The dual-specificity MAP kinase phosphatases: critical roles in development and cancer.

Intracellular signaling by mitogen-activated protein (MAP) kinases (MAPK) is involved in many cellular responses and in the regulation of various physiological and pathological conditions. Tight control of the localization and duration of extracellular-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), or p38 MAPK activity is thus a fundamental aspect of cell biology. Several members of the dual-specificity phosphatase (DUSPs) family are able to dephosphorylate MAPK isoforms with different specificity, cellular, and tissue localization. Understanding how these phosphatases are themselves regulated during development or in physiological and pathological conditions is therefore fundamental. Over the years, gene deletion and knockdown studies have completed initial in vitro studies and shed a new light on the global and specific roles of DUSPs in vivo. Whereas DUSP1, DUSP2, and DUSP10 appear as crucial players in the regulation of immune responses, other members of the family, like the ERK-specific DUSP6, were shown to play a major role in development. Recent findings on the involvement of DUSPs in cancer progression and resistance will also be discussed.

Post-translational regulation of the ERK phosphatase DUSP6/MKP3 by the mTOR pathway.

MAP kinases phosphatases (MKPs) belong to the dual-specificity phosphatase family (DUSP) and dephosphorylate phosphothreonine and phosphotyrosine within MAP kinases. We had previously shown that DUSP6/MKP-3 was phosphorylated and degraded upon growth factor stimulation, in a MEK-dependent manner. Here we show that another pathway involved in growth factor signaling, the PI3K/mTOR signaling pathway, accounts for a part of the phosphorylation and degradation of DUSP6 induced by serum growth factors, as evidenced by experiments using pharmacological inhibitors of PI3 kinase and mammalian target of rapamycin (mTOR). Moreover, specific agonists of the mTOR pathway, such as amino acids or insulin/IGF-1, which do not activate extracellular signal regulated kinases (ERKs) in our cellular model, were also able to induce the phosphorylation and degradation of DUSP6. However, a basal activity of MEK was required for the mTOR pathway-mediated phosphorylation to occur. Mutagenesis studies identified serine 159 within DUSP6 as the target of the mTOR pathway. The ERK phosphatase DUSP6 may thus constitute a novel branch-point of the crosstalk between two major signaling pathways induced by growth factors, the MEK/ERK pathway and the PI3K/mTOR pathway.

Inducible expression of a MAP kinase phosphatase-3-GFP chimera specifically blunts fibroblast growth and ras-dependent tumor formation in nude mice.

The p42/p44 mitogen activated protein kinase (MAPK) pathway participates in a wide range of cellular programs including proliferation, migration, differentiation, and survival. Specific pharmacological inhibitors, like PD98059 and U0126, are often used to inhibit p42/p44 MAPK signaling. However, these inhibitors are not appropriate to study the function of these kinases in whole organisms. We thus developed an inducible system designed to inhibit p42/p44 MAPK activity through the expression of a phosphatase specific for these two kinases, the MAPK phosphatase 3 (MKP-3). A fibroblast cell line was established in which MKP-3 expression is controlled by tetracycline. Tetracycline-induced MKP-3 resulted in partial de-phosphorylation of p42/p44 MAPKs in serum-stimulated cells. However, we could improve MKP-3 stability and thereby the rate of MAPK de-phosphorylation, when the C-terminal end of MKP-3 was fused to the green fluorescent protein (GFP). Importantly, the fusion of GFP to MKP-3 did not alter the specificity of the phosphatase towards its MAPK substrates. We further show that conditional expression of MKP-3-GFP in this fibroblast cell line results in the inhibition of: (a) the phosphorylation of the p42/p44 MAPK substrates Elk1 and HIF-1alpha, (b) vascular endothelial growth factor (VEGF), cyclin D1, and c-fos gene transcription in response to MAPK pathway activation, and (c) cell proliferation. Finally, the MKP-3-GFP inducible cell line was transformed by Ha-ras and injected into nude mice. Treatment of mice with the tetracycline analog doxycycline resulted in a large delay in tumor emergence and growth as compared to the untreated control group, indicating that MKP-3-GFP activity is maintained in vivo. Altogether, these results show that inducible expression of MKP-3-GFP constitutes a valuable tool to study the role of p42/p44 MAPKs in various cellular responses in both cultured cell and animal models, a tool that may also be used to block unwanted cell growth in pathological conditions.

