Table of contents

MAPK and embryonic stem cells

Embryonic stem (ES) cells can be derived and propagated from multiple strains of mouse and rat through application of small-molecule inhibitors of the fibroblast growth factor (FGF)/Erk pathway and of glycogen synthase kinase 3. These conditions shield pluripotent cells from differentiation-inducing stimuli. This study investigated the effect of these inhibitors on the development of pluripotent epiblast in intact pre-implantation embryos. Blockade of Erk signalling from the 8-cell stage was found not impede blastocyst formation but suppresses development of the hypoblast. The size of the inner cell mass (ICM) compartment is not reduced, however. Throughout the ICM, the epiblast-specific marker Nanog is expressed, and in XX embryos epigenetic silencing of the paternal X chromosome is erased. Epiblast identity and pluripotency were confirmed by contribution to chimaeras with germline transmission. These observations indicate that segregation of hypoblast from the bipotent ICM is dependent on FGF/Erk signalling and that in the absence of this signal, the entire ICM can acquire pluripotency. Furthermore, the epiblast does not require paracrine support from the hypoblast. Thus, naive epiblast and ES cells are in a similar ground state, with an autonomous capacity for survival and replication, and high vulnerability to Erk signalling. The relationship between naive epiblast and ES cells was probed directly. Dissociated ICM cells from freshly harvested late blastocysts gave rise to up to 12 ES cell clones per embryo when plated in the presence of inhibitors. It is proposed that ES cells are not a tissue culture creation, but are essentially identical to pre-implantation epiblast cells (Nichols, 2009).

MAPK and gastrulation

Mesoderm induction is a critical early step in vertebrate development, involving changes in gene expression and morphogenesis. In Xenopus, normal mesoderm formation depends on signalling through the fibroblast growth factor (FGF) tyrosine kinase receptor. One important signalling pathway from receptor tyrosine kinases involves p21ras. Ras associates with the serine kinase c-Raf-1 in a GTP-dependent manner, and this complex phosphorylates and activates MAPK/ERK kinase (MEK), a protein kinase with dual specificity. MEK then activates p42mapk and (at least in mammals) p44mapk, both members of the mitogen-activated protein (MAP) kinase family. FGF activates MAP kinase during mesoderm induction: the use of dominant-negative constructs suggests that mesoderm induction by FGF requires both Ras and Raf. However, these experiments do not reveal whether Ras and Raf do act through MAP kinase to induce mesoderm or whether another pathway, such as the phosphatidylinositol 3-kinase cascade, is involved. It is shown that expression of active forms of MEK or of MAP kinase induces ventral mesoderm of the kind elicited by FGF. Overexpression of a Xenopus MAP kinase phosphatase blocks mesoderm induction by FGF, and causes characteristic defects in mesoderm formation in intact embryos, whereas inhibition of the P13 kinase and p70 S6 kinase pathways has no effect on mesoderm induction by FGF. FGF induces different types of mesoderm in a dose-dependent manner; strikingly, this is mimicked by expressing different levels of activated MEK. Together, these experiments demonstrate that activation of MAP kinases is necessary and sufficient for mesoderm formation (Umbhauer, 1995).

The role of MAP kinase was examined during mesoderm induction and axial patterning in Xenopus embryos. MAP Kinase Phosphatase (MKP-1) was used to inactivate endogenous MAP kinase and was found to prevent the induction of early and late mesodermal markers by both FGF and activin. In whole embryos, MKP-1 disrupts posterior axial patterning, generating a phenotype similar to that obtained with a dominant inhibitory FGF receptor. Overexpression of either constitutively active MAP kinase or constitutively active MAP kinase kinase (MEK) is sufficient to induce Xbra expression, while only constitutively active MEK is able to significantly induce expression of muscle actin. When MAP kinase phosphorylation is used as a sensitive marker of FGF receptor activity in vivo, this activity is found to persist at a low and relatively uniform level throughout blastula stage embryos. The finding that a low level of MAP kinase phosphorylation exists in unstimulated animal caps and is absent in caps overexpressing a dominant inhibitory FGF receptor provides a basis for the observation that overexpression of this receptor inhibits activin induction. These results indicate that FGF-dependent MAP kinase activity plays a critical role in establishing the responsiveness of embryonic tissues to mesoderm inducers (LaBonne, 1995).

The transcriptional activity of a set of genes, which are all expressed in overlapping spatial and temporal patterns within the Spemann organizer of Xenopus embryos, can be modulated by peptide growth factors. Xegr-1, a zinc finger protein-encoding gene, has been identified as a novel member of this group of genes. The spatial expression characteristics of Xegr-1 during gastrulation are most similar to those of Xbra (see Drosophila Brachyenteron). Making use of animal cap explants, analysis of the regulatory events that govern induction of Xegr-1 gene activity reveals that, in sharp contrast to transcriptional regulation of Xbra, activation of Ets-serum response factor (SRF) transcription factor complexes is required and sufficient for Xegr-1 gene expression. The Ets-SRF complexes are known to act downstream of the MAP kinase pathway, and in the case of Xegr-1 the complex is shown to function downstream of FGF signaling. The finding that Xegr-1 activation requires Ets-SRF complexes provides the first indication for Ets-SRF complexes binds to serum response elements that are activated during gastrulation. MAP kinase signaling cascades can induce and sustain expression of both Xegr-1 and Xbra. Ectopic Xbra is found to induce Xegr-1 transcription by an indirect mechanism that appears to operate via primary activation of fibroblast growth factor secretion. These findings define a cascade of events that links Xbra activity to the activation of FGF signaling and the subsequent signal-regulated control of Xegr-1 transcription in the context of early mesoderm induction in Xenopus laevis (Panitz, 1998).

The activity and function of mitogen-activated protein kinase (MAPK) has been examined during neural specification in Xenopus. Ectodermal MAPK activity increases between late blastula and midgastrula stages. At midgastrula, MAPK activity in both newly induced neural ectoderm and ectoderm overexpressing the anterior neural inducer noggin is 5-fold higher than in uninduced ectoderm. Overexpression of MAPK phosphatase-1 (MKP-1) in ectoderm inhibits MAPK activity and prevents neurectoderm-specific gene expression when the ectoderm is recombined with dorsal mesoderm or treated with fibroblast growth factor (FGF). Neurectoderm-specific gene expression is observed, however, in ectoderm overexpressing both noggin and MKP-1. To evaluate the role of MAPK in posterior regionalization, ectodermal isolates were treated with increasing concentrations of FGF and assayed for MAPK activity and neurectoderm-specific gene expression. Although induction of posterior neural ectoderm by FGF is accompanied by an elevation of MAPK activity, relative MAPK activity associated with posterior neural fate is no higher than that of ectoderm specified to adopt an anterior neural fate. Thus, increasingly posterior neural fates are not correlated with quantitative increases in MAPK activity. Because MAPK has been shown to down-regulate Smad1, MAPK may disrupt bone morphogenetic protein 4 (BMP-4) signaling during neural specification. These results suggest that MAPK plays an essential role in the establishment of neural fate in vivo (Uzgare, 1998).

FGF signaling has been implicated in germ layer formation and axial determination. An antibody specific for the activated form of mitogen-activated protein kinase (MAPK) was used to monitor FGF signaling in vivo during early Xenopus development. Activation of MAPK in young embryos is abolished by injection of a dominant negative FGF receptor (XFD) RNA, suggesting that MAPK is activated primarily by FGF in this context. A transition from cytoplasmic to nuclear localization of activated MAPK occurs in morula/blastula stage embryo animal and marginal zones coinciding with the proposed onset of mesodermal competence. It is also possible that the subcellular localization of activated MAPK is part of the actual 'switch' which, once turned on by a putative developmental timer, will allow activated MAPK to activate the FGF signaling pathway as required to respond to mesodermal induction. In Drosophila, a similar phenomenon occurs in the EGFR-dependent and Sevenless-dependent activation of MAPK in which activated MAPK is observed only in the cytoplasm for 2 and 6 hours, respectively, before translocation to the nucleus. These results suggest that an additional regulated step is present in these RTK pathways (Curran, 2000).

Activated MAPK delineates the region of the dorsal marginal zone before blastopore formation and persists in this region during gastrulation, indicating an early role for FGF signaling in dorsal mesoderm. Activated MAPK is also found in posterior neural tissue from late gastrulation onward. Inhibition of FGF signaling does not block posterior neural gene expression (HoxB9) or activation of MAPK; however, inhibition of FGF signaling does cause a statistically significant decrease in the level of activated MAPK. These results point toward the involvement of other receptor tyrosine kinase signaling pathways in posterior neural patterning (Curran, 2000).

The loss of expression of activated MAPK in postinvolution mesoderm may indicate that a specific downregulation of FGF signaling is required for full differentiation of particular mesodermal fates. Activated MAPK expression is lost after the tissue passes over the blastopore lip during involution, though it is maintained in the developing notochord. eFGF and Xbra have similar expressions. Cells overexpressing FGFR1 can not differentiate into myoblasts and FGF signaling can block the differentiation of those cells into muscle. Thus, the downregulation of activated MAPK (and FGF signaling) in more lateral and anterior mesoderm following involution may be necessary for further mesodermal differentiation to proceed. This explanation is also consistent with the evidence for the role of FGF in the maintenance of Xbra expression. A downregulation of FGF signaling following the involution of the anterior mesoderm would eliminate Xbra in that tissue as well. It is not clear how FGF signaling is maintained in some mesoderm and not in others (Curran, 2000).

Knowledge of when and where signaling pathways are activated is crucial for understanding embryonic development. This study systematically analyzes and compares the signaling pattern of four major pathways by localization of the activated key components ß-catenin (Wnt proteins), MAPK (tyrosine kinase receptors/FGF), Smad1 (BMP proteins) and Smad2 (Nodal/activin/Vg1). The distribution of these components has been determined at 18 consecutive stages in Xenopus development, from early blastula to tailbud stages. The image obtained is that of very dynamic and widespread activities, with very few inactive regions. Signaling fields can vary from large gradients to restricted areas with sharp borders. They do not respect tissue boundaries. This direct visualization of active signaling verifies several predictions inferred from previous functional data. It also reveals unexpected signal patterns, pointing to some poorly understood aspects of early development. In several instances, the patterns strikingly overlap, suggesting extensive interplay between the various pathways. To test this possibility, maternal ß-catenin signaling has been manipulated and the effect on the other pathways in the blastula embryo has been determined. The patterns of P-MAPK, P-Smad1 and P-Smad2 are indeed strongly dependent on ß-catenin at this stage: their dorsal accumulation is absent in UV-irradiated embryos. The highest levels are then found symmetrically in the vegetal-equatorial region, similar to ß-catenin. Upon LiCl treatment, P-MAPK and P-Smad2 are strongly activated also in the ventral side. A similar activation is observed at the site of ß-catenin overexpression. Despite extensive colocalization, P-MAPK and P-Smad2 activation appear, nevertheless, spatially more restricted than ß-catenin: in all conditions, high P-MAPK is limited to a broad equatorial ring, while P-Smad2 activation is most prominent in the vegetal hemisphere. These differences obviously reflect the differential distribution of other determinants, which limit activation of P-MAPK to the marginal zone and Smad2 to the vegetal pole. P-Smad1 has an opposite polarity, i.e. weakest in the dorsal animal region. In UV-irradiated embryos, P-Smad1 is also activated on the dorsal side. LiCl treatment or ventral ß-catenin overexpression causes a significant decrease in the ventral side. In conclusion, these data show that maternal ß-catenin signaling is an important factor in controlling intensity and pattern of the other pathways at blastula stages. While other parameters regulate the latitude of the activation fields, ß-catenin can entirely account for the dorsoventral polarity. Mechanistically, ß-catenin probably contributes to Smad2 activation by stimulating Xnrs expression. How ß-catenin controls MAPK and Smad1 remains to be investigated (Schohl, 2002).

In the sea urchin embryo, the skeleton of the larva is built from a population of mesenchymal cells known as the primary mesenchyme cells (PMCs). These derive from the large micromeres that originate from the vegetal pole at fourth cleavage. At the blastula stage, the 32 cells of this lineage detach from the epithelium and ingress into the blastocoel by a process of epithelial-mesenchymal transition. Shortly before ingression, there is a transient and highly localized activation of the MAP-kinase ERK in the micromere lineage. Ingression of the PMCs requires the activity of ERK, MEK and Raf, and depends on the maternal Wnt/ß-catenin pathway. Dissociation experiments and injection of mRNA encoding a dominant-negative form of Ras indicates that this activation is probably cell autonomous. The transcription factors Ets1 and Alx1 were identified as putative targets of the phosphorylation by ERK. Both proteins contain a single consensus site for phosphorylation by the MAP kinase ERK. In addition, the Ets1 protein sequence contains a putative ERK docking site. Overexpression of ets1 by injection of synthetic mRNA in the egg causes a dramatic increase in the number of cells becoming mesenchymal at the blastula stage. This effect could be largely inhibited by treating embryos with the MEK inhibitor U0126. Moreover, mutations in the consensus phosphorylation motif substituting threonine 107 by an aspartic or an alanine residue resulted, respectively, in a constitutively active form of Ets1 that could not be inhibited by U0126 or in an inactive form of Ets1. These results show that the MAP kinase pathway, working through phosphorylation of Ets1, is required for full specification of the PMCs and their subsequent transition from epithelial to mesenchymal state (Röttinger, 2004).

MAPK and somitogenesis

The temporal and spatial regulation of somitogenesis requires a molecular oscillator, the segmentation clock. Through Notch signaling, the oscillation in cells is coordinated and translated into a cyclic wave of expression of hairy-related and other genes. The wave sweeps caudorostrally through the presomitic mesoderm (PSM) and finally arrests at the future segmentation point in the anterior PSM. By experimental manipulation and analyses in zebrafish somitogenesis mutants, a novel component involved in this process has been found. The level of Fgf/MAPK activation (highest in the posterior PSM) serves as a positional cue within the PSM that regulates progression of the cyclic wave and thereby governs the positions of somite boundary formation (Sawada, 2001).

Modulating Fgf signaling resulted in alterations in somite size. Detailed analyses of gene expressions in manipulated wild-type and mutant embryos reveal a novel function of Fgf/MAPK signaling in the PSM: the maintenance of cells in an immature state that allows the her1 wave to sweep through the PSM. Suppression of Fgf signaling posteriorizes the domain shift of her1 expression, as well as the expression of other segmentation genes such as mesp, a bHLH transcription factor crucial for segmentation initiation, and paraxial protocadherin. This leads to a posterior shift in segment border formation and larger somites. These results are complementary to those obtained with transplantation of Fgf beads, strengthening the idea that an Fgf signal determines the position of segment border formation by negatively regulating the maturation of the PSM. Since Fgf signal is known to have profound effects on many developmental processes such as cell growth and maintenance of progenitor cells, it is possible that manipulation of an Fgf signal locally changes the cell number in the PSM by regulating cell proliferation and/or cell migration within the mesoderm (axial, paraxial and lateral plate mesoderm). This could cause alterations in somite size. However, no such effect was observed in manipulated PSM, indicating that an Fgf signal in the PSM simply regulates the maturation status of cells without affecting cell proliferation or migration (Sawada, 2001).

