Role Of MAP Kinase Signaling Research Articles

Tie2Cre B-Rafflox/flox Mice Reveal a Role for B-Raf in Tpo-Mediated Thrombopoiesis and Survival of Mature Megakaryocytes in Adult Hematopoiesis.

Thrombopoietin (Tpo) is the primary cytokine regulator of megakaryocytopoiesis. Tpo engagement of its receptor, Mpl, activates the classic MAP kinase (Raf/MEK/ERK) pathway, but the biological role of MAP kinase signaling in megakaryocytopoiesis remains poorly defined. Raf family kinases control signal flow through the classic MAP kinase pathway from activated cytokine receptors. We therefore undertook a genetic approach to understand the role of MAP kinase signaling and to identify which Raf family member is important for megakaryocytopoiesis. Using data from in vitro B-raf-/- ES cell cultures, fetal liver hematopoietic cells from mid-gestation B-raf-/- embryos, and B-raf-/- chimeric mice, we recently reported that B-Raf acts in a cell autonomous manner to quantitatively affect megakaryocytopoiesis. However, limitations of the chimeric mouse model, and mid-gestation lethality of B-raf-/- embryos precluded a detailed mechanistic understanding of B-Raf activity or the evaluation of B-Raf in adult megakaryocytopoiesis. We therefore generated tissue-restricted B-Raf deficient mice by crossing B-rafflox/flox mice with mice expressing Cre recombinase controlled by the Tie2 promoter/enhancer. The Tie2Cre+/B-rafflox/flox mice were born with normal Mendelian genetics and without gross abnormalities. Circulating leukocytes demonstrated complete recombination of the floxed B-Raf allele, and western blots showed undetectable B-Raf expression in platelet, spleen, and thymus lysates, consistent with complete hematopoietic Cre-mediated recombination. Steady state platelet counts were not altered in the B-Raf deficient animals at baseline: Tie2Cre+/B-rafflox/flox mice = 788 +/−57 x 103/mm3; Tie2Cre-/B-rafflox/flox mice 800 +/−40 x 103/mm3 (p=0.76, n=4). However, Tie2Cre+/B-rafflox/flox mice had a markedly impaired platelet count rise following Tpo injection, with peak counts of 3,375 +/−752 x 103/mm3 compared with 5,320 +/−606 x 103/mm3 for Tie2Cre-/B-rafflox/flox mice (p=0.0147, n=4) at 6 days post injection. In vitro expansion of CD41+ cells from Tie2Cre+/B-rafflox/flox bone marrow was only a third that of control mice, suggesting that the impaired in vivo platelet rise following Tpo reflects, at least in part, a decreased expansion of megakaryocyte lineage cells. Day 4 Tie2Cre+/B-rafflox/flox bone marrow cultures also yielded decreased low (2N-8N) and high (>32N) ploidy CD41+ cells compared with marrow from Tie2Cre-/B-rafflox/flox mice, while intermediate (16N-32N) ploidy CD41+ megakaryocytes were relatively preserved. TUNEL analysis revealed increased apoptotic death of the high ploidy (>32N) cells, a second possible mechanism contributing to the impaired platelet rise following Tpo injection. Together, our data demonstrate that the B-Raf/MAP kinase pathway is required for normal adult Tpo-induced thrombopoiesis through its effect on megakaryocyte lineage expansion and apoptotic cell death of mature megakaryocytes. Additional analysis is now underway to more fully define the role of B-Raf in megakaryocytopoiesis, including detailed biochemical studies to determine how the absence of B-Raf impacts intracellular signaling during this complex developmental process.

Apoptosis of cerebellar granule cells induced by organotin compounds found in drinking water: Involvement of MAP kinases

Mono- and dialkyl organotin compounds are used primarily as heat stabilizers in polyvinyl chloride (PVC) plastics. Recently, monomethyltin (MMT), dimethyltin (DMT), monobutyltin (MBT), and dibutyltin (DBT) have been detected in water from homes and businesses served by PVC pipes. While trialkyl organotins such as trimethyltin (TMT) and triethyltin (TET) are well known neurotoxicants, the toxicity of the mono- and dialkyl organotins is not well described. The present study compared the cytotoxicity of organotins found in drinking water with the known neurotoxicant TMT in primary cultures of cerebellar granule cells, and examined the role of MAP kinase signaling in organotin-induced cell death. Twenty-four hour exposure to TMT resulted in a concentration-dependent decrease in cell viability with an EC 50 of 3 μM. Exposure to MMT, DMT, and MBT at concentrations up to 10 μM had no effect. DBT, however, was very potent, and decreased cell viability with an EC 50 of 0.3 μM. Staining of organotin-treated cerebellar granule cells with the nuclear dye Syto-13 revealed that TMT and DBT, but not MMT, DMT, or MBT, produced condensation and fragmentation of chromatin characteristic of apoptosis. TMT- and DBT-induced apoptosis was confirmed using TUNEL staining and measurement of PARP cleavage. Activation of MAP kinase pathways was examined after 6 h of exposure to the organotins which induced apoptosis. Both TMT and DBT activated ERK1/2, but only TMT activated the JNK/c-Jun and p38 pathways. Pharmacologic blockade of JNK/c-Jun and p38 activation significantly decreased apoptosis produced by TMT, but not by DBT. These results show that DBT is a potent neurotoxicant in vitro, but unlike TMT, does not induce cell death via activation of MAP kinase signaling.

A Mitogen-Activated Protein Kinase Pathway Essential for Mating and Contributing to Vegetative Growth in Neurospora crassa

MAP kinases homologous to Saccharomyces cerevisiae Fus3p/Kss1p have been identified in plant pathogenic fungi and are required for pathogenicity and sexual reproduction. To better understand the role of MAP kinase signaling in Neurospora crassa, and to identify downstream target genes of the pathway, we isolated, cloned, and disrupted the FUS3 homolog mak-2. Ste12p is a transcription factor target of Fus3p that activates genes of the mating pathway in yeast, and we also characterized the N. crassa STE12 homolog pp-1. The mak-2 and pp-1 mutants have reduced growth rate, produce short aerial hyphae, and fail to develop protoperithecia. In addition, ascospores carrying null mutations of either gene are inviable. Subtractive cloning was used to isolate genes having reduced expression in the mak-2 mutant. Expression of some of these genes is protoperithecia specific and three of them are part of a gene cluster potentially involved in the production of a polyketide secondary metabolite. Microarray analysis was used to extend the analysis of gene expression in mak-2 and pp-1 mutants. The role of the MAP kinase pathway in both sexual and asexual development as well as secondary metabolism is consistent with the dual regulation of the mating process and pathogencity observed in fungal pathogens.

Cell cycle arrest and release in starfish oocytes and eggs

Immature starfish oocytes are arrested at the prophase of meiosis I. Once released from this arrest, meiotic maturation proceeds to the completion of meiosis II in the absence of fertilization, resulting in a second arrest at the female pronucleus stage. This article discusses how Cdc2 kinase is activated in response to the hormone 1–methyladenine at the release from the meiotic prophase arrest, and how MAP kinase is involved in the arrest at the female pronucleus stage. The experimental findings reveal both generality and divergence in the initial Cdc2 kinase activation pathway and in the role of MAP kinase signaling.