Defects in adhesion and migration, but not in proliferation and differentiation, of embryonic stem cells upon replacement of integrin subunit beta1A by beta1D.

Beta1D is a skeletal muscle-specific splice variant of the beta1 integrin subunit, while beta1A integrin subunit has a wide tissue distribution. We have previously shown that replacement of beta1A by beta1D by homologous recombination (knockin) in all mouse tissues was embryonic lethal. Through two successive rounds of homologous recombination, we have now produced embryonic stem (ES) cells expressing beta1D instead of beta1A, and analyzed the ability of beta1D to support ES cell differentiation in vitro and in teratomas in vivo. Beta1D knockin (KI) ES cells grew at a similar rate but as more compact colonies than the beta1A-expressing cells. Increased cell cohesiveness, however, did not appear to involve changes in cadherin activity. Although in both beta1A and beta1D-KI ES cells only one beta1 allele is active; the expression of beta1 integrins in the beta1D-KI ES cells was reduced by 50%, compared with that in the beta1A-expressing cells; this correlated with impaired adhesive and migratory capacities. It appeared that during in vitro cardiac differentiation, in spite of a slight delay in the induction of two cardiac-specific transcripts, the alpha- and beta-myosin heavy chains, contracting cardiomyocytes were detected in similar numbers and at the same time in embryoid bodies (EB) derived from beta1D-KI and from beta1A cells. Furthermore, replacement of beta1A by beta1D in ES cells did not affect neurite differentiation in embryoid bodies in the presence of retinoic acid suggesting that beta1D supports neurogenesis. However, the impaired migration of other cells from the EB, including endodermal cells, prevented the normal outgrowth of neurites in beta1D-KI EB. Finally, injection of beta1D-KI ES cells in the flank of syngeneic mice gave rise to fully developed teratomas containing simple and pluristratified epithelia, muscle, cartilage, blood vessels, and tissues from the neural lineage. These results show that the muscle-specific splice variant beta1D, in spite of its specific cytoplasmic domain, supports the differentiation of many cell types. This further suggests that the embryonic lethality in the beta1D-KI embryos was mainly due to the different ability of beta1 A and beta1D to mediate cell adhesion and migration.

Induction of cell scattering by expression of beta1 integrins in beta1-deficient epithelial cells requires activation of members of the rho family of GTPases and downregulation of cadherin and catenin function.

