, which is constitutively active due to activation function-1, increased AR transactivation. However, the glutamine tracts had no influence on activation function-1 activity, suggesting that the glutamine tracts modulate the binding of AR to DNA. Q5, like polyQ, was found to be involved in the interaction between the NH
2- and COOH-terminal regions of AR (N–C interaction). These results indicate that the inhibitory effects of polyQ and Q5 on AR transactivation are the due, at least in part, to their negative regulation of N–C interaction.
Bile acid sequestrants are used as medicinal drugs to treat dyslipidemia and type 2 diabetes. We found that cholestyramine, a bile acid sequestrant, increases cecal short-chain fatty acid (SCFA) ...production and intestinal immunoglobulin A (IgA) in C57BL/6J mice. In a 12-week high-fat diet study, feeding cholestyramine (2% (w/w)) significantly promoted C2-C4 SCFAs in the cecum by approximately 1.6-fold and fecal IgA by 1.8-fold. In an 8-week normal-fat diet study, feeding cholestyramine (1 and 2%) increased the cecal propionic acid content by approx. 2.0-fold. Fecal IgA was also significantly increased at 4 weeks (1%: 1.7-fold; 2%: 2.1-fold) and 8 weeks (1%: 1.8-fold; 2%: 2.0-fold) in the normal-fat diet study. These results indicate that bile acid sequestrants may exert their physiological functions, such as intestinal IgA production, through SCFA-dependent signaling pathways.
We constructed a dominant negative form of human hypoxia-inducible factor (HIF)-2alpha, HIF-2alphaDoN, which inhibited HIF transcriptional activity induced by hypoxia and by HIF-2alpha. HIF-2alphaDoN ...formed a complex with HIF-1 beta and interacted with DNA containing hypoxia response elements (HREs). Thus, the complex appears to inhibit the binding of HIF-2 to HREs, and HIF-2alphaDoN might provide a useful therapeutic tool for HIF-2alpha-related diseases.
Lactic acid bacterium-containing fermentates provide beneficial health effects by regulating the immune response. A naturally fermented vegetable beverage, a traditional Japanese food, reportedly ...provides health benefits; however, the beneficial function of its bacteria has not been clarified. Apilactobacillus kosoi is the predominant lactic acid bacterium in the beverage. Using murine Peyer's patch cells, we compared the immunoglobulin A (IgA)-inducing activity of
10H
to those of 29 other species of lactic acid bacteria and found that species belonging to the genus
(
10H
, A. apinorum JCM30765
, and A. kunkeei JCM16173
) possessed significantly higher activity than the others. Thereafter, lipoteichoic acids (LTAs), important immunostimulatory molecules of Gram-positive bacteria, were purified from the three
species, and their IgA-inducing activity was compared to those of LTAs from Lactiplantibacillus plantarum JCM1149
and a probiotic strain, Lacticaseibacillus rhamnosus GG. The results revealed that LTAs from
species had significantly higher activity than others. We also compared the LTA structure of
10H
with that of
JCM1149
and L. rhamnosus GG. Although d-alanine or both d-alanine and carbohydrate residues were substituents of free hydroxyl groups in the polyglycerol phosphate structure in LTAs from strains JCM1149
and GG, d-alanine residues were not found in LTA from strain 10H
by
H nuclear magnetic resonance (NMR) analysis. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis of the glycolipid structure of LTA revealed that LTA from strain 10H
contained dihexosyl glycerol, whereas trihexosyl glycerol was detected in LTAs from other strains. These structural differences may be related to differences in IgA-inducing activity.
The components of lactic acid bacteria that exert immunostimulatory effects are of increasing interest for therapeutic and prophylactic options, such as alternatives to antibiotics, cognitive enhancements, and vaccine adjuvants. LTAs act as immunostimulatory molecules in the host innate immune system by interacting with pattern recognition receptors. However, as LTA structures differ among species, detailed knowledge of the structure-function relationship for immunostimulatory effects is required. Comparisons of the IgA-inducing activity of LTAs have demonstrated that LTAs from the genus
possess distinctive activities to stimulate mucosal immunity. The first analysis of the LTA structure from the genus
suggests that it differs from structures of LTAs of related species of lactic acid bacteria. This knowledge is expected to aid in the development of functional foods containing lactic acid bacteria and pharmaceutical applications of immunostimulatory molecules from lactic acid bacteria.
