Related Experiment Videos
Role of double-stranded RNA-activated protein kinase R (PKR) in deoxynivalenol-induced ribotoxic stress response
Hui-Ren Zhou1, Allan S Lau, James J Pestka
1Departments of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824-1224, USA.
Abstract:
Trichothecene mycotoxins and other protein synthesis inhibitors activate mitogen-activated protein kinase (MAPKs) via a mechanism that has been termed the "ribotoxic stress response." MAPKs are believed to mediate the leukocyte apoptosis that is observed following experimental exposure to these chemical agents in vitro and in vivo. The purpose of this research was to test the hypothesis that double-stranded, RNA-activated protein kinase R (PKR) is a critical upstream mediator of the ribotoxic stress response induced by the trichothecene deoxynivalenol (DON) and other translational inhibitors. DON was found to readily induce phosphorylation of JNK 1/2, ERK 1/2, and p38 in the murine macrophage RAW 264.7 cell line, within 5 min of culture addition, in a concentration-dependent fashion. Effects were maximal from 15 to 30 min and lasted up to 6 h. The translational inhibitors anisomycin and emetine also had similar effects when added to cultures at equipotent concentrations to DON. DON rapidly activated PKR within 1 to 5 min, as evidenced by autophosphorylation and by phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha). Interestingly, the latter effect was associated with rapid degradation of eIF2alpha. Pretreatment of RAW 264.7 cells with two inhibitors of PKR, 2-aminopurine (2-AP) or adenine (Ad), markedly impaired MAPK phosphorylation in RAW 264.7 cells according to the following rank order JNK>p38>ERK. The capacity of DON to induce MAPK phosphorylation was also markedly suppressed in a stable transformant of the human promonocytic U-937 cell line containing an antisense PKR expression vector. This suppression followed a rank order of JNK>p38>ERK in this PKR-deficient cell line when compared to control cells transfected with vector only. Apoptosis induction by DON and two other translational inhibitors, anisomycin and emetine, was almost completely abrogated in PKR-deficient cells. Together, the results indicate that PKR plays a critical upstream role in the ribotoxic stress response inducible by translational inhibitors.
Insights
Protein kinase R (PKR) is crucial for the ribotoxic stress response, mediating mitogen-activated protein kinase (MAPK) activation and apoptosis induced by deoxynivalenol (DON) and other protein synthesis inhibitors.
Area of Science:
- Cellular Biology
- Molecular Toxicology
- Immunology
Background:
- Protein synthesis inhibitors, including trichothecene mycotoxins like deoxynivalenol (DON), trigger a cellular defense mechanism known as the ribotoxic stress response.
- Mitogen-activated protein kinases (MAPKs) are key signaling molecules activated during this response and are implicated in mediating apoptosis following exposure to these toxins.
- The precise upstream mediators of this response, particularly the role of double-stranded RNA-activated protein kinase R (PKR), remain to be fully elucidated.
Purpose of the Study:
- To investigate the hypothesis that protein kinase R (PKR) is a critical upstream regulator of the ribotoxic stress response.
- To determine if PKR mediates the activation of mitogen-activated protein kinases (MAPKs) induced by deoxynivalenol (DON) and other translational inhibitors.
- To assess the role of PKR in the induction of apoptosis by these agents.
Main Methods:
- Utilized the murine macrophage cell line RAW 264.7 and a human promonocytic cell line (U-937) with suppressed PKR expression.
- Administered deoxynivalenol (DON), anisomycin, and emetine to cell cultures and assessed MAPK phosphorylation (JNK, ERK, p38) and PKR activation (autophosphorylation, eIF2alpha phosphorylation).
- Employed PKR inhibitors (2-aminopurine, adenine) and antisense PKR expression vectors to evaluate the functional role of PKR in MAPK activation and apoptosis.
Main Results:
- Deoxynivalenol (DON) rapidly induced phosphorylation of JNK, ERK, and p38 in RAW 264.7 cells in a concentration- and time-dependent manner.
- DON treatment led to rapid activation of PKR, evidenced by autophosphorylation and eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation, followed by eIF2alpha degradation.
- Inhibition or deficiency of PKR significantly impaired DON-induced MAPK phosphorylation (JNK>p38>ERK) and almost completely abrogated apoptosis induced by DON and other translational inhibitors.
Conclusions:
- Protein kinase R (PKR) plays a critical upstream role in the ribotoxic stress response induced by translational inhibitors like deoxynivalenol.
- PKR activation is essential for the subsequent phosphorylation of MAPKs (JNK, p38, ERK) and the induction of apoptosis.
- These findings highlight PKR as a key mediator in cellular responses to protein synthesis-inhibiting toxins.