Hematopoietic cytokines play a key regulatory role in myeloid differentiation by stimulating the proliferation of certain precursors, inducing the differentiation of others, and activating mature cells. G-CSF and GM-CSF are the most important cytokines directing myeloid differentiation. Two key steps in the maturation of these cells are the acquisition of cell surface receptors and of the components for the downstream signaling pathways. Two myeloid cell lines can serve as models for these signaling events: 1) EML is a multipotent stem cell factor- dependent cell line immortalized with a dominant negative retinoic acid receptor alpha. Although they express the GM-CSF receptor, uninduced EML cells will not respond to GM-CSF. Induction with IL-3/ATRA induces the cells to differentiate and to become GM-CSF responsive. Over- expression of the oncogene EVI-1 in these cells blocks this acquisition of GM-CSF responsiveness. 2) 32 Dcl3 cells proliferate and differentiate in response to G-CSF. Transfection of a derivative line, 32D.C10 (which lacks G-CSFR), with a receptor carrying a Y764F mutation renders the cells unable to proliferate in response to G-CSF, although they will differentiate with this induction. We propose to use these cell lines as models for analyzing the molecular events required for early myeloid differentiation. To date, signaling pathways that mediate G- or GM-CSF-induced myeloid differentiation remain to be identified. Nonetheless, it is clear that differentiation-specific signaling results in the activation of key regulatory genes, such as C/ebpalpha. We hypothesize that C/ebpalpha is ne of a set of regulatory genes, many of which have yet to be identified, that represent nuclear targets for differentiation-inducing signals that initiate at the membrane. We hypothesize that these genes provide a molecular point of entry into understanding the process of myeloid differentiation. We propose: 1. To determine at which point in the induction of differentiation by EML the GM-CSF signaling pathway becomes primed, to investigate the mechanism by which the pathway becomes responsive to the proliferative signal transduced by GM-CSF, and to characterize the block in the pathway induced by EVI-1 over-expression. 2. To identify the set of genes that are up-regulated by the """"""""differentiation arm"""""""" of the G-CSF signaling. 3. To establish the functional role of downstream genes identified in Aims 1 and 2 by over-expression in EML and 32D. The phenotype of transduced EML cells will be examined for the ability to bypass the need for early priming of cytokine response; transfected 32D cells will be assayed for separation of proliferative and differentiative responses to G-CSF.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL063357-04
Application #
6647337
Study Section
Project Start
2002-08-01
Project End
2003-07-31
Budget Start
Budget End
Support Year
4
Fiscal Year
2002
Total Cost
Indirect Cost
Name
Yale University
Department
Type
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Wilson, Michael; Tsakraklides, Vasiliki; Tran, Minh et al. (2016) EVI1 Interferes with Myeloid Maturation via Transcriptional Repression of Cebpa, via Binding to Two Far Downstream Regulatory Elements. J Biol Chem 291:13591-607
Khanna-Gupta, Arati (2013) Bone Marrow Failure Syndromes: The Ribosomopathies. J Bone Marrow Res 1:
Khanna-Gupta, Arati; Abayasekara, Nirmalee; Levine, Michelle et al. (2012) Up-regulation of translation eukaryotic initiation factor 4E in nucleophosmin 1 haploinsufficient cells results in changes in CCAAT enhancer-binding protein ? activity: implications in myelodysplastic syndrome and acute myeloid leukemia. J Biol Chem 287:32728-37
Khanna-Gupta, Arati (2011) Regulation and deregulation of mRNA translation during myeloid maturation. Exp Hematol 39:133-41
Halene, Stephanie; Gao, Yuan; Hahn, Katherine et al. (2010) Serum response factor is an essential transcription factor in megakaryocytic maturation. Blood 116:1942-50
Lee, Han M; Zhang, Hui; Schulz, Vincent et al. (2010) Downstream targets of HOXB4 in a cell line model of primitive hematopoietic progenitor cells. Blood 116:720-30
Halene, Stephanie; Gaines, Peter; Sun, Hong et al. (2010) C/EBPepsilon directs granulocytic-vs-monocytic lineage determination and confers chemotactic function via Hlx. Exp Hematol 38:90-103
Rabinovich, Peter M; Komarovskaya, Marina E; Wrzesinski, Stephen H et al. (2009) Chimeric receptor mRNA transfection as a tool to generate antineoplastic lymphocytes. Hum Gene Ther 20:51-61
Cheng, Ee-Chun; Luo, Qing; Bruscia, Emanuela M et al. (2009) Role for MKL1 in megakaryocytic maturation. Blood 113:2826-34
Friedman, Rachel S C; Krause, Diane S (2009) Regeneration and repair: new findings in stem cell research and aging. Ann N Y Acad Sci 1172:88-94

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