Overexpression and activation of the RON receptor tyrosine kinase in a panel of human colorectal carcinoma cell lines

RON is a receptor tyrosine kinase belonging to the MET proto-oncogene family. The purposes of this study are to determine the expression and activation of RON in a panel of human colon carcinoma cell lines. Western blotting showed that RON is barely detectable in normal and SV-40-transformed colon epithelial cells, but highly expressed and constitutively activated in several colon carcinoma cell lines including Colo201, HT-29, HCT116, and SW837. Moreover, a novel RON variant with a molecular mass of 160 kDa (RONDelta160) was identified from HT-29 cells. The cDNA encoding RONDelta160 has an in-frame deletion of 109 amino acids in the extracellular domain of the RON beta chain, which is caused by splicing out of two exons in the RON mRNA. No mutations were found in the kinase domain of the RON gene in five carcinoma cell lines screened. By expressing RON in colon epithelial cells, we found that RON activation increases cell motile-invasive activities and protects cells against apoptotic death. These data suggest that RON expression and activation are deregulated in colon carcinoma cell lines. By abnormal activation of RON, this receptor and its variant may regulate motile-invasive phenotypes of certain colon carcinoma cells in vivo.     

Roles of Axin in the Wnt signalling pathway

The Wnt signalling pathway is conserved in various species from worms to mammals, and plays important roles in development, cellular proliferation, and differentiation. The molecular mechanisms by which the Wnt signal regulates cellular functions are becoming increasingly well understood. Wnt stabilizes cytoplasmic beta-catenin, which stimulates the expression of genes including c-myc, c-jun, fra-1, and cyclin D1. Axin, newly recognized as a component of the Wnt signalling pathway, negatively regulates this pathway. Other components of the Wnt signalling pathway, including Dvl, glycogen synthase kinase-3beta, beta-catenin, and adenomatous polyposis coli, interact with Axin, and the phosphorylation and stability of beta-catenin are regulated in the Axin complex. Thus, Axin acts as a scaffold protein in the Wnt signalling pathway, thereby regulating cellular functions.     

The effects of antipsychotics on beta-catenin, glycogen synthase kinase-3 and dishevelled in the ventral midbrain of rats

Protein kinase B and glycogen synthase kinase-3 have been identified as susceptibility genes for schizophrenia and altered protein and mRNA levels have been detected in the brains of schizophrenics post-mortem. Recently, we reported that haloperidol, clozapine and risperidone alter glycogen synthase kinase-3 and beta-catenin protein expression and glycogen synthase kinase-3 phosphorylation levels in the rat prefrontal cortex and striatum. In the current study, beta-catenin, adenomatous polyposis coli, Wnt1, dishevelled and glycogen synthase kinase-3 were examined in the ventral midbrain and hippocampus using western blotting. In addition, beta-catenin and GSK-3 were examined in the substantia nigra and ventral tegmental area using confocal and fluorescence microscopy. The results indicate that repeated antipsychotic administration results in significant elevations in glycogen synthase kinase-3, beta-catenin and dishevelled-3 protein levels in the ventral midbrain and hippocampus. Raclopride causes similar changes in beta-catenin and GSK-3 in the ventral midbrain, suggesting that D2 dopamine receptor antagonism mediated the changes observed following antipsychotic administration. In contrast, amphetamine, a drug capable of inducing psychotic episodes, had the opposite effect on beta-catenin and GSK-3 in the ventral midbrain. Collectively, the results suggest that antipsychotics may exert their beneficial effects through modifications to proteins that are associated with the canonical Wnt pathway.     