Adhesion receptors, which connect cells to each other and to the surrounding extracellular matrix (ECM), play a crucial role in the control of tissue structure and of morphogenesis. In this work, we have studied how intercellular adhesion molecules and beta1 integrins influence each other using two different beta1-null cell lines, epithelial GE11 and fibroblast-like GD25 cells. Expression of beta1A or the cytoplasmic splice variant beta1D, induced the disruption of intercellular adherens junctions and cell scattering in both GE11 and GD25 cells. In GE11 cells, the morphological change correlated with the redistribution of zonula occluden (ZO)-1 from tight junctions to adherens junctions at high cell confluency. In addition, the expression of beta1 integrins caused a dramatic reorganization of the actin cytoskeleton and of focal contacts. Interaction of beta1 integrins with their respective ligands was required for a complete morphological transition towards the spindle-shaped fibroblast-like phenotype. The expression of an interleukin-2 receptor (IL2R)-beta1A chimera and its incorporation into focal adhesions also induced the disruption of cadherin-based adhesions and the reorganization of ECM-cell contacts, but failed to promote cell migration on fibronectin, in contrast to full-length beta1A. This indicates that the disruption of cell-cell adhesion is not simply the consequence of the stimulated cell migration. Expression of beta1 integrins in GE11 cells resulted in a decrease in cadherin and alpha-catenin protein levels accompanied by their redistribution from the cytoskeleton-associated fraction to the detergent-soluble fraction. Regulation of alpha-catenin protein levels by beta1 integrins is likely to play a role in the morphological transition, since overexpression of alpha-catenin in GE11 cells before beta1 prevented the disruption of intercellular adhesions and cell scattering. In addition, using biochemical activity assays for Rho-like GTPases, we show that the expression of beta1A, beta1D, or IL2R-beta1A in GE11 or GD25 cells triggers activation of both RhoA and Rac1, but not of Cdc42. Moreover, dominant negative Rac1 (N17Rac1) inhibited the disruption of cell-cell adhesions when expressed before beta1. However, all three GTPases might be involved in the morphological transition, since expression of either N19RhoA, N17Rac1, or N17Cdc42 reversed cell scattering and partially restored cadherin-based adhesions in GE11-beta1A cells. Our results indicate that beta1 integrins regulate the polarity and motility of epithelial cells by the induction of intracellular molecular events involving a downregulation of alpha-catenin function and the activation of the Rho-like G proteins Rac1 and RhoA.

Cre-loxP-mediated inactivation of the alpha6A integrin splice variant in vivo: evidence for a specific functional role of alpha6A in lymphocyte migration but not in heart development.

Two splice variants of the alpha6 integrin subunit, alpha6A and alpha6B, with different cytoplasmic domains, have previously been described. While alpha6B is expressed throughout the development of the mouse, the expression of alpha6A begins at 8.5 days post coitum and is initially restricted to the myocardium. Later in ontogeny, alpha6A is found in various epithelia and in certain cells of the immune system. In this study, we have investigated the function of alpha6A in vivo by generating knockout mice deficient for this splice variant. The Cre- loxP system of the bacteriophage P1 was used to specifically remove the exon encoding the cytoplasmic domain of alpha6A in embryonic stem cells, and the deletion resulted in the expression of alpha6B in all tissues that normally express alpha6A. We show that alpha6A-/- mice develop normally and are fertile. The substitution of alpha6A by alpha6B does not impair the development and function of the heart, hemidesmosome formation in the epidermis, or keratinocyte migration. Furthermore, T cells differentiated normally in alpha6A-/- mice. However, the substitution of alpha6A by alpha6B leads to a decrease in the migration of lymphocytes through laminin-coated Transwell filters and to a reduction of the number of T cells isolated from the peripheral and mesenteric lymph nodes. Lymphocyte homing to the lymph nodes, which involves various types of integrin-ligand interactions, was not affected in the alpha6A knockout mice, indicating that the reduced number of lymph node cells could not be directly attributed to defects in lymphocyte trafficking. Nevertheless, the expression of alpha6A might be necessary for optimal lymphocyte migration on laminin in certain pathological conditions.

Adhesion complexes formed by OVCAR-4 cells on laminin 1 differ from those observed on fibronectin.

Cell adhesion to laminin 1 or to fibronectin is mediated by distinct sets of integrins and is differentially regulated by protein kinase C (PKC). It suggests that upon integrin ligation to laminin 1 or to fibronectin different intracellular signaling pathways could be activated. we have therefore investigated the formation of signaling complexes induced during cell adhesion to laminin 1 or to fibronectin. Following cell adhesion to laminin 1 the re-arrangement of the cytoskeleton was slower than that observed on fibronectin and it was activated by treating the cells with H-7, an inhibitor of PKC. Conversely, treatment of laminin-adhering cells with a PKC activator resulted in a rapid disorganization of the actin cyto skeleton while a similar treatment had no effect on fibronectin-adhering cells. These results suggested that the structural organization of the adhesion complexes might be substrate-specific and might correspond to a different arrangement of cytoskeletal and/or cytoplasmic proteins. Reflection interference contrast microscopy (RICM) images revealed that cell-substratum contacts formed on laminin 1 were not well differentiated in contrast to those developed on fibronectin. However, immunofluorescence staining revealed a similar organisation of actin microfilaments, talin and phosphotyrosyl-containing proteins on both substrates. In contrast, differences were observed for vinculin distribution within cells spread on fibronectin or on laminin 1. Following cell adhesion to fibronectin most of the vinculin appeared as thick patches at the tips of the actin stress fibers while in laminin-adhering cells vinculin was recruited into thin streaks localized at the end of only some actin stress fibers.

