Gisèle Cherqui

Insulin and IGF-1 stimulate the β-catenin pathway through two signalling cascades involving GSK-3β inhibition and Ras activation

We examined the interplay between the insulin/IGF-1- and β-catenin-regulated pathways, both of which are suspected to play a role in hepatocarcinogenesis. Insulin and IGF-1 stimulated the transcription of a Lef/Tcf-dependent 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-3β (GSK-3β) since the effects of insulin and IGF-1 were inhibited by dominant-negative mutants of PI 3-K or Akt and an uninhibitable GSK-3β. Together with inhibiting GSK-3β, insulin and IGF-1 increased the cytoplasmic levels of β-catenin. The PI 3-K/Akt/GSK-3β pathway was not the sole to mediate insulin and IGF-1 stimulation of Lef/Tcf-dependent transcription. The Ras signalling pathway was also required as (i) the stimulatory effects 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-3β to stimulate Lef/Tcf-dependent transcription. This study provides the first evidence that insulin and IGF-1 stimulate the β-catenin pathway through two signalling cascades bifurcating downstream of PI 3-K and involving GSK-3β inhibition and Ras activation. These findings demonstrate for the first time the ability of insulin and IGF-1 to activate the β-catenin pathway in hepatoma cells and thereby provide new insights into the role of these factors in hepatocarcinogenesis.

Smad7 inhibits the survival nuclear factor κB and potentiates apoptosis in epithelial cells

In this study, we examined the effect of the stable expression of Smad7 in two different cell lines on apoptosis induced by various stimuli including TGF-β, serum withdrawal, loss of cell adhesion (anoikis) and TNF-α. Smad7 increased TGF-β-mediated apoptosis in Mv1Lu cells as well as anoikis and/or serum withdrawal-induced apoptosis in Mv1Lu and MDCK cells. Smad7 markedly decreased the activity of the survival NF-κB transcription factor in MDCK cells. Interestingly, the stable expression of oncogenic Ras in MDCK cells which suppressed Smad7 inhibition of NF-κB also suppressed Smad7 potentiation of serum withdrawal-induced apoptosis and anoikis. In addition, Smad7 inhibited TNF-α stimulation of NF-κB and increased TNF-α-mediated apoptosis in MDCK cells. Our results provide the first evidence that Smad7 induces sensitization of cells to different forms of cell death. They moreover demonstrate that Smad7 inhibits the survival NF-κB factor, providing a potential mechanism whereby Smad7 potentiates cell death.

Downregulation of the colon tumour-suppressor homeobox gene Cdx-2 by oncogenic ras.

Constitutive activation of the ras proto-oncogene is a frequent and early event in colon cancers, but the downstream nuclear targets are not fully understood. The Cdx-1 and Cdx-2 homeobox genes play crucial roles in intestinal cell proliferation and differentiation. In addition, Cdx-2 is a colonic tumour-suppressor gene, whereas Cdx-1 has oncogenic potential. Here, we show that constitutive activation of ras alters Cdx-1 and Cdx-2 expression in human colonic Caco-2 and HT-29 cells that harbour a normal ras proto-oncogene. Oncogenic ras downregulates Cdx-2 through activation of the PKC pathway and a decline in activity of the Cdx-2 promoter AP-1 site. This decline results from a PKC-dependent decrease in the relative expression of c-Jun, an activator of Cdx-2 transcription, compared to c-Fos, an inhibitor of Cdx-2. Unlike Cdx-2, Cdx-1 is upregulated by oncogenic ras and this effect is mediated by activation of the MEK1 pathway. These results indicate that oncogenic ras activation has opposite effects on Cdx-1 and Cdx-2 expression through distinct signalling pathways and they provide the first evidence for a functional link between ras activation and the downregulation of the Cdx-2 tumour-suppressor gene in colon cancer cells.