Altered adhesion features and signal transduction in NRK-49F cells transformed by down-regulation of lysyl oxidase

Lysyl oxidase (LOX) down-regulation induced an oncogenic phenotype in NRK-49F. This event was accompanied by a constitutive activation of ras oncogene and down-regulation of PDGF beta receptor, among other important phenotypic and molecular modifications. In the present paper we show that ras activation is not accompanied by a constitutive activation of the MAP kinases as expected. Surprisingly, even if MAPK-independent, ras activation was accompanied by a constitutive Ser(63) and Ser(73) phosphorylation of c-jun, a further downstream target of ras. Although rare, this ras alternative pathway has been described. Since ras alone is seldom able to trigger cell transformation and the transformed phenotype showed clearly an abnormal adhesion pattern, we investigated the main molecules involved in cell-cell adhesion. In fact, we found that beta-catenin was up-regulated, escaping the glycogen synthase kinase-3 beta (GSK-3 beta) control, through unclear mechanisms. Its nuclear accumulation was accompanied by an up-regulation of cyclin D1, as classically described in the activation of the Wnt/beta-catenin signal pathway. We believe that the resulting up-regulation of cyclin D1 acted in synergy with ras to induce the cell transformation.     

Differentiation-inducing factor-1 suppresses gene expression of cyclin D1 in tumor cells

To determine the mechanism by which differentiation-inducing factor-1 (DIF-1), a morphogen of Dictyostelium discoideum, inhibits tumor cell proliferation, we examined the effect of DIF-1 on the gene expression of cyclin D1. DIF-1 strongly reduced the expression of cyclin D1 mRNA and correspondingly decreased the amount of beta-catenin in HeLa cells and squamous cell carcinoma cells. DIF-1 activated glycogen synthase kinase-3beta (GSK-3beta) and inhibition of GSK-3beta attenuated the DIF-1-induced beta-catenin degradation, indicating the involvement of GSK-3beta in this effect. Moreover, DIF-1 reduced the activities of T-cell factor (TCF)/lymphoid enhancer factor (LEF) reporter plasmid and a reporter gene driven by the human cyclin D1 promoter. Eliminating the TCF/LEF consensus site from the cyclin D1 promoter diminished the effect of DIF-1. These results suggest that DIF-1 inhibits Wnt/beta-catenin signaling, resulting in the suppression of cyclin D1 promoter activity.     

Beta-catenin triggers nuclear factor kappaB-dependent up-regulation of hepatocyte inducible nitric oxide synthase

Disruption of cell-to-cell contacts, as observed in many pathophysiological conditions, prime hepatocytes for compensatory hyperplastic response that involves induction of several genes, including proto-oncogenes and other gene targets of beta-catenin signaling pathway. By using cultured hepatocytes and experimental models of adherens junction disruption we have investigated changes in beta-catenin subcellular localization and their relationships with inducible nitric oxide synthase (iNOS) expression. Two experimental models were employed: (a) rat hepatocytes obtained by collagenase liver perfusion within the first 48 h of culture; (b) 48-h old cultured hepatocytes, transiently transfected or not with a plasmid encoding for dominant/negative inhibitory kappa B-alpha, exposed to ethylene glycol-bis-(2-aminoethylether)-N,N,N',N'-tetraacetic acid/LiCl treatment. beta-Catenin signaling and cellular localization, iNOS expression and nuclear factor kappaB involvement, were investigated using morphological, cell and molecular biology techniques. E-cadherin-mediated disruption of cell-to-cell contacts induces early beta-catenin translocation from membrane to cytoplasm and nuclear compartments, events that are followed by up-regulation of c-myc, cyclin D1 and beta-transducin repeat-containing protein expression. This, in turn, resulted eventually in iNOS induction that was mechanistically related to nuclear factor kappaB activation, as unequivocally shown in cells expressing dominant negative inhibitory kappa B-alpha. Our data indicate that E-cadherin disassembly and concomitant inactivation of glycogen synthase kinase-3beta result in nuclear factor kappaB-dependent induction of iNOS in hepatocytes.     

A GSK3-binding peptide from FRAT1 selectively inhibits the GSK3-catalysed phosphorylation of axin and beta-catenin.

