Regulation of c-Jun NH2-terminal kinases in isolated canine gastric parietal cells.
c-Jun NH2-terminal kinases (JNKs) are protein kinases that are activated by a wide variety of extracellular signals. This study investigated the expression and regulation of JNKs in isolated gastric canine parietal cells. Western blot analysis of cell lysates from highly purified (>95%) parietal cells with an antibody recognizing JNK1 and to a lesser degree JNK2 revealed the presence of two bands of 46 and 54 kDa, respectively. JNK1 activity was quantitated by immunoprecipitation and in-gel kinase assays. Of the different agents tested, carbachol was the most potent inducer of JNK1 activity, whereas histamine and epidermal growth factor induced weaker responses. The proinflammatory cytokine tumor necrosis factor-alpha stimulated JNK1 but had no effect on extracellular signal-regulated kinase (ERK2) induction, suggesting that activation of JNK1 might represent an important event in mediation of the inflammatory response in the stomach. The action of carbachol was dose (0.1-100 microM) and time dependent, with a maximal stimulatory effect (fourfold) detected after 30 min of incubation and sustained for 2 h. Addition of the specific protein kinase C (PKC) inhibitor GF109203X did not affect the stimulatory action of carbachol. The intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid-AM inhibited carbachol induction of JNK1 activity by 60%. Thapsigargin (1 microM), an intracellular Ca2+-rising agent, induced JNK1 activity more than threefold. Carbachol activation of JNK1 resulted in induction of c-Jun (protein) transcriptional activity and in stimulation of parietal cell mRNA content of c-jun. In conclusion, our data indicate that carbachol induces JNK activity in gastric parietal cells via intracellular Ca2+-dependent, PKC-independent pathways, leading to induction of c-jun gene expression via phosphorylation and transcriptional activation of c-Jun.
Nagahara
A
A
Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0682, USA.
Wang
L
L
Del Valle
J
J
Todisco
A
A
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000096:biosynthesis / Q000235:genetics*; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004285:Dogs; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005748:Gastric Fundus; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D016758:Genes, jun / Q000187:drug effects; D006632:Histamine / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases; D008301:Maleimides / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1; D048056:Mitogen-Activated Protein Kinase 9; D020928:Mitogen-Activated Protein Kinases; D010295:Parietal Cells, Gastric / Q000187:drug effects / Q000201:enzymology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011494:Protein Kinases / Q000096:biosynthesis / Q000235:genetics*; D016755:Proto-Oncogene Proteins c-jun / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D019284:Thapsigargin / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D011994:Recombinant Proteins; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D004815:Epidermal Growth Factor; D019284:Thapsigargin; D006632:Histamine; D002217:Carbachol; D011494:Protein Kinases; D048056:Mitogen-Activated Protein Kinase 9; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases; C070515:bisindolylmaleimide I