The data are largely consistent with the 'clock-and-wavefront' model in which a cyclic wave operates in conjunction with a maturation wavefront that gradually moves posteriorly, resulting in arrest of the cyclic wave and initiation of segment furrow formation. Fgf/MAPK signaling negatively regulates the wavefront activity and restricts it to the anterior PSM that is devoid of MAPK activation. In zebrafish, the essential components of a conserved somite-making mechanism, the segmentation clock and wavefront are Notch- and fused somites-dependent, respectively. Zebrafish after eight/deltaD mutation desychronizes the oscillation wave, while, in the absence of Fused somites, the anterior PSM fails to acquire the wavefront activity. How could Fgf/MAPK signal interact with these components? In fact, it has been reported that the Ras/MAPK pathway interacts with the Notch pathway in C. elegans vulval development and malignant transformation of cultured cells. However, no interaction between Fgf/MAPK and Notch or Fss pathways could be demonstrated in this study: modulating Fgf signaling exerts identical effects on wild-type and after eight/DeltaD or fused somites mutants in terms of gene expression. Furthermore, the patterns of ERK activation and fgf8 expression in the PSM is not affected by after eight/DeltaD or fused somites mutations. Thus, it is concluded that the activation and action of Fgf/MAPK signaling in the PSM are not mediated by Notch or Fss pathway (Sawada, 2001).

The fact that four to five somites are normally formed after SU5402 treatment indicates that the positioning of furrow formation is already specified or Fgf insensitive at least at the position -IV to -V in the PSM. The result also indicates that ERK activation in segmented somites is not involved in segment border formation. Interestingly, the Fgf-sensitive region corresponds approximately to the heat-shock sensitive zone in zebrafish; that is, the initial defects in the segmental pattern of somite boundaries are observed five somites caudal to the forming somite at the time of heat shock. These data suggest that position -IV to -V represents a position at which the level of Fgf/MAPK activation drops below a threshold, rendering the cells competent to maturation signals. In support of this, transplanted Fgf8 beads exert their effects only when they are located in the Fgf-negative anterior PSM. Importantly, the relative position of MAPK activation domain to the newly formed somite is kept constant in the PSM as the embryos extend. These observations are consistent with the idea that the level of Fgf/MAPK activation serves as a positional cue within the PSM (Sawada, 2001).

MAPK and the immune response

There is remarkable conservation in the recognition of pathogen-associated molecular patterns (PAMPs) by innate immune responses of plants, insects and mammals. An Arabidopsis thaliana leaf cell system has been developed based on the induction of early-defense gene transcription by flagellin, a highly conserved component of bacterial flagella that functions as a PAMP in plants and mammals. A complete plant MAP kinase cascade (MEKK1, MKK4/MKK5 and MPK3/MPK6) and WRKY22/WRKY29 transcription factors has been identified that functions downstream of the flagellin receptor FLS2, a leucine-rich-repeat (LRR) receptor kinase. Activation of this MAPK cascade confers resistance to both bacterial and fungal pathogens, suggesting that signaling events initiated by diverse pathogens converge into a conserved MAPK cascade (Asai, 2002).

Engagement of the T-cell receptor (TCR) with cognate ligands provokes different outcomes depending on the developmental stage of the T cell and on the properties of the ligand. In immature thymocytes TCR stimulation may result in maturation (positive selection) or death (negative selection), whereas in mature T cells it may induce proliferation, death or unresponsiveness. The role of the MAP kinase (MAPK) cascade in T cell maturation was examined by expressing a catalytically inactive form of MAPK kinase (MEK-1) in thymocytes, thereby blocking MAPK activation. Positive selection of these cells is inhibited but negative selection and TCR-induced proliferation are unaffected. These results indicate that the intracellular signals regulating lineage commitment in T cells parallel those in photoreceptor cell specification in Drosophila and vulval cell differentiation in C. elegans, suggesting that general rules for cell-type specification could apply among all metazoans (Alberola-Ila, 1995).

T cells activated by antigen receptor stimulation in the absence of accessory cell-derived costimulatory signals lose the capacity to synthesize the growth factor interleukin-2 (IL-2), a state called clonal anergy. An analysis of CD3- and CD28-induced signal transduction reveals reduced ERK and JNK enzyme activities in murine anergic T cells. The amounts of ERK and JNK proteins were unchanged, and the kinases can be fully activated in the presence of phorbol ester. Dephosphorylation of the calcineurin substrate NFATp (preexisting nuclear factor of activated T cells) also remains inducible. These results suggest that a specific block in the activation of ERK and JNK contributes to defective IL-2 production in clonal anergy (Li, W., 1996).

