G PROTEIN-COUPLED RECEPTOR KINASE 3 (GRK3) REGULATES G PROTEIN-COUPLED RECEPTORS ON MURINE BONE MARROW NICHE MESENCHYMAL STEM CELLS AND HEMATOPOIETIC STEM-PROGENITOR CELLS

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  • March 20, 2019
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  • Brozowski, Jaime
    • Affiliation: School of Medicine, Department of Microbiology and Immunology
Abstract
  • The bone marrow microenvironment, termed niche, supports hematopoietic cell development and thus, is vital for establishment of the immune system. Within the niche reside bone marrow mesenchymal stem cells (BmMSCs) that surround the hematopoietic stem-progenitor cells (HSPCs) to support their development, maintenance, and function; however, the intracellular regulatory mechanisms of BmMSCs and HSPCs are still being defined. The goal of this dissertation work is to provide further insight into the regulatory mechanisms that modulate functionality of BmMSCs and HSPCs. Our data suggest G protein-coupled receptor kinase 3 (GRK3) functions as a negative regulator of G protein-coupled receptors (GPCRs) on BmMSCs and HSPCs. BmMSCs isolated from GRK3-deficient (Grk3-/-) mice have enhanced proliferation and osteogenic differentiation ex vivo compared to wildtype (WT) BmMSCs. Grk3-/- BmMSC cultures also have higher levels of CXCL12, an essential chemokine for HSPC development, and interestingly, Grk3-/- mice have increased hematopoietic cell numbers isolated from the bone marrