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Early Activation of MAPK p44/42 Is Partially Involved in DON-Induced Disruption of the Intestinal Barrier Function and Tight Junction Network

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ABSTRACT

Deoxynivalenol (DON), produced by the plant pathogens Fusariumgraminearum and Fusarium culmorum, is one of the most common mycotoxins, contaminating cereal and cereal-derived products. Although worldwide contamination of food and feed poses health threats to humans and animals, pigs are particularly susceptible to this mycotoxin. DON derivatives, such as deepoxy-deoxynivalenol (DOM-1), are produced by bacterial transformation of certain intestinal bacteria, which are naturally occurring or applied as feed additives. Intestinal epithelial cells are the initial barrier against these food- and feed-borne toxins. The present study confirms DON-induced activation of MAPK p44/42 and inhibition of p44/42 by MAPK-inhibitor U0126 monoethanolate. Influence of DON and DOM-1 on transepithelial electrical resistance (TEER), viability and expression of seven tight junction proteins (TJ), as well as the potential of U0126 to counteract DON-induced effects, was assessed. While DOM-1 showed no effect, DON significantly reduced TEER of differentiated IPEC-J2 and decreased expression of claudin-1 and -3, while leaving claudin-4; ZO-1, -2, and -3 and occludin unaffected. Inhibition of p44/42 counteracted DON-induced TEER decrease and restored claudin-3, but not claudin-1 expression. Therefore, effects of DON on TEER and claudin-3 are at least partially p44/42 mediated, while effects on viability and claudin-1 are likely mediated via alternative pathways.

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U0126 monoethanolate (U0126) (10 µM) counteracted deoxynivalenol (DON)-induced p44/42 mitogen activated protein kinase (MAPK) (Erk1/2), but not p38 MAPK activation. Differentiated IPEC-J2 were either pretreated with U0126 (10 µM) or complete cultivation medium before addition of DON (20 µM) for 1 h. Alternatively, differentiated IPEC-J2 were treated with deepoxy-deoxynivalenol (DOM-1) (100 µM) for 1 h. Phosphorylation of p44/42 MAPK (ERK1/2) (p-p44/42) and p38 MAPK (p-p38) was determined by (A) immunoblotting and (B) densitometry after normalization with endogenous signals (total p44/42 (t-p44/42) or total p38 (t-p38)) and ß-actin. Data were normalized to control and represent mean ± SD, n = 4. Statistically significant differences (p < 0.05) are indicated by different letters (a,b).
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toxins-08-00264-f002: U0126 monoethanolate (U0126) (10 µM) counteracted deoxynivalenol (DON)-induced p44/42 mitogen activated protein kinase (MAPK) (Erk1/2), but not p38 MAPK activation. Differentiated IPEC-J2 were either pretreated with U0126 (10 µM) or complete cultivation medium before addition of DON (20 µM) for 1 h. Alternatively, differentiated IPEC-J2 were treated with deepoxy-deoxynivalenol (DOM-1) (100 µM) for 1 h. Phosphorylation of p44/42 MAPK (ERK1/2) (p-p44/42) and p38 MAPK (p-p38) was determined by (A) immunoblotting and (B) densitometry after normalization with endogenous signals (total p44/42 (t-p44/42) or total p38 (t-p38)) and ß-actin. Data were normalized to control and represent mean ± SD, n = 4. Statistically significant differences (p < 0.05) are indicated by different letters (a,b).

Mentions: The ability of DON (20 µM) to trigger early activation of p44/42 MAPK (ERK1/2) and p38 MAPK signaling in differentiated IPEC-J2 was tested via immunoblotting (Figure 2). Treatment of IPEC-J2 with 20 µM DON for 1 h led to a significant activation of p44/42 MAPK (2.567 ± 0.33, p = 0.025) and p38 MAPK (4.11 ± 0.56, p = 0.000). DOM-1 (100 µM) did not affect the phosphorylation status of either MAPK protein. Pretreatment of differentiated IPEC-J2 with MAPK inhibitor U0126 (10 µM), followed by a 1 h DON exposure in the presence of U0126 (10 µM), prevented DON-induced p44/42 MAPK (ERK1/2) phosphorylation. DON-induced phosphorylation of p38 MAPK (3.15 ± 0.31, p = 0.000) was not affected by U0126 and remained significantly elevated compared to the control.


