ABERRANT SUBCELLULAR TARGETING OF THE G185R NEUTROPHIL ELASTASE MUTANT ASSOCIATED WITH SEVERE CONGENITAL NEUTROPENIA INDUCES PREMATURE APOPTOSIS OF DIFFERENTIATING PROMYELOCYTES & EXPRESSION AND FUNCTION OF THE TRANSIENT RECEPTOR POTENTIAL 2 (TRPM2) ION CHANNEL IN DENDRITIC CELLS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Pam Massullo, M.S. ******* The Ohio State University 2007 Approved by Dissertation Committee: ________________________ Advisor Belinda R. Avalos, M.D., Advisor ________________________ Santiago Partida-Sanchez, Ph.D., Co-Advisor Co-Advisor James C. Lang, Ph.D. Molecular, Cellular, and Developmental Biology John M. Robinson, Ph.D. Graduate Program ABSTRACT Part I : Severe congenital neutropenia (SCN) is a bone marrow failure disorder usually diagnosed in the first year of life and characterized by extremely low numbers of peripheral blood neutrophils, a myeloid maturation arrest in the bone marrow, and recurrent infections. Despite dramatic improvements in survival and quality of life with granulocyte colony-stimulating factor (G-CSF) therapy, patients with SCN have a life-long increased risk of developing leukemia. Mutations in the ELA2 gene encoding neutrophil elastase (NE) are present in most patients with SCN. However, the mechanisms by which these mutations cause neutropenia remain unknown. To investigate the effects of mutant NE expression on granulopoiesis, we used the HL-60 promyelocytic cell line retrovirally transduced with the G185R NE mutant that is associated with a severe SCN phenotype. We show that the mutant enzyme accelerates apoptosis of differentiating but not of proliferating cells. Using metabolic labeling, confocal immunofluorescence microscopy, and immunoblot analysis of subcellular fractions, we also demonstrate that the G185R mutant is abnormally processed and localizes predominantly to the nuclear and plasma membranes rather than to the cytoplasmic compartment observed with the wild-type (WT) enzyme. Expression ii of the G185R mutant appeared to alter the subcellular distribution and expression of adaptor protein 3 (AP3), which traffics proteins from the trans-Golgi apparatus to the endosome. These observations provide further insight into potential mechanisms by which NE mutations cause neutropenia and suggest that abnormal protein trafficking and accelerated apoptosis of differentiating myeloid cells contribute to the severe SCN phenotype resulting from the G185R mutation. In the subset of patients with SCN transforming to acute myeloid leukemia (AML), mutations that truncate the cytoplasmic tail of the G-CSF receptor (G- CSFR) have been detected. We identified a novel mutation in the extracellular portion of the G-CSFR within the WSXWS motif in a patient with SCN without AML who was refractory to G-CSF treatment. The mutation affected a single allele and introduced a premature stop codon that deletes the distal extracellular region and the entire transmembrane and cytoplasmic portions of the G-CSFR. Subsequent reports have demonstrated that this mutant decreases the surface expression of the wild-type receptor and thereby inhibits proliferative signaling by the wild-type G-CSFR, suggesting a common mechanism underlying G-CSF refractoriness in SCN patients. NE is a serine protease stored in the primary granules of neutrophils that proteolytically cleaves multiple cytokines and cell surface proteins on release from activated neutrophils. Recent reports of mutations in the gene encoding this enzyme in some patients with neutropenic syndromes prompted us to investigate iii whether G-CSF or its receptor G-CSFR were also substrates for NE. Previous research in the laboratory demonstrated that NE enzymatically degrades both G- CSF and the G-CSFR, strongly arguing in favor of a catalytic mechanism. We show that NE abrogates proliferative signals generated by the G-CSFR in myeloid progenitor cells, as indicated by the decreased numbers and size of CFU-GM arising from marrow progenitors pre-treated with NE. These findings provide additional insights into mechanisms by which G-CSF/G-CSFR interactions may be modulated. Collectively, our data indicate that the G185R NE mutant that is associated with the most severe phenotype in SCN is missorted to the plasma membrane and that the normal or WT NE can degrade and inactivate both G- CSF and the G-CSFR. This suggests that aberrant interaction of NE mutants with membrane proteins critical for the survival of maturating myeloid cells is the pathophysiologic mechanism leading to neutropenia. iv Part II : Dendritic cells (DCs) orchestrate immunity by amplifying innate and initiating adaptive immune responses. DCs traffic in response to chemokines and inflammatory mediators. Although