Alois Kozubík

Aryl hydrocarbon receptor-mediated disruption of contact inhibition is associated with connexin43 downregulation and inhibition of gap junctional intercellular communication

The aryl hydrocarbon receptor (AhR) contributes to the control of cell-to-cell communication, cell adhesion, migration or proliferation. In the present study, we investigated the regulation of connexin43 (Cx43) and Cx43-mediated gap junctional intercellular communication (GJIC) during the AhR-dependent disruption of contact inhibition in non-tumorigenic liver epithelial cells. The contact inhibition of cell proliferation is a process restricting the cell division of confluent non-transformed cells, which is frequently abolished in cancer cells; however, the mechanisms contributing to its disruption are still only partially understood. Disruption of contact inhibition, which was induced by toxic AhR ligands 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or polycyclic aromatic hydrocarbons in epithelial WB-F344 cells, reduced Cx43 protein levels, possibly via enhanced proteasomal degradation, significantly decreased the amount of gap junction plaques and downregulated GJIC, in an AhR-dependent manner. Although both intracellular and membrane Cx43 pools were markedly reduced in cells released from contact inhibition by TCDD, siRNA-mediated Cx43 knock-down was not sufficient to stimulate proliferation in contact-inhibited cells. Our data suggest that downregulation of Cx43/GJIC in non-transformed epithelial cells is an inherent part of disruption of contact inhibition, which occurs at the post-transcriptional level. This process runs in parallel with alterations of other forms of cell-to-cell communication, thus suggesting that toxic AhR agonists may simultaneously abrogate contact inhibition and reduce GJIC, two essential mechanisms linked to deregulation of cell-to-cell communication during tumor promotion and progression.

Receptor tyrosine kinases activate canonical WNT/β-catenin signaling via MAP kinase/LRP6 pathway and direct β-catenin phosphorylation.

Receptor tyrosine kinase signaling cooperates with WNT/β-catenin signaling in regulating many biological processes, but the mechanisms of their interaction remain poorly defined. We describe a potent activation of WNT/β-catenin by FGFR2, FGFR3, EGFR and TRKA kinases, which is independent of the PI3K/AKT pathway. Instead, this phenotype depends on ERK MAP kinase-mediated phosphorylation of WNT co-receptor LRP6 at Ser1490 and Thr1572 during its Golgi network-based maturation process. This phosphorylation dramatically increases the cellular response to WNT. Moreover, FGFR2, FGFR3, EGFR and TRKA directly phosphorylate β-catenin at Tyr142, which is known to increase cytoplasmic β-catenin concentration via release of β-catenin from membranous cadherin complexes. We conclude that signaling via ERK/LRP6 pathway and direct β-catenin phosphorylation at Tyr142 represent two mechanisms used by various receptor tyrosine kinase systems to activate canonical WNT signaling.

Mitogen-activated protein kinases promote WNT/beta-catenin signaling via phosphorylation of LRP6.

ABSTRACT LDL-related protein 6 (LRP6) is a coreceptor of WNTs and a key regulator of the WNT/β-catenin pathway. Upon activation, LRP6 is phosphorylated within its intracellular PPPS/TP motifs. These phosphorylated motifs are required to recruit axin and to inhibit glycogen synthase kinase 3 (GSK3), two basic components of the β-catenin destruction complex. On the basis of a kinome-wide small interfering RNA (siRNA) screen and confirmative biochemical analysis, we show that several proline-directed mitogen-activated protein kinases (MAPKs), such as p38, ERK1/2, and JNK1 are sufficient and required for the phosphorylation of PPPS/TP motifs of LRP6. External stimuli, which control the activity of MAPKs, such as phorbol esters and fibroblast growth factor 2 (FGF2) control the choice of the LRP6-PPPS/TP kinase and regulate the amplitude of LRP6 phosphorylation and WNT/β-catenin-dependent transcription. Our findings suggest that cells not only recruit one dedicated LRP6 kinase but rather select their LRP6 kinase depending on cell type and the external stimulus. Moreover, direct phosphorylation of LRP6 by MAPKs provides a unique point for convergence between WNT/β-catenin signaling and mitogenic pathways.

