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A Novel Zak Knockout Mouse with a Defective Ribotoxic Stress Response

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−/− 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.


BMDMs from C57BL/6 zak−/− mice do not induce the RSR or RSR-associated gene expression. Panel A: Western blot for phosphorylated JNK1/2 and p38 performed following intoxication of wild-type zak+/+ (wt) or zak−/− BMDMs with 1 μg/mL (~17 μM) or 10 ng/mL (~0.17 μM) ricin for 4 h in serum-free BM culture media. Band intensity for phosphorylated JNK1/2 and p38 was normalized against that for GAPDH loading controls. Normalized band intensity is shown for phosphorylated JNK1/2 (white bars) and phosphorylated p38 (black bars). Band intensity was measured using ImageJ 1.46r software (National Insitutes of Health, Bethesda, MD, USA). Panel B: qRT-PCR was used to detect zak, c-jun, and cxcl-1 mRNAs from wild-type or zak−/− BMDMs. β-actin was used as a reference gene. Ricin intoxication was performed in BM culture media with the addition of 10 ng/mL (~0.17 μM) ricin for a period of 4 h. The white and black bars represent datum from wt and zak−/− BMDMs, respectively. The graphs were generated from three technical replicates for each target mRNA. For Panels 1A and 1B, numerical data were processed using Microsoft Excel for Mac, Version 14.6.5, 2011 (Microsoft Corporation, Cambridge, MA, USA) and Prism for Mac, Version 5.0d, 2010 (GraphPad Software, Inc., La Jolla, CA 92037 USA), respectively.
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toxins-08-00259-f001: BMDMs from C57BL/6 zak−/− mice do not induce the RSR or RSR-associated gene expression. Panel A: Western blot for phosphorylated JNK1/2 and p38 performed following intoxication of wild-type zak+/+ (wt) or zak−/− BMDMs with 1 μg/mL (~17 μM) or 10 ng/mL (~0.17 μM) ricin for 4 h in serum-free BM culture media. Band intensity for phosphorylated JNK1/2 and p38 was normalized against that for GAPDH loading controls. Normalized band intensity is shown for phosphorylated JNK1/2 (white bars) and phosphorylated p38 (black bars). Band intensity was measured using ImageJ 1.46r software (National Insitutes of Health, Bethesda, MD, USA). Panel B: qRT-PCR was used to detect zak, c-jun, and cxcl-1 mRNAs from wild-type or zak−/− BMDMs. β-actin was used as a reference gene. Ricin intoxication was performed in BM culture media with the addition of 10 ng/mL (~0.17 μM) ricin for a period of 4 h. The white and black bars represent datum from wt and zak−/− BMDMs, respectively. The graphs were generated from three technical replicates for each target mRNA. For Panels 1A and 1B, numerical data were processed using Microsoft Excel for Mac, Version 14.6.5, 2011 (Microsoft Corporation, Cambridge, MA, USA) and Prism for Mac, Version 5.0d, 2010 (GraphPad Software, Inc., La Jolla, CA 92037 USA), respectively.

Mentions: Based on these promising findings, we backcrossed the zak−/− mice over 11 generations onto a C57BL/6 genetic background, and mated the resulting heterozygote zak+/− mice to generate a congenic C57BL/6 zak−/− population. The C57BL/6 zak−/− strain displayed no defect in fecundity or overall growth and development. However, compared to that of wild-type C57BL/6 mice (i.e., zak+/+), BMDMs from the zak−/− C57BL/6 mice demonstrated a distinct absence of the RSR following ricin treatment, as measured by absent or decreased phosphorylation of p38 and JNK1/2 (Figure 1A). These results mirror previous studies in HCT-8 and Vero cells, where pretreatment with the ZAK-specific inhibitor DHP-2 or siRNA knockdown of zak blocked or decreased ricin-induced p38 and JNKs activation [29].


A Novel Zak Knockout Mouse with a Defective Ribotoxic Stress Response
BMDMs from C57BL/6 zak−/− mice do not induce the RSR or RSR-associated gene expression. Panel A: Western blot for phosphorylated JNK1/2 and p38 performed following intoxication of wild-type zak+/+ (wt) or zak−/− BMDMs with 1 μg/mL (~17 μM) or 10 ng/mL (~0.17 μM) ricin for 4 h in serum-free BM culture media. Band intensity for phosphorylated JNK1/2 and p38 was normalized against that for GAPDH loading controls. Normalized band intensity is shown for phosphorylated JNK1/2 (white bars) and phosphorylated p38 (black bars). Band intensity was measured using ImageJ 1.46r software (National Insitutes of Health, Bethesda, MD, USA). Panel B: qRT-PCR was used to detect zak, c-jun, and cxcl-1 mRNAs from wild-type or zak−/− BMDMs. β-actin was used as a reference gene. Ricin intoxication was performed in BM culture media with the addition of 10 ng/mL (~0.17 μM) ricin for a period of 4 h. The white and black bars represent datum from wt and zak−/− BMDMs, respectively. The graphs were generated from three technical replicates for each target mRNA. For Panels 1A and 1B, numerical data were processed using Microsoft Excel for Mac, Version 14.6.5, 2011 (Microsoft Corporation, Cambridge, MA, USA) and Prism for Mac, Version 5.0d, 2010 (GraphPad Software, Inc., La Jolla, CA 92037 USA), respectively.
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toxins-08-00259-f001: BMDMs from C57BL/6 zak−/− mice do not induce the RSR or RSR-associated gene expression. Panel A: Western blot for phosphorylated JNK1/2 and p38 performed following intoxication of wild-type zak+/+ (wt) or zak−/− BMDMs with 1 μg/mL (~17 μM) or 10 ng/mL (~0.17 μM) ricin for 4 h in serum-free BM culture media. Band intensity for phosphorylated JNK1/2 and p38 was normalized against that for GAPDH loading controls. Normalized band intensity is shown for phosphorylated JNK1/2 (white bars) and phosphorylated p38 (black bars). Band intensity was measured using ImageJ 1.46r software (National Insitutes of Health, Bethesda, MD, USA). Panel B: qRT-PCR was used to detect zak, c-jun, and cxcl-1 mRNAs from wild-type or zak−/− BMDMs. β-actin was used as a reference gene. Ricin intoxication was performed in BM culture media with the addition of 10 ng/mL (~0.17 μM) ricin for a period of 4 h. The white and black bars represent datum from wt and zak−/− BMDMs, respectively. The graphs were generated from three technical replicates for each target mRNA. For Panels 1A and 1B, numerical data were processed using Microsoft Excel for Mac, Version 14.6.5, 2011 (Microsoft Corporation, Cambridge, MA, USA) and Prism for Mac, Version 5.0d, 2010 (GraphPad Software, Inc., La Jolla, CA 92037 USA), respectively.
Mentions: Based on these promising findings, we backcrossed the zak−/− mice over 11 generations onto a C57BL/6 genetic background, and mated the resulting heterozygote zak+/− mice to generate a congenic C57BL/6 zak−/− population. The C57BL/6 zak−/− strain displayed no defect in fecundity or overall growth and development. However, compared to that of wild-type C57BL/6 mice (i.e., zak+/+), BMDMs from the zak−/− C57BL/6 mice demonstrated a distinct absence of the RSR following ricin treatment, as measured by absent or decreased phosphorylation of p38 and JNK1/2 (Figure 1A). These results mirror previous studies in HCT-8 and Vero cells, where pretreatment with the ZAK-specific inhibitor DHP-2 or siRNA knockdown of zak blocked or decreased ricin-induced p38 and JNKs activation [29].

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−/− 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.