2017 — 2018 |
Davis, Kara Lynn Spitzer, Matthew Wu, Joy Y |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Single-Cell High-Dimensional Characterization of the Bone Marrow Microenvironment in Health and Disease
Project Summary In the bone marrow, hematopoiesis is dependent upon support from the surrounding microenvironment, or niche. The bone marrow microenvironment is complex, with hematopoietic and mesenchymal cell populations interacting to influence the formation of hematopoietic cells, blood vessels and bone. In order to understand the molecular and cellular mechanisms by which multiple cell populations support hematopoiesis, it would be helpful to analyze a large number of cell types simultaneously. Mass cytometry (CyTOF) is a novel technique in which flow cytometry is performed using antibodies coupled to rare earth metal isotopes rather than fluorochromes, followed by mass spectrometry. Unimpeded by spectral overlap, CyTOF allows for the analysis of >40 simultaneous parameters. Using CyTOF we have recently published reference maps of the major hematopoietic cell populations in mouse and human bone marrow. We now propose to expand these to include the mesenchymal populations. Our central hypothesis is that alterations in the distribution and function of mesenchymal populations reciprocally influence bone marrow hematopoiesis. As a corollary, we suggest that bone pathologies affecting mesenchymal cells, such as osteoporosis, will necessarily perturb hematopoietic cell development and function. The parathyroid hormone (PTH) receptor (PTH1R) is a G protein coupled receptor whose signaling in bone has profound effects on bone formation and hematopoiesis. To investigate how PTH signaling alters the bone marrow microenvironment we propose the following aims: In Aim 1 we will expand the reference map of hematopoietic cells in murine bone marrow to include mesenchymal populations by incorporating antibodies to identify endothelial cells, mesenchymal stem/progenitor cells, and osteoblasts. We will further identify major cell type-specific cytokines and signaling pathways stimulated by PTH. In Aim 2 we will examine how bone marrow populations are altered by disruption of PTH/PTH1R/Gs? signaling. We have demonstrated that mice lacking the Gs? subunit, which mediates many PTH-dependent actions in bone, in osteoblast progenitors (Gs?-OsxKO mice) exhibit severe osteoporosis, loss of B lymphocyte precursors, and a failure to increase bone mass in response to PTH. By comparing the frequencies of hematopoietic and mesenchymal populations in Gs?-OsxKO bone marrow to the reference map generated in Aim 1, we will reveal how the absence of Gs? signaling in osteoblasts impacts the bone marrow ecosystem. We will also examine the alterations in cytokine production and PTH-dependent signaling within the bone marrow of Gs?-OsxKO mice. In Aim 3 we will generate a reference map of the human bone marrow microenvironment. Understanding the structure of normal mesenchymal populations in human bone marrow is foundational to examining alterations in disease, and such a reference map will be of value in studying the role of bone marrow microenvironment in human disease, aging, and in response to bone- and blood-targeting medications.
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0.97 |
2018 |
Wu, Joy Y |
R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Role of the Parathyroid Hormone Receptor in Osteoblast Support of Erythropoiesis
Project Summary Erythrocytes play a crucial role in the delivery of oxygen to meet the metabolic needs of tissues. Erythroid development occurs within the bone marrow, but when bleeding or erythrocyte destruction leads to a need for increased erythrocyte production (erythropoiesis), the spleen can become a secondary site for stress erythropoiesis. Within the bone marrow, the role of the osteoblast lineage in supporting hematopoietic stem cells and differentiation of hematopoietic lineages including B lymphocytes is now well established. Recent studies have expanded the contributions of the osteoblast lineage to the support of erythropoiesis as well, as osteoblasts are a potential source of the critical erythrocyte-regulating hormone erythropoietin (Epo). We have been interested in the role of signaling downstream of the parathyroid hormone receptor (PTH1R), a G protein- coupled receptor, in osteoblasts in regulating osteoblast support of erythropoiesis. In mice lacking PTH1R in the skeleton (PTH1R-OsxKO mice), we find a dramatic loss of erythrocytes in the spleen. The mechanisms that govern the migration of erythroid progenitors and erythroblasts from the bone marrow to the spleen in times of stress are largely undefined. Since these mice carry an osteoblast-specific deletion of PTH1R, the most likely model is that bone marrow erythropoiesis is sufficient at steady state, but unable to provide sufficient erythroid progenitors to the spleen during stress erythropoiesis. However, we find cells descended from osteoprogenitors in the spleen, therefore an impaired spleen environment in PTH1R-OsxKO mice may also be at work. Consistent with a defect in osteoblastic regulation of erythropoiesis, in preliminary studies we find a significant decrease in expression of Epo mRNA in bones of PTH1R-OsxKO mice. We hypothesize that PTH1R signaling in osteoblast progenitors regulates bone marrow erythroid development and trafficking to the spleen. In this proposal we will systematically analyze the following steps in PTH1R-OsxKO mice: 1) bone marrow commitment, differentiation and proliferation of erythroid progenitors and erythroblasts; 2) exit of marrow erythroblasts from the bone marrow to enter the peripheral circulation; 3) homing of circulating erythroid progenitors to the spleen, followed by engraftment and expansion within the splenic microenvironment. Together the accomplishment of the proposed studies will pinpoint the site(s) of dysregulation of spleen hematopoiesis in PTH1R-OsxKO mice, and may shed light upon the mechanisms that govern stress erythropoiesis as well as further clarifying a role for osteoblasts in the support of bone marrow erythropoiesis.
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0.97 |
2019 — 2021 |
Wu, Joy Y |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Interactions of Pth and Wnt Signaling in Bone Formation
Project Summary Osteoporosis is common and devastating, as 20% of adults with a hip fracture die within 1 year and another 50% never walk independently again. Medications to promote bone formation might be a superior approach to treating osteoporosis. Recombinant parathyroid hormone (PTH) and anti-sclerostin antibody increase bone formation by targeting PTH receptor (PTH1R) and Wnt signaling pathways, respectively. However, these medications require injections and can only be used for up to two years. In mice, combined treatment with PTH and anti-sclerostin antibody is more effective than either medication alone. Understanding the mechanisms by which combined PTH1R and Wnt signaling increase bone mass could lead to novel treatments to decrease fracture risk. We will test our central hypothesis that PTH1R is required for Wnt to fully stimulate bone formation. Mechanistically, we propose that PTH1R is required to stabilize the Wnt effector b-catenin in osteoprogenitors, which in turn maximally stimulates expression of the osteoblast gene program. We will use two approaches to activate Wnt signaling in mice lacking PTH1R in bone: pharmacologically with a novel water soluble Wnt surrogate, and genetically by knocking out the Wnt inhibitor sclerostin. We propose to use two innovative methods to overcome current barriers to understanding how PTH and Wnt signaling interact in bone. First, we will use mass cytometry (CyTOF) to analyze expression of >40 parameters, allowing us to distinguish mesenchymal stem cells, osteoprogenitors and osteoblasts, and to simultaneously examine the effects of PTH1R and Wnt signaling in these populations. Second, we will use single-cell RNA-sequencing to evaluate PTH1R and Wnt signaling in osteoprogenitors. We have preliminary data that in the absence of PTH1R signaling in osteoprogenitors, increased Wnt signaling fails to increase bone. In Specific Aim 1 we will determine whether intact PTH1R signaling is required for Wnt-dependent bone formation by pharmacological and genetic activation of Wnt signaling in 1 month-old control (PTH1ROsxWT) and PTH1ROsxKO mice. We will assess bone formation by histology, quantitative histomorphometry, and micro-computed tomography (µCT). In Specific Aim 2 we will determine whether PTH1R is required for Wnt signaling to increase osteoprogenitor numbers by performing mass cytometry on bone cells of mice from Aim 1, using antibody panels to distinguish mesenchymal stem cells, osteoprogenitors and osteoblasts. We will simultaneously evaluate PTH1R and Wnt signaling in each population. We will use single-cell RNA-sequencing (scRNA-seq) to determine whether PTH1R is required for Wnt signaling to increase osteoblast gene programs in osteoprogenitors. In Aim 3 we will validate our findings in adult PTH1ROsxWT and PTH1ROsxKO mice treated with Wnt surrogate ligand, using µCT, histomorphometry, mass cytometry and scRNA-seq. Successful completion of these aims will provide more detailed understanding of the mechanisms by which PTH1R and Wnt cooperate to increase bone formation.
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0.97 |