Bile acid sequestrants are used as medicinal drugs to treat dyslipidemia and type 2 diabetes. We found that cholestyramine, a bile acid sequestrant, increases cecal short-chain fatty acid (SCFA) ...production and intestinal immunoglobulin A (IgA) in C57BL/6J mice. In a 12-week high-fat diet study, feeding cholestyramine (2% w/w) significantly promoted C2-C4 SCFAs in the cecum by ~1.6-fold and fecal IgA by 1.8-fold. In an 8-week normal-fat diet study, feeding cholestyramine (1% and 2%) increased the cecal propionic acid content by ~2.0-fold. Fecal IgA was also significantly increased at 4 weeks (1%: 1.7-fold; 2%: 2.1-fold) and 8 weeks (1%: 1.8-fold; 2%: 2.0-fold) in the normal-fat diet study. These results indicate that bile acid sequestrants may exert their physiological functions, such as intestinal IgA production, through SCFA-dependent signaling pathways.
► Hypoxia up-regulates the expression of GAPDH gene in breast cancer cells. ► The novel HRE is functional in breast cance cells, but not in prostate cancer cells. ► HIF-1α binds to the novel HRE of ...the GAPDH gene in hypoxic breast cancer cells. ► The GC-box adjacent to the novel HRE is essential for HIF-1 activation. ► Binding of Sp1 to the GC-box is required for activation of HIF-1 on the novel HRE.
Hypoxia up-regulates the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in a cell type-specific manner. It is unknown whether this occurs in breast cancer. Here, we report that hypoxia up-regulates the GAPDH gene expression through breast cancer-specific molecular mechanisms in MCF-7 cells. Mutation analysis identified a novel hypoxia response element (HRE), in addition to the HRE found previously in prostate cancer LNCaP cells. Knockdown and overexpression of hypoxia-inducible factor (HIF)-1α indicated that HIF-1 contributed to the up-regulation of GAPDH gene expression by hypoxia. Although chromatin immunoprecipitation (ChIP) and plasmid immunoprecipitation analyses revealed the presence of HIF-1α on the novel HRE in both hypoxic cell lines, a mutation in either the novel HRE or its 3′-flanking GC-box resulted in a reduction of hypoxia-increased GAPDH promoter activity only in MCF-7 cells. ChIP analysis showed that Sp1 bound to the GC-box in MCF-7 cells, but not in LNCaP cells, in normoxia and hypoxia. Knockdown of Sp1 reduced hypoxia-increased promoter activity and expression level of GAPDH in MCF-7 cells. These results indicate that in MCF-7 cells, the activation of HIF-1 on the novel HRE contributes to the breast cancer-specific hypoxic induction of GAPDH gene expression and absolutely depends on the presence of Sp1 on the GC-box.
Peroxiredoxin 4 (PRDX4), a secretory protein that is preferentially retained in the endoplasmic reticulum (ER), is encoded by a gene located on the X chromosome and highly expressed in colonic ...tissue. In this study, we investigated the role of PRDX4 by means of male PRDX4-knockout (PRDX4-/y) mice in the development of intestinal inflammation using a dextran sulfate sodium (DSS)-induced colitis model.
Acute colitis was induced with DSS (2.5% in drinking water) in wild-type (WT) and PRDX4-/y male C57BL/6 mice. Histological and biochemical analyses were performed on the colonic tissues.
PRDX4 was mainly localized in the colonic epithelial cells in WT mice. The disease activity index (DAI) scores of PRDX4-/y mice were significantly higher compared to those of WT mice. Specifically, PRDX4-/y mice showed marked body weight loss and shortening of colon length compared to WT mice, whereas the myeloperoxidase levels were increased in PRDX4-/y compared to WT mice. In addition, the mRNA expression levels of TNF-α and IFN-γ were significantly higher in the colonic mucosa of PRDX4-/y compared to WT mice. Moreover, the levels of CHOP and activated caspase 3 were higher in the colonic tissues of PRDX4-/y compared to WT mice following treatment with DSS. The ER also showed greater expansion in PRDX4-/y than WT mice, which was consistent with severe ER stress under PRDX4 deficiency.
Our results demonstrated that the lack of PRDX4 aggravated the colonic mucosal damage induced by DSS. Because PRDX4 functions as an ER thiol oxidase as well as an antioxidant, DSS induced oxidative damage and ER stress to a greater degree in PRDX4-/y than WT mice. These findings suggest that PRDX4 may represent a novel therapeutic molecule in intestinal inflammation.
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•ER-retained peroxiredoxin 4 (PRDX4) protein is highly expressed in the colon.•PRDX4 localizes to colonic epithelial cells.•PRDX4-/y mice show high disease severity of colitis.•The oxidative damage and ER stress are higher in PRDX4-/y than WT mice.•PRDX4 may represent a novel therapeutic molecule in intestinal inflammation.
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