Abstract
BackgroundThe MPL protein is a major regulator of megakaryopoiesis and platelet formation as well as stem cell regulation. Aberrant MPL and downstream Jak/STAT signaling results in the development of the Myeloproliferative Neoplasms (MPN). The pathogenetic and phenotypic features of the classical MPNs cannot be explained by the known mutations and genetic variants associated with the disease.MethodsIn order to identify potential pathways involved in MPN development, we have performed a functional screen using retroviral insertional mutagenesis in cells dependent on MPL activation. We have used viral transduction and plasmid transfections to test the effects of candidate gene overexpression on growth and differentiation of megakaryocytic cells. The shRNA approach was used to test for the effects of candidate gene downregulation in cells. All effects were tested with candidate gene alone or in presence of hematopoietic relevant kinases in the growth medium. We assayed the candidate gene cellular localization in varying growth conditions by immunofluorescence. Flow Cytometry was used for testing of transduction efficiency and for sorting of positive cells.ResultsWe have identified the DLGAP1 gene, a member of the Scribble cell polarity complex, as one of the most prominent positive candidates. Analyses in hematopoietic cell lines revealed DLGAP1 centrosomal and cytoplasmic localization. The centrosomal localization of DLGAP1 was cell cycle dependent and hematopoietic relevant tyrosine kinases: Jak2, SRC and MAPK as well as the CDK1 kinase promoted DLGAP1 dissociation from centrosomes. DLGAP1 negatively affected the growth rate of MPL dependent hematopoietic cells and supported megakaryocytic cells polyploidization, which was correlated with its dissociation from centrosomes.ConclusionsOur data support the conclusion that DLGAP1 is a novel, potent factor in MPL signaling, affecting megakaryocytic growth and differentiation, relevant to be investigated further as a prominent candidate in MPN development.
Highlights
The MPL protein is a major regulator of megakaryopoiesis and platelet formation as well as stem cell regulation
Retroviral insertions into the fourth intron of discs-large associated protein 1 (DLGAP1) result in MPL dependent cell proliferation We used the Murine Stem Cell Virus (MSCV) based retroviral construct containing additional sequences of an artificial signaling protein based on the MPL receptor [20] to identify genes that cooperate with MPL signaling
DLGAP1 expression was upregulated in human myeloid malignancies in published data sets from high throughput gene expression studies Since K562 cells arose from a CML associated erythroleukemia, we examined relative levels of DLGAP1 mRNA in a database at the FHCRC of patient samples in all the phases of CML as well as normal CD34 cells
Summary
The MPL protein is a major regulator of megakaryopoiesis and platelet formation as well as stem cell regulation. Aberrant MPL and downstream Jak/STAT signaling results in the development of the Myeloproliferative Neoplasms (MPN). The thrombopoietin receptor (myeloproliferative leukemia protein-MPL) activates the major signaling pathway that regulates megakaryocyte development and platelet production. Abnormal MPL signaling leads to the development of Myeloproliferative Neoplasms (MPN), a group of clonal stem cell disorders characterized by an overproduction of mature myeloid cells with a tendency to transform to acute myeloid leukemia (AML). Mutations in JAK2, MPL and in the calreticulin (CALR) genes have been identified as a central feature in most of these cases [3]. These three mutations result in direct or indirect dysregulation of JAK2 signaling with constitutive activation of cytokine dependent JAK-STAT/PI3K/AKT downstream signaling pathways.
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