The induction of T cell proliferation requires signals from the TCR and a co-receptor molecule, such as CD28, that activate parallel and partially cross-reactive signaling pathways that involve the Rolled homolog ERK. These pathways are disrupted by agonists that utilize adenylate cyclase (Drosophila homolog: rutabaga) and cAMP-dependent protein kinase A (Drosophila homolog: PKA). The adenylate cyclase activator, forskolin, inhibits anti-CD3-induced shift in Lck electrophoretic mobility, suggesting an intervention at TCR-coupled phosphoinositide turnover that precedes the activation o PKC. Forskolin also inhibits PKC downstream events, such as c-jun (Drosophila homolog: DJun) expression, which is critical for the activation process of T cells. A large part of the anti-CD3-induced ERK activation is PKC dependent. Both PKC-dependent and -independent activation of ERK are sensitive to inhibition by forskolin or a cell-permeable cAMP analogue (Tamir, 1996).

CD40 is a 45- to 50-kDa transmembrane glycoprotein that plays an important role in B cell proliferation, survival, memory, and Ig isotype switching. In comparison to signaling via the B cell Ag receptor (BCR), CD40 cross-linking is less effective at activating protein tyrosine kinases. Interestingly, however, CD40 engagement results in the phosphorylation of both extracellular signal-regulated protein kinase (ERK) and the Ras guanine nucleotide exchange factor, Son of sevenless. In addition, both ERK and c-Jun NH2-terminal kinase (Drosophila homolog: Basket/JNK) activities are increased after both CD40 and BCR ligation. Treatment of cells with phorbol ester as well as inhibitors of protein kinase C abrogated these signaling events after BCR treatment; however, no effect is seen on CD40-mediated activation of ERK or c-Jun NH2-terminal kinase, suggesting that the BCR and CD40 differentially utilize protein kinase C to couple with these signaling pathways (Li, Y., 1996).

Mitogen-activated protein kinase (MAPK) extracellular signal-regulated protein kinase (ERK) cascade is involved in CD40 signaling in mouse B cells. Analysis of ERK activities in a B cell lymphoma line, which shows an increase in DNA synthesis or arrest of the cell cycle by cross-linking of CD40 or surface IgM (sIgM) cross-linking, respectively, indicates that one of the ERK isoforms, ERK2, was preferentially and rapidly activated after CD40 cross-linking. The CD40-mediated ERK2 activation is comparable to that after sIgM stimulation, although the activity was reduced toward the basal level within several minutes after stimulation. In contrast, ERK1 and ERK2 are activated to a similar extent by sIgM cross-linking, and the activities remained stable for at least 10 min. Furthermore, similar features of differential activation of ERK isoforms are observed in normal resting B cells in CD40 and sIgM signaling. These results suggest divergent regulatory pathways for ERK1 and ERK2 activation, and they support the notion that CD40 signaling may utilize a limited set of elements in the ERK cascade. Co-stimulation of cells with anti-CD40 mAb rescues the cells from anti-IgM-mediated apoptosis, whereas this co-stimulation results in activation of ERK isoforms comparable to that in sIgM stimulation, without a synergistic effect. This result indicates the dominance of ERK activation in sIgM signaling over that of CD40, and it suggests that ERK activation may not be linked to the biological effect that CD40 stimulation in this cell line (Kashiwada, 1996).

The chemotactic peptide f-Met-Leu-Phe (fMLP) stimulates leukocyte functions through binding and activation of a specific G-protein-coupled formyl peptide receptor (FPR). Stimulation of neutrophils with fMLP induces the activation of ERK1 and ERK2. Stimulation of transfected cells with fMLP results in increased tyrosine phosphorylation and activation of ERK1 and ERK2 and the activation of MEK, the MAP kinase/ERK kinase. The activation of both ERKs and MEK is inhibited by pertussis toxin, indicating that activation is dependent upon a Gi/Go-like protein that couples to the receptor. Unlike neutrophils, FPR-transfected fibroblasts do not express the Src-related kinase Lyn. In the absence of Lyn, fMLP stimulation does not result in an increased tyrosine phosphorylation of the adapter protein SHC, whereas it is still able to induce MAP kinase activation. These data suggest that Lyn and SHC are not the only upstream signals for activation of the MAP kinase/ERK pathway by fMLP (Torres, 1996).

The p42 and p44 mitogen-activated protein kinases (MAPKs), also called Erk2 and Erk1, respectively, have been implicated in proliferation as well as in differentiation programs. The specific role of the p44 MAPK isoform in the whole animal has been evaluated by generation of p44 MAPK-deficient mice by homologous recombination in embryonic stem cells. The p44 MAPK-/- mice are viable, fertile, and of normal size. Thus, p44 MAPK is apparently dispensable and p42 MAPK (Erk2) may compensate for its loss. However, in p44 MAPK-/- mice, thymocyte maturation beyond the CD4+CD8+ stage is reduced by half, with a similar diminution in the thymocyte subpopulation expressing high levels of T cell receptor (CD3high). In p44 MAPK-/- thymocytes, proliferation in response to activation with a monoclonal antibody to the T cell receptor in the presence of phorbol myristate acetate is severely reduced even though activation of p42 MAPK is more sustained in these cells. The p44 MAPK apparently has a specific role in thymocyte development. These findings indicate that there may be a physiological distinction between p42 and p44 MAPK isoforms (Pages, 1999).

