Progress 01/01/10 to 12/31/14
Outputs
Target Audience: The target audience for our work includes the members of the field of hematology, and in particular, researchers and clinicians who are interested in the mechanisms that regulate the erythroid development. The goal of our work and the work of the field in general is to develop new treatments for anemia. Thus, another audience would be pharmaceutical companies developing new drugs to treat anemia and other hematological diseases. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? In the past year, I organized a professional development session for students in the Genetics Graduate program at Penn State, which included two students working on this project. Two speakers were brought in: Dr. Omid Harandi, a postdoctoral Fellow at the Dana Farber Cancer Institute at Harvard Medical School, and Dr. Srinivas K. Chunduru from Exilion Pharmaceuticals. Our lab supports five graduate students, one post-doctoral fellows, five undergraduate students, and one rotating graduate position. How have the results been disseminated to communities of interest? In the past year, I have presented our work at the American Society for Hematology (ASH) Annual Conference and the 15th annual International Conference on Chronic Myeloid Leukemia in Estoril, Portugal. My students have presented their work at the 19th Meeting on Hemoglobin Switching at Oxford University. In addition, I have presented our work at invited seminars at the University of Wisconsin, Department of Nutritional Sciences; University of Wisconsin, School of Medicine Blood Research Group; and the Penn State Hershey Medical School Department of Pharmacology. Results have also been disseminated via publications (see list). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This is the final report, so I will summarize our findings over the entire project period. The first goal of the project was to characterize the role of hedgehog (HH) signaling in the development of stress erythroid progenitors. Our work has demonstrated that HH signaling plays a key role in the development of new erythrocytes following bone marrow transplant (BMT). Stress erythropoiesis is essential to generate new erythrocytes in the immediate post-transplant period, which maintains survival of the transplant recipient until donor stem cells can engraft in the bone marrow. Using mutations in the HH signaling pathway or chemical inhibitors of HH signaling, we show that blocking HH signaling compromises stress erythropoiesis to such an extent that the recipient is unable to generate sufficient erythrocytes to survive. Our data show that HH signaling plays two roles: First, HH signaling restricts the developmental potential of stem cells to the erythroid or red blood cell lineage; Second, HH signaling promotes the rapid expansion of self-renewing stress erythroid progenitors. Analysis of targets of HH signaling gave us insight it the mechanisms by which HH signaling regulates this process. A key aspect of the restriction to the erythroid lineage is the induction of Sox 3 expression. Sox3 is a transcription factor. Mutation of Sox3 blocks the development of stress erythroid progenitors. Another target of HH signaling is the receptor Lgr4, which responds to the ligand R-spondin1. We propose that Lgr4 signaling synergizes with canonical Wnt signaling to drive the self-renewal of early stress erythroid progenitors. We are presently analyzing the effects of mutating Lgr4 expression on stress erythropoiesis. In addition, HH signaling cooperates with other signaling pathways regulated by bone morphogenetic protein 4 (BMP4) and growth and differentiation factor 15 (GDF15) to induce the expression of a naturally-occurring, truncated form of the Stk receptor known as Sf-Stk. I will discuss Sf-Stk’s role in stress erythropoiesis in the accomplishments under the third goal. The identification of these target genes has allowed us to develop an outline of a gene regulatory network that will guide future experiments. The work on HH signaling will be reported in a manuscript that is in preparation. The second goal of the project was to analyze the role of GDF15-dependent signaling in stress erythropoiesis. GDF15 is a growth factor that is a member of the TGF-b family of growth factors. Our data show that mutation of GDF15 compromises stress erythropoiesis. GDF15 signaling plays an essential role in the regulating the expression of BMP4. Previous work from the lab showed that the BMP4 expression in the spleen is regulated by tissue hypoxia. The primary response to hypoxia is the activation of the hypoxia inducible transcription factor Hif2a. Under normal oxygen conditions Hif2a is unstable and the protein is rapidly degraded. The turnover of Hif2a is mediated by a VHL-dependent ubiquitin ligase. The Hif2a-dependent hypoxia response is designed to be a transient response. In fact, one of the targets of Hif2a regulation is VHL, which is part of a negative feedback loop that attenuates the hypoxia response. GDF15 maintains