Ethanol induced NF-{kappa}B activation protects against cell injury in cultured rat gastric mucosal epithelium.
Ethanol is a well-established irritant inducing inflammation in gastric mucosa, but the effects at the cellular level remain unclear. This study investigates NF-kappaB activation in gastric mucosal cells by ethanol and assesses the effects of heat shock pretreatment in this ulcerogenic situation. Rat gastric mucosal epithelia were exposed to ethanol for different time periods. Heat shock was induced by incubating the cells at 42 degrees C for 1 h prior to the experiments. For evaluation of NF-kappaB activation, the nuclear fraction of the cell lysates was analyzed with an EMSA or an ELISA-based assay. Caspase-3 (a promoter of apoptosis) activity was measured with a time-resolved fluorescence based assay, cell viability with a tetrazolium assay, and cell membrane integrity with a LDH assay. Ethanol (1-5%) induced NF-kappaB activation, reaching a maximum after 3 h, and also led to moderately increased COX-2 expression. Heat shock pretreatment and the intracellular calcium chelator BAPTA were able to inhibit ethanol-induced NF-kappaB activation. Heat shock pretreatment decreased ethanol-induced caspase-3 activation, decreased cell membrane damage, and retained cellular viability. Inhibition of NF-kappaB activation by NEMO-binding peptide, by decreasing RelA expression, or by inhibiting COX-2 activity by CAY-14040 promoted the effects of ethanol, such as increased caspase-3 activity and decreased cell viability. In conclusion, ethanol induces NF-kappaB activation via a calcium-dependent pathway and induces COX-2 expression. Inhibition of the NF-kappaB activation or COX-2 activity potentiates apoptosis and cell damage induced by ethanol, suggesting a protective role for NF-kappaB activation and COX-2 expression.
Mustonen
Harri
H
Dept. of Surgery, Helsinki Univ. Central Hospital, Box 700, 00029 HUS, Helsinki, Finland. harri.mustonen@helsinki.fi
Hietaranta
Antti
A
Puolakkainen
Pauli
P
Kemppainen
Esko
E
Paimela
Hannu
H
Kiviluoto
Tuula
T
Kivilaakso
Eero
E
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D051546:Cyclooxygenase 2 / Q000378:metabolism; D052246:Cyclooxygenase 2 Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D000431:Ethanol / Q000633:toxicity*; D005753:Gastric Mucosa / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D006360:Heat-Shock Proteins / Q000378:metabolism; D006419:Heme Oxygenase (Decyclizing) / Q000378:metabolism; D006358:Hot Temperature; D007509:Irritants / Q000633:toxicity*; D007555:Isoxazoles / Q000494:pharmacology; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D013450:Sulfones / Q000494:pharmacology; D013997:Time Factors; D051996:Transcription Factor RelA / Q000378:metabolism
C501048:3-(4-methylsulfonylphenyl)-4-phenyl-5-trifluoromethylisoxazole; D002614:Chelating Agents; D052246:Cyclooxygenase 2 Inhibitors; D006360:Heat-Shock Proteins; D007509:Irritants; D007555:Isoxazoles; D016328:NF-kappa B; D010455:Peptides; D034741:RNA, Small Interfering; C498145:Rela protein, rat; D013450:Sulfones; D051996:Transcription Factor RelA; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D000431:Ethanol; D004533:Egtazic Acid; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D006419:Heme Oxygenase (Decyclizing); C491523:Hmox1 protein, rat; D051546:Cyclooxygenase 2; C489691:Ptgs2 protein, rat; D053148:Caspase 3; D002118:Calcium