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)/stres s-activated protein kinase cascade to stimulate or inhibit DNA synthes is in primary cultures of adult rat hepatocytes was examined. Treatmen t of hepatocytes with media containing hyperosmotic glucose (75 mM fin al), tumor necrosis factor alpha (TNF alpha, 1 ng/ml final), and hepat ocyte growth factor (HGF, 1 ng/ml final) caused activation of JNK1. Gl ucose, TNF alpha, 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 aden oviruses coupled to constructs to express either dominant negatives Ra s(N17), Rac1(N17), Cdc42(N17), SEK1(-), or JNK1(-) blunted the abiliti es of glucose, TNF alpha, or HGF to increase JNK1 activity, to increas e phosphorylation of c-Jun at serine 63, and to stimulate DNA synthesi s. Furthermore, infection of hepatocytes by a recombinant adenovirus e xpressing a dominant-negative c-run mutant (TAM67) also blunted the ab ilities of glucose, TNF alpha, and HGF to stimulate DNA synthesis. The se data demonstrate that multiple agonists stimulate DNA synthesis in primary cultures of hepatocytes via a Ras/Rac1/Cdc42/SEK/JNK/c-Jun pat hway. Glucose and HGF treatments reduced glycogen synthase kinase 3 (G SK3) activity and increased c-Jun DNA binding. Go-infection of hepatoc ytes with recombinant adenoviruses to express dominant-negative forms of PI3 kinase (p110 alpha/p110 gamma) increased basal GSK3 activity, b locked the abilities of glucose and HGF treatments to inhibit GSK3 act ivity, and reduced basal c-Jun DNA binding. However, expression of dom inant-negative PI3 kinase (p110 alpha/p110 gamma) neither significantl y blunted the abilities of glucose and HGF treatments to increase c-Ju n DNA binding, nor inhibited the ability of these agonists to stimulat e DNA synthesis. These data suggest that signaling by the JNK/stress-a ctivated protein kinase cascade, rather than by the PI3 kinase cascade , plays the pivotal role in the ability of agonists to stimulate DNA s ynthesis in primary cultures of rat hepatocytes.