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A Novel Zak Knockout Mouse with a Defective Ribotoxic Stress Response

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ABSTRACT

Ricin activates the proinflammatory ribotoxic stress response through the mitogen activated protein 3 kinase (MAP3K) ZAK, resulting in activation of mitogen activated protein kinases (MAPKs) p38 and JNK1/2. We had a novel zak−/− mouse generated to study the role of ZAK signaling in vivo during ricin intoxication. To characterize this murine strain, we intoxicated zak−/− and zak+/+ bone marrow–derived murine macrophages with ricin, measured p38 and JNK1/2 activation by Western blot, and measured zak, c-jun, and cxcl-1 expression by qRT-PCR. To determine whether zak−/− mice differed from wild-type mice in their in vivo response to ricin, we performed oral ricin intoxication experiments with zak+/+ and zak−/− mice, using blinded histopathology scoring of duodenal tissue sections to determine differences in tissue damage. Unlike macrophages derived from zak+/+ mice, those derived from the novel zak−/− strain fail to activate p38 and JNK1/2 and have decreased c-jun and cxcl-1 expression following ricin intoxication. Furthermore, compared with zak+/+ mice, zak−/− mice have decreased duodenal damage following in vivo ricin challenge. zak−/− mice demonstrate a distinct ribotoxic stress–associated phenotype in response to ricin and therefore provide a new animal model for in vivo studies of ZAK signaling.

No MeSH data available.


C57BL/6 backcrossed zak−/− mice have a lower duodenal pathology score than zak+/+ (wt) mice following oral intoxication with ricin. Mice were gavaged with 10 mg/kg ricin (oral murine LD50 ~10–30 mg/kg [23,33,34]) or vehicle (PBS) and sacrificed ~22 h following gavage. Histopathology scoring as defined in Methods was performed on H&E stained duodenal sections. The number of animals in each group are as follows: wt gavaged with vehicle = 8; zak−/− gavaged with vehicle = 6; wt gavaged with ricin = 10; zak−/− gavaged with ricin = 8. Horizontal lines mark the mean score for each treatment group. Data reflect a compilation of two independent experiments. * Indicates significance p < 0.05 by one-way ANOVA and Tukey’s multiple comparison test. Statistical analysis was performed using Prism for Mac, Version 5.0d, 2010 (GraphPad Software, Inc., La Jolla, CA 92037 USA).
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toxins-08-00259-f002: C57BL/6 backcrossed zak−/− mice have a lower duodenal pathology score than zak+/+ (wt) mice following oral intoxication with ricin. Mice were gavaged with 10 mg/kg ricin (oral murine LD50 ~10–30 mg/kg [23,33,34]) or vehicle (PBS) and sacrificed ~22 h following gavage. Histopathology scoring as defined in Methods was performed on H&E stained duodenal sections. The number of animals in each group are as follows: wt gavaged with vehicle = 8; zak−/− gavaged with vehicle = 6; wt gavaged with ricin = 10; zak−/− gavaged with ricin = 8. Horizontal lines mark the mean score for each treatment group. Data reflect a compilation of two independent experiments. * Indicates significance p < 0.05 by one-way ANOVA and Tukey’s multiple comparison test. Statistical analysis was performed using Prism for Mac, Version 5.0d, 2010 (GraphPad Software, Inc., La Jolla, CA 92037 USA).

Mentions: Because the ribotoxic stress response is associated with both inflammatory and apoptotic responses [12,31], we hypothesized that a deficiency in this innate response to ribosomal damage would result in reduced ricin-associated tissue damage. Therefore, we employed a mouse model of oral ricin intoxication described by Yoder et al. [23] and also by Flora et al. [32]. Following oral ricin administration we found that zak−/− mice did indeed have a decrease in duodenal pathology compared to congenic wild-type zak+/+ mice (Figure 2). The data suggests that the zak−/− mice have a distinct ZAK-associated phenotype, possibly resulting in the observed decrease in epithelial abnormalities during ricin intoxication. Interestingly, vehicle-treated zak−/− mice demonstrated increased histopathologic abnormalities compared with vehicle-treated zak+/+ mice.


A Novel Zak Knockout Mouse with a Defective Ribotoxic Stress Response
C57BL/6 backcrossed zak−/− mice have a lower duodenal pathology score than zak+/+ (wt) mice following oral intoxication with ricin. Mice were gavaged with 10 mg/kg ricin (oral murine LD50 ~10–30 mg/kg [23,33,34]) or vehicle (PBS) and sacrificed ~22 h following gavage. Histopathology scoring as defined in Methods was performed on H&E stained duodenal sections. The number of animals in each group are as follows: wt gavaged with vehicle = 8; zak−/− gavaged with vehicle = 6; wt gavaged with ricin = 10; zak−/− gavaged with ricin = 8. Horizontal lines mark the mean score for each treatment group. Data reflect a compilation of two independent experiments. * Indicates significance p < 0.05 by one-way ANOVA and Tukey’s multiple comparison test. Statistical analysis was performed using Prism for Mac, Version 5.0d, 2010 (GraphPad Software, Inc., La Jolla, CA 92037 USA).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037485&req=5

toxins-08-00259-f002: C57BL/6 backcrossed zak−/− mice have a lower duodenal pathology score than zak+/+ (wt) mice following oral intoxication with ricin. Mice were gavaged with 10 mg/kg ricin (oral murine LD50 ~10–30 mg/kg [23,33,34]) or vehicle (PBS) and sacrificed ~22 h following gavage. Histopathology scoring as defined in Methods was performed on H&E stained duodenal sections. The number of animals in each group are as follows: wt gavaged with vehicle = 8; zak−/− gavaged with vehicle = 6; wt gavaged with ricin = 10; zak−/− gavaged with ricin = 8. Horizontal lines mark the mean score for each treatment group. Data reflect a compilation of two independent experiments. * Indicates significance p < 0.05 by one-way ANOVA and Tukey’s multiple comparison test. Statistical analysis was performed using Prism for Mac, Version 5.0d, 2010 (GraphPad Software, Inc., La Jolla, CA 92037 USA).
Mentions: Because the ribotoxic stress response is associated with both inflammatory and apoptotic responses [12,31], we hypothesized that a deficiency in this innate response to ribosomal damage would result in reduced ricin-associated tissue damage. Therefore, we employed a mouse model of oral ricin intoxication described by Yoder et al. [23] and also by Flora et al. [32]. Following oral ricin administration we found that zak−/− mice did indeed have a decrease in duodenal pathology compared to congenic wild-type zak+/+ mice (Figure 2). The data suggests that the zak−/− mice have a distinct ZAK-associated phenotype, possibly resulting in the observed decrease in epithelial abnormalities during ricin intoxication. Interestingly, vehicle-treated zak−/− mice demonstrated increased histopathologic abnormalities compared with vehicle-treated zak+/+ mice.

View Article: PubMed Central - PubMed

ABSTRACT

Ricin activates the proinflammatory ribotoxic stress response through the mitogen activated protein 3 kinase (MAP3K) ZAK, resulting in activation of mitogen activated protein kinases (MAPKs) p38 and JNK1/2. We had a novel zak&minus;/&minus; mouse generated to study the role of ZAK signaling in vivo during ricin intoxication. To characterize this murine strain, we intoxicated zak&minus;/&minus; and zak+/+ bone marrow&ndash;derived murine macrophages with ricin, measured p38 and JNK1/2 activation by Western blot, and measured zak, c-jun, and cxcl-1 expression by qRT-PCR. To determine whether zak&minus;/&minus; mice differed from wild-type mice in their in vivo response to ricin, we performed oral ricin intoxication experiments with zak+/+ and zak&minus;/&minus; mice, using blinded histopathology scoring of duodenal tissue sections to determine differences in tissue damage. Unlike macrophages derived from zak+/+ mice, those derived from the novel zak&minus;/&minus; strain fail to activate p38 and JNK1/2 and have decreased c-jun and cxcl-1 expression following ricin intoxication. Furthermore, compared with zak+/+ mice, zak&minus;/&minus; mice have decreased duodenal damage following in vivo ricin challenge. zak&minus;/&minus; mice demonstrate a distinct ribotoxic stress&ndash;associated phenotype in response to ricin and therefore provide a new animal model for in vivo studies of ZAK signaling.

No MeSH data available.