Molecular pathology of the fibroblast growth factor family

The human fibroblast growth factor (FGF) family contains 22 proteins that regulate a plethora of physiological processes in both developing and adult organism. The mutations in the FGF genes were not known to play role in human disease until the year 2000, when mutations in FGF23 were found to cause hypophosphatemic rickets. Nine years later, seven FGFs have been associated with human disorders. These include FGF3 in Michel aplasia; FGF8 in cleft lip/palate and in hypogonadotropic hypogonadism; FGF9 in carcinoma; FGF10 in the lacrimal/salivary glands aplasia, and lacrimo‐auriculo‐dento‐digital syndrome; FGF14 in spinocerebellar ataxia; FGF20 in Parkinson disease; and FGF23 in tumoral calcinosis and hypophosphatemic rickets. The heterogeneity in the functional consequences of FGF mutations, the modes of inheritance, pattern of involved tissues/organs, and effects in different developmental stages provide fascinating insights into the physiology of the FGF signaling system. We review the current knowledge about the molecular pathology of the FGF family. Hum Mutat 30:1–11, 2009.

Inhibitors of lipoxygenase metabolism exert synergistic effects with retinoic acid on differentiation of human leukemia HL-60 cells

The interaction between drugs suppressing the production of arachidonic acid metabolites and inducers of granulocytic differentiation, i.e., all-trans retinoic acid and dimethyl sulphoxide (DMSO) was investigated using the human myeloid leukemia HL-60 cell line. The experiments were designed as a complete factorial combination of treatments and used chemiluminescence as a marker of cell oxidative burst (level of differentiation). It was clearly demonstrated that two structurally different inhibitors of 5-lipoxygenase metabolism, i.e., 3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-di methyl propanoic acid (MK-886) and esculetin, significantly potentiated the HL-60 cell differentiation induced by retinoic acid or DMSO. Detailed mathematical evaluation of the results revealed the synergistic character of the interaction. The most significant effects were achieved with a combination of 5-lipoxygenase inhibitors and low doses of retinoic acid. These results were confirmed by analysis of cell morphology and expression of cell surface antigen CD11b after treatment of the cells with selected concentrations of agents. In contrast to those on differentiation, no additional effects of MK-886 or esculetin on cell proliferation (cell number and cell cycle parameters) and apoptosis were observed. An inhibitor of cyclooxygenases, indomethacin, affected neither cell proliferation nor differentiation of cells. The results implied that either modulation of 5-lipoxygenase metabolism or a certain type of imbalance in arachidonic acid metabolism could modulate the effects of retinoic acid or DMSO on myeloid cell differentiation.

The Interplay of the Aryl Hydrocarbon Receptor and β-Catenin Alters Both AhR-Dependent Transcription and Wnt/β-Catenin Signaling in Liver Progenitors

β-catenin is a key integrator of cadherin-mediated cell-cell adhesion and transcriptional regulation through the Wnt/β-catenin pathway, which plays an important role in liver biology. Using a model of contact-inhibited liver progenitor cells, we examined the interactions of Wnt/β-catenin signaling with the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, which mediates the toxicity of dioxin-like compounds, including their effects on development and hepatocarcinogenesis. We found that AhR and Wnt/β-catenin cooperated in the induction of AhR transcriptional targets, such as Cyp1a1 and Cyp1b1. However, simultaneously, the activation of AhR led to a decrease of dephosphorylated active β-catenin pool, as well as to hypophosphorylation of Dishevelled, participating in regulation of Wnt signaling. A sustained AhR activation by its model ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), led to a downregulation of a number of Wnt/β-catenin pathway target genes. TCDD also induced a switch in cytokeratin expression, where downregulation of cytokeratins 14 and 19 was accompanied with an increased cytokeratin 8 expression. Together with a downregulation of additional markers associated with stem-like phenotype, this indicated that the AhR activation interfered with differentiation of liver progenitors. The downregulation of β-catenin was also related to a reduced cell adhesion, disruption of E-cadherin-mediated cell-cell junctions and an increased G1-S transition in liver progenitor cell line. In conclusion, although β-catenin augmented the expression of selected AhR target genes, the persistent AhR activation may lead to downregulation of Wnt/β-catenin signaling, thus altering differentiation and/or proliferative status of liver progenitor cells.

Breast cancer-specific mutations in CK1ε inhibit Wnt/β-catenin and activate the Wnt/Rac1/JNK and NFAT pathways to decrease cell adhesion and promote cell migration

