Abstract

This proposal focuses on the mechanism of action of the hormone erythropoietin (EPO) in erythroid cells.
The aim of the laboratory has shifted from the structure of the EPO receptor (EPOR) to the posttranslational modification of the EPOR, signaling of proliferation and viability, and the mechanisms that active apoptosis. The hypothesis that an erythroid cell can become more or less responsive to EPO due to phosphorylation of serine residues of the EPOR will be tested. This possibility is important in determining if the EPOR and related family of receptors can be modulated through intracellular signaling to become hyper-responsive to hormones and growth factors in cancers and hyper-proferative diseases. EPOR mediates its actions by interacting with the tyrosine protein kinase, JAK2. Preliminary evidence shows that the EPOR phosphorylated on serine residues selectively binds to the JAK2 kinase in vivo and in vitro. Cells that are 100-fold more responsive to EPO have a large fraction of JAK2 preassociated with the EPOR.
The first aim of this proposal will be to test the interaction of JAK2 in cells with varied responsiveness to EPO in vivo and in vitro. Biochemical experiments will determine which sites are phosphorylated. EPOR molecules in which the serines are phosphorylated will deleted will be tested for ability to interact with JAK2 and will be tested for function.
The second aim will be to test our hypothesis that there are two distinct signaling pathways in erythroid cells that separately control proliferation and viability (protection from apoptosis). The signals mediated by EPO that protect cells from apoptosis by maintaining Bcl-XI expression and phosphorylation of BAD will be studied. PI3- kinase will be tested for roles in proliferation and survival. Studies using dominant negative and estrogen activated forms of MAP kinase will test the hypothesis that MAP kinase is important in proliferation. The role of autocrine TNF alpha secretion in erythroid cells on proliferation and activation of JNK will be studied. The role of JNK in proliferation will be tested by dominant negative forms.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK039781-11A2
Application #
6129983
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Badman, David G
Project Start
1987-09-30
Project End
2004-03-31
Budget Start
2000-05-15
Budget End
2001-03-31
Support Year
11
Fiscal Year
2000
Total Cost
$178,920
Indirect Cost

  Institution

Name
Virginia Commonwealth University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
City
Richmond
State
VA
Country
United States
Zip Code
23298

  Publications

Abutin, Randolph M; Chen, Jingchun; Lung, Tina K et al. (2009) Erythropoietin-induced phosphorylation/degradation of BIM contributes to survival of erythroid cells. Exp Hematol 37:151-8
Gewirtz, David A; Di, Xu; Walker, Teneille D et al. (2006) Erythropoietin fails to interfere with the antiproliferative and cytotoxic effects of antitumor drugs. Clin Cancer Res 12:2232-8
Jacobs-Helber, Sarah M; Sawyer, Stephen T (2004) Jun N-terminal kinase promotes proliferation of immature erythroid cells and erythropoietin-dependent cell lines. Blood 104:696-703
Chen, Jingchun; Jacobs-Helber, Sarah M; Barber, Dwayne L et al. (2004) Erythropoietin-dependent autocrine secretion of tumor necrosis factor-alpha in hematopoietic cells modulates proliferation via MAP kinase--ERK-1/2 and does not require tyrosine docking sites in the EPO receptor. Exp Cell Res 298:155-66
Jacobs-Helber, Sarah M; Roh, Kwan-Ho; Bailey, Daniel et al. (2003) Tumor necrosis factor-alpha expressed constitutively in erythroid cells or induced by erythropoietin has negative and stimulatory roles in normal erythropoiesis and erythroleukemia. Blood 101:524-31
Jacobs-Helber, Sarah M; Abutin, Randolph M; Tian, Cuixia et al. (2002) Role of JunB in erythroid differentiation. J Biol Chem 277:4859-66
Koury, Mark J; Sawyer, Stephen T; Brandt, Stephen J (2002) New insights into erythropoiesis. Curr Opin Hematol 9:93-100
Jacobs-Helber, S M; Ryan, J J; Sawyer, S T (2000) JNK and p38 are activated by erythropoietin (EPO) but are not induced in apoptosis following EPO withdrawal in EPO-dependent HCD57 cells. Blood 96:933-40
Sawyer, S T; Jacobs-Helber, S M (2000) Unraveling distinct intracellular signals that promote survival and proliferation: study of erythropoietin, stem cell factor, and constitutive signaling in leukemic cells. J Hematother Stem Cell Res 9:21-9
Lawson, A E; Bao, H; Wickrema, A et al. (2000) Phosphatase inhibition promotes antiapoptotic but not proliferative signaling pathways in erythropoietin-dependent HCD57 cells. Blood 96:2084-92

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