MAPK, development and differentiation

In 3T3-L1 fibroblasts, Ras proteins mediate both insulin-induced differentiation to adipocytes and activation of cytosolic serine/threonine kinases, including Raf-1 kinase, mitogen-activated protein kinase (MAPK), and Rsk. Insulin- and Ras-induced activation of MAPK is not required for the differentiation process, and in fact antagonizes it. The treatment of 3T3-L1 preadipocytes with MEK-specific inhibitor PD98059 blocks insulin- and Ras-induced MAPK activation but has no effect on (or only slightly enhances) adipocytic differentiation. Tumor necrosis factor alpha (TNF-alpha), an inhibitor of insulin-stimulated adipogenesis, activates MAPK in 3T3-L1 cells. PD98059 treatment blocks MAPK activation by TNF-alpha and reverses the blockade of adipogenesis mediated by low TNF-alpha concentrations. 3T3-L1 transfectants containing hyperactivated MEK1 or overexpressed MAPK display impaired adipocytic differentiation. PD98059 treatment also reverses the blockade of differentiation in MEK1 transfectants. These results indicate that MAPK does not promote but can contribute to inhibition of the process of adipocytic differentiation of 3T3-L1 cells (Font de Mora, 1997).

The two MAP kinases JNK and ERK direct distinct cellular activities, even though they share a number of common substrates, including several transcription factors. JNK and ERK signaling were compared during PC12 cell differentiation; an investigation was carried out to determine how activation of c-Jun by the MAPKs contributes to this cellular response. Exposure to nerve growth factor, or expression of constitutively active MEK1 -- two treatments that cause differentiation of PC12 cells into a neuronal phenotype -- results in activation of ERK-type MAP kinases and phosphorylation of c-Jun on several sites, including Ser63 and Ser73. Constitutively activated c-Jun, which mimics the MAPK-phosphorylated form of the protein, can induce neuronal differentiation of PC12 cells independent of upstream signals. Conversely, expression of dominant-negative c-Jun bZIP prevents neurite outgrowth induced by activated MEK1. Activation of MEKK1, which stimulates the JNK pathway, is not sufficient for PC12 cell differentiation but can induce apoptosis. However, neurite outgrowth is triggered when c-Jun is co-expressed with activated MEKK1 or SEK1. Consistently, MEK-induced ERK activation in PC12 cells induces c-Jun expression, while JNK signaling does not. Therefore, dual input of expression and phosphorylation of c-Jun provided by the ERK pathway is required to direct neuronal differentiation in PC12 cells (Leppa, 1998).

Human epidermis contains two types of proliferative keratinocyte: the stem cell, which has a high self-renewal capacity and low probability of terminal differentiation, and the transit-amplifying cell, the daughter of a stem cell that is destined to differentiate within about three to five rounds of division. Keratinocytes with characteristics of stem cells persist in culture and can be distinguished from transit-amplifying cells on the basis of the type of colony they form (2, 3). Colonies founded by stem cells are able to self-renew, whereas transit-amplifying colonies contain fewer than 30-40 cells, all of which undergo terminal differentiation. Human epidermal stem cells express higher levels of beta1 integrins and are more adhesive than the keratinocytes that are destined to differentiate. To investigate whether high beta1 integrin expression and adhesiveness are essential for maintaining keratinocytes in the stem cell compartment, a dominant-negative beta1 integrin mutant, CD8beta1, was introduced into cultured human keratinocytes, thereby interfering with beta1 integrin function. Surface beta1 integrin levels, adhesiveness, and mitogen-activated protein (MAP) kinase activation on fibronectin were reduced, and exit from the stem cell compartment was stimulated. Adhesiveness and proliferative potential are restored by overexpressing wild-type beta1 integrin or by constitutive MAP kinase activation. Conversely, a dominant-negative MAP kinase kinase 1 mutant decreases adhesiveness and stem cell number in the absence of CD8beta1. MAP kinase activation by alpha6beta4-mediated adhesion and mitogens is normal in CD8beta1 cells, and constitutive MAP kinase activation does not affect adhesion and proliferation of control keratinocytes. It is concluded that beta1 integrins and MAP kinase cooperate to maintain the epidermal stem cell compartment in vitro (Zhu, 1999).

