The Ras/Rac1/Cdc42/SEK/JNK/c-Jun cascade is a key pathway by which agonists stimulate DNA synthesis in primary cultures of rat hepatocytes.
The ability of signaling via the JNK (c-Jun NH2-terminal kinase)/stress-activated protein kinase cascade to stimulate or inhibit DNA synthesis in primary cultures of adult rat hepatocytes was examined. Treatment of hepatocytes with media containing hyperosmotic glucose (75 mM final), tumor necrosis factor alpha (TNFalpha, 1 ng/ml final), and hepatocyte growth factor (HGF, 1 ng/ml final) caused activation of JNK1. Glucose, TNFalpha, or HGF treatments increased phosphorylation of c-Jun at serine 63 in the transactivation domain and stimulated hepatocyte DNA synthesis. Infection of hepatocytes with poly-L-lysine-coated adenoviruses coupled to constructs to express either dominant negatives Ras N17, Rac1 (N17), Cdc42 (N17), SEK1-, or JNK1- blunted the abilities of glucose, TNFalpha, or HGF to increase JNK1 activity, to increase phosphorylation of c-Jun at serine 63, and to stimulate DNA synthesis. Furthermore, infection of hepatocytes by a recombinant adenovirus expressing a dominant-negative c-Jun mutant (TAM67) also blunted the abilities of glucose, TNFalpha, and HGF to stimulate DNA synthesis. These data demonstrate that multiple agonists stimulate DNA synthesis in primary cultures of hepatocytes via a Ras/Rac1/Cdc42/SEK/JNK/c-Jun pathway. Glucose and HGF treatments reduced glycogen synthase kinase 3 (GSK3) activity and increased c-Jun DNA binding. Co-infection of hepatocytes with recombinant adenoviruses to express dominant- negative forms of PI3 kinase (p110alpha/p110gamma) increased basal GSK3 activity, blocked the abilities of glucose and HGF treatments to inhibit GSK3 activity, and reduced basal c-Jun DNA binding. However, expression of dominant-negative PI3 kinase (p110alpha/p110gamma) neither significantly blunted the abilities of glucose and HGF treatments to increase c-Jun DNA binding, nor inhibited the ability of these agonists to stimulate DNA synthesis. These data suggest that signaling by the JNK/stress-activated protein kinase cascade, rather than by the PI3 kinase cascade, plays the pivotal role in the ability of agonists to stimulate DNA synthesis in primary cultures of rat hepatocytes.
Auer
K L
KL
Department of Radiation Oncology, Medical College of Virginia, Richmond, Virginia 23298-0058, USA.
Contessa
J
J
Brenz-Verca
S
S
Pirola
L
L
Rusconi
S
S
Cooper
G
G
Abo
A
A
Wymann
M P
MP
Davis
R J
RJ
Birrer
M
M
Dent
P
P
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D038341:Glycogen Synthase Kinases; D006160:Guanosine Triphosphate / Q000378:metabolism; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D020764:cdc42 GTP-Binding Protein; D018631:ras Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; D016755:Proto-Oncogene Proteins c-jun; D014409:Tumor Necrosis Factor-alpha; D017228:Hepatocyte Growth Factor; D006160:Guanosine Triphosphate; D004247:DNA; D011494:Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D038341:Glycogen Synthase Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein; D018631:ras Proteins; D005947:Glucose