BMP4 expression during the recovery from anemia by inhibiting the expression of VHL. GDF15 dependent signaling activates the transcription factor Smad2/3. During the recovery from anemia, Smad2/3 binds to a site in the VHL gene and recruits a transcriptional repressor complex, which inhibits the expression of VHL. In the absence of GDF15, VHL expression shuts down the Hif2a-dependent expression of BMP4, which is an essential signal that regulates the expansion and differentiation of stress erythroid progenitors. A manuscript describing this work is being revised for re-submission. The third goal of this projectwas to characterize the role of SF-Stk signaling stress erythropoiesis. Our previous work showed that HH, BMP4 and GDF15 together up-regulated the expression of Sf-Stk in stress erythroid progenitors. Mutation of Sf-Stk leads to defects in the recovery from anemia. Our analysis showed that in control mice, stress erythroid progenitors first expand to generate a large pool of progenitors. During this expansion, the cells are unable to differentiate into erythrocytes. Then a switch occurs that causes the progenitors to acquire the ability to differentiate, which then occurs in a synchronous wave of differentiation. Sf-Stk mutant mice differentiate early. The problem with that defect is that they are unable to generate sufficient erythrocytes to survive a bone marrow transplant. We have identified a number of downstream targets of Sf-Stk: They include Grb2 and Gab2. These signaling proteins are required for the activation of Stat3, which in turn, leads to the expression of the transcription factor Pu.1. The ultimate activation of Pu.1 expression is important for the self-renewal of early stress erythroid progenitors. Mutations in Grb2 and Gab2 exhibit defects similar to the Sf-Stk mutation, which further supports their role downstream of the Sf-Stk in this process. We are now exploring the mechanisms by which Sf-Stk regulates differentiation of stress erythroid progenitors. A manuscript describing the phenotype of Sf-Stk mutant mice is in preparation.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Gandhi UH, Kaushal N, Hegde S, Finch ER, Kudva AK, Kennett MJ, Jordan CT, Paulson RF, Prabhu KS. 2014. Selenium suppresses leukemia through the action of endogenous eicosenoids. Cancer Research 74:3890-3901.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Paulson RF. 2014. Targeting a new regulator of erythropoiesis to alleviate anemia. Nature Medicine. 20:334-335.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Anderson NM, Javadi M, Berndl E, Berberovic Z, Bailey ML, Huang K, Flenniken AM, Osborne LR, Adamson SL, Rossant J, Carter-Su C, Wang C, McNagny KM, Paulson RF, Minden MD, Stanford WL, Barber DL. 2013. ENU mutagenesis identifies a novel platelet phenotype in a loss of function Jak2 allele. PLoS ONE 8:e75472.
- Type:
Theses/Dissertations
Status:
Submitted
Year Published:
2014
Citation:
Xiang, J. 2014. Regulation of stress erythropoiesis: Interaction between the microenvironment and stress erythroid progenitors. Ph.D. Thesis. The Pennsylvania State University. 184 pp.
- Type:
Theses/Dissertations
Status:
Submitted
Year Published:
2014
Citation:
Redkar, S. 2014. Stress erythropoiesis: Lessons from the murine model and the current understanding of in the human system. M.S. Paper. The Pennsylvania State University. 16 pp.
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: The work in the laboratory is designed to advance the field of hematology research and in particular provide new data that could be used to develop new therapies for anemia. The target audience is, therefore, other members of field and researchers working on similar problems. In addition, our work is intended for clinicians who treat anemia patients, and pharmaceutical companies working on developing new drugs to treat anemia. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? One of my graduate students, Laura Bennett, was chosen for an NIH T32 training grant in Animal Models of Inflammation. Ms. Bennett has received training in a number of new research techniques as proscribed by the training curriculum of the Training grant. I participated in the training grant as well by teaching a short course in Experimental Methods in Hematopoiesis. How have the results been disseminated to communities of interest? Results from our work were reported at the Red Cells Gordon Conference in July 2013. In addition, our was presented in invited seminars at the New York Blood Center and Albert Einstein College of Medicine in March of 2013. What do you plan to do during the next reporting period to accomplish the goals? The first priority is to publish our manuscripts. The second priority is to continue the study of and expand our knowledge of the mechanisms that regulate stress erythropoiesis during the recovery from anemia. We have completed most of the experiments described in the original project and are now extending those findings to refine the mechanism of regulation to the molecular level.