The Axin-dependent phosphorylation of beta-catenin catalysed by glycogen synthase kinase-3 (GSK3) is inhibited during embryogenesis. This protects beta-catenin against ubiquitin-dependent proteolysis, leading to its accumulation in the nucleus, where it controls the expression of genes important for development. Frequently rearranged in advanced T-cell lymphomas 1 (FRAT1) is a mammalian homologue of a GSK3-binding protein (GBP), which appears to play a key role in the correct establishment of the dorsal-ventral axis in Xenopus laevis. Here, we demonstrate that FRATtide (a peptide corresponding to residues 188-226 of FRAT1) binds to GSK3 and prevents GSK3 from interacting with Axin. FRATtide also blocks the GSK3-catalysed phosphorylation of Axin and beta-catenin, suggesting a potential mechanism by which GBP could trigger axis formation. In contrast, FRATtide does not suppress GSK3 activity towards other substrates, such as glycogen synthase and eIF2B, whose phosphorylation is independent of Axin but dependent on a 'priming' phosphorylation. This may explain how the essential cellular functions of GSK3 can continue, despite the suppression of beta-catenin phosphorylation.     

Casein kinase II phosphorylation of E-cadherin increases E-cadherin/beta-catenin interaction and strengthens cell-cell adhesion

Beta-catenin, a member of the Armadillo repeat protein family, binds directly to the cytoplasmic domain of E-cadherin, linking it via alpha-catenin to the actin cytoskeleton. A 30-amino acid region within the cytoplasmic domain of E-cadherin, conserved among all classical cadherins, has been shown to be essential for beta-catenin binding. This region harbors several putative casein kinase II (CKII) and glycogen synthase kinase-3beta (GSK-3beta) phosphorylation sites and is highly phosphorylated. Here we report that in vitro this region is indeed phosphorylated by CKII and GSK-3beta, which results in an increased binding of beta-catenin to E-cadherin. Additionally, in mouse NIH3T3 fibroblasts expression of E-cadherin with mutations in putative CKII sites resulted in reduced cell-cell contacts. Thus, phosphorylation of the E-cadherin cytoplasmic domain by CKII and GSK-3beta appears to modulate the affinity between beta-catenin and E-cadherin, ultimately modifying the strength of cell-cell adhesion.     

TM4SF1, a binding protein of DVL2 in hepatocellular carcinoma,positively regulates beta-catenin/TCF signalling

The interaction between Axin and DVL2 is critical for the breaking down of the beta-catenin destruction complex and the activation of the Wnt/beta-catenin cascade. However, this biological process remains poorly understood. In the present study, TM4SF1 was identified as the interacting partner of DVL2 and positively regulated as Wnt/beta-catenin signalling by strengthening the DVL2-Axin interaction. The expression levels of TM4SF1 were elevated in hepatocellular carcinoma (HCC) and were induced by Kras signalling. The overexpression of TM4SF1 promoted the growth and motility of HCC cells, and up-regulated the target genes (Axin2 and cyclin D1). The down-regulation of TM4SF1 impaired the capability of HCC cells for growth, migration and metastasis. In addition, the down-regulation of TM4SF1 promoted the ubiquitination of beta-catenin. In summary, these results reveal the oncogenic functions of TM4SF1 in HCC progression and suggest that TM4SF1 might be a target for treatment.     

Formation of the head organizer in hydra involves the canonical Wnt pathway

Stabilization of beta-catenin by inhibiting the activity of glycogen synthase kinase-3beta has been shown to initiate axis formation or axial patterning processes in many bilaterians. In hydra, the head organizer is located in the hypostome, the apical portion of the head. Treatment of hydra with alsterpaullone, a specific inhibitor of glycogen synthase kinase-3beta, results in the body column acquiring characteristics of the head organizer, as measured by transplantation experiments, and by the expression of genes associated with the head organizer. Hence, the role of the canonical Wnt pathway for the initiation of axis formation was established early in metazoan evolution.