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Activation of the MAP kinase pathway by c-Kit is PI-3 kinase dependent in hematopoietic progenitor/stem cell lines
Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
Umeå University, Faculty of Medicine, Medical Biosciences, Pathology.
Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
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2004 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 104, no 1, 51-57 p.Article in journal (Refereed) Published
Abstract [en]

The Steel factor (SF) and its receptor c-Kit play a critical role for various cell types at different levels in the hematopoietic hierarchy. Whether similar or distinct signaling pathways are used upon c-Kit activation in different cell types within the hematopoietic hierarchy is not known. To study c-Kit signaling pathways in the hematopoietic system we have compared c-Kit downstream signaling events in SF-dependent hematopoietic stem cell (HSC)–like cell lines to those of mast cells. Both Erk and protein kinase B (PKB)/Akt are activated by ligand-induced activation of the c-Kit receptor in the HSC-like cell lines. Surprisingly, phosphoinositide-3 (PI-3) kinase inhibitors block not only PKB/Akt activation but also activation of Raf and Erk. SF-induced activation of Ras is not affected by inhibition of PI-3 kinase. In mast cells and other more committed hematopoietic precursors, the activation of Erk by SF is not PI-3 kinase dependent. Our results suggest that a molecular signaling switch occurs during differentiation in the hematopoietic system whereby immature hematopoietic progenitor/stem cells use a PI-3 kinase–sensitive pathway in the activation of both Erk and PKB/Akt, which is then switched upon differentiation to the more commonly described PI-3 kinase–independent mitogen-activated protein (MAP) kinase pathway.

Place, publisher, year, edition, pages
2004. Vol. 104, no 1, 51-57 p.
Keyword [en]
1-Phosphatidylinositol 3-Kinase/antagonists & inhibitors/*metabolism, Animals, Cell Line, Chromones/pharmacology, Enzyme Activation, Enzyme Inhibitors/pharmacology, Femur/cytology, Hematopoietic Stem Cells/cytology/*enzymology, MAP Kinase Signaling System/*physiology, Mice, Mice; Inbred C57BL, Mitogen-Activated Protein Kinases/metabolism, Morpholines/pharmacology, Phosphorylation, Protein-Serine-Threonine Kinases/metabolism, Proto-Oncogene Proteins/metabolism, Proto-Oncogene Proteins c-akt, Proto-Oncogene Proteins c-kit/*metabolism, Proto-Oncogene Proteins c-raf/metabolism, Stem Cell Factor/metabolism, ras Proteins/metabolism
Identifiers
URN: urn:nbn:se:umu:diva-15405DOI: 10.1182/blood-2003-07-2554PubMedID: 14996702OAI: oai:DiVA.org:umu-15405DiVA: diva2:155077
Available from: 2008-01-11 Created: 2008-01-11 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Receptor tyrosine kinase c-Kit signalling in hematopoietic progenitor cells
Open this publication in new window or tab >>Receptor tyrosine kinase c-Kit signalling in hematopoietic progenitor cells
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The receptor tyrosine kinase (RTK) c-Kit is expressed in hematopoietic stem and progenitor cells, mast cells and in several non-hematopoietic tissues. In the hematopoietic system c-Kit and its ligand Steel Factor (SF, aka Stem Cell Factor) are critical for proliferation, survival and differentiation. Mutations in either receptor or ligand lead to lethal anaemia, hematopoietic stem cell defects, mast cell deficiency and a series of non-hematological defects.

The aims of the studies included in this thesis are to describe the signalling pathways downstream c-Kit in hematopoietic stem/progenitor cells and to further analyse the role of c-Kit signalling in fundamental biological functions.

To study c-Kit signalling in the hematopoietic system we have employed hematopoietic stem cell-like cell lines which share many properties with primary hematopoietic stem cells in vitro and in vivo, including surface markers, multipotentiality, capacity for self-renewal and long term repopulation. In paper I we demonstrate that upon SF activation the RTK c-Kit is autophosphorylated and downstream signalling mediators are transiently activated. Surprisingly we find that the c-Kit mediated activation of the MAPK pathway is dependent on the activation of phosphoinositide 3-kinase (PI3K) in hematopoietic progenitor cells and that differentiation of these progenitors to mast cells results in a signalling switch where Raf activation changes from PI3K dependent to PI3K independent. We here establish that PI3K activity is required for viability and proliferation of hematopoietic progenitor cells. In paper II we studied the conventional protein kinase C (cPKC) involvement in c-Kit signalling. We observe that the cPKCs can phosphorylate c-Kit on serine 746 and that this phosphorylation negatively regulates the activation of the receptor. We demonstrate that inhibition of this negative phosphorylation results in dramatically increased protein kinase B (PKB) activation and as a consequence inhibition of cPKCs rescues cells from starvation induced apoptosis. Moreover we exhibit that the cPKCs are necessary for full activation of extracellular signal-regulated kinase (Erk) and that impaired PKC activity leads to hampered proliferation. In paper III we demonstrate that in addition to the cPKCs also the novel PKC is required for Erk activation and proliferation. Furthermore we present results indicating that PKC negatively regulates differentiation of bone marrow.