The dominant-negative function of chimeras between the beta1 cytoplasmic domain and the extracellular domain of a nonintegrin protein could be caused by competition with endogenous integrins for intracellular factors. Alternatively, the chimeras might deliver a signal that reduces the affinity of the endogenous integrins for their ligands. The data favor a competition model in which beta1 signaling through MAPK is impaired. CD8beta1 expression results in reduced signaling to the MAPK cascade when keratinocytes are plated on fibronectin but such expression has no effect on MAPK activation in response to alpha6beta4-mediated adhesion or mitogens. Bypassing the beta1 integrin signaling defect by introduction of constitutively activated MAPKK1 into CD8beta1-expressing cells restores adhesiveness and increases the proportion of stem cells. Conversely, a dominant-negative MAPKK1 mutant is sufficient to reduce the adhesiveness and proliferative potential of keratinocytes in the absence of CD8beta1. The ability of the wild-type chicken beta1 construct to rescue proliferative potential and MAPK activation in CD8beta1-expressing cells argues against an affinity modulation signal from CD8beta1 and suggests that the strength of the MAPK signal depends on the ratio of functional (i.e., wild-type) to mutant (i.e., CD8beta1) receptors (Zhu, 1999).

There are at least three mechanisms by which beta1 integrin-mediated adhesion can activate MAPK. In the first two, MAPK is activated via Ras, either through FAK or Shc. In keratinocytes, FAK phosphorylation is not inhibited by CD8beta1, as observed when similar dominant-negative beta1 constructs are expressed in other cell types. The Shc-mediated pathway involves the interaction of the transmembrane and juxtamembrane extracellular domains of integrin alpha subunits with caveolin, and it is difficult to envisage how this process would be affected by overexpression of the beta1 cytoplasmic domain. The data presented in this paper favor a third mechanism that is independent of Ras and FAK. It should also be noted that in some contexts MAPK can down-regulate beta1 integrin function (Zhu, 1999).

The results show that a signaling pathway involving beta1 integrins and MAPK controls epidermal stem cell fate in vitro and raise two important questions: why are high integrin levels required in order for a keratinocyte to remain a stem cell, and how are those levels controlled? Because ligand binding suppresses overt terminal differentiation within the basal layer of the epidermis, high surface levels of beta1 integrins could protect stem cells from differentiation. Because the beta1 integrins have a pericellular distribution in stem and transit-amplifying cells, the proportion of surface integrins in contact with the basement membrane will be similar in both cell populations. It therefore seems likely that it is the absolute number of occupied receptors that is important for the protective effect (Zhu, 1999).

The extent to which stem cell behavior is preprogrammed or environmentally regulated has long been a subject for debate. Although epidermal stem cell number is subject to autoregulation, it is believed that environmental factors, specifically the composition of the basement membrane, could also be key determinants. Stimulatory or inhibitory input into the beta1 integrin/MAPK pathway at different levels could provide a mechanism by which the environment influences the proliferative capacity of basal keratinocytes. ECM proteins can modulate beta1 integrin expression and activation, and local variation in the composition of the basement membrane could thus play a role in establishing and maintaining the patterned distribution of stem cells within the epidermal basal layer (Zhu, 1999).

Epidermal growth factor (EGF) stimulates branching morphogenesis of the fetal mouse submandibular gland (SMG): the EGF receptor (EGFR) is localized principally, if not exclusively, on the epithelial components of the fetal SMG. The EGFR is a receptor tyrosine kinase, and after binding of its ligand, it triggers several intracellular signaling cascades, among them the one activating the mitogen-activated protein kinases (MAPK) ERK-1/2. An investigation was carried out to see whether EGF utilizes the ERK-1/2 signaling cascade to stimulate branching morphogenesis in the fetal mouse SMG. SMG rudiments were collected as matched pairs at E14, E16, and E18, placed into wells of defined medium (BGJb), and exposed to EGF for 5 or 30 min. EGF induces the appearance of multiple bands of phosphotyrosine-containing proteins, including bands at 170 kDa and 44 kDa/42 kDa, presumably corresponding to the phosphorylated forms of EGFR and ERK-1/2, respectively. Other blots show the specific appearance of the phosphorylated EGFR and of phospho-ERK-1/2 in response to EGF. Immunohistochemical staining for phosphotyrosine increases at the plasma membrane after EGF stimulation for 5 or 30 min. Diffuse cytoplasmic staining for MEK-1/2 (the MAPK kinase that activates ERK-1/2) increases near the cell membrane after EGF stimulation. Phospho-ERK-1/2 localizes in the nuclei of a few epithelial cells after EGF for 5 min, but in the nuclei of many cells after EGF for 30 min. PD98059, an inhibitor of phosphorylation and activation of MEK-1/2, by itself inhibits branching morphogenesis and, furthermore, decreases the stimulatory effect of EGF on branching. Western blots confirm that this inhibitor blocks phosphorylation of ERK-1/2 in fetal SMGs exposed to EGF. These results show that components of the ERK-1/2 signaling cascade are present in epithelial cells of the fetal SMG, that they are activated by EGF, and that inhibition of this cascade perturbs branching morphogenesis. However, EGF does not cause phosphorylation of two other MAPKs (SAPK/JNK or p38MAPK) in fetal SMGs. These results imply that the ERK-1/2 signaling is responsible, at least in part, for the stimulatory effect of EGF on branching morphogenesis of the fetal mouse SMG (Kashimata, 2000).