IntroductionBreast cancer is one of the most common types of cancer in women. One of the genes that were found mutated in breast cancer is casein kinase 1 epsilon (CK1ε). Because CK1ε is a crucial regulator of the Wnt signaling cascades, we determined how these CK1ε mutations interfere with the Wnt pathway and affect the behavior of epithelial breast cancer cell lines.MethodsWe performed in silico modeling of various mutations and analyzed the kinase activity of the CK1ε mutants both in vitro and in vivo. Furthermore, we used reporter and small GTPase assays to identify how mutation of CK1ε affects different branches of the Wnt signaling pathway. Based on these results, we employed cell adhesion and cell migration assays in MCF7 cells to demonstrate a crucial role for CK1ε in these processes.ResultsIn silico modeling and in vivo data showed that autophosphorylation at Thr 44, a site adjacent to the breast cancer point mutations in the N-terminal lobe of human CK1ε, is involved in positive regulation of the CK1ε activity. Our data further demonstrate that, in mammalian cells, mutated forms of CK1ε failed to affect the intracellular localization and phosphorylation of Dvl2; we were able to demonstrate that CK1ε mutants were unable to enhance Dvl-induced TCF/LEF-mediated transcription, that CK1ε mutants acted as loss-of-function in the Wnt/β-catenin pathway, and that CK1ε mutants activated the noncanonical Wnt/Rac-1 and NFAT pathways, similar to pharmacological inhibitors of CK1. In line with these findings, inhibition of CK1 promoted cell migration as well as decreased cell adhesion and E-cadherin expression in the breast cancer-derived cell line MCF7.ConclusionsIn summary, these data suggest that the mutations of CK1ε found in breast cancer can suppress Wnt/β-catenin as well as promote the Wnt/Rac-1/JNK and Wnt/NFAT pathways, thus contributing to breast cancer development via effects on cell adhesion and migration. In terms of molecular mechanism, our data indicate that the breast cancer point mutations in the N-terminal lobe of CK1ε, which are correlated with decreased phosphorylation activities of mutated forms of CK1ε both in vitro and in vivo, interfere with positive autophosphorylation at Thr 44.

The planar cell polarity pathway drives pathogenesis of chronic lymphocytic leukemia by the regulation of B-lymphocyte migration.

The planar cell polarity (PCP) pathway is a conserved pathway that regulates cell migration and polarity in various contexts. Here we show that key PCP pathway components such as Vangl2, Celsr1, Prickle1, FZD3, FZD7, Dvl2, Dvl3, and casein kinase 1 (CK1)-ε are upregulated in B lymphocytes of patients with chronic lymphocytic leukemia (CLL). Elevated levels of PCP proteins accumulate in advanced stages of the disease. Here, we show that PCP pathway is required for the migration and transendothelial invasion of CLL cells and that patients with high expression of PCP genes, FZD3, FZD7, and PRICKLE1, have a less favorable clinical prognosis. Our findings establish that the PCP pathway acts as an important regulator of CLL cell migration and invasion. PCP proteins represent an important class of molecules regulating pathogenic interaction of CLL cells with their microenvironment.

Activation of the aryl hydrocarbon receptor is the major toxic mode of action of an organic extract of a reference urban dust particulate matter mixture: The role of polycyclic aromatic hydrocarbons

Many of the toxic and carcinogenic effects of urban air pollution have been linked to polycyclic aromatic hydrocarbons (PAHs) adsorbed to airborne particulate matter (PM). The carcinogenic properties of PAHs in complex organic mixtures derived from PM have been chiefly attributed to their mutagenicity. Nevertheless, PAHs are also potent activators of the aryl hydrocarbon receptor (AhR), which may contribute to their nongenotoxic effects, including tumor promotion. As the genotoxicity of carcinogenic PAHs in complex mixtures derived from urban PM is often inhibited by other mixture constituents, the AhR-mediated activity of urban PM extracts might significantly contribute to the carcinogenic activity of such mixtures. In the present study, we used an organic extract of the urban dust standard reference material, SRM1649a, as a model mixture to study a range of toxic effects related to DNA damage and AhR activation. Both the organic extract and its neutral aromatic fraction formed a low number of DNA adducts per nucleotide in the liver epithelial WB-F344 cells model, without inducing DNA damage response, such as tumor suppressor p53 activation and apoptosis. In contrast, we found that this extract, as well as its neutral and polar fractions, were potent inducers of a range of AhR-mediated responses, including induction of the AhR-mediated transcription, such as cytochrome P450 1A1/1B1 expression, and the AhR-dependent cell proliferation. Importantly, these toxic events occurred at doses one order of magnitude lower than DNA damage. The AhR-mediated activity of the neutral fraction was linked to PAHs and their derivatives, as polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls were only minor contributors to the overall AhR-mediated activity. Taken together, our data suggest that more attention should be paid to the AhR-dependent nongenotoxic events elicited by urban PM constituents, especially PAHs and their derivatives.

TGF-β1-induced EMT of non-transformed prostate hyperplasia cells is characterized by early induction of SNAI2/Slug

Epithelial–mesenchymal transition (EMT) underlying cancer cell invasion and metastasis has been thoroughly studied in prostate cancer. Although EMT markers have been clinically observed in benign prostate hyperplasia, molecular events underlying the onset and progression of EMT in benign prostate cells have not been described.