In conclusion, with the studies in this thesis we display details in the signalling pathways induced upon RTK c-Kit activation and we demonstrate that c-Kit has significant effects on hematopoietic cell-physiology.

Place, publisher, year, edition, pages
Umeå: Medicinsk biovetenskap, 2006. 60 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1058
Keyword
RTK, c-Kit, MAPK, PI3K, PKC, hematopoietic cells, proliferation, differentiation, apoptosis
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-888 (URN)91-7264-180-0 (ISBN)
Public defence
2006-10-27, Major Groove, 6L, umeå universitet, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2006-10-10 Created: 2006-10-10 Last updated: 2009-10-02Bibliographically approved
2. The role of Lhx2 in the hematopoietic stem cell function, liver development and disease
Open this publication in new window or tab >>The role of Lhx2 in the hematopoietic stem cell function, liver development and disease
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

During embryonic development, generation of functional organs is dependent on proper interactions between different cell types. Elucidation of the mechanisms operating during organ formation might provide insights into the origin of many pathological disorders in the adult. Gene inactivation studies in mice have provided invaluable tool to study the function of genes critical for morphogenesis of distinct organs. A LIM-homeodomain transcription factor Lhx2 has previously been reported to play a role in fetal liver development and hematopoiesis, as its inactivation leads to lethal anemia due to underdeveloped liver. This thesis focuses on the function of Lhx2 in the development of these two organ systems.

Reciprocal signaling between ventral foregut endoderm and mesenchyme of the septum transversum regulates the liver formation, expansion and differentiation. A fully formed liver is composed of endoderm-derived hepatocytes and cholangiocytes and a variety of mesenchyme-derived cell types, such as endothelial cells and hepatic stellate cells. In early stages of liver development Lhx2 is expressed in the liver-associated septum transversum mesenchyme, a part of which becomes integrated into the liver organ and develops into hepatic stellate cells. Functional Lhx2 expression in the hepatic mesenchyme is necessary for normal liver outgrowth and differentiation. Loss of Lhx2 from developing hepatic stellate cells leads to their activation and excessive deposition of collagen fibres, resulting in hepatic fibrosis and severely distorted liver architecture. Transfection of Lhx2 to human stellate cell line downregulates genes associated with stellate cell activation and fibrogenesis. Thus, Lhx2 is the first gene identified to negatively regulate events leading to hepatic fibrosis. Elucidation of the molecular mechanisms involved in this process might therefore be instrumental for the development of novel therapies useful in treatment of this disorder.

Fetal liver is also a major site of hematopoiesis in the embryo and provides physiological conditions necessary for the efficient expansion of hematopoietic stem cells (HSCs). The hematopoietic defect observed in Lhx2-deficient embryos is cell-nonautonomous, indicating that Lhx2 might control secreted factors involved in the self-renewal of HSCs. This putative second role of Lhx2 has been investigated by analyzing the mechanism whereby Lhx2 expression generates in vitro self-renewing HSC-like cell lines. Interestingly, in agreement with the cell nonautonomous phenotype of the lethal anemia in Lhx2-/- embryos, the mechanism of self-renewal is dependent on Lhx2 expression and occurs via secreted factor(s). Identification of these factor(s) might potentially allow ex vivo expansion of HSCs for therapeutic purposes.

The Lhx2-immortalized HSC-like cell lines share many basic features with HSCs and self-renew in vitro in presence of Steel factor (SF). SF/c-Kit signaling mediates a wide variety of biological activities in cells at many different levels in the hematopoietic hierarchy. We used the HSC-like cell lines as an in vitro model system to compare signal transduction pathways from c-Kit receptor in stem cells versus differentiated hematopoietic cells. HSCs require PI-3K dependent activation of Raf1-Mek-Erk cascade for their survival and self-renewal in response to SF, whereas activation of Erk is PI-3K independent in committed myeloid and mast cells. Thus, the mode of SF/c-Kit signaling is dependent on the differentiation status of the cells.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi, 2004. 108 p.
Keyword
Cell and molecular biology, liver fibrosis, liver development, Lhx2, hematopoietic stem cells, Cell- och molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Cellbiology
Identifiers
urn:nbn:se:umu:diva-373 (URN)91-7305-748-7 (ISBN)
Public defence
2004-12-10, Major Groove, 6L, Norrlands Universitetssjukhus, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2004-11-19 Created: 2004-11-19 Last updated: 2010-08-02Bibliographically approved

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