mkp-1 encoding mitogen-activated protein kinase phosphatase 1, a verotoxin1 responsive gene, detected by differential display reverse transcription-PCR in Caco-2 cells

The major cytotoxic effect of the verotoxins (VTs) produced by strains of V T-producing Escherichia coli is the inhibition of host-cell protein synthes is, but VTs are also suspected to play a role in apoptotic cell signaling a nd cytokine release. Four differentially expressed genes, including mkp-1 ( encoding mitogen-activated protein kinase phospatase 1), were detected by d ifferential 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 muta tions 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 w as essential for mkp-1 induction. Increased amounts of phosphorylated c-Jun protein were also found with wtVT1 and anisomycin. Although the precise me chanism 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 signa l cascade of MKP-1. This is the first report of detection by DD RT-PCR of c ellular genes induced by bacterial toxins.