Inactivation of hepatocyte nuclear factor-4{alpha} mediates alcohol-induced downregulation of intestinal tight junction proteins

Abstract

Chronic alcohol exposure has been shown to increase the gut permeability in the distal intestine, in part, through induction of zinc deficiency. The present study evaluated the molecular mechanisms whereby zinc deficiency mediates alcohol-induced intestinal barrier dysfunction. Examination of zinc finger transcription factors in the gastrointestinal tract of mice revealed a prominent distribution of hepatocyte nuclear factor-4alpha (HNF-4alpha). HNF-4alpha exclusively localizes in the epithelial nuclei and exhibited an increased abundance in mRNA and protein levels in the distal intestine. Chronic alcohol exposure to mice repressed the HNF-4alpha gene expression in the ileum and reduced the protein level and DNA binding activity of HNF-4alpha in all of the intestinal segments with the most remarkable changes in the ileum. Chronic alcohol exposure also decreased the mRNA levels of tight junction proteins, particularly in the ileum. Caco-2 cell culture studies were conducted to determine the role of HNF-4alpha in regulation of the epithelial tight junction and barrier function. Knockdown of HNF-4alpha in Caco-2 cells decreased the mRNA and protein levels of tight junction proteins in association with disruption of the epithelial barrier. Alcohol treatment inactivated HNF-4alpha, which was prevented by N-acetyl-cysteine or zinc. The link between zinc and HNF-4alpha function was confirmed by zinc deprivation, which inhibited HNF-4alpha DNA binding activity. These results indicate that inactivation of HNF-4alpha due to oxidative stress and zinc deficiency is likely a novel mechanism contributing to the deleterious effects of alcohol on the tight junctions and the intestinal barrier function.

Fig. 1.

Distribution of hepatocyte nuclear factor-4α (HNF-4α) in the gastrointestinal tract of mice. HNF-4α was detected by immunohistochemical staining. Exclusive localization of HNF-4α in the nuclei of epithelial cells was found in all of the intestinal segments, but not in the stomach. Scale bars: 20 μM.

Fig. 2.

Protein and mRNA levels of HNF-4α in the gastrointestinal tract of mice. A: immunoblot analysis of HNF-4α protein levels. The immunoblot bands were quantified by densitometry analysis, and the HNF-4α-to-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratios were calculated. B: real-time RT-PCR assay of HNF-4α mRNA levels. The gene expressions, normalized to β-actin, were expressed as relative changes, setting the values of duodenum as one. Results are means ± SD (n = 3 experiments in A, n = 4 in B). Significant differences (P < 0.05) between intestinal segments were determined by ANOVA and are indicated by different letters. D, duodenum; J, jejunum; I, ileum; Ce, cecum; Co, colon.

Fig. 3.

Effects of chronic alcohol exposure on intestinal HNF-4α in mice. A: HNF-4α function was assessed by measuring DNA binding activity with a Trans-AM HNF Family Transcription Factor ELISA Kit. EtOH, ethanol; Ctrl, control. B: HNF-4α protein levels were measured by immunoblot analysis. The immunoblot bands were quantified by densitometry analysis. C: HNF-4α mRNA levels were measured by real-time RT-PCR. The gene expressions, normalized to β-actin, were expressed as relative changes, setting the values of duodenum as one. Results are means ± SD (n = 6 in A; n = 3 in B; n = 4 in C). Significant differences (P < 0.05) among intestinal segments were determined by ANOVA and are indicated by different letters. *Significant differences (P < 0.05) between alcohol-fed and pair-fed mice for each intestinal segment determined by t-test.

Fig. 4.

Effects of chronic alcohol exposure on expression of tight junction proteins in mice. A: mRNA levels of tight junction proteins. The mRNA levels of claudin-1, occludin, and ZO-1 were measured by real-time RT-PCR. The gene expressions, normalized to β-actin, were expressed as relative changes, setting the values of duodenum as one. Results are means ± SD (n = 4). Significant differences (P < 0.05) among intestinal segments were determined by ANOVA and are indicated by different letters. *Significant differences (P < 0.05) between alcohol-fed and pair-fed mice for each intestinal segment determined by t-test. B: correlations between mRNA levels of HNF-4α and tight junction proteins. Correlation coefficients were used to determine linear association between intestinal HNF-4α and tight junction protein mRNA variables.

Fig. 5.

Effects of HNF-4α small-interfering RNA (siRNA) transfection on the epithelial barrier of Caco-2 cells. Caco-2 cells were transfected with human HNF-4α siRNA or control siRNA on day 21 for 24 h. A: immunoblot analysis of HNF-4α protein levels. The immunoblot bands were quantified by densitometry analysis. B: immunocytochemical localization of HNF-4α. The nuclei were counterstained by propidium iodide (PI). C: epithelial barrier function. The epithelial barrier function of insert-cultured Caco-2 cells was assessed by measuring the transepithelial electrical resistance (TEER) with an epithelial voltohm-meter and the paracellular FD-4 penetration from the apical to the basolateral compartments. Results are means ± SD (n = 3 in A; n = 8 in C). *Significant differences (P < 0.05) between control siRNA and HNF-4α siRNA detected by t-test (A). *Significant differences (P < 0.05) between time points determined by ANOVA (C).

Fig. 6.

Effects of HNF-4α siRNA transfection on tight junction proteins. Caco-2 cells were transfected with HNF-4α siRNA for 24 h. A: real-time RT-PCR assay of gene expression of tight junction proteins. The mRNA levels, normalized to β-actin, were expressed as relative changes, setting the values of controls as one. B: immunocytochemical staining of occludin. C: immunoblot analysis of occludin protein levels. The immunoblot bands were quantified by densitometry analysis. Results are means ± SD (n = 4 in A; n = 3 in C). *Significant differences (P < 0.05) between control siRNA and HNF-4α siRNA detected by t-test (A and C).

Fig. 7.

Effects of alcohol on HNF-4α protein and function in Caco-2 cells. Caco-2 cells were exposed to 5% alcohol for 24 h, and NAC and zinc were supplemented to determine the role of oxidative stress and zinc deprivation. A: HNF-4α function was assessed by measuring HNF-4α DNA binding activity with a Trans-AM HNF Family Transcription Factor ELISA kit. B: immunoblot assay of HNF-4α protein levels. The immunoblot bands were quantified by densitometry analysis. Results are means ± SD (n = 6 in A; n = 3 in B). Significant differences (P < 0.05) between time points were determined by ANOVA and are indicated by different letters in A.

Fig. 8.

Effects of zinc deprivation on HNF-4α protein and function in Caco-2 cells. Caco-2 cells were treated on day 21 with N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) at 2, 3, and 4 μM or 4 μM TPEN plus 100 μM zinc for 24 h. A: HNF-4α function was assessed by measuring HNF-4α DNA binding activity with a Trans-AM HNF Family Transcription Factor ELISA kit. B: immunoblot assay of HNF-4α protein levels. The immunoblot bands were quantified by densitometry analysis. Results are means ± SD (n = 6 in A; n = 3 in B). Significant differences (P < 0.05) between time points were determined by ANOVA and are indicated by different letters.