Control of thrombopoietin-induced megakaryocytic differentiation by the mitogen-activated protein kinase pathway

Thrombopoietin (TPO) is the major regulator of both growth and differentiation of megakaryocytes. We previously showed that both functions can be generated by TPO in the megakaryoblastic cell line UT7, in which murine Mpl was introduced, and are independently controlled by distinct regions of the cytoplasmic domain of Mpl. Particularly, residues 71 to 94 of this domain (deleted in the mutant mpl delta3) were found to be required for megakaryocytic maturation but dispensable for proliferation. We show here that TPO-induced differentiation in UT7 cells is tightly dependent on a strong, long-lasting activation of the mitogen-activated protein kinase (MAPK) pathway. Indeed, (i) in UT7-mpl cells, TPO induced a strong activation of extracellular signal-regulated kinases (ERK) which was persistent until at least 4 days in TPO-containing medium; (ii) a specific MAPK kinase (MEK) inhibitor inhibited TPO-induced megakaryocytic gene expression; (iii) the Mpl mutant mpl delta3, which displayed no maturation activity, transduced only a weak and transient ERK activation in UT7 cells; and (iv) TPO-induced megakaryocytic differentiation in UT7-mpl delta3 cells was partially restored by expression of a constitutively activated mutant of MEK. The capacity of TPO to trigger a strong and prolonged MAPK signal depended on the cell in which Mpl was introduced. In BAF3-mpl cells, TPO triggered a weak and transient ERK activation, similar to that induced in UT7-mpl delta3 cells. In these cells, no difference in MAPK activation was found between normal Mpl and mpl delta3. Thus, depending on the cellular context, several distinct regions of the cytoplasmic domain of Mpl and signaling pathways may contribute to generate quantitative variations in MAPK activation.     

Substitution of an aspartic acid results in constitutive activation of c-kit receptor tyrosine kinase in a rat tumor mast cell line RBL-2H3

The c-kit protooncogene encodes a receptor tyrosine kinase that mediates signals required for differentiation, proliferation and survival of mast cells. We have already shown the constitutive activation of c-kit receptor tyrosine kinase (KIT) in a human mast cell leukemia line (HMC-1) and a murine mastocytoma cell line (P-815). We here examined whether such constitutive activation of KIT occurred in the rat tumor mast cell line RBL-2H3 as well, which is frequently used as a tool for studying functions of mast cells. In RBL-2H3 cells, KIT was constitutively phosphorylated on tyrosine and activated in the absence of autocrine production of its ligand, stem cell factor (SCF). Sequencing analysis revealed that one of c-kit genes of RBL-2H3 cells had a point mutation, resulting in amino acid substitution of Tyr for Asp in codon 817. When rat wild-type c-kit cDNA and mutant-type c-kit cDNA encoding KITTyr817 were transfected into cells of a human embryonic kidney cell line (293T), only mutant form KITTyr817 was constitutively phosphorylated on tyrosine and activated in the absence of SCF. Since mutations at the same Asp codon constitutively activated KIT in all the human HMC-1, murine P-815, and rat RBL-2H3 cell lines, and since the incorporation of antisense oligonucleotides of c-kit messenger RNA significantly suppressed the proliferation of RBL-2H3 cells, the activating mutations in the Asp codon of the c-kit gene appeared to be involved in neoplastic growth of mast cells.     

Triple projections of neurones located in S1 and S2 segments of the cat spinal cord to the C6 segment, the cerebellum and the reticular formation

We have assessed five signal transduction pathways to determine the role each might play in the malignant transformation of mammary epithelium initiated by neu, heregulin/NDF, TGFalpha, v-Ha-ras and c-myc in transgenic mice. The study involves a molecular and pharmacologic assessment of Erk/MAP kinase, Jnk/SAP kinase, PI 3-kinase, protein kinase C, and the Src-related kinases Lck and Fyn. Our results indicate that oncogenes capable of transforming mammary gland epithelium activate and require specific signal transduction pathways. For example, mammary tumors initiated by neu, v-Ha-ras and c-myc have high levels of active Erk/MAP kinase and their anchorage independent growth is strongly inhibited by PD098059, an inhibitor of Mek/ MAP kinase kinase. By contrast, Erk/MAP kinase activity is weak in tumors initiated by TFGalpha and heregulin/NDF and the corresponding cell lines are not growth inhibited by PD098059. Similarly, PI 3-kinase is strongly activated in neu, TGFalpha and heregulin/NDF initiated tumor cell lines, but not in c-myc or v-Ha-ras initiated tumor cell lines. The anchorage independent growth of all these tumor cell lines are, however, inhibited by the specific PI 3-kinase inhibitor LY294001. Further illustrating this oncogene-based specificity, PP1, a specific inhibitor of the Src-like kinases, Lck and Fyn, blocks anchorage-independent cell growth only in the TGFalpha initiated mammary tumor cell line. Taken together with additional observations, we conclude that certain oncogenes reliably require the recruitment/activation of specific signal transduction pathways. Such specific relationships between the initiating oncogene and a required pathway may reflect a direct activating effect or the parallel activation of a pathway that is a necessary oncogenic collaborator for transformation in the mammary gland. The work points to a molecular basis for targeting therapy when an initiating oncogene can be implicated; for example, because of amplification, increased expression, genetic alteration, or heritable characteristics.     

Constitutive activation of Mek1 by mutation of serine phosphorylation sites

A variety of extracellular signals lead to the phosphorylation and activation of mitogen-activated protein kinases (MAP kinases). An activator of MAP kinases, Mek1, phosphorylates MAP kinases at threonine and tyrosine residues and is itself phosphorylated at serine-218 and -222 by the protooncogene product Raf-1. By introducing negatively charged residues that may mimic the effect of phosphorylation at positions 218 and 222, we have activated the capacity of Mek1 to phosphorylate MAP kinase by > 100-fold. The most effective activation by a single substitution resulted from the introduction of aspartate at position 218, whereas the introduction of either aspartate or glutamate at position 222 was ineffective. Expression of the activated Mek1 phosphorylation-site mutants in COS-7 cells led to the activation of MAP kinase in the cells and resulted in an increase in the mass of the transfected COS-7 cell population, suggesting an important role of Mek1 in the transduction of mitogenic signals.     

Phosphoinositide 3-kinase induces scattering and tubulogenesis in epithelial cells through a novel pathway

Hepatocyte growth factor/scatter factor (HGF/SF) treatment of the Madin-Darby canine kidney epithelial cell line causes scattering of cells grown in monolayer culture and the formation of branching tubules by cells grown in collagen gels. HGF/SF causes prolonged activation of both the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase 2 (ERK2) and the phosphoinositide 3-OH kinase (PI 3-kinase) target protein kinase B (PKB)/Akt; inhibition of either the MAP kinase pathway by the MAP kinase/ERK kinase inhibitor PD98059 or the PI 3-kinase pathway by LY294002 blocks HGF/SF induction of scattering, although in morphologically distinct ways. Expression of constitutively activated PI 3-kinase, Ras, or R-Ras will cause scattering, but activated Raf will not, indicating that activation of the MAP kinase pathway is not sufficient for this response. Downstream of PI 3-kinase, activated PKB/Akt and Rac are both unable to induce scattering, implicating a novel pathway. Scattering induced by Ras or PI 3-kinase is sensitive to PD98059, as well as to LY294002, suggesting that basal MAP kinase activity is required, but not sufficient, for the scattering response. Induction of MDCK cell tubulogenesis in collagen gels by HGF/SF is inhibited by PD98059; expression of activated Ras and Raf causes disorganized growth in this system, but activated PI 3-kinase or R-Ras causes branching tubule formation similar to that seen with HGF/SF treatment. These data indicate that multiple signaling pathways acting downstream of Met and Ras are needed for these morphological effects; scattering is induced primarily by the PI 3-kinase pathway, which acts through effectors other than PKB/Akt or Rac and requires at least basal MAP kinase function. Elevated PI 3-kinase activity induces tubulogenesis, but total inhibition and excess activation of the MAP kinase pathway both oppose this effect.     

Ganglioside GM1 activates the mitogen-activated protein kinase Erk2 and p70 S6 kinase in U-1242 MG human glioma cells

Gangliosides are implicated in the regulation of cellular proliferation as evidenced by differences in ganglioside composition associated with malignant transformation and density of cells in culture, as well as their inhibitory effects when added to cells growing in culture. Exogenously added gangliosides have a bimodal effect on proliferation in U-1242 MG glioma cells, inhibiting DNA synthesis in growing cells and stimulating it in quiescent cells. We investigated the mechanisms involved in stimulation of DNA synthesis using [3H]thymidine incorporation and immune complex kinase assays to identify responsible signal transduction pathways. Treatment of quiescent U-1242 MG cells with GM1 caused activation of the mitogen-activated protein (MAP) kinase isoform Erk2. Pretreatment with the specific MAP kinase kinase inhibitor PD98059 prevented the GM1-stimulated Erk2 activation and GM1-stimulated DNA synthesis. GM1 treatment stimulated another distinct signaling pathway leading to activation of p70 S6 kinase (p70s6k), and this was prevented by pretreatment with rapamycin. Rapamycin also inhibited GM1-stimulated DNA synthesis. Activation of both pathways and stimulation of DNA synthesis were inhibited by forskolin treatment; however, GM1 had no effect on cyclic AMP levels. Platelet-derived growth factor also activated both Erk2 and p70s6k but did not cause DNA synthesis, suggesting that GM1 may stimulate additional cascades, which also contribute to GM1-mediated DNA synthesis.     

Binding and regulation of thrombopoietin to human megakaryocytes

Thrombopoietin (TPO, c-Mpl ligand) is considered to play an important role in the regulation of megakaryocytopoiesis and platelet production by activating the cytokine receptor c-Mpl. We have examined the binding of 125I-TPO to the human megakaryocytic cell line, CMK, and to primary human megakaryocytes. Scatchard analysis of TPO binding to its cognate receptor in megakaryocytic cells suggested the existence of a single class of c-Mpl receptors. CMK cells exhibited 1223 receptors per cell with a dissociation constant (Kd) of Kd = 223 pM, whereas primary human megakaryocytes exhibited 12140 receptors per cell and a dissociation constant of Kd = 749 pM. The pretreatment of CMK cells and primary bone marrow megakaryocytes with TPO resulted in a decreased binding of TPO to the c-Mpl receptors. This down-regulation was observed within 3 h and was not inhibited by cycloheximide. Phorbol ester, an activator of protein kinase C, also inhibited TPO binding to the c-Mpl receptors by reducing the number of these receptors. The pretreatment of CMK cells with IL-3, IL-6 and DMSO, all of which induced the differentiation of CMK cells, did not affect the binding of TPO to the c-Mpl receptors. These results suggest an additional mechanism, where protein kinase C may help to regulate the binding of TPO to these cells.     

Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells

Nerve growth factor (NGF) induces sustained activation of classical MAP kinase (MAPK, also known as ERK) and neuronal differentiation in PC12 cells, whereas epidermal growth factor (EGF) induces transient activation of ERK/MAPK and stimulates proliferation of the cells. Although previous studies showed that sustained activation of ERK/MAPK is important for neuronal differentiation of the cells, a recent report revealed that inhibition of the sustained phase of ERK/MAPK activation alone does not block neurite outgrowth caused by NGF. These results suggest requirement for an additional signaling pathway(s) triggered by NGF in neuronal differentiation. Here we show that NGF induces sustained activation of p38, a subfamily member of the MAPK superfamily, and that inhibition of the p38 pathway blocks neurite outgrowth in PC12 cells. Surprisingly, expression of constitutively active MAPK/ERK kinase (MAPKK, also known as MEK) results in p38 activation as well as ERK/MAPK activation, and a p38 inhibitor blocks neurite outgrowth caused by the constitutively active MAPKK/MEK. Moreover, constitutive activation of p38 is able to induce neurite outgrowth when combined with EGF treatment. These results reveal an essential role of p38 in neuronal differentiation in PC12 cells.     

