Publication Date

2015-06-16

Availability

Open access

Embargo Period

2015-06-16

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2015-06-02

First Committee Member

Joshua M. Hare

Second Committee Member

Abigail S. Hackam

Third Committee Member

Herman Cheung

Fourth Committee Member

Justin M. Percival

Fifth Committee Member

Jonathan S. Stamler

Abstract

Osteogenesis and adipogenesis are two closely linked processes that share the same precursor, bone marrow mesenchymal stem cells (MSCs). MSCs are a rare population of non-hematopoietic stromal cells, present in bone marrow and almost every type of connective tissue. It is crucial to understand the signals that govern the specificity of MSC differentiation. Adipogenic transcription factor PPARγ can stimulate adipogenesis while inhibit osteogenesis. However, the role of potential regulators of PPARγ activity in this differentiation shift remains unknown. One potential regulator is nitric oxide (NO). NO enhances adipogenesis of human preadipocytes while decreasing cell proliferation. Studies in mice with a targeted deletion of NOS1, NOS2 or NOS3 manifest distinct bone effects. These observations suggest that NO may play an important role in regulating the balance between adipocyte and osteoblast differentiation. NO exerts its bioactivity not only by the production via nitric oxide synthases but also by the enzymatic degradation via S-nitrosylation. S-nitrosoglutathione (GSNO) exists in equilibrium with protein SNOs and can be degraded by the ubiquitously expressed GSNO reductase (GSNOR). By reducing the intracellular concentration of GSNO, GSNOR indirectly regulates SNOs and thus serves as a key denitrosylase. Impaired denitrosylation, as manifested in GSNOR–/– mice, alters multiple stem cell behaviors, including hematopoetic stem cell number and MSCs endothelial differentiation. We have obtained GSNOR-deficient (GSNOR–/–) mice that proves to be a powerful tool to modulate endogenous SNO levels. MSCs have been isolated and characterized from bone marrow of wild type mice and GSNOR–/– mice, which will allow us to check the MSCs behavior under different levels of NO and SNO. Our data reveal that GSNOR modulates PPARγ S-nitrosylation without affecting overall abundance of this transcription factor. Nitrosylated PPARγ has diminished transcriptional activity which is associated with reduced adipocyte differentiation and increased osteoblast formation. We further identified Cystein 139 as the target of PPARγ S-nitrosylation. Thus, S-nitrosylation participates in lineage bifurcation between adipocytes and osteoblasts. Further characterization of GSNOR–/– mice shows that they have lower body weight and bone density, with higher bone formation and bone resorption rate. Our results also suggest that GSNOR may contribute to parathyroid hormone regulation and calcium/phosphate urinary excretion, which in turn can affect the phenotype of GSNOR–/– mice. Together, these findings may provide mechanistic support for therapeutic strategies designed to offset disorders characterized by pathologic bone loss and/or excessive adipogenesis.

Keywords

Mesenchymal Stem Cells; S-nitrosylation; Differentiation

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