Ubiquitin-proteasome inhibitor enhances tumour necrosis factor-alpha-induced apoptosis in rat gastric epithelial cells.
BACKGROUND
Tumour necrosis factor (TNF-alpha) is a candidate factor for involvement in inflammation-mediated gastric mucosal injury. However, the effect of this cytokine on gastric epithelial cells has been poorly investigated. In the present study, we examined whether gastric epithelial cells are resistant to TNF-alpha-induced apoptosis, and whether this resistance is related to ubiquitin-proteasome-associated nuclear factor-kappaB (NF-kappaB) activation.


METHODS
The rat gastric mucosal cell line RGM-1 was grown in DMEM/F12 medium supplemented with 10% FCS. Confluent monolayers of cells were pretreated or not for 60 min with PSI, a peptide aldehyde known to specifically inhibit the chymotrypsin-like activity of 26S proteasome. Cells were subsequently stimulated with recombinant rat TNF-alpha and their viability was determined by WST-1 assay. Apoptosis was confirmed by fluorescence microscopy after staining with Hoechst 33342 and propidium iodide, and DNA fragmentation was determined by flow cytometry using an APO-BRDU kit. IkappaB-alpha and the p65 binding subunit of NF-kappaB were detected by Western blots.


RESULTS
Twenty-four-hour incubation with TNF-alpha alone or PSI alone did not affect the cell viability of RGM-1 cells. Pretreatment with PSI significantly enhanced the level of apoptosis induced by TNF-alpha. In RGM-1 cells treated with TNF-alpha, cytoplasmic IkappaB-alpha decreased and p65 in nuclear extracts increased markedly 30 min after cytokine stimulation. Pretreatment with PSI at 12.5 micromol/L blocked these TNF-alpha-induced changes.


CONCLUSIONS
PSI enhances TNF-alpha-induced apoptosis through inhibition of NF-kappaB activation in RGM-1 cells.
Naito
Y
Y
First Department of Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Japan. ynaito@koto.kpu-m.ac.jp
Handa
O
O
Takagi
T
T
Ishikawa
T
T
Imamoto
E
E
Nakagawa
S
S
Yamaguchi
T
T
Yoshida
N
N
Matsui
H
H
Yoshikawa
T
T
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002135:Calcium-Binding Proteins; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003546:Cysteine Endopeptidases; D004268:DNA-Binding Proteins / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D005753:Gastric Mucosa / Q000166:cytology / Q000378:metabolism*; D020672:I-kappa B Proteins; D008562:Membrane Glycoproteins / Q000378:metabolism; D008858:Microscopy, Phase-Contrast; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D050863:Synaptotagmin I; D050857:Synaptotagmins; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D025801:Ubiquitin / Q000037:antagonists & inhibitors*
D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D008562:Membrane Glycoproteins; D009097:Multienzyme Complexes; D016328:NF-kappa B; D009419:Nerve Tissue Proteins; C000606206:Nfkbia protein, rat; D009842:Oligopeptides; D050863:Synaptotagmin I; D014409:Tumor Necrosis Factor-alpha; D025801:Ubiquitin; C090535:benzyloxycarbonyl-isoleucyl-glutamyl(O-tert-butyl)-alanyl-leucinal; D050857:Synaptotagmins; D000072000:NF-KappaB Inhibitor alpha; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex