Progress 07/01/99 to 06/30/04
Outputs
Overall aims of this project were to advance a molecular understanding of erythropoietin (Epo) dependent erythropoiesis. Significance lies in 1/ improving a basic understanding how how the Epo receptor, and ~ 30 related (& clinically important) cytokine receptors relay signals for progenitor cell proliferation and survival and 2/ the potential to discover novel regulatory molecules within erythroid progenitor cells as candidate targets for new anti-anemia pharmaceutical agents. In work published this year we described first a specific and essential role for a defined Epo receptor intracellular domain (and residue, Tyr343) during Epo's co-signaling with a synergistic cytokine, stem cell factor (Li et al., J Biol Chem). Second, using a knock-in mouse model we reported on defects in the in vivo signaling of a (phospho)tyrosine-null Epo receptor form, as well as its the apparent failure of an Epo/EpoR/Jak2 - only signaling axis to support stress-induced extramedullary
erythropoiesis (Li et al. BLOOD). This latter finding is of further potential significance in that it suggests the the basic biological processes of steady-state vs stress erythropoiesis (ie, red blood cell formation durin anemia) may differ in important and genetically definable ways.
Impacts
Overall goals are to define cell and molecular mechanisms by which the novel dual-specificity kinase, DYRK3, acts to regulate red blood cell development.
Publications
- Li K., Menon, M., Karur, V., Hegde, S., and Wojchowski, D. M. 2003. Attenuated signaling by a phosphotyrosine-null Epo receptor form in primary erythroid progenitor cells. Blood 102:3147-3153.
- Li, K., Miller, C., Hegde, S., and Wojchowski, D. M. 2003. Roles for an Epo Receptor Tyr-343 Stat5 pathway in proliferative co-signaling with Kit. Journal of Biological Chemistry. 278:40702-40709.
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Progress 01/01/02 to 12/31/02
Outputs
Research efforts in 2002 continued to focus on key regulators of red blood cell development (erythropoiesis) and now channel into three separable programs: 1) mechanisms of eythropoietin (Epo) action, 2) mechanisms of Epo and stem cell factor (SCF) co-signaling of progenitor cell proliferation, and 3) roles for the erythroid-restricted protein kinase, Dyrk3, during late erythroid development. Epo is the primary hormonal regulator of red cell production; SCF and its receptor (Kit) are required for erythropoiesis and Kit mutations are linked to blood cell, germ cell and GI cancers; and Dyrk3 is a novel dual-specificity kinase that may act as a suppressor of late erythroid development. Relevance of investigations to human and animal health is direct in that anemia of chronic disease (and chemotherapy) is pervasive, but can be treated effectively by Epo therapy. In part, this project seeks to define critical regulators of Epo-dependent red cell production certain of which
may be valuable as new targets for small molecule inhibitors/activators. In studies of Epo action, Epo receptor knock-in mouse models have been adopted and used to discover important roles for select cytoplasmic phosphotyrosine sites and linked pathways in supporting late erytrhoid progenitor cell mitogenesis, amd survival (Li et al, submitted). Studies of Epo and SCF receptor co-function have defined an important role for an Epo-activated Stat5 transcription factor (Li et al, in preparation) and for a SCF activated Src family kinase (Sahu et al, in preparation) in Epo receptor and Kit co-signaling. Studies of Dyrk3 are newly sponsored (NIH 12/02 - 12/07) and point to inhibitory roles for this cytoplasmic kinase during late erythropoiesis. In addition, one paper on Dyrk3 coupling to a CREB transcription factor has been published this year (Li et al) and manuscripts on dyrk3 gene regulation and Dyrk3 modulation of programmed cell death are in preparation. In 2003, efforts will continue
in each of the above three veins of investigation.
Impacts
Relevance of investigations to human and animal health is direct in that anemia of chronic disease (and chemotherapy) is pervasive, but can be treated effectively by Epo therapy. In part, this project seeks to define critical regulators of Epo-dependent red cell production certain of which may be valuable as new targets for small molecule inhibitors/activators. Such therapeutics have the potential to markedly improve clinical conditions associated with renal disease, chronic infection (eg, AIDS), marrow transplantation and chemotherapy (as primary examples).
Publications
- Li, K.,Zhao, S. Q., Vinit, K., and Wochowski, D. M. 2002. DYRK3 activation, engagement of PKA and modulation of progenitor cell survival. J. Biol. Chem. 277:47052-60
- Miller, C. P., Heilman, D., and Wojchowski, D. M. 2002. Erythropoietin receptor-dependent erythroid colony-formin-unit development: Capacities of Y343 and phosphotyrosine-null receptor forms. BLOOD 99:898-904
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Progress 01/01/01 to 12/31/01
Outputs
Efforts continue to focus on cell - intrinsic mechanisms that regulate red blood cell development. In completed work we first have used a transgenic mouse model to reveal that the essential activity of Epo during red blood cell production depends only upon a quite limited cytoplasmic subdomain of the Epo receptor. Second, we also have discovered a novel dual -specificity kinase (DYRK3) that is expressed selectively in erythroid progenitor cells. Interestingly, experiments using antisense oligonucletides to block DYRK3 expression suggest that this kinase functions as an important suppressor of red blood cell production. Based on these findings, a collaborative program has been established to further investigate DYRK3 (and the prospective utility of small molecule inhibitors of DYRK3 in the context of the treatment of anemias). An application to NIH for additional funding for these experiments currently is being prepared.
Impacts
Work continues to reveal important details concerning how Epo (the prime hormonal regulator of red blood cell production) functions in concert with its single transmembrane receptor. This work, as well as our new work with DYRK3 kinase, promise to uncover new and potentially important targets for small molecule targeting. Id eas, targets which when inhibited (or activated) by pharmaceutical agents, stimulate red cell production and relieve anemia associated with chronic disease, renal disease, chemotherapy and AIDS.
Publications
- Geiger, J.N., Knudsen, G.T., Pandit, A., Panek, L., Yoder, M. Lord, K., Creasy, C.L., Burns, B.M., Dillon, S., and Wojchowski, D.M. 2001. mDYRK3 kinase is expressed selectively in late erythroid progenitor cells and attenuates CFUe development. BLOOD 97:901-910.
- Pircher, T.J., Geiger, J.N., Zhang, D., Miller, C.P., Gaines, P., and Wojchowski, D.M. 2001. Integrative signaling by minimal erythropoietin receptor forms and c-Kit. J Biol Chem 276:8995-9002.
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Progress 01/01/00 to 12/31/00
Outputs
Progress over the past year includes: 1) the development of a uniquely advantageous primary cell model for studies of erythropoietin (Epo) action - Here, thiamphenicol is used to convert murine spleen into a highly erythroid organ and a 300-fold increase in frequencies of Epo-responsive erythroid progenitor cells is achieved; 2) the development of transgenic mice expressing novel minimal Epo receptor forms as conditionally activatable chimeras - Here, new insights are being gained into the key necessary-and-sufficient cellular mediators of Epo-dependent red cell production; 3) the discovery of new routes of regulated expression & action of the essential erythroid transcription factor GATA1 - Here, GATA1 is revealed to auto-regulate its expression via a novel intronic set of cis elements, and to also act in a dominant fashion to affect commitment to megakaryocytic development (platelet production); and 4) the discovery that select immediate response genes function as
co-targets for integrated signals from the Epo receptor, and the cell surface receptor for stem cell factor (another cytokine who's action is critical for efficient red cell production).
Impacts
The impact of the above progress (items 1-4) is summarized here. First, our development of a system for the preparation and purification of useful numbers of developmentally staged erythroid progenitor cells not only provides an avenue for directly advancing a molecular understanding of Epo action, but also is already proving useful in direct tests of small syntheic angonists / antagonists designed to mimic Epo's action (Epo presently is used world-wide to treat anemias associated with renal failure, chemotherapy, & AIDS for example, but is very costly). Second, the transgenic mice models we have developed promise to advance our basic understanding of how (hematopoietic) cytokines regulate blood cell growth & survival, and are of potential impact in the rationale design of new anti-cancer therapies.
Publications
- Seshasayee, D., Geiger, J.N., Gaines, P., Wojchowski, D.M. 2000. Intron 1 elements promote erythroid-specific GATA1 gene expression. J. Biol. Chem. 275:22969-22977.
- Pircher, T.J., Zhao, S., Geiger, J.N., Joneja, B., Wojchowski, D.M. 2000. Pim1 kinase selectively inhibits apoptosis due to genotoxic stress. Oncogene 19:3684-3692.
- Gregory, R.C., Lord, K.A., Panek, L.B., Gaines, P., Dillon, S.B., Wojchowski, D.M. 2000. Subtraction cloning and initial characterization of novel Epo immediate response genes. Cytokine 12:845-857.
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Progress 01/01/99 to 12/31/99
Outputs
During the first 8 months of this renewed project, progress has been made in three areas. 1) Several novel Epo target genes have been cloned by subtraction, and genomic approaches are in place to define others. The goal here is to dissect action mechanisms for this prime hormonal regulator of red blood cell production, in part, towards the development small molecule activators of key Epo response pathways. 2) The DNA binding protein GATA-1 has been discovered to autoregulate its expression via elements within intron 1 of the GATA-1 gene. GATA-1 is a dominantly acting transcription factor required for the development of not only red cells, but platelets. 3) The action of GATA-1 in activating the expression of the platelet progenitor marker, alphaIIb, has been shown to depend upon a direct co-factor Friend of GATA1, or FOG. FOG also is required for platelet development, and these findings lend insight into action mechanisms. Overall, this progress continues to advance
an understanding of mechanisms that regulate the growth and development of blood cells within these clinically important lineages.
Impacts
This research should identify new targets for pharmaceutical agents that enhance (Epo-dependent) red blood cell production. Such agents would directly aid the treatment of numerous diseases with associated anemias.
Publications
- Miller, C.P., Noguchi, C., and Wojchowski, D.M. 1999. A minimal cytoplasmic subdomain of the erythropoietin receptor mediates erythroid and megakaryocytic cell development. Blood. 95:3381-3388.
- Wojchowski, D.M., Gregory, R.C., Miller, C.P., Pandit, A.K., and Pircher, T.J. 1999. Signal transduction in the erythropoietin receptor system. Experimental Cell Research. 252:143-156.
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