MAPKs are crucially involved in the regulation of growth and differentiation of a variety of cells. To elucidate the role of MAPKs in keratinocyte differentiation, activation of ERK, JNK, and p38 in response to stimulation with extracellular calcium was analyzed. Evidence is provided that calcium-induced differentiation of keratinocytes is associated with rapid and transient activation of the Raf/MEK/ERK pathway. Stimulation of keratinocytes with extracellular calcium results in activation of Raf isozymes and their downstream effector ERK within 10-15 min, but does not increase JNK or p38 activity. Calcium-induced ERK activation differs in kinetics from mitogenic ERK activation by epidermal growth factor and can be modulated by alterations of intracellular calcium levels. Interestingly, calcium stimulation leads to down-regulation of Ras activity at the same time that ERK activation is initiated. Expression of a dominant-negative mutant of Ras also does not significantly impair calcium-induced ERK activation, indicating that calcium-mediated ERK activation does not require active Ras. Despite the transient nature of ERK activation, calcium-induced expression of the cyclin-dependent kinase inhibitor p21/Cip1 and the differentiation marker involucrin is sensitive to MEK inhibition, which suggests a role for the Raf/MEK/ERK pathway in early stages of keratinocyte differentiation (Schmidt, 2000).

In amphibian development, muscle is specified in the dorsal lateral marginal zone (DLMZ) of the gastrula embryo. Two critical events specify the formation of skeletal muscle: the expression of the myogenic transcription factor, XMyoD, and the secretion of bone morphogenetic protein (BMP) antagonists by the adjacent Spemann organizer. Inhibition of BMP signaling during early gastrula stages converts XMyoD protein into an instructive differentiation factor in the DLMZ. Yet, the intracellular signaling factors connecting BMP antagonism and activation of XMyoD remain unknown. BMP antagonism induces the activity of mitogen-activated protein kinase (MAPK), and the activity of MAPK is necessary for muscle-specific differentiation. Treatment of gastrula-stage DLMZ explants with MAPK pathway inhibitors ventralizes mesoderm and prevents muscle differentiation. Expression of XMyoD in ventral mesoderm weakly induces muscle formation; however, the coexpression of a constitutively active MEK1 with XMyoD efficiently induces muscle differentiation. Activation of the MAPK pathway does not induce the transcription of XMyoD, but increases its protein levels and transcriptional activity. Thus, MAPK activation is subsequent to BMP antagonism, and participates in the dorsalization of mesoderm by converting the XMyoD protein into a potent differentiation factor (Zetser, 2001).

During mammalian palatal fusion, the medial edge epithelial (MEE) cells must stop DNA synthesis prior to the initial contact of opposing palatal shelves and thereafter selectively disappear from the midline. Exogenous EGF has been shown to inhibit the cessation of DNA synthesis and induce cleft palate; however, the precise intracellular mechanism has not been determined. It was hypothesized that EGF signaling acting via ERK1/2 would maintain MEE DNA synthesis and cell proliferation and consequently inhibit the process of palatal fusion. Palatal shelves from E13 mouse embryos were maintained in organ cultures and stimulated with EGF. EGF-treated palates fail to fuse with intact MEE and had significant ERK1/2 phosphorylation. Both EGF-induced ERK1/2 phosphorylation and BrdU-incorporation localize in the nucleus of MEE cells. Subsequent inhibition assays using U0126, a specific inhibitor of ERK1/2 phosphorylation, were conducted. U0126 inhibits EGF-induced ERK1/2 phosphorylation in a dose-dependent manner and consequently MEE cells stop proliferation. The threshold of ERK1/2 inactivation to stop MEE DNA synthesis coincides with the level required to rescue the EGF-induced cleft palate phenotype. These results indicate that EGF-induced inhibition of palatal fusion is dependent on nuclear ERK1/2 activation and that this mechanism must be tightly regulated during normal palatal fusion (Yamamoto, 2003).

Cells in the early vertebrate somite receive cues from surrounding tissues, which are important for their specification. A number of signalling pathways involved in somite patterning have been described extensively. By contrast, the interactions between cells from different regions within the somite are less well characterised. This study demonstrates that myotomally derived FGFs act through the MAPK signal transduction cascade and in particular, ERK1/2 to transcriptionally activate expression of bHLH factor scleraxis in a population of mesenchymal progenitor cells in the dorsal sclerotome. Levels of active, phosphorylated ERK protein in the developing somite are crucial for the expression of scleraxis and Mkp3. MKP3 is a dual specificity phosphatase and a specific antagonist of ERK MAP kinases and in somites Mkp3 transcription depends on the presence of active ERK. Therefore, MKP3 and ERK MAP kinase constitute a negative feedback loop activated by FGF in sclerotomal progenitor cells. It is proposed that tight control of ERK signalling strength by MKP3 is important for the appropriate regulation of downstream cellular responses, including the activation of scleraxis. Increased or decreased levels of phosphorylated ERK result in the loss of scleraxis transcripts and the loss of distal rib development, highlighting the importance of the MKP3-ERK-MAP kinase mediated feedback loop for cell specification and differentiation (Smith, 2005).

Table of contents

rolled/MAPK: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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