Lck associates with and is activated by Kit in a small cell lung cancer cell line: inhibition of SCF-mediated growth by the Src family kinase inhibitor PP1

At least 70% of small cell lung cancers (SCLCs) express the Kit receptor tyrosine kinase and its ligand, stem cell factor (SCF). In an effort to define the signal transduction pathways activated by Kit in SCLC, we focused on Src family kinases and, in particular, Lck, a Src-related tyrosine kinase that is expressed in hemopoietic cells and certain tumors, including SCLC. SCF treatment of the H526 cell line induced a physical association between Kit and Lck that, in vitro, was dependent on phosphorylation of the juxtamembrane domain of Kit. Stimulation of Kit with recombinant SCF resulted in a rapid 3-6-fold increase in the specific activity of Lck, which was similar in magnitude to the activation of Lck resulting from the cross-linking of the T-cell receptor complex of Jurkat cells. Lck activity peaked by 5 min after SCF addition, and the elevated activity persisted for at least 30 min in the presence of SCF, with kinetics similar to the activation of mitogen-activated protein kinase. PP1, an inhibitor of Src family kinases with selectivity for Lck, completely inhibited SCF-mediated growth but had little effect on insulin-like growth factor-I-mediated growth. PP1 antagonized both SCF-mediated proliferation and inhibition of apoptosis. PP1 had no effect on Kit kinase activity but was shown to block total Lck activity by at least 90% by immune complex kinase assay. Low levels of Src, Hck, and Yes were also expressed in the H526 cell line; only Yes showed a consistent increase in specific activity, which was also inhibited by PP1 following SCF treatment. These data demonstrate that, in the H526 SCLC cell line, Lck and, possibly, Yes are downstream of Kit in a signal transduction pathway; the inhibition by PP1 of SCF-mediated proliferation and inhibition of apoptosis suggests that Src family kinases are intermediates in the signaling pathways that regulate these processes.     

Prostaglandin D synthase in human megakaryoblastic cells

The cytosol fraction of human platelets did not convert prostaglandin (PG) H2 to PGD2. However, a homogenate of human megakaryoblastic CMK cells (precursor cells of platelets) produced PGD2 from PGH2. The PGD synthase activity was localized in the cytosol of CMK cells, and absolutely required glutathione. The catalytic properties and Western and Northern blottings indicated that the enzyme was PGD synthase of the hematopoietic type rather than the lipocalin type. When CMK cells were differentiated to megakaryocytes with phorbol ester along with induction of cyclooxygenase-1, the PGD synthase activity increased about 2-fold for 2 days and then decreased. In another human megakaryoblastic cell line, Dami, the PGD synthase increased about 10-fold by the addition of phorbol ester. Thus, the PGD synthase, which was undetectable in platelets, appeared during differentiation of megakaryoblasts to megakaryocytes.     

