ORCID

http://orcid.org/0000-0002-6234-7005

Date of Award

Spring 5-15-2021

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Developmental, Regenerative, & Stem Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

For a cell to function properly, it must be able to interact with and respond to environmental cues; however, expression of surface molecules, proteins, and receptors is not always sufficient to execute a cellular response. Proper organization of the plasma membrane is necessary to facilitate these highly regulated protein interactions, such that a cell can respond to stressors, growth factors, and other signaling molecules. Tetraspanins are a family of transmembrane proteins which help correctly orient surface molecules on the cell membrane, often through tetraspanin enriched microdomains, a membrane structure similar to lipid rafts. As a family, tetraspanins are known to regulate cell activation, migration, adhesion, and apoptosis, but the mechanism by which these effects occur is largely unknown. CD53 is a tetraspanin that is exclusively expressed on hematopoietic cells and has been previously implicated in lymphocyte activation and function; nevertheless, no mechanism to explain the function of CD53 nor the binding partners with which CD53 interacts have been identified. Elucidating the function and signaling pathways of CD53 will provide insight into how this protein is able to organize the plasma membrane and facilitate interaction with the cellular environment. To investigate the role of CD53, we generated a Cd53-/- mouse and studied the effects on hematopoietic cells. Cd53 is upregulated on mouse and human hematopoietic stem cells (HSCs) following multiple methods of induced cellular stress. While loss of CD53 does not impair HSC expansion or homing to the bone marrow, Cd53-/- HSCs have a significant functional impairment upon transplantation, which is most severe in granulocyte-colony stimulating factor (G-CSF) mobilized splenic HSCs. Through transcriptomic analysis, we identified the DREAM complex pathway as a potential mediator of this functional impairment, with Cd53-/- HSCs having significantly dysregulated DREAM complex target gene expression. Further investigation into the role of CD53 in HSC cycling revealed that CD53 protects HSCs from over-cycling and possible exhaustion, with Cd53-/- HSCs having increased cell cycling in response to G-CSF treatment compared to WT HSCs and markedly reduced tolerance to serial 5-FU treatment. Together, these data suggest that CD53 is vital to protecting HSCs during stress by maintaining cell quiescence.

While characterizing this stem cell phenotype, we observed a significant reduction in the number of B cells in the peripheral blood of Cd53-/- mice. During normal B lymphopoiesis, CD53 expression increases through B cell maturation, consistent with activation of EBF1, which has been shown to directly target and enhance Cd53 expression. Through association with IL-7Ra, CD53 maintains signaling through the PI3K and STAT5 pathways to support early B cell growth, promote B cell differentiation, and reduce B cell death. Mixed bone marrow chimeras reveal that CD53 functions cell autonomously during early B cell development. Analysis of bone marrow B cell development demonstrates that this loss of B cells originates with early B cell progenitors, which display reduced IL-7Ra expression and signaling. Specifically, we observe reduced PI3K and STAT5 activation in pre-pro- and pro-B cells in the absence of CD53, with a consequent increase in apoptosis in these populations. Finally, co-immunoprecipitation and proximity ligation studies demonstrate a physical interaction between CD53 and IL-7Ra, suggesting that these proteins associate at the cell surface to maintain homeostatic early B cell signaling necessary for normal B cell development.

In addition to a role in normal B cell development, CD53 also affects malignant B cells. Our data identify the tetraspanin CD53 as an important and upregulated surface protein during malignant transformation of both mouse and human B cell acute lymphoblastic leukemias (B ALLs). Using the Eu-Myc mouse model, we found CD53 to be expendable for mouse survival, but required for leukemia bone marrow involvement in Eu-Myc-driven B-ALLs. While there was equal distribution of malignant cells in the spleen, lymph nodes, and blood, only Eu-Myc+; Cd53+/+ cells took permanent residence in the bone marrow, with Eu-Myc+; Cd53-/- cells largely remaining in the periphery. This bone marrow homing defect is cell-intrinsic, as even when transplanted into WT recipients, Eu-Myc+; Cd53-/- cells still did not reside or expand within the bone marrow. Further studies confirmed that CD53 promotes the homing of both normal and malignant B cells to the bone marrow, in part, by supporting CXCR4 intracellular signaling. These data support a model where CD53 is important for malignant B cell homing to the bone marrow and protection from chemotherapy.

Overall, these studies identify and expand on previously unknown roles for CD53 in HSC quiescence, B cell development, and malignant homing and expansion. Through association with IL-7 receptor, CXCR4, and other proteins, CD53 is able to regulate cellular function by membrane organization and protein stabilization. Future studies will continue to identify binding partners of CD53 in different cell types and characterize the implications of perturbation of CD53 expression. Because CD53 expression is restricted to hematopoietic cells, it is a promising drug target with hopefully few off-target effects, especially in the fields of bone marrow transplantation and B ALL treatment.

Language

English (en)

Chair and Committee

Laura G. Schuettpelz

Committee Members

Mary Dinauer, S C. Morley, Jeff Bednarski, Charles Kaufman,

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