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Rifampin and digoxin induction of MDR1 expression and function in human intestinal (T84) epithelial cells.

ABSTRACT: Oral drug bioavailability is limited by intestinal expression of P-glycoprotein (MDR1, Pgp, ABCB1) whose capacity is regulated via nuclear receptors e.g. the pregnane X receptor (PXR, SXR, NR1I2). In order to study dynamic regulation of MDR1 transport capacity we have identified the T84 epithelial cell-line as a model for human intestine co-expressing MDR1 with PXR. The ability of rifampin, a known PXR agonist and digoxin, a model MDR1 substrate, to regulate MDR1 expression and transport activity has been tested, in these T84 cells.Transport was assayed by bi-directional [(3)H]-digoxin transepithelial fluxes across epithelial layers of T84 cells seeded onto permeable filter supports following pre-exposure to rifampin and digoxin. Quantitative real-time PCR, Western blotting and immunocytochemistry were used to correlate induction of MDR1 transcript and protein levels with transport activity.Rifampin exposure (10 microM, 72 hours) increased MDR1 transcript levels (3.4 fold), MDR1 total protein levels (4.4 fold), apical MDR1 protein (2.7 fold) and functional activity of MDR1 (1.2 fold). Pre-incubation with digoxin (1 microM, 72 hours) potently induced MDR1 transcript levels (92 fold), total protein (7 fold), apical MDR1 protein (4.7 fold) and functional activity (1.75 fold). Whereas PXR expression was increased by rifampin incubation (2 fold), digoxin reduced PXR expression (0.3 fold).Chronic digoxin pre-treatment markedly upregulates MDR1 expression and secretory capacity of T84 epithelia. Digoxin-induced changes in MDR1 levels are distinct from PXR-mediated changes resulting from rifampin exposure.

SUBMITTER: Haslam IS

PROVIDER: S-EPMC2438975 | biostudies-other | 2008 May

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:Although systemic hypertension is a risk factor of age-related macular degeneration, antihypertensive medications do not affect the risk of the disease. One condition that induces hypertension is high intake of dietary salt resulting in increased blood osmolarity. In order to prove the assumption that, in addition to hypertension, high osmolarity may aggravate neovascular retinal diseases, we determined the effect of extracellular hyperosmolarity on the expression of angiogenic cytokines in cultured human retinal pigment epithelial (RPE) cells. METHODOLOGY/PRINCIPAL FINDINGS:Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Hypoxia and oxidative stress were induced by the addition of the hypoxia mimetic CoCl2 and H2O2, respectively. Alterations in gene expression were determined with real-time RT-PCR. Secretion of bFGF was evaluated by ELISA. Cell viability was determined by trypan blue exclusion. Nuclear factor of activated T cell 5 (NFAT5) expression was knocked down with siRNA. Hyperosmolarity induced transcriptional activation of bFGF, HB-EGF, and VEGF genes, while the expression of other cytokines such as EGF, PDGF-A, TGF-?1, HGF, and PEDF was not or moderately altered. Hypoxia induced increased expression of the HB-EGF, EGF, PDGF-A, TGF-?1, and VEGF genes, but not of the bFGF gene. Oxidative stress induced gene expression of HB-EGF, but not of bFGF. The hyperosmotic expression of the bFGF gene was dependent on the activation of p38?/? MAPK, JNK, PI3K, and the transcriptional activity of NFAT5. The hyperosmotic expression of the HB-EGF gene was dependent on the activation of p38?/? MAPK, ERK1/2, and JNK. The hyperosmotic expression of bFGF, HB-EGF, and VEGF genes was reduced by inhibitors of TGF-?1 superfamily activin receptor-like kinase receptors and the FGF receptor kinase, respectively. Hyperosmolarity induced secretion of bFGF that was reduced by inhibition of autocrine/paracrine TGF-?1 signaling and by NFAT5 siRNA, respectively. Hyperosmolarity decreased the viability of the cells; this effect was not altered by exogenous bFGF and HB-EGF. Various vegetable polyphenols (luteolin, quercetin, apigenin) inhibited the hyperosmotic expression of bFGF, HB-EGF, and NFAT5 genes. CONCLUSION:Hyperosmolarity induces transcription of bFGF and HB-EGF genes, and secretion of bFGF from RPE cells. This is in part mediated by autocrine/paracrine TGF-?1 and FGF signaling. It is suggested that high intake of dietary salt resulting in osmotic stress may aggravate neovascular retinal diseases via stimulation of the production of angiogenic factors in RPE cells, independent of hypertension.

Project description:Campylobacter concisus infections of the gastrointestinal tract can be accompanied by diarrhea and inflammation, whereas colonization of the human oral cavity might have a commensal nature. We focus on the pathophysiology of C. concisus and the effects of different clinical oral and fecal C. concisus strains on human HT-29/B6 colon cells. Six oral and eight fecal strains of C. concisus were isolated. Mucus-producing HT-29/B6 epithelial monolayers were infected with the C. concisus strains. Transepithelial electrical resistance (R(t)) and tracer fluxes of different molecule size were measured in Ussing chambers. Tight junction (TJ) protein expression was determined by Western blotting, and subcellular TJ distribution was analyzed by confocal laser-scanning microscopy. Apoptosis induction was examined by TUNEL-staining and Western blot of caspase-3 activation. All strains invaded confluent HT-29/B6 cells and impaired epithelial barrier function, characterized by a time- and dose-dependent decrease in R(t) either after infection from the apical side but even more from the basolateral compartment. TJ protein expression changes were sparse, only in apoptotic areas of infected monolayers TJ proteins were redistributed. Solely the barrier-forming TJ protein claudin-5 showed a reduced expression level to 66±8% (P<0.05), by expression regulation from the gene. Concomitantly, Lactate dehydrogenase release was elevated to 3.1±0.3% versus 0.7±0.1% in control (P<0.001), suggesting cytotoxic effects. Furthermore, oral and fecal C. concisus strains elevated apoptotic events to 5-fold. C. concisus-infected monolayers revealed an increased permeability for 332 Da fluorescein (1.74±0.13 vs. 0.56±0.17 10(-6) cm/s in control, P<0.05) but showed no difference in permeability for 4 kDa FITC-dextran (FD-4). The same was true in camptothecin-exposed monolayers, where camptothecin was used for apoptosis induction.In conclusion, epithelial barrier dysfunction by oral and fecal C. concisus strains could mainly be assigned to apoptotic leaks together with moderate TJ changes, demonstrating a leak-flux mechanism that parallels the clinical manifestation of diarrhea.

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Rifampin and digoxin induction of MDR1 expression and function in human intestinal (T84) epithelial cells.

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