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mkp-1 encoding mitogen-activated protein kinase phosphatase 1, a verotoxin 1 responsive gene, detected by
S Kojima1, I Yanagihara, G Kono
1Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka Suita-City, Osaka, Japan 565-0871, USA.
Abstract:
The major cytotoxic effect of the verotoxins (VTs) produced by strains of VT-producing Escherichia coli is the inhibition of host-cell protein synthesis, but VTs are also suspected to play a role in apoptotic cell signaling and cytokine release. Four differentially expressed genes, including mkp-1 (encoding mitogen-activated protein kinase phospatase 1), were detected by differential display reverse transcription-PCR (DD RT-PCR) stimulated by VT1 in Caco-2 cells. Northern blot analysis showed the induction of mkp-1 mRNA 6 h after VT1 stimulation. Neither mutant VT1 (mutVT1), harboring two mutations in the A subunit (E167Q-R170L), nor cycloheximide induced mkp-1 mRNA, but mkp-1 mRNA was detected with both wild-type VT1 (wtVT1) and anisomycin, a 28S rRNA inhibitor. Therefore, we concluded that the A subunit of VT1 was essential for mkp-1 induction. Increased amounts of phosphorylated c-Jun protein were also found with wtVT1 and anisomycin. Although the precise mechanism of induction of MKP-1 is unknown, we hypothesized that 28S rRNA not only was a sensor for ribotoxic stress, but also was involved in the signal cascade of MKP-1. This is the first report of detection by DD RT-PCR of cellular genes induced by bacterial toxins.
Insights
Verotoxins (VTs) from E. coli induce mkp-1 gene expression in Caco-2 cells. The VT1 A subunit is essential for this induction, suggesting a role for 28S rRNA in the signaling pathway.
Area of Science:
- Microbiology
- Molecular Biology
- Cell Biology
Background:
- Verotoxins (VTs) from Escherichia coli primarily inhibit host-cell protein synthesis.
- VTs are also implicated in apoptotic signaling and cytokine release.
- Understanding cellular responses to bacterial toxins is crucial.
Purpose of the Study:
- To identify cellular genes induced by VT1 in Caco-2 cells using differential display reverse transcription-PCR (DD RT-PCR).
- To investigate the role of the VT1 A subunit in gene induction.
- To explore the potential involvement of 28S rRNA in the signaling pathway.
Main Methods:
- Differential display reverse transcription-PCR (DD RT-PCR) to detect gene expression changes.
- Northern blot analysis to confirm mRNA induction.
- Treatment with wild-type VT1 (wtVT1), mutant VT1 (mutVT1), cycloheximide, and anisomycin.
Main Results:
- Four differentially expressed genes, including mkp-1 (mitogen-activated protein kinase phosphatase 1), were identified.
- mkp-1 mRNA was induced by wtVT1 and anisomycin, but not by mutVT1 or cycloheximide.
- Phosphorylated c-Jun protein increased with wtVT1 and anisomycin treatment.
Conclusions:
- The A subunit of VT1 is essential for the induction of mkp-1.
- 28S rRNA may act as a sensor for ribotoxic stress and participate in the MKP-1 signaling cascade.
- This study is the first to report the detection of toxin-induced cellular genes via DD RT-PCR.