INSULIN ACTIVATES NUCLEAR FACTOR KAPPA B IN MAMMALIAN CELLS THROUGH A RAF-1-MEDIATED PATHWAY

We examined the effect of insulin on nuclear factor κB (NF-κB) activity in Chinese hamster ovary (CHO) cells overexpressing wild-type (CHO-R cells) or -defective insulin receptors mutated at Tyr1162 and Tyr1163 autophosphorylation sites (CHO-Y2 cells). In CHO-R cells, insulin caused a specific, time-, and concentration-dependent activation of NF-κB. The insulin-induced DNA-binding complex was identified as the p50/p65 heterodimer. Insulin activation of NF-κB: 1) was related to insulin receptor number and tyrosine kinase activity since it was markedly reduced in parental CHO cells which proved to respond to insulin growth factor-1 and phorbol 12-myristate 13-acetate (PMA) activation, and was dramatically decreased in CHO-Y2 cells; 2) persisted in the presence of cycloheximide and was blocked by pyrrolidine dithiocarbamate, aspirin and sodium salicylate, three compounds interfering with IκB degradation and/or NF-κB•IκB complex dissociation; 3) was independent of both PMA-sensitive and atypical (ζ) protein kinases C; and 4) was dependent on Raf-1 kinase activity since insulin-stimulated NF-κB DNA binding activity was inhibited by 8-bromo-cAMP, a Raf-1 kinase inhibitor. Moreover, insulin activation of NF-κB-driven luciferase reporter gene expression was blocked in CHO-R cells expressing a Raf-1 dominant negative mutant. This is the first evidence that insulin activates NF-κB in mammalian cells through a post-translational mechanism requiring both insulin receptor tyrosine kinase and Raf-1 kinase activities.

Enterocytic differentiation of the human Caco‐2 cell line correlates with alterations in integrin signaling

We previously reported that the enterocytic differentiation of human colonic Caco‐2 cells correlated with down‐regulation of fibronectin (FN) and laminin (LN), two extracellular matrix components interacting with cell surface integrin receptors. We now investigated whether Caco‐2 cell differentiation was associated with alterations in integrin signaling with special interest in the expression and activity of focal adhesion kinase (FAK) and mitogen‐activated protein (MAP) kinase. The differentiation of Caco‐2 cells was associated with: (1) down‐regulation of β1 integrin expression at the mRNA and protein levels; (2) increased FAK expression together with decreased FAK autophosphorylation; (3) decreased FAKs ability to associate with PI3‐kinase and pp60c‐src; and (4) increased MAP kinase expression along with decreased MAP activity. In addition, we show that FAK and MAP kinase belong to distinct integrin signaling pathways and that both pathways remain functional during Caco‐2 cell differentiation since the coating of differentiating cells on FN and LN but not on polylysine increased the tyrosine phosphorylation of FAK and of its endogenous substrate paxillin, and stimulated MAP kinase activity. In conclusion, our results provide evidence that FAK and MAP kinase, two signaling molecules activated independently by β1 integrins in Caco‐2 cells, undergo alterations of both expression and activity during the enterocytic differentiation of this cell line. J Cell Physiol 177:618–627, 1998.

Insulin-Mediated Cell Proliferation and Survival Involve Inhibition of c-Jun N-terminal Kinases through a Phosphatidylinositol 3-Kinase- and Mitogen-Activated Protein Kinase Phosphatase-1-Dependent Pathway1

We previously reported that long term treatment with insulin led to sustained inhibition of c-Jun N-terminal kinases (JNKs) in CHO cells overexpressing insulin receptors. Here we investigated the signaling molecules involved in insulin inhibition of JNKs, focusing on phosphatidylinositol 3-kinase (PI 3-K) and mitogen-activated protein kinase phosphatase-1 (MKP-1). In addition, we examined the relevance of JNK inhibition for insulin-mediated proliferation and survival. Insulin inhibition of JNKs was mediated by PI 3-K, as it was blocked by wortmannin and LY294002 and required the de novo synthesis of a phosphatase(s), as it was abolished by orthovanadate and actinomycin D. MKP-1 was a good candidate because 1) insulin stimulation of MKP-1 expression correlated with insulin inhibition of JNKs; 2) insulin stimulation of MKP-1 expression, like insulin inhibition of JNKs, was mediated by PI 3-K; and 3) the transient expression of an antisense MKP-1 RNA reduced the insulin inhibitory effect on JNKs. The overexp...

Reduced protein kinase C activity in sporadic Alzheimer's disease fibroblasts

A decrease in the protein kinase C immunoreactivity and an altered protein phosphorylation have been reported in patients with Alzheimers disease, but discordant results have been obtained from determinations of protein kinase C activity. By assaying the calcium- and phospholipid-dependent phosphorylation of a lysine-rich histone after detergent extraction, we have determined the total protein kinase C activity in fibroblasts from patients with sporadic Alzheimers disease, age-matched controls and young subjects. The activity was not significantly different between young and aged controls, whereas it was significantly lower (0.70 +/- 0.12 vs 1.16 +/- 0.23 nmol/min/mg protein, P less than 0.01) in the patients. The total amount of protein kinase C estimated from the binding of phorbol dibutyrate to intact cells was also significantly lower (1.70 +/- 0.41 vs 2.48 +/- 0.54 pmol/mg protein, P less than 0.01). This decrease in protein kinase C activity suggests that abnormal protein phosphorylation might play a role in the pathogenesis of the disease.