most chemokine receptor stimulation in DCs is 2+ 2+ accompanied by intracellular Ca release and Ca influx, the identity and functions of the ion channels responsible remains largely unknown. Here we aimed to investigate the expression and function of the transient receptor potential (melastatin-related) 2 (TRPM2) ion channel. TRPM2 is a Ca 2+ - permeable channel with unique gating behavior, as direct binding of ADPR, the main catalytic product from the ectoenzyme CD38, evokes channel opening. Ca 2+ -mobilizing metabolites produced by CD38 are essential for DC migration. Multiple cell types were examined to find a model in which to study CD38 derived Ca 2+ -mobilizing metabolites and TRPM2. Using a newly generated TRPM2 antibody and RT-PCR the expression of TRPM2 was confirmed in primary hematopoietic cells and cell lines. TRPM2 currents were demonstrated by electrophysiology experiments. Initial experiments to knockdown TRPM2 protein expression were performed in primary DCs. Migration to a variety of chemokines was examined in the presence of inhibitors to CD38-derived Ca 2+ metabolites. We propose a model where CD38 metabolites activate TRPM2, leading to increased plasma membrane permeability, Ca 2+ influx, and chemotaxis. This data provides further insights into mechanisms of ADPR-gated TRPM2 activation, and advances our understanding of how inflammatory signals, such as chemokines, modulate immunity, as DC trafficking is critical for efficacious immune responses. v Dedicated to my parents vi ACKNOWLEDGMENTS I would like to express my deep appreciation to my advisor, Dr. Belinda Avalos, for her inspiration, friendship, and support. I am fortunate to have worked in her laboratory. I am indebted to my co-advisor, Dr. Santiago Partida-Sanchez, for giving me the opportunity to work in his laboratory, for the freedom to explore science, intellectual support, encouragement, and patience. I am indebted to Dr. Jas Lang and Dr. John Robinson for volunteering their time to serve on my committee, for generously sharing of lab equipment, and for critical review of this dissertation. I am grateful to my past and current lab member Jing, Tammy, Harivadan, and Adriana. Your friendship and support have made the lab a stimulating environment to work over the years. I wish to thank Dr. Larry Druhan and Dr. Melissa Hunter for being generous with their time and providing technical assistance and expertise. Your advice and insight were invaluable during my time in the laboratory. I would like to convey my gratitude to Dr David Bisaro, director of the MCDB program, for his support during my transition between laboratories. Special thanks to Jan Zinaich of the MCDB program for all of her help throughout my time at OSU. vii I want to thank Dr. Tom Knobloch for sharing lab equipment; Dr. Andrea Fleig and Ingo Lang for collaboration on the electrophysiology experiments; Dr. David Williams for providing a plasmid, Dr. Matthew Kennedy and Dr Kenneth Rock for sharing cell lines; Dr. Bruce Bunnell for generating the retrovirus; and Dr. Clay Marsh for letting me complete experiments in his laboratory. Lastly, I would like to say thank you to my wonderful family. To my parents, Elio and Mary, for their undying love, support, and guidance. To my husband, Matthew, you are my best friend and I love you. viii VITA March 1, 1977……………………..Born – Youngstown, Ohio 1999………………………..….…...Bachelor of Science Youngstown State University Youngstown, Ohio 1999-2001………………..…….….Graduate Research and Teaching Associate Youngstown State University Youngstown, Ohio 2004……………………..……........Master of Science Youngstown State University Youngstown, Ohio 2001-2006……………..………......Graduate Research Associate The Ohio State University Columbus, Ohio PUBLICATIONS 1. Massullo P , Sumoza-Toledo A, Bhagat H, Partida-Sanchez S. TRPM channels, calcium and redox sensors during innate immune responses. Semin Cell Dev Biol. 2006;17(6):654-666. 2. Massullo P , Druhan LJ, Bunnell BA, Hunter MG, Robinson JM, Marsh CB, Avalos BR. Aberrant subcellular targeting of the G185R neutrophil elastase mutant associated with severe congenital neutropenia induces premature apoptosis of differentiating promyelocytes. Blood, 2005; 105(9):3397-3404. 3. Druhan LJ, Ai J, Massullo P , Kindwall-Keller T, Ranalli MA, Avalos BR. Novel mechanism for G-CSF refractoriness in patients with severe congenital neutropenia. Blood, 2005; 105(2):584-591. ix 4. Hunter MG, Druhan LJ, Massullo PR , Avalos BR. Proteolytic cleavage of granulocyte colony-stimulating factor and its receptor by neutrophil elastase induces growth inhibition and decreased cell surface expression of the granulocyte colony-stimulating factor receptor. American Journal of Hematology,