The cytoplasmic domain of alpha 6A integrin subunit is an in vitro substrate for protein kinase C.

We have investigated whether the cytoplasmic domain of alpha 6A integrin subunit can be phosphorylated by Ser/Thr kinases using synthetic peptides as in vitro substrates. This domain was phosphorylated by protein kinase C (PKC) and cyclic AMP-dependent kinase (protein kinase A, PKA) but not by mitogen-activated protein kinase. While Ser1041 has been shown to be phosphorylated in PMA-stimulated cells in vitro, Ser1048 was phosphorylated by PKA. Furthermore pharmacological agents which induce a rise in cyclic AMP concentration failed to stimulate the phosphorylation of the alpha 6A cytoplasmic domain in intact cells. These results suggest that PKC, but not PKA, is involved in the physiological phosphorylation of the alpha 6A integrin subunit.

Cellular interactions with the extracellular matrix are coupled to diverse transmembrane signaling pathways.

Extracellular matrix (ECM) glycoproteins such as laminin, fibronectin, or collagen IV play a major role in cell behavior regulation. The molecular mechanisms taking place at the interface between the ECM and the cell surface are now rather well defined; however, very little is known about intracellular signals induced by these interactions. In order to get insights into the transduction pathways involved in cell-ECM interactions we have investigated the effects of several intracellular kinase inhibitors. Calmodulin-dependent kinase inhibitors, W-7 and sphingosine, have negative effects on cell-matrix interactions. They inhibit adhesion of several cell lines to laminin (IC50 = 4-10 microM), fibronectin and collagen IV (IC50 = 7-25 microM). The effects are immediate, reversible, and also cell specific, certain combinations of cell line-substrate being irresponsive to these inhibitors. In contrast, two inhibitors, H-7 and staurosporine, for which protein kinase C is a common target, increase two- to fourfold the attachment of HT1080, OVCAR-4, and B16F10 cells to laminin but not to fibronectin. Another inhibitor, HA-1004, known to inhibit protein kinase A at low concentrations, has an activating effect only at high concentration (> 200 microM) when it becomes an inhibitor of protein kinase C. These inhibitors are without effect on RuGli and Saos-2 cell adhesion on the three substrates. Altogether these results suggest that calmodulin-dependent kinases and protein kinase C could be separately involved in ECM-induced cellular responses. However, the effects of kinase inhibitors are substrate-specific and cell type-specific, suggesting that the intracellular signals induced by the extracellular matrix vary with the nature of integrin involved in signal transmission.

IL-5-induced IgA synthesis by LPS-stimulated mouse B cells is prevented by protein kinase C inhibitors.

We have previously demonstrated that activation of protein kinase C (PKC) by phorbol esters induces selectively IgA synthesis by mouse B cells. In this study, we investigated the effects of a number of protein kinase inhibitors on IgA secretion induced by a recombinant murine IL-5 in LPS-stimulated mouse B cells. The results show that PKC inhibitors, such as sphingosine (SPH), staurosporine (STP) and H-7, blocked IL-5-induced IgA synthesis; the protein kinase A inhibitor HA-1004 and the inhibitor of calcium/calmodulin dependent protein kinase W-7 had no effect on IgA secretion induced by IL-5. The proliferation of the IL-5 sensitive B13 cell line in response to IL-5 was also inhibited by addition of SPH or STP or H-7. The data suggest an involvement of the PKC pathway in IL-5-induced B cell differentiation into IgA secreting cells.