Dual effect of metformin in cultured rat hepatocytes: potentiation of insulin action and prevention of insulin-induced resistance.

The ability of the biguanide hypoglycemic agent metformin to improve the acute effects of insulin on glucose and/or lipid metabolism was investigated in both insulin-responsive and insulin-resistant cultured rat hepatocytes: (1) metformin (20 micrograms/mL, 16 hours) increased the insulin-dependent stimulation of glycogen and lipid synthesis through an exclusive enhancement of the responsiveness without modification of the cell sensitivity to the hormone; (2) metformin neither altered basal glycogenesis from [U-14C]glucose and basal lipogenesis from [1-14C]acetate nor insulin binding. These results indicate the ability of this drug to selectively potentiate the acute action of insulin at a postreceptor step in normal liver cells. A prolonged incubation with insulin (16 hours, 5 x 10(-7) mol/L) led the hepatocytes to a state of resistance evidenced by a 50% decrease in their maximal responsiveness and sensitivity to a subsequent acute stimulation by the hormone, as assessed on lipogenesis. Addition of metformin (20 micrograms/mL) during the overnight incubation of hepatocytes with insulin prevented the decrease in cell responsiveness and sensitivity to the hormone for the stimulation of lipogenesis, thus showing that metformin was able to hamper the development of the resistant state to the hormone in this pathway. These results strongly suggest that metformin improves type 2 diabetes through an effect at the hepatic level on both insulin action and insulin-induced resistance.

Down-regulation of NF-κB activity and NF-κB p65 subunit expression by ras and polyoma middle T oncogenes in human colonic Caco-2 cells

The products of ras and src proto-oncogenes are frequently activated in a constitutive state in human colorectal cancer. In this study we attempted to establish whether the tumorigenic progression induced by oncogenic activation of p21ras or pp60c-src in human colonic cells is associated with alterations of the activity and expression of nuclear factor κB (NF-κB), a transcription factor suspected to participate in the development of cancer. To this end, we used Caco-2 cells made highly tumorigenic by transfection with an activated Val-12 human Ha-ras gene or with the polyoma middle T (PyMT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity. Compared with control vector-transfected Caco-2 cells, both oncogene-transfected cell lines exhibited: (i) decreased constitutive NF-κB DNA-binding activity and NF-κB-mediated reporter gene expression, without alteration of their response to TNF-α for activation of these parameters; (ii) reduced NF-κB cytosolic stores along with a decreased p65 expression due, at least in part, to destabilization of p65 mRNA; (iii) a decrease in adhesion to extracellular matrix component-coated substrata which was partially corrected when stimulating NF-κB transcriptional activity with TNF-α. These results indicate that the tumorigenic progression induced by oncogenic p21ras or PyMT/pp60c-src in human colonic Caco-2 cells is associated with a down-regulation of p65 expression and NF-κB activity which could be responsible for the reduced adhesive properties of these cells after oncogene transfection.

Protein kinase C and insulin receptor β-subunit serine phosphorylation in cultured foetal rat hepatocytes

In digitonin-permeabilized cultured foetal hepatocytes, insulin receptor beta-subunit was highly phosphorylated on serine residues in the presence of [gamma-32P]ATP and Ca2+, a process enhanced after short exposure to insulin with no detectable insulin receptor autophosphorylation. By contrast with this situation, experiments performed with isolated foetal insulin receptors revealed an insulin stimulation of both serine phosphorylation and tyrosine autophosphorylation. In permeabilized cells, insulin receptor beta-subunit phosphorylation was increased after a 2-min exposure to phorbol 12-myristate 13-acetate (PMA) prior to applying the permeabilization/phosphorylation step, while it was inhibited by chronic treatment with PMA leading to protein kinase C (PKC) down modulation. The PKC specific inhibitor, GF109203X, strikingly reduced basal and insulin-enhanced phosphorylation of insulin receptor beta-subunit in permeabilized cells, but failed to exert any effect with isolated receptors. Labelling of glycogen from [U-14C]glucose determined 1 h after a 10-min transitory exposure to insulin and/or modulators of PKC activity showed that PMA prevented insulin glycogenic response, whereas GF109203X was ineffective. Thus, although not directly responsible for insulin receptor serine phosphorylation in cultured foetal hepatocytes, PKC physiologically regulates this process which may inhibit insulin receptor tyrosine kinase activity. This regulation is independent of the antagonistic effect of PMA-activated PKC on insulin glycogenic response.