Outside-In signaling of soluble and solid-phase fibrinogen through integrin alphaIIbbeta3 is different and cooperative with each other in a megakaryoblastic leukemia cell line, CMK

The function and the outside-in signaling pathways of alphaIIbbeta3 were examined in relation to cell adhesion using a megakaryoblastic leukemia cell line, CMK. After 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, the cells adhered to the culture plate and underwent megakaryocytic differentiation with expression of alphaIIbbeta3. Binding of soluble fibrinogen to the cells via alphaIIbbeta3 was dependent on cell adhesion. Cell detaching reduced the affinity of this integrin for soluble fibrinogen, although its surface expression was almost unchanged. In contrast, detached cells became tightly adherent to the fibrinogen-coated plate (solid-phase fibrinogen). The same ligand, fibrinogen, present either in soluble or solid-phase form, triggered differential signaling pathways mediated by alphaIIbbeta3. By the stimulation with soluble fibrinogen, Syk was tyrosine-phosphorylated but FAK was dephosphorylated, whereas solid-phase fibrinogen promptly caused tyrosine phosphorylation of FAK followed by delayed phosphorylation of Syk. In addition, the binding of soluble fibrinogen to the cells adherent to fibrinogen-coated plate resulted in tyrosine phosphorylation of integrin beta3 and a complex formation of integrin beta3 with Syk. This implies the cooperation of both soluble and solid-phase fibrinogen-mediated signaling pathways.     

Persistent activation of mitogen-activated protein kinases p42 and p44 and ets-2 phosphorylation in response to colony-stimulating factor 1/c-fms signaling

An antibody that specifically recognized phosphothreonine 72 in ets-2 was used to determine the phosphorylation status of endogenous ets-2 in response to colony-stimulating factor 1 (CSF-1)/c-fms signaling. Phosphorylation of ets-2 was detected in primary macrophages, cells that normally express c-fms, and in fibroblasts engineered to express human c-fms. In the former cells, ets-2 was a CSF-1 immediate-early response gene, and phosphorylated ets-2 was detected after 2 to 4 h, coincident with expression of ets-2 protein. In fibroblasts, ets-2 was constitutively expressed and rapidly became phosphorylated in response to CSF-1. In both cell systems, ets-2 phosphorylation was persistent, with maximal phosphorylation detected 8 to 24 h after CSF-1 stimulation, and was correlated with activation of the CSF-1 target urokinase plasminogen activator (uPA) gene. Kinase assays that used recombinant ets-2 protein as a substrate demonstrated that mitogen-activated protein (MAP) kinases p42 and p44 were constitutively activated in both cell types in response to CSF-1. Immune depletion experiments and the use of the MAP kinase kinase inhibitor PD98059 indicate that these two MAP kinases are the major ets-2 kinases activated in response to CSF-1/c-fms signaling. In the macrophage cell line RAW264, conditional expression of raf kinase induced ets-2 expression and phosphorylation, as well as uPA mRNA expression. Transient assays mapped ets/AP-1 response elements as critical for basal and CSF-1-stimulated uPA reporter gene activity. These results indicate that persistent activation of the raf/MAP kinase pathway by CSF-1 is necessary for both ets-2 expression and posttranslational activation in macrophages.     

Rap1 mediates sustained MAP kinase activation induced by nerve growth factor

Activation of mitogen-activated protein (MAP) kinase (also known as extracellular-signal-regulated kinase, or ERK) by growth factors can trigger either cell growth or differentiation. The intracellular signals that couple growth factors to MAP kinase may determine the different effects of growth factors: for example, transient activation of MAP kinase by epidermal growth factor stimulates proliferation of PC12 cells, whereas they differentiate in response to nerve growth factor, which acts partly by inducing a sustained activation of MAP kinase. Here we show that activation of MAP kinase by nerve growth factor involves two distinct pathways: the initial activation of MAP kinase requires the small G protein Ras, but its activation is sustained by the small G protein Rap1. Rap1 is activated by CRK adaptor proteins and the guanine-nucleotide-exchange factor C3G, and forms a stable complex with B-Raf, an activator of MAP kinase. Rap1 is required for at least two indices of neuronal differentiation by nerve growth factor: electrical excitability and the induction of neuron-specific genes. We propose that the activation of Rap1 by C3G represents a common mechanism to induce sustained activation of the MAP kinase cascade in cells that express B-Raf.