We examined the interplay between the insulin/IGF-1-and b-catenin-regulated pathways, both of which are suspected to play a role in hepatocarcinogenesis. Insulin and IGF-1 stimulated the transcription of a Lef/Tcfdependent luciferase reporter gene by 3 ± 4-fold in HepG2 cells. This stimulation was mediated through the activation of phosphatidylinositol 3-kinase (PI 3-K)/Akt and the inhibition of glycogen synthase kinase-3b (GSK3b) since the eects of insulin and IGF-1 were inhibited by dominant-negative mutants of PI 3-K or Akt and an uninhibitable GSK-3b. Together with inhibiting GSK-3b, insulin and IGF-1 increased the cytoplasmic levels of bcatenin. The PI 3-K/Akt/GSK-3b pathway was not the sole to mediate insulin and IGF-1 stimulation of Lef/Tcfdependent transcription. The Ras signalling pathway was also required as (i) the stimulatory eects of insulin and IGF-1 were inhibited by dominant-negative Ras or the MEK1 inhibitor PD98059 and (ii) activated Ha-Ras or constitutively active MEK1 synergized with catalytically inactive GSK-3b to stimulate Lef/Tcf-dependent transcription. This study provides the ®rst evidence that insulin and IGF-1 stimulate the b-catenin pathway through two signalling cascades bifurcating downstream of PI 3-K and involving GSK-3b inhibition and Ras activation. These ®ndings demonstrate for the ®rst time the ability of insulin and IGF-1 to activate the b-catenin pathway in hepatoma cells and thereby provide new insights into the role of these factors in hepatocarcinogenesis. Oncogene (2001) 20, 252 ± 259.

Stimulation of macrophages with endotoxin and/or cytokines is responsible for the expression of the inducible isoform of nitric oxide synthase (iNOS). Because macrophages are exposed to low pH within the microenvironment of inflammatory lesions, the potential role of acidic pH as an additional regulator of iNOS was investigated. Substitution of the culture medium of rat peritoneal macrophages at pH 7.4 with medium at pH 7.0 up-regulated iNOS activity, as reflected by a 2.5-fold increase in nitrite accumulation. The increase in iNOS activity was associated with a similar increase in iNOS mRNA expression that reflected an increase in iNOS mRNA synthesis rather than stability. Low environmental pH-induced iNOS gene transcription involved the activation of nuclear factor-B (NF-B) transcription factor since exposure of macrophages to low environmental pH both increased NF-B binding activity in the nucleus and enhanced NF-B-driven reporter gene expression. In addition, treatment of macrophages with pyrrolidine dithiocarbamate or n-acetyl-leucinyl-leucinyl-norleucinal, two drugs preventing NF-B translocation to the nucleus, canceled low pH-induced nitrite accumulation. The overall mechanism required the synthesis of tumor necrosis factor ␣ (TNF␣). Indeed, 1) elevated TNF␣ bioactivity was observed in the medium of macrophages exposed to pH 7.0, and 2) incubation of macrophages with a neutralizing anti-TNF␣ antibody impaired both NF-B activation and nitrite accumulation in response to acid challenge. In summary, exposure of macrophages to acidic microenvironment in inflammatory lesions leads to the up-regulation of iNOS activity through the activation of NF-B.Acidosis is a hallmark of both ischemia and inflammation processes. The decrease of pH in tissue ischemia is secondary to the release of H ϩ during ATP hydrolysis and to the accumulation of CO 2 (1). The acidic environment in inflammatory lesions and abscesses (2) is due to increased metabolic acid generation during cell activation. This originates primarily from the hexose monophosphate shunt, by the dissociation of hydrated CO 2 (3).In most cases, acidosis occurs along with nitric oxide (NO) 1 generation. In ischemia, NO generation is due in one part to the acidification and reduction of the large pool of nitrite present within the tissue (4). In inflammatory processes, macrophage exposure to bacterial lipopolysaccharide (LPS) or cytokines such as tumor necrosis factor ␣ (TNF␣) and interferon-␥ (IFN-␥) causes the expression of the inducible isoform of NO synthase (NOS II or iNOS) that is responsible for high output production of NO (5). The expression of iNOS is regulated mainly at the transcriptional level. Analyses of the murine iNOS promoter have shown the presence of numerous consensus sequences for the binding of transcription factors (6, 7), of which nuclear factor-B (NF-B) (8), interferon regulatory factor-1 (IRF-1) (9), and signal transducer and activator of transcription (STAT) 1␣ (10) are functionally important for iNOS induction. NF-B is composed of a p...

We previously reported that insulin activates nuclear factor B (NF-B) in Chinese hamster ovary (CHO)-R cells overexpressing wild-type insulin receptors (IRs) through a pathway requiring IR tyrosine kinase and Raf-1 kinase activities. We now investigated whether the activation of NF-B by insulin could serve an antiapoptotic function. Insulin (10 ؊9 -10 ؊7 M) inhibited apoptosis induced by serum withdrawal in CHO-R cells in a concentration-dependent manner. Insulin antiapoptotic signaling: (i) was dependent on IR number and IR tyrosine kinase activity since it was reduced in parental CHO cells and was abolished in CHO-Y2 cells overexpressing IRs mutated at Tyr 1162/1163 ; (ii) was, like insulin activation of NF-B, dependent on Raf-1 but not on mitogen-activated protein kinase activity since both processes were decreased by the dominant-negative Raf-1 mutant Raf-C4 whereas they persisted in mitogen-activated protein kinase-depleted cells; and (iii) required NF-B activation since it was decreased by proteasome inhibitors and the dominant-negative IB-␣ (A32/36) mutant and was mimicked by overexpression of the NF-B c-Rel subunit. We also show that insulin antiapoptotic signaling but not insulin activation of NF-B involved phosphatidylinositol 3-kinase (PI 3-kinase), as supported by the inhibition of the former but not of the latter process by the PI 3-kinase inhibitor LY294002. Inhibition of both NF-B and PI 3-kinase totally abolished insulin antiapoptotic signaling. Thus insulin exerts a specific antiapoptotic function which is dependent on IR tyrosine kinase activity and is mediated by both a Raf-1-dependent pathway that leads to NF-B activation and a PI 3-kinase-dependent pathway.

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