Date of Completion

1-30-2017

Embargo Period

7-28-2017

Keywords

Fracture, Periosteum, PI3 kinase, mesenchymal stem cells, osterix, myeloid macrophages osteoclast precursors

Major Advisor

Archana Sanjay

Associate Advisor

Peter Maye

Associate Advisor

Barbara Kream

Associate Advisor

Blanka Rogina

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Campus Access

Abstract

The periosteum contains mesenchymal progenitors required for bone repair. The signaling pathways regulating periosteal response to fracture are largely unknown. Phosphatidylinositol-3 Kinase (PI3K) is a lipid kinase activated by various signaling pathways. Cbl, an E3 ubiquitin ligase and major adaptor protein, can modulate PI3K activity. Substitution of tyrosine 737 to phenylalanine (YF) in Cbl abolishes interaction between Cbl and PI3K. Previously, Dr. Sanjay’s lab showed that YF mice displayed increased bone volume under homeostatic conditions and formed a larger bony callus during fracture repair. The goal of this study was to further investigate the role of PI3K signaling upon initiation of fracture healing. Here we present evidence that absence of PI3K regulation by Cbl leads to robust periosteal thickening with increased proliferation of periosteal cells. YF periosteal progenitors also showed augmented osteogenic differentiation. Our studies revealed that increased stability and nuclear localization of Osterix observed in YF periosteal cells may explain this enhanced osteogenic differentiation since expression of Osterix transcriptional target genes is increased.

The impact of aberrant PI3K signaling on macrophages and osteoclast precursors (OCPs) in fractured periosteum is not well unexplored. Recent studies have highlighted important functions for macrophages and OCPs in regulating bone formation and osteogenic differentiation. Here we analyzed the effect of PI3K activity modulation by Cbl on macrophages and/or OCPs upon initiation of fracture healing. We found increased myeloid precursors in YF bone marrow (BM) and periosteum and observed an enhanced responsiveness to M-CSF signaling in BM macrophages (BMMs). We developed a novel technique for culturing periosteum derived macrophages (POMs) which may be utilized to better understand their interaction with periosteal osteoprogenitors. Histological analysis of fractured femurs revealed an increase in myeloid lineage cells in expanded YF periosteum upon bone injury. Further analysis of these cells is required to delineate the macrophage and OCP populations present.

Overall, our findings highlight a novel role for Cbl and PI3K in modulating the periosteal osteogenic and myeloid response during early stages of fracture repair. Detailed understanding of cellular and molecular mechanisms regulating skeletal repair is essential to identify targets and create new tools to improve bone repair.

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