Angiotensin II stimulates calcium-dependent activation of c-Jun N-terminal kinase.
In GN4 rat liver epithelial cells, angiotensin II (Ang II) and other agonists which activate phospholipase C stimulate tyrosine kinase activity in a calcium-dependent, protein kinase C (PKC)-independent manner. Since Ang II also produces a proliferative response in these cells, we investigated downstream signaling elements traditionally linked to growth control by tyrosine kinases. First, Ang II, like epidermal growth factor (EGF), stimulated AP-1 binding activity in a PKC-independent manner. Because increases in AP-1 can reflect induction of c-Jun and c-Fos, we examined the activity of the mitogen-activated protein (MAP) kinase family members Erk-1 and -2 and the c-Jun N-terminal kinase (JNK), which are known to influence c-Jun and c-Fos transcription. Ang II stimulated MAP kinase (MAPK) activity but only approximately 50% as effectively as EGF; again, these effects were independent of PKC. Ang II also produced a 50- to 200-fold activation of JNK in a PKC-independent manner. Unlike its smaller effect on MAPK, Ang II was approximately four- to sixfold more potent in activating JNK than EGF was. Although others had reported a lack of calcium ionophore-stimulated JNK activity in lymphocytes and several other cell lines, we examined the role of calcium in GN4 cells. The following results suggest that JNK activation in rat liver epithelial cells is at least partially Ca(2+) dependent: (i) norepinephrine and vasopressin hormones that increase inositol 1,4,5-triphosphate stimulated JNK; (ii) both thapsigargin, a compound that produces an intracellular Ca(2+) signal, and Ca(2+) ionophores stimulated a dramatic increase in JNK activity (up to 200-fold); (iii) extracellular Ca(2+) chelation with ethylene glycol tetraacetic acid (EGTA) inhibited JNK activation by ionophore and intracellular chelation with 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl-ester (BAPTA-AM) partially inhibited JNK activation by Ang II or thapsigargin; and (iv) JNK activation by Ang II was inhibited by pretreatment of cells with thapsigargin and EGTA, a procedure which depletes intracellular Ca(2+) stores. JNK activation following Ang II stimulation did not involve calmodulin; either W-7 nor calmidizolium, in concentrations sufficient to inhibit Ca(2+)/calmodulin-dependent kinase II, blocked JNK activation by Ang II. In contrast, genistein, in concentrations sufficient to inhibit Ca(2+)-dependent tyrosine phosphorylation, prevented Ang II and thapsigargin-induced JNK activation. In summary, in GN4 rat liver epithelial cells, Ang II stimulates JNK via a novel Ca(2+)-dependent pathway. The inhibition by genistein suggest that Ca(2+)-dependent tyrosine phosphorylation may modulate the JNK pathway in a cell type-specific manner, particularly in cells with a readily detectable Ca(2+)-regulated tyrosine kinase.
Zohn
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Department of Pharmacology, University of North Carolina at Chapel Hill 27599, USA.
Yu
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Li
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Cox
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Earp
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D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000502:physiology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004848:Epithelium / Q000201:enzymology; D019833:Genistein; D007529:Isoflavones / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D011493:Protein Kinase C / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism
D002147:Calmodulin; D004791:Enzyme Inhibitors; D007529:Isoflavones; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D000804:Angiotensin II; D019833:Genistein; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium