Diana Canals Hernaez
Doctor of Philosophy in Medical Genetics (PhD)
Investigating the role of podocalyxin in promoting tumor growth and metastasis, and its value as a therapeutic target in carcinoma
Dr. Kelly McNagny obtained his Ph.D. in Cellular Immunology at the U. of Alabama at Birmingham in 1990. There he worked with Dr. Max D. Cooper (Howard Hughes Medical Institute, National Academy of Sciences) and his research focused on cell surface proteins expressed by preB cells that regulate B cell maturation and homing. He then moved to the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany where he performed his postdoctoral studies in the lab of Dr. Thomas Graf from 1991 to 1996. There his work focused on transcriptional control of hematopoietic stem cell maturation and cell fate. He performed some of the first studies to identify transcription factors that regulate the gene expression and differentiation of eosinophils, which are known to play a major role in allergic and asthmatic responses. In addition, he identified a number of novel hematopoietic stem cell surface proteins and began analyzing their function. He continued his studies at the EMBL as a semi-independent, Visiting Scientist from 1996 to 1998 prior to starting his own laboratory at The Biomedical Research Centre, at UBC where his currently a full professor in Medical Genetics and interim co-director of The BRC.
His laboratory has followed two primary interests: 1) the transcription factor networks that regulate fate determination in various cells that make blood, and 2) the cell surface proteins expressed by hematopoietic stem cells that and allow them to communicate with their microenvironment. In this regard, his lab has identified a novel family of hematopoietic cell surface proteins, called the CD34 family, and shown that these are essential for a number of developmentally important processes. Through gene knockout studies he has shown that these molecules act as a type of molecular “Teflon” to make cells more mobile and invasive and also facilitate chemotaxis. He has delineated the function of these molecules in diverse set of biological processes including: 1) gut and kidney formation, 2) vascular permeability, 3) mucosal inflammatory disease, 4) stem cell homing and migration, and 5) epithelial tumor progression.
Dr. McNagny has received a number of awards for his work at UBC including a CIHR Scholarship, a Michael Smith Foundation for Health Research Senior Scholarship and the 2002 Showell-Pfizer Junior Faculty Award from the American Association for Immunology. He is a member of the Stem Cell Network Centre of Excellence (past member of the Stem Cell Policy Committee and Research Management Committee and current Sub-chair of the Training and Education Committee), and a member of the AllerGen Network Centre of Excellence (Research Management Committee and Co-Chair of the Biomarkers Program).
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Fibrosis is the result of dysregulated tissue regeneration and is characterized by excessive accumulation of matrix proteins that become detrimental to tissue function. Type 2 immunity has long been associated with fibrotic scarring because of its role in wound healing and parasite-initiated tissue remodeling. Our objective was to examine two components of this inflammatory pathway that could potentially be modulated to limit fibrosis, namely RORα, a nuclear receptor required for ILC2 development, and CD34, a sialomucin involved in trafficking of eosinophils and mast cells to peripheral tissues. Using a model of infection-induced chronic gut inflammation, we demonstrate that Rora-deficient mice are protected from fibrosis; infected intestinal tissues displayed diminished pathology and attenuated collagen deposition. Although Rora is known for its role in ILC2s, we found that Salmonella-induced fibrosis was independent of eosinophils, STAT6 signaling and Th2 cytokines arguing that ILC2s are dispensable in this disease model. Instead, we observed reduced levels of ILC3- and T cell-derived IL-17A and IL-22 in infected tissues. Furthermore, using Rorasg/sg/Rag1-/- bone marrow chimeric mice, we found that restoring ILC function was sufficient to re-establish IL-17A and IL-22 production and a profibrotic phenotype. Our findings suggest that RORα-dependent ILC3 functions are pivotal in mediating gut fibrosis and they offer an avenue for therapeutic intervention in Crohn’s-like diseases. CD34 has been shown to drive lung inflammation and colitis by coordinating immune cell recruitment. However CD34 is also expressed by multiple non-hematopoietic subsets including endothelial and mesenchymal cells. To assess CD34 function in pulmonary repair, we induced lung injury by bleomycin administration. We found that Cd34-/- mice displayed severe weight loss and early mortality compared to WT controls. CD34-deficient animals developed severe interstitial edema and endothelial delamination, indicating impaired endothelial function. Chimeric Cd34-/- mice reconstituted with WT hematopoietic cells exhibited early mortality compared to WT mice reconstituted with Cd34-/- cells thus confirming this to be a non-hematopoietic defect. Lastly, CD34-deficient mice were more sensitive to lung damage caused by influenza infection, displaying greater weight loss and more extensive pulmonary remodeling. These results suggest that CD34 plays a protective role in maintaining vascular integrity in response to lung damage.
Mucosal surfaces present an important barrier between the host and environment. Maintenance of barrier function requires intricate cross-talk between a diverse array of immune cells and the epithelia, acting synergistically to respond to harmful antigens and maintain tolerance to innocuous antigens. In this thesis I utilized an array of transgenic animals to explore the cellular and molecular mechanisms that initiate adaptive immune responses in the lung and gut mucosa.Recently, innate lymphoid cells have been characterized for their role in maintaining barrier immunity. Group 2 innate lymphoid cells (ILC2s) colonize the lung and provide a rapid source of IL-5 and IL-13 in a T and B cell independent manner in response to protease antigens. Using ILC2-deficient mice, I examined the role of these cells in mucosal inflammation using mouse models of allergic asthma and hypersensitivity pneumonitis (HP). ILC2s were critical in initiation of a Th2 response to locally, but not systemically delivered allergens and were completely dispensable for Th1 and Th17 dependent responses. The PI3K pathway plays an important role in regulating leukocyte activation, survival, migration and cytokine release. It is negatively regulated by the lipid phosphatase Ship1, and Ship1-/- mice develop a wide array of hematological disorders leading to a reduced lifespan. The severe phenotype associated with loss of Ship1 throughout the immune systems masks subtler roles it plays in specific leukocyte subsets. Using a conditional deletion approach, I examined the role of Ship1 in T cells, B cells and dendritic cells (DCs) in mouse models of allergic asthma and helminth infection. While loss of Ship1 in B cells did not influence susceptibility to a HDM model of allergic asthma, loss of Ship1 in either the T cells or DCs protected from disease development due to an immune skewing to a Th1 response. Additionally, loss of Ship1 in DCs rendered mice susceptible to infection with the intestinal helminth Trichuris muris, further highlighting this Th1 immune skewing.
Despite the widespread use of CD34-family sialomucins (CD34, podocalyxin (Podxl) andendoglycan) as vascular endothelial cell (EC) markers, there is remarkably little known oftheir role in vascular development and functions with the exception of vessel lumenformation in the developing mouse embryo (Podxl) and vessel patency during tumorangiogenesis and inflammation (CD34). Because germ-line deletion of Podxl in mice causesperinatal death, we generated mice that conditionally delete Podxl in vascular endothelialcells (PodxlΔEC mice) to study the role of podocalyxin in adult mouse vessels. AlthoughPodxlΔEC adult mice are viable and thrive, we discovered increased basal and inflammationinducedpulmonary vascular permeability. Furthermore, PodxlΔEC mouse adult lungs displayairspace enlargement with increased collagen deposition and exhibit a gene expressionprofile similar to regenerating lung. To study whether endothelial cell morphology influencesthe defective lung architecture and the vascular permeability phenotype in PodxlΔEC mice, weisolated primary vascular ECs from lung tissue. PodxlΔEC ECs display enhanced adhesion tofibronectin (FN) in a static adhesion assay. When plated on matrix-coated transwells,PodxlΔEC EC spread normally on FN but display defective spreading on laminin and collagenI. Thus, expression of Podxl in EC is required for normal lung architecture and function inadult mice and adhesion of EC to extracellular matrix components.Although its expression has not been well characterized, in humans, endoglycan expressionhas been reported in vascularized tissues. In mouse blood vessels, we showed that vascularsmooth muscle cells (vSMC), but not EC, express the highest levels of endoglycan. Using amouse aortic smooth muscle line (MOVAS-1) we found that forced expression ofendoglycan enhances basal but not platelet derived growth factor (PDGF)ββ-dependentvSMC migration in vitro. Further studies to understand the role of endoglycan in primaryvSMC showed that endoglycan is upregulated with differentiation to a contractile phenotype,but is not influenced by inflammatory stimuli or mitogenic factors. The findings of this thesis suggest that the CD34 family regulates vascular development andfunction with a role for podocalyxin in EC-matrix adhesion relevant to normal lung functionand a role for endoglycan in SMC differentiation and migration.
No abstract available.
Overall, we aimed to discover more about mast cell physiology, focusing on their homeostatic regulation in vivo, their activation in vitro and in allergic disease, their gene expression patterns, and their surface antigens. In our first study, our objective was to establish the function of Src homology 2-containing inositol 5’-phosphatase (SHIPI), in mast cells in vivo. SHIP1 inhibits immune receptor signaling throughhydrolysis of the phosphatidylinositol-3 kinase (P13K) product Pl-3,4,5-P₃ ,forming P1-3,4-P₂. In mast cells, SHIPI represses FcεRI- and cytokine-mediated activation in vitro, but little is known regarding the function of SHIPI in mast cells in vivo, or the susceptibility of Shipl⁻/⁻ mice to mast cell-associated diseases. We found that ⁻ mice have systemic mast cell hyperplasia, increased serum levels of IL-6, TNF, and IL-5, and a heightened anaphylactic response. Further, by reconstituting mast cell-deficient mice with Ship1⁺/⁺ or Shipl⁻/⁻ mast cells, we found that the above defects were due to loss of SHIPI in mast cells. Additionally, we found that micereconstituted with Shipl⁻/⁻ mast cells suffered worse allergic asthma pathology than those reconstituted with Ship1⁺/⁺ mast cells. In summary, our data show that SHIPI represses allergic inflammation and mast cell hyperplasia in vivo, and that SHIPI exerts these effects specifically in mast cells. In our second study we compared Lin⁻Sca-1⁺c-kit⁺ (LSK) cells, which are highlyenriched for hematopoietic stem cells (HSC), and mast cells, using microarray expression analysis, and identified prion protein (PrPC) as a potentially novel marker of mast cells. Upon further investigation, we found that PrPC (1) is expressed on the surface of human and mouse mast cells, both in vitro and in vivo; (2) is not required for mast cell differentiation or tissue homeostasis; (3) is released by mast cells atsteady state and rapidly upon activation; and (4) is released in response to mast cell dependent allergic inflammation in vivo. Since mast cells are long-lived and known to traffic to the brain and central nervous system (CNS), our observations could have important implications for the transmission and pathology of prion diseases. Further, mast cells could be a unique system to investigate PrPc’s normal function.
CD34 and its relatives, Podocalyxin and Endoglycan, comprise of a family ofsurface sialomucins expressed by hematopoietic stem/progenitor cells, and vascularendothelia. Recent data suggest that they serve as either pro- or anti-adhesion moleculesdepending on their cellular context and their post-translational modifications. We wereinterested in identifying Podocalyxin ligands and their cellular distribution andunderstanding the role of these factors in signaling, adhesion and migration. Using both alambda phage screen assay and mass spectrometry, we identified the Na⁺/H⁺ exchangerregulatory factor-i (NHERF-l) as a selective ligand for Podocalyxin and Endoglycan butnot for the closely related CD34. Furthermore, we showed that NHERF-1 is expressedby all, lineage⁻, Sca-1⁺ and c-kit⁺ (LSK) cells, which are known to express Podocalyxinand have long-term repopulating characteristics of hematopoietic stem cells. In addition,upon IL-3 stimulation of a factor dependent cell line (FDC-P 1) these proteins re-localizeand co-localize in an asymmetrical pattern. By using a lentiviral based shRNA system tosilence Podocalyxin and NHERF- i proteins, we observed that migration across stromalmonolayer towards a CXCL12 and SCF gradient is significantly impeded in cells thatlack Podocalyxin but not NHERF-1. Following in vitro stimulation with a combinationof CXCL12 and SCF we observed that Podocalyxin co-associates with CXCR4.Furthermore, cells lacking Podocalyxin have decreased phospho-AKT, a key signalingmolecule downstream of c-kit and CXCR4 receptors. Taken together, our data supportsthe conclusion that Podocalyxin co-association with CXCR4 modulates downstreamsignaling to efficiently regulate HSC homing.
The CD34-family sialomucin, podocalyxin (Podxl), is broadly expressed on the luminal face of blood vessels in adult mammals; however, its biological function on vascular endothelial cells (vEC) is not well-defined. Here, we reveal specific functions for podocalyxin in maintaining endothelial barriers using HUVEC monolayers as a model in vitro. Detailed analysis of barrier HUVEC characteristics using electrical cell-substrate impedance sensing (ECIS) and live cell imaging revealed essential roles for podocalyxin in maintaining cell-cell and cell-matrix interactions. Thus, podocalyxin-deficient HUVEC fail to form a functional barrier when plated on several extracellular matrix (ECM) substrates. Regardless of ECM substrate, these monolayers lack adherens junctions and focal adhesions; and display a disorganized cortical actin cytoskeleton. To explore an in vivo function of podocalyxin, we conditionally deleted Podxl in vEC using the Tie2Cre strain (PodxlΔTie2Cre). Although we did not detect altered permeability in naïve mice at steady state, systemic priming with lipopolysaccharides (LPS) disrupted the blood-brain barrier (BBB) in PodxlΔTie2Cre but not WT mice. To study the potential consequence of this BBB breach, we used a selective agonist of PAR-1, a thrombin receptor expressed by neurons and glial cells. As a polar peptide, the PAR-1 agonist (TFLLRN), is normally excluded from CNS parenchyma by the BBB. In response to systemic administration of TFLLRN, LPS-primed PodxlΔTie2Cre mice experienced a dramatic behavioral change marked by a severely dampened neurological electrical activity. We conclude that podocalyxin expression by CNS vECs is required to maintain BBB integrity under inflammatory conditions. Supplementary materials available at: http://hdl.handle.net/2429/68998
No abstract available.
SHIP1 (SH2 domain containing inositol-5ʼ-phosphatase) is a negative regulator of thephosphatidylinositol-3 kinase (PI3K) pathway. SHIP1 is expressed in hematopoietic cells,and mediates its effect by hydrolyzing PIP₃, an important second messenger generated bythe PI3K pathway. In this way, SHIP attenuates a variety of signaling cascades includingthose mediating cell survival and proliferation. Due to its importance in regulating immunecell signaling, SHIP1 is an attractive therapeutic target.In this thesis, I explored the role of SHIP1 in the context of rheumatoid arthritis, anautoimmune inflammatory disease. To this end, I employed the K/BxN serum transfer modelof rheumatoid arthritis. This disease model is auto-antibody driven and lymphocyteindependent, and thus facilitates characterization of the effector phase of disease, a processthat relies on components of the innate immune system. Arthritis was dramaticallyexacerbated in global SHIP1 knock-out mice, as evidenced by changes in ankle thickness,clinical scoring and histological analysis. Heterozygous SHIP1 mice also displayedincreased disease severity in comparison to wild type litter mates, possibly due to anexpanded population of circulating neutrophils, that increases with age. Since naive globalSHIP1 knock-out mice exhibit a range of hematopoietic abnormalities, to elucidate the cellintrinsic contribution of SHIP1 ablation to the disease phenotype, I induced K/BxN mediatedarthritis in mice with lineage restricted deletion of SHIP1. Mice with a neutrophil/macrophage-restricted loss of SHIP1 (LysMcre), like global SHIP1 knock-out mice,displayed an alternatively activated ʻM2ʼ biased macrophage phenotype, and developedexacerbated disease. Neutrophil-restricted loss of SHIP1 (GEcre) was also sufficient toexacerbate disease and resulted in earlier disease onset. In order to identify how the loss ofSHIP1 in neutrophils results in heightened disease severity, I performed a series of in vitroexperiments utilizing polymorphonuclear leukocytes freshly isolated from bone marrow.While we cannot exclude that SHIP1 may be playing additional roles in neutrophil functions,I report that SHIP1 plays a role in attenuating responsiveness of neutrophils to GPCR andFcγR ligation, two families of receptors that are necessary for induction and amplification ofrheumatoid arthritis in the K/BxN serum transfer model.