mkp-1 encoding mitogen-activated protein kinase phosphatase 1, a verotoxin 1 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 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.
Kojima
S
S
Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka Suita-City, Osaka, Japan 565-0871, USA.
Yanagihara
I
I
Kono
G
G
Sugahara
T
T
Nasu
H
H
Kijima
M
M
Hattori
A
A
Kodama
T
T
Nagayama
K I
KI
Honda
T
T
D000818:Animals; D000841:Anisomycin / Q000494:pharmacology; D001427:Bacterial Toxins / Q000378:metabolism* / Q000494:pharmacology; D018938:Caco-2 Cells; D018797:Cell Cycle Proteins; D054638:Dual Specificity Phosphatase 1; D015870:Gene Expression; D006801:Humans; D017874:Immediate-Early Proteins / Q000096:biosynthesis / Q000235:genetics*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis / Q000235:genetics*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D012333:RNA, Messenger; D012339:RNA, Ribosomal, 28S / Q000037:antagonists & inhibitors; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D022622:Shiga Toxin 1
D001427:Bacterial Toxins; D018797:Cell Cycle Proteins; D017874:Immediate-Early Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D012339:RNA, Ribosomal, 28S; D022622:Shiga Toxin 1; D000841:Anisomycin; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C517019:DUSP1 protein, human; D054638:Dual Specificity Phosphatase 1; D017027:Protein Tyrosine Phosphatases