Gene regulation of intestinal porcine epithelial cells IPEC-J2 is dependent on the site of deoxynivalenol toxicological action

Abstract

The intestinal epithelial cell layer represents the border between the luminal and systemic side of the gut. The decision between absorption and exclusion of substances is the quintessential function of the gut and varies along the gut axis. Consequently, potentially toxic substances may reach the basolateral domain of the epithelial cell layer via blood stream. The mycotoxin deoxynivalenol (DON) is a Fusarium derived secondary metabolite known to enter the blood stream and displaying a striking toxicity on the basolateral side of polarised epithelial cell layers in vitro. Here we analysed potential mechanisms of apical and basolateral DON toxicity reflected in the gene expression. We used the jejunum-derived, polarised intestinal porcine epithelial cell line IPEC-J2 as an in vitro cell culture model. Luminal and systemic DON challenge of the epithelial cell layer was mimicked by a DON application from the apical or basolateral compartment of membrane inserts for 72 h. We compared the genome-wide gene expression of untreated and DON-treated IPEC-J2 cells with the GeneChip® Porcine Genome Array of Affymetrix. Low basolateral DON (200 ng/mL) application triggered 10 times more gene transcripts in comparison to the corresponding apical application (2539 versus 267) despite the intactness of the challenged cell layer as measured by transepithelial electrical resistance. Analysis of the regulated genes by bioinformatic resource DAVID identified several groups of biochemical pathways modulated by concentration and orientation of DON application. Selected genes representing pathways of the cellular metabolism, information processing and structural design were analysed in detail by quantitative PCR. Our findings clearly show that apical and basolateral challenge of epithelial cell layers trigger different gene response profiles paralleled with a higher susceptibility towards basolateral challenge. The evaluation of toxicological potentials of mycotoxins should take this difference in gene regulation dependent on route of application into account.

Figure 1. Response of transepithelial electrical resistance (TEER) to apical and basolateral deoxynivalenol (DON) application in polarised IPEC-J2 cells.

Cells were grown for 7 d on membrane inserts (0 h) and incubated than for 72 h with DON (0, 200 or 2000 ng/mL) from apical or basolateral side. TEER values are given as kOhm per insert (membrane area 4.5 cm²) with 1 kOhm being the level of confluence. Mean±SEM from 5 separate experiments. Significant differences to untreated control were calculated by ANOVA and Dunnett's post hoc test (** p≤0.01).

Figure 2. Number of regulated IPEC-J2 genes after 72 h apical and basolateral DON treatment analysed by microarray.

Membrane cultures were treated with 200 and 2000 ng/mL DON from apical and basolateral side for 72 h. Isolated mRNA was analysed with GeneChip® Porcine Genome Array. Number of significantly (p<0.05) up- or down-regulated genes in comparison to untreated control genes are given. Values represent numbers of annotated and non-annotated genes of three independent experiments.

Figure 3. Genes commonly regulated in different treatment groups (Venn diagram).

Number of genes commonly regulated in response to (A) 200 ng/mL apical or basolateral DON application, (B) 200 or 2000 ng/mL apical DON application, (C) 200 or 2000 ng/mL basolateral DON application and (D) 2000 ng/mL apical or basolateral DON application.

Figure 4. Expression of selected genes measured by microarray and qPCR in membrane cultured IPEC-J2 cells regulated in all DON treatments.

Regulation of transcripts of (A) TRA1 and (B) CAV2 in response to apical and basolateral DON (200 and 2000 ng/mL) treatment. mRNA levels are given as fold increase or decrease over untreated control. Bars represent the means of 3 (microarray) and 5 (qPCR) independent experiments (±SEM). Significant differences to untreated control were calculated by ANOVA and Dunnett's post hoc test (* p≤0.05; ** p≤0.01).

Figure 5. Expression of selected metabolic genes measured by microarray and qPCR in membrane cultured IPEC-J2 cells in response to apical and basolateral DON application.

Regulation of transcripts of (A) PDHA1, (B) SDHB and (C) CYC1 in response to apical and basolateral DON (200 and 2000 ng/mL) treatment. mRNA levels are given as fold increase over untreated control. Bars represent means of 3 (microarray) and 5 (qPCR) independent experiments (±SEM). Significant differences to untreated control were calculated by ANOVA and Dunnett's post hoc test (* p≤0.05; ** p≤0.01).

Figure 6. Expression of selected genes of the genetic information flow measured by microarray and qPCR in membrane cultured IPEC-J2 cells in response to apical and basolateral DON application.

Regulation of transcripts of (A) RPL10A and (B) LIG1 in response to apical and basolateral DON (200 and 2000 ng/mL) treatment. mRNA levels are given as fold increase over untreated control. Bars represent means of 3 (microarray) and 5 (qPCR) independent experiments (±SEM). Significant differences to untreated control were calculated by ANOVA and Dunnett's post hoc test (* p≤0.05; ** p≤0.01).

Figure 7. Expression of selected genes of cellular processes by microarray and qPCR in membrane cultured IPEC-J2 cells in response to apical and basolateral DON application.

Regulation of transcripts of (A) LAMP2 (lysosomes), (B) CDKN1A (cell cycle), (C) CTNNB1 (focal adhesion) and (D) CLDN3 (tight junction) in response to apical and basolateral DON (200 and 2000 ng/mL) treatment. The mRNA levels are given as fold increase over untreated control. Bars represent means of 3 (microarray) and 5 (qPCR) independent experiments (±SEM). Significant differences to untreated control were calculated by ANOVA and Dunnett's post test (* p≤0.05; ** p≤0.01).