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A Dissertation Entitled Erk1/2 Signaling Pathway And A Dissertation Entitled Erk1/2 Signaling Pathway and Transcriptional Repressor Gfi1 in the Regulation of Neutrophil versus Monocyte Development in Response to G-CSF and M-CSF by Nan Hu Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology _________________________________________ Dr. Fan Dong, Committee Chair _________________________________________ Dr. Lirim Shemshedini, Committee Member _________________________________________ Dr. Deborah Chadee, Committee Member _________________________________________ Dr.Stanislaw Stepkowski, Committee Member _________________________________________ Dr. Kam Yeung, Committee Member _________________________________________ Dr. Patricia R. Komuniecki, Dean College of Graduate Studies The University of Toledo August 2015 Copyright 2015, Nan Hu This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Erk1/2 Signaling Pathway and Transcriptional Repressor Gfi1 in the Regulation of Neutrophil versus Monocyte Development in Response to G-CSF and M-CSF by Nan Hu Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology The University of Toledo August 2015 All lineages of mature blood cells are derived from the hematopoietic stem cells which reside in the bone marrow. Blood cell development, a process called hematopoiesis, is regulated by different mechanisms. At the cellular level, multiple transcription factors are critical for blood cell development. For example, the ratio of c/EBP to PU.1 is important for the direction of differentiation. High c/EBP to PU.1 ratio favors neutrophil differentiation whereas low c/EBP to PU.1 ratio leads to monocyte/macrophage differentiation. In addition to transcription factors, hematopoietic cytokines also play important roles in hematopoiesis. Granulocyte-colony stimulating factor (G-CSF) acts on the lineage of granulocytes while macrophage-colony stimulating factor (M-CSF) acts on the monocytic lineage. Hematopoietic cytokines are known to stimulate cell proliferation and survival, but whether these cytokines actively induce the differentiation of hematopoietic progenitor cells is still controversial. Our lab has previously shown that murine myeloblastic 32D cells transfected with wild type G-CSF receptor differentiated into neutrophils after G-CSF treatment, but cells transfected with the truncated G-CSFR lacking the three C-terminal tyrosine residues (Tyr 729, 744, 769) iii failed to differentiate. To address whether the three tyrosine residues are involved in granulocytic differentiation, 32D cells were transfected with a mutant G-CSFR (mA) in which the three tyrosine residues have been mutated to phenylalanine. Interestingly, 32D/mA cells showed monocytic differentiation in response to G-CSF, as evidenced by attachment to culture flasks, morphological features and expression of monocytic differentiation markers F4/80, Mmp-12 and M-CSF. By individually mutating each of the three tyrosine residues, we demonstrate that Tyr 729 of G-CSFR is required for the neutrophil differentiation in response to G-CSF. We further demonstrate that Erk1/2 activation upon G-CSF treatment is prolonged in 32D/Y729F and FDCPmix/Y729F cells as compared to WT cells and inhibition of Erk1/2 pathway using specific inhibitors and Erk1/2 knockdown partially reversed monocytic differentiation of 32D/Y729F and FDCPmix/Y729F cells. Together, these results indicate that Tyr729 of the G-CSFR negatively regulates Erk1/2 activation and that mutation of Tyr729 leads to prolonged Erk1/2 activation. Furthermore, Erk1/2 activation upon M-CSF treatment is prolonged in Lin- primary bone marrow cells as compared to G-CSF treatment. Mek1/2 inhibitors favor neutrophil development at the expense of monocytic development of Lin- bone marrow cells upon M-CSF treatment. To find out the downstream targets of Erk1/2, we examined expressions of c-Fos, Egr1 and phosphorylation of c-Fos which are important for monocytic differentiation. Both 32D/Y729F cells and FDCPmix/Y729F cells showed prolonged induction of c-Fos and Egr1, which is consistent with stronger induction of TRE3-tk-Luc (containing 3 repeats of AP1 binding elements) and EBS24-Luc (containing 4 repeats of Egr1 binding sequence). We further showed that c-Fos or Egr1 knockdown restored neutrophil differentiation of 32D/Y729F cells and FDCPmix/Y729F cells, along iv with repression of Mmp-12 and M-CSF. Together we have shown that Tyr729 is essential for instructing neutrophil development, and activation of Erk1/2 regulates neutrophil versus monocyte development through Egr1 and c-Fos. Gfi1 is a transcriptional repressor that plays an important role in hematopoiesis. Gfi1 favors neutrophil development and antagonize monocyte/macrophage development. It has been shown that Gfi1 represses M-CSF, M-CSF receptor and Egr2 expression which are important for macrophage differentiation. Gfi1 also represses PU.1 transcription and further inhibits PU.1 activity through protein-protein interaction. However, it is still incompletely understood how Gfi1 regulates neutrophil versus monocyte cell fate choice. Here we demonstrate that Gfi1 represses Egr1 and c-Fos to block macrophage development in both 32D and FDCP-mix A4 cell. Gfi1 knockout mice lack neutrophils and have elevated number of abnormal monocyte progenitors. We have also shown that mRNA levels of Egr1 and c-Fos were elevated and Erk1/2 activation was stronger in Gfi1-/- BM cells compared to Gfi1+/+ cells. To address whether strong activation of Erk1/2 is associated with the abnormal differentiation of Gfi1-/- BM cells through Egr1 and c-Fos, Gfi1-/- BM cells were treated with Mek1/2 inhibitors U0126 and PD0325901. Egr1 and c-Fos mRNA levels were repressed significantly. Interestingly, U0126 and PD0325901 also restored neutrophil development of Gfi1-/- BM cells in response to G-CSF. Together, our data showed a new mechanism of the action of Gfi1 in myelopoeisis that Gfi1 represses Egr1 and c-Fos expression through direct promoter binding and the abnormal differentiation of Gfi1-/- BM cells is associated with strong activation of Erk1/2. v This work is dedicated to my mother, Peizhi Kong, for her love and sacrifice. Acknowledgements I wish to thank my advisor, Dr. Fan Dong, for introducing me to the field of hematology, his guidance in my research and giving me the opportunity to work on very interesting projects. Gratitude also goes to my committee members, Dr. Lirim Shemshedini, Dr. Deborah Chadee, Dr. Stanislaw Stepkowski and Dr. Kam Yeung, for their help and insights. I also would like to thank the people I have been working with in the past five years, Dr. Yaling Qiu for teaching me experiments and making the lab feel like home, Srimathi Karsturirangan, Yangyang Zhang and previous lab member Dr. Pei Du. It is a pleasure to work with them. I am eternally gratefully to my mother, Peizhi Kong, for her greatest love, sacrifice and support. Without her love, I would not achieve anything. I also would like to thank my husband, Jonathan McAtee, for his love and continuous encouragement, my daughter, Allison McAtee, for bringing the joy of being a parent, and all my family for loving and supporting me. Last but not the least, I wish to thank all my friends for being supportive and being there for me. vii Table of Contents Abstract ............................................................................................................................. iii Acknowledgements ......................................................................................................... vii Table of Contents ........................................................................................................... viii List of Figures ................................................................................................................... xi List of Abbreviations ..................................................................................................... xiii 1 Introduction .....................................................................................................................1 1.1 Hematopoiesis .................................................................................................1 1.2 Hematopoietic cytokine and cytokine receptors .............................................3 1.3 Granulocyte Colony Stimulating Factor (G-CSF) Receptor ...........................5 1.4 Granulocyte Colony Stimulating Factor (G-CSF) Receptor signaling ...........8 1.5 Hematopoietic transcription factors ................................................................9 1.6 Growth factor independent 1 (Gfi1) .............................................................11 1.7 Egr1 and c-Fos ..............................................................................................13 2 Material and Methods ................................................................................................15 2.1 Cell lines and cell culture ..............................................................................15 2.2 Construction of plasmids ..............................................................................16 2.3 Transfection and generation of stable cell lines ............................................16 2.4 Flow cytometry .............................................................................................16 2.5 Western
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