Axelle Cadoret

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.

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.

Hepatocyte proliferation during liver regeneration is impaired in mice with liver-specific IGF-1R knockout

Recent evidence indicates that growth hormone (GH) is involved in liver regeneration. To test whether insulin‐like growth factor I (IGF‐I) mediates this effect, we studied liver regeneration induced by partial hepatectomy in liver‐specific IGF type 1 receptor knockout (LIGFREKO) mice. The absence of IGF‐1R caused a significant decrease in hepatocyte proliferation in males (−52%), but not in females, as assessed by Ki67 immunohistochemistry. Cyclin D1 and cyclin A protein levels in the livers of LIGFREKO males were only half those in controls, indicating that cyclin induction during liver regeneration is dependent on IGF‐1R signaling. Analyzing the signaling cascade initiated by IGF‐1R, we observed a lack of IRS‐1 induction in LIGFREKO livers. In contrast, the induction of IRS‐2 synthesis was similar in LIGFREKO and control groups, suggesting the existence of differential regulation of IRS synthesis during liver regeneration. Regenerating livers from LIGFREKO animals also showed significantly less activated ERKs than controls. Our findings demonstrate that IGF‐1R makes a significant contribution to liver regeneration. Using the LIGFREKO model, we provide new evidence that IGF‐1R/IRS‐1/ERK signaling may be the intracellular pathway controlling the cell cycle via cyclin D1 and cyclin A in the regenerating liver.

Origins and functions of liver myofibroblasts.

Myofibroblasts combine the matrix-producing functions of fibroblasts and the contractile properties of smooth muscle cells. They are the main effectors of fibrosis in all tissues and make a major contribution to other aspects of the wound healing response, including regeneration and angiogenesis. They display the de novo expression of α-smooth muscle actin. Myofibroblasts, which are absent from the normal liver, are derived from two major sources: hepatic stellate cells (HSCs) and portal mesenchymal cells in the injured liver. Reliable markers for distinguishing between the two subpopulations at the myofibroblast stage are currently lacking, but there is evidence to suggest that both myofibroblast cell types, each exposed to a particular microenvironment (e.g. hypoxia for HSC-MFs, ductular reaction for portal mesenchymal cell-derived myofibroblasts (PMFs)), expand and exert specialist functions, in scarring and inflammation for PMFs, and in vasoregulation and hepatocellular healing for HSC-MFs. Angiogenesis is a major mechanism by which myofibroblasts contribute to the progression of fibrosis in liver disease. It has been clearly demonstrated that liver fibrosis can regress, and this process involves a deactivation of myofibroblasts, although probably not to a fully quiescent phenotype. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.

Distinct proteomic features of two fibrogenic liver cell populations: Hepatic stellate cells and portal myofibroblasts

In chronic liver diseases, the accumulation of extracellular matrix leading to fibrosis is caused by myofibroblasts, the origins of which are debatable. We performed a comparative proteomic study to identify markers and gain insight into distinct functions of myofibroblasts derived either from hepatic stellate cells (HSCs) or from portal mesenchymal cells. After isolation from normal liver and culture in similar conditions, myofibroblastic HSCs (MF‐HSCs) presented enlarged cytoplasms whereas portal myofibroblasts (PMFs) were more proliferative, and formed more stress fibers. The two cell types were subjected to comparative analyses by 2‐D MS/MS. Six proteins were overexpressed in PMFs, with myofibroblast‐related typical functions. Among them, cofilin‐1 showed the greatest difference in expression and a lower pI than expected. Immunoblot demonstrated higher levels of phosphorylation, a modification of the protein implicated in stress fiber formation. Eleven proteins, mostly involved in stress response, were overexpressed in MF‐HSCs. Cytoglobin had the highest level of overexpression, as confirmed by reverse transcription quantitative real‐time PCR, immunoblot and immunocytochemical analyses. These results identify cytoglobin as the best marker for distinguishing MF‐HSCs from PMFs and suggest different functions for the two cell populations in the liver wound healing response, with a prominent role for PMFs in scar formation.

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...

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.

Vitamin D nuclear receptor deficiency promotes cholestatic liver injury by disruption of biliary epithelial cell junctions in mice.