Early Activation of MAPK p44/42 Is Partially Involved in DON-Induced Disruption of the Intestinal Barrier Function and Tight Junction Network
U0126 monoethanolate (U0126) (10 µM) counteracted deoxynivalenol (DON)-induced p44/42 mitogen activated protein kinase (MAPK) (Erk1/2), but not p38 MAPK activation. Differentiated IPEC-J2 were either pretreated with U0126 (10 µM) or complete cultivation medium before addition of DON (20 µM) for 1 h. Alternatively, differentiated IPEC-J2 were treated with deepoxy-deoxynivalenol (DOM-1) (100 µM) for 1 h. Phosphorylation of p44/42 MAPK (ERK1/2) (p-p44/42) and p38 MAPK (p-p38) was determined by (A) immunoblotting and (B) densitometry after normalization with endogenous signals (total p44/42 (t-p44/42) or total p38 (t-p38)) and ß-actin. Data were normalized to control and represent mean ± SD, n = 4. Statistically significant differences (p < 0.05) are indicated by different letters (a,b).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037490&req=5

toxins-08-00264-f002: U0126 monoethanolate (U0126) (10 µM) counteracted deoxynivalenol (DON)-induced p44/42 mitogen activated protein kinase (MAPK) (Erk1/2), but not p38 MAPK activation. Differentiated IPEC-J2 were either pretreated with U0126 (10 µM) or complete cultivation medium before addition of DON (20 µM) for 1 h. Alternatively, differentiated IPEC-J2 were treated with deepoxy-deoxynivalenol (DOM-1) (100 µM) for 1 h. Phosphorylation of p44/42 MAPK (ERK1/2) (p-p44/42) and p38 MAPK (p-p38) was determined by (A) immunoblotting and (B) densitometry after normalization with endogenous signals (total p44/42 (t-p44/42) or total p38 (t-p38)) and ß-actin. Data were normalized to control and represent mean ± SD, n = 4. Statistically significant differences (p < 0.05) are indicated by different letters (a,b).
Mentions: The ability of DON (20 µM) to trigger early activation of p44/42 MAPK (ERK1/2) and p38 MAPK signaling in differentiated IPEC-J2 was tested via immunoblotting (Figure 2). Treatment of IPEC-J2 with 20 µM DON for 1 h led to a significant activation of p44/42 MAPK (2.567 ± 0.33, p = 0.025) and p38 MAPK (4.11 ± 0.56, p = 0.000). DOM-1 (100 µM) did not affect the phosphorylation status of either MAPK protein. Pretreatment of differentiated IPEC-J2 with MAPK inhibitor U0126 (10 µM), followed by a 1 h DON exposure in the presence of U0126 (10 µM), prevented DON-induced p44/42 MAPK (ERK1/2) phosphorylation. DON-induced phosphorylation of p38 MAPK (3.15 ± 0.31, p = 0.000) was not affected by U0126 and remained significantly elevated compared to the control.

View Article: PubMed Central - PubMed

ABSTRACT

Deoxynivalenol (DON), produced by the plant pathogens Fusariumgraminearum and Fusarium culmorum, is one of the most common mycotoxins, contaminating cereal and cereal-derived products. Although worldwide contamination of food and feed poses health threats to humans and animals, pigs are particularly susceptible to this mycotoxin. DON derivatives, such as deepoxy-deoxynivalenol (DOM-1), are produced by bacterial transformation of certain intestinal bacteria, which are naturally occurring or applied as feed additives. Intestinal epithelial cells are the initial barrier against these food- and feed-borne toxins. The present study confirms DON-induced activation of MAPK p44/42 and inhibition of p44/42 by MAPK-inhibitor U0126 monoethanolate. Influence of DON and DOM-1 on transepithelial electrical resistance (TEER), viability and expression of seven tight junction proteins (TJ), as well as the potential of U0126 to counteract DON-induced effects, was assessed. While DOM-1 showed no effect, DON significantly reduced TEER of differentiated IPEC-J2 and decreased expression of claudin-1 and -3, while leaving claudin-4; ZO-1, -2, and -3 and occludin unaffected. Inhibition of p44/42 counteracted DON-induced TEER decrease and restored claudin-3, but not claudin-1 expression. Therefore, effects of DON on TEER and claudin-3 are at least partially p44/42 mediated, while effects on viability and claudin-1 are likely mediated via alternative pathways.

No MeSH data available.


Related in: MedlinePlus