Impacts
What was accomplished under these goals?
The project has three objectives. Following are the accomplishments for each: (1) Characterize the role of Hedgehog signaling in the development of stress erythroid progenitors. We have determined the role for Hedgehog signaling in the expansion of stress erythroid progenitors, which occurs early during recovery from anemia. We have identified target genes that are activated by Hedgehog signaling and play a key role in this process of expansion. A manuscript describing this work is in preparation for publication. (2) Analysis of the role of GDF15 signaling in stress erythropoiesis. Our work has identified a key target gene that is repressed by GDF15-dependent signaling. This gene, VHL, regulates hypoxia-dependent responses in cells. These data demonstrate a new mechanism for regulating hypoxia-dependent gene expression during recovery from anemia. A manuscript describing this work will be submitted in the near future. (3) Analysis of Sf-Stk signaling during the recovery from anemia. Our data show that Sf-Stk is a gene whose expression is induced by Hedgehog signaling. It plays a key role in expanding early progenitors and preventing their differentiation. Again, this work will be detailed in the manuscript of Hedgehog signaling mentioned in (2) above.
Publications
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Anemia is a debilitating disease that causes significant morbidity and mortality. In addition it has a profound negative impact on quality of life. Our work focuses on understanding the mechanisms by which new erythrocytes are rapidly made at times of anemic stress. In healthy individuals, the bone marrow constantly produces new erythrocytes that are needed to replace worn out erythrocytes. In response to anemic stress, the body induces a physiological response designed to increase oxygen delivery to the tissues. An increase in erythropoiesis is integral to that response. At these times stress erythropoiesis predominates. Stress erythropoiesis is able to rapidly generate large numbers of new erythrocytes much faster than steady state bone marrow erythropoiesis. My laboratory demonstrated that stress erythropoiesis utilizes unique progenitor populations and this process is regulated by signals that are distinct from the signals that are associated with steady state erythropoiesis. In the past year, we refined our model. Using Friend virus induced erythroleukemia as an experimental system, we identified four distinct progenitor populations that expand and ultimately differentiate into new erythrocytes in response to anemic stress. Our present work is focused on characterizing these progenitors and using that information to develop potential therapies for anemia. PARTICIPANTS: Robert F. Paulson is the principal investigator on the project. His role is two-fold, he is involved in the development of experimental strategies and the management of the project. Dr. Paulson also is actively involved in data interpretation and the writing of manuscripts. Five Graduate students are involved in this work, Shailaja Hegde, Sneha Hariharan, Dai-Chen Wu, Laura Bennett and Jie Xiang are all graduate students working on experiments that support the project. Each is performing experiments that will be reported in their doctoral dissertations. Because of the role of graduate students in performing the experiments, the project should also be considered for training purposes as well as the goals of the project. TARGET AUDIENCES: The results of our experiments are reported in peer reviewed journals and at presentations at scientific meetings. The target audience for these reports is scientists studying similar problems. In a broader sense our work provides a basic framework for the development of new therapies for anemia and leukemia. Therefore, pharmaceutical companies and clinicians would also be part of the target audience PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
My research is leading to a better understanding of how the body responds to anemic stress. This work provides is the initial stage of a larger research program designed to develop effective treatments for anemia. The impact of my research is measured by our publications and the acceptance of our models in the larger Hematology field.
Publications
- Hegde, S., P. Hankey, and R. F. Paulson. 2012. Self renewal of leukemia stem cells in Friend virus induced erythroleukemia requires proviral insertional activation of Spi1 and Hedgehog signaling, but not mutation of p53. Stem Cells 30:121-130.
- Paulson, R. F. 2011. Erythropoiesis lagging pIgA1 steps in to assist Epo. Nature Medicine 17:1346-1348.
- Anderson, N., Z. Berberovic, E. Berndl, M. L. Bailey, A. M. Flenniken, L. R. Osborne, S. L. Adamson, J. Rossant, C. Wang, M. D. Minden, K. M. McNagny, R. F. Paulson, D. L. Barber, and W. L. Stanford. 2012. Cytopenia induction by 5-Fluorouracil identifies thrombopoietic mutants in sensitized ENU mutagenesis screens. Experimental Hematology 40:48-60.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Our work focuses on the mechanisms that regulate the rapid production of new erythrocytes at times of acute need. Throughout our lifetimes, our bone marrow generates new erythrocytes at a constant rate however there are times when bone marrow erythropoiesis cannot keep pace with the need for new red cells. At these times stress erythropoiesis predominates. This process has a greater capacity than steady state erythropoiesis to rapidly generate new erythrocytes. We have discovered that stress erythropoiesis relies on a dedicated stress response pathway that utilizes novel progenitor cells and signals. Using a mouse model for bone marrow transplant, we have identified a population of stress erythroid progenitor cells that exhibit stem cell like properties. Our analysis shows that these cells will generate only erythrocytes when transplanted into mice, meaning that they are erythroid restricted. In addition, they also have the ability to self renew and are maintained long term when transplanted into recipient mice. Based on these observations, we have termed these progenitor cells stress erythroid stem cells. Our recent work is investigating the mechanisms by which these cell self renew. The rationale for this experiment is that if we understand these mechanisms in more detail then we may be able to manipulate the cells in culture and use them for therapeutic purposes. This analysis of self renewal also supports another project in the laboratory which concerns the analysis of leukemia stem cells in Friend virus induced erythroleukemia. Friend virus leukemia virus activates the stress erythropoiesis pathway and the stress erythroid progenitors are the targets of the virus. Recently we showed that Friend virus leukemia stem cells correspond to the self renewing population of stress erythroid stem cells. Friend virus leukemia stem cells are easy to grow in culture and we are using them as a model system to study self renewal. This work has led to the identification of specific pathways involved in self renewal and in collaboration with Dr. Sandeep Prabhu we have identified a compound that blocks the self renewal of leukemia stem cells. Overall this work has lead to the identification of novel compound that blocks self renewal of leukemia stem cells and helped to identify new pathways that regulate self renewal in stress erythroid stem cells. These findings will aid in the development of new therapies for anemia and the treatment of leukemia. PARTICIPANTS: Robert F. Paulson is the principal investigator on the project. He is involved in the conception of the experiments and the management of the project. In addition, he is involved in data interpretation and the writing of manuscripts. Ms. Shailaja Hegde acts both as a research technician and as a graduate student on the project. She has performed experiments and helped manage the project. Lei Shi, Dai-Chen Wu, Laura Bennett and Jie Xiang are all graduate students working on experiments that support the project. There work will also be reported in their doctoral dissertations. Given that students are involved in this work, the project should also be considered for training purposes as well as the goals of the project. TARGET AUDIENCES: The results of our experiments are reported in peer reviewed journals and at presentations at scientific meetings. The target audience for these reports is scientists studying similar problems. In a broader sense our work provides a basic framework for the development of new therapies for anemia and leukemia. Therefore, pharmaceutical companies and clinicians would also be part of the target audience PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
My research has defined a new adult stem cell population and further elucidated the mechanisms that regulate stress erythropoiesis. This work provides a foundation for future translational work designed to more effectively treat leukemia and anemia. The impact of my research is measured by our publications and the acceptance of our models in the larger Hematology field.
Publications
- Kaushal, N., S. Hegde, J. A. Lumadue, R. F. Paulson, and K. S. Prabhu. 2011. The regulation of erythropoiesis by selenium in mice. Antioxidant and Redox Signaling 14:1403-1412.
- Hegde, N. V., E. L. Unger, G. L. Jensen, P. A. Hankey, and R. F. Paulson. 2011. Interrelationships between tissue iron status and erythropoiesis during postweaning development following neonatal iron deficiency in rats. American Journal of Physiology Gastrointestinal and Liver Physiology 300:G470-476.
- Paulson, R. F., L. Shi, and D-C. Wu. 2011 Stress Erythropoiesis: New signals and new stress progenitor cells. Current Opinion in Hematology 18:139-145.
- Trompouki, E., T. V. Bowman, L. N. Lawton, Z. P. Fan, D-C. Wu, A. DiBiase, C. S. Martin, J. N. Cech, A. K. Sessa, J. L. Leblanc, P. Li, E. Durand, C. Mosimann, G. C. Heffner, G. Q. Daley, R. F. Paulson, R. A. Young, and L. I. Zon. 2011. Lineage regulators direct BMP and Wnt pathways to cell-specific programs during differentiation and regeneration. Cell (In Press).
- Hegde, S. N., N. Kaushal, R. C. Kodihalli, C. Chiaro, K. T. Hafer, U. H. Gandhi, J. T. Thompson, J. P. Vanden Heuval, M. J. Kennett, P. Hankey, R. F. Paulson, and K. S. Prabhu. 2011. Delta12-prostaglandin J3, an omega-3 fatty acid-derived endogenous metabolite, selectively ablates leukemia stem cells in mice. Blood (In Press).
- Shi, L. 2011. Role of Short-form Stk signaling in stress erythropoiesis. Ph.D. Thesis. The Pennsylvania State University, University Park, PA. 114 pp.
- Wu, D-C. 2011. From exploring roles of signals in stress erythropoiesis to in vitro expanding stress erythroid progenitors that alleviate anemia. Ph.D. Thesis. The Pennsylvania State University, University Park, PA. 168 pp.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Work in my laboratory focuses on the study of stress erythropoiesis. Steady state erythropoiesis is primarily homeostatic, generating new erythrocytes at constant rate to replace worn out erythrocytes. The situation is different at times of acute anemia where rapid production of new erythrocytes is needed for survival. At these time stress erythropoiesis predominates. My laboratory identified a novel stress erythropoiesis pathway that relies on signals and progenitor cells that are distinct from steady state erythropoiesis. Stress erythropoiesis utilizes Hedgehog and BMP4 dependent signaling to promote the rapid expansion of a specialized population of stress erythroid progenitors. In the past year, we demonstrated that a key component in the regulation of this response in low oxygen levels or hypoxia in the tissues. Hypoxia dependent gene transcription regulates the expression of BMP4. This finding was significant because inappropriate activation of this pathway could lead to elevated erythrocyte counts which could lead to cardiovascular complications. The regulation of bMP4 by hypoxia ensures that stress erythropoiesis only occurs at times of anemic stress. In addition, we characterized the stress erythroid progenitor cell populations that respond to BMP4 dependent signals at times of acute anemia. These results showed that immature stress erythroid progenitors have the unique ability to function as an erythroid lineage restricted stem cell. Our findings show that these cells could be used a means for cellular based therapy for anemia patients. Overall, our work has established a model for stress erythropoiesis that has identified several potential mechanisms for therapeutic intervention. We are presently testing these possibilities in animal models of anemia. PARTICIPANTS: Robert F. Paulson is the Principal Investigator on the project. He is responsible for the day to management of the project. In addition his duties include experimental design, data analysis and manuscript preparation. Two Graduate students, Dai-Chen Wu and Lei Shi, have contributed to the project. In consultation with Dr. Paulson, they designed and performed experiments that contributed to this work. Shailaja Hegde is a Research technician in the laboratory who also performed experiments that contributed to this project and helped manage the project. The experiments performed by the graduate students will be reported in their doctoral dissertations. In that sense, this project should also be considered for training purposes as well as the goals of the project. TARGET AUDIENCES: The results of this project are reported in peer reviewed publications and presentations at scientific meetings, which are targeted to an audience of scientists studying similar questions. Our work provides a foundation for development of new therapeutic modalities to treat anemia. Therefore, in a broader sense, the audience for our work therefore would also include Pharmaceutical companies and clinicians. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our work has lead to a comprehensive model for stress erythropoiesis. This work lays the groundwork for developing translational approaches to treat chronic anemias, which are worldwide health problems. The impact of our work is evident in the citations of our publications and the incorporation of our model into the mainstream thinking in Hematology.
Publications
- Harandi, O., S. Hegde, D. Wu, D. Mckeone, and R. F. Paulson. 2010. Murine erythroid short term radioprotection requires a BMP4 dependent, self renewing population of stress erythroid progenitors. Journal of Clinical Investigation (In Press).
- Wu, D. and R. F. Paulson. 2010 Hypoxia regulates BMP4 expression in the murine spleen during recovery from acute anemia. PLoS ONE 5:e11303.
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