Alterations in apical junctional complexes (AJCs) have been reported in genetic or acquired biliary diseases. The vitamin D nuclear receptor (VDR), predominantly expressed in biliary epithelial cells in the liver, has been shown to regulate AJCs. The aim of our study was thus to investigate the role of VDR in the maintenance of bile duct integrity in mice challenged with biliary‐type liver injury. Vdr−/− mice subjected to bile duct ligation (BDL) displayed increased liver damage compared to wildtype BDL mice. Adaptation to cholestasis, ascertained by expression of genes involved in bile acid metabolism and tissue repair, was limited in Vdr−/− BDL mice. Furthermore, evaluation of Vdr−/− BDL mouse liver tissue sections indicated altered E‐cadherin staining associated with increased bile duct rupture. Total liver protein analysis revealed that a truncated form of E‐cadherin was present in higher amounts in Vdr−/− mice subjected to BDL compared to wildtype BDL mice. Truncated E‐cadherin was also associated with loss of cell adhesion in biliary epithelial cells silenced for VDR. In these cells, E‐cadherin cleavage occurred together with calpain 1 activation and was prevented by the silencing of calpain 1. Furthermore, VDR deficiency led to the activation of the epidermal growth factor receptor (EGFR) pathway, while EGFR activation by EGF induced both calpain 1 activation and E‐cadherin cleavage in these cells. Finally, truncation of E‐cadherin was blunted when EGFR signaling was inhibited in VDR‐silenced cells. Conclusion: Biliary‐type liver injury is exacerbated in Vdr−/− mice by limited adaptive response and increased bile duct rupture. These results indicate that loss of VDR restricts the adaptation to cholestasis and diminishes bile duct integrity in the setting of biliary‐type liver injury. (Hepatology 2013;58:1401–1412)

c-myc-induced hepatocarcinogenesis in the absence of IGF-I receptor

Numerous tumours, including hepatocarcinomas, produce IGFs, and some depend on these growth factors in a paracrine or autocrine fashion. We have shown that c‐myc‐induced experimental hepatocarcinogenesis is associated with enhanced production of IGF‐II. To assess the role of the IGF‐I receptor (IGF‐IR) in hepatocarcinogenesis, we generated conditional mutant mice that overexpressed c‐myc and were knocked out for IGF‐IR specifically in the liver. We compared these mice with littermate controls that also overexpressed c‐myc but had wild‐type IGF‐IR alleles. We found that the pretumoral phase, induced by early c‐myc expression and characterised by increased cell proliferation, was largely unaffected by the lack of IGF‐IR. To our further surprise, hepatocellular carcinomas (HCCs) lacking IGF‐IR readily developed and progressed at the same rate as control HCCs. At 9 months, all c‐myc transgenic mice displayed well‐differentiated multifocal tumours, regardless of whether their livers—and their tumours—were able to produce IGF‐IR. Levels of IRS‐1 and IRS‐2 were elevated in all tumours in the presence or absence of IGF‐IR, suggesting that the signalling pathway downstream of IGF‐IR is activated via IGF‐IR‐independent mechanisms in HCC. In conclusion, the deregulation of IGF signalling pathways, which often occurs during liver tumorigenesis, does not necessarily require IGF‐IRs, and hepatic IGF‐IR alone may not play a determinant role in c‐myc‐induced hepatocarcinogenesis.

Oncogene-induced up-regulation of Caco-2 cell proliferation involves IGF-II gene activation through a protein kinase C-mediated pathway

We previously reported that ras and polyoma middle T (PyMT), a constitutive activator of the src proto-oncogene product, up-regulated Caco-2 cell proliferation along with protein kinase C (PKC) alpha expression and PKC activity. We aimed to investigate whether oncogene-induced up-regulation of Caco-2 cell proliferation involved stimulation of the autocrine IGF-II/IGF-I receptor (IGF1R) loop described in these cells and if so, to analyse the role of overexpressed and activated PKC. Compared with control vector transfected Caco-2 cells, ras- and PyMT-transfected cells exhibited increased expression of the 6.0 and 4.8 kb IGF-II transcripts. This was due to increased activity of the P3 and P4 promoters of the IGF-II gene which correlated with increased expression and DNA-binding activity of Sp1, a transcription factor interacting with several specific sites in P3 and P4 promoters. Oncogene-transfected cells displayed enhanced autocrine IGF-II production, which was fully responsible for the oncogene-induced increase in their proliferation since this increase was blunted by anti-human IGF-II and IGF1R (αIR3) antibodies. PKC mediated oncogene activation of the IGF-II gene presumably through action on Sp1 since (i) PKC activation by phorbol 12-myristate 13-acetate increased Sp1 expression, P3 and P4 activity and IGF-II mRNA in control but not in oncogene-transfected cells; and (ii) PKC inhibition by the PKC inhibitor Gö6976 reduced Sp1, P3 and P4 activity and IGF-II mRNA in all three cell lines. This is the first evidence that ras- and PyMT/src oncogenes up-regulate Caco-2 cell proliferation through a PKC-mediated pathway which stimulates IGF-II gene transcription and thereby increases autocrine IGF-II production. The mechanisms underlying IGF-II gene activation by PKC most probably involve action on Sp1.