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.

Ephemerally expressed wild-type and mutant steroid hormone receptors are equally able to influence expression of transient or resident templates.

We have tested transiently expressed mutant and chimeric glucocorticoid receptors (GR) for their ability to influence transcription of either a co-transfected or a stably integrated reporter gene. To the latter purpose we have generated a cell line harbouring 2 chromosomally anchored copies of the well-characterized mouse mammary tumor virus (MMTV) promoter/enhancer region fused to the bacterial beta-galactosidase gene (LacZ). We were particularly interested in verifying whether some earlier characterized dominant negative GR mutants would still act the same way on chromosomal targets. We show that trans-regulation (activation/-repression) of the chromosomally anchored reporter is qualitatively and quantitatively indistinguishable from trans-regulation obtained with transient co-transfection. In parallel, we also tested ephemerally expressed wild-type progesterone receptor (PR) and androgen receptor (AR) for their capacity of acting on either transient or resident MMTV reporter templates. Also in this case we show that activation of chromosomally anchored or transiently co-transfected reporter by both these steroid hormone receptors is qualitatively and quantitatively indistinguishable. These results outline that newly expressed trans-effectors may exert their specific function independently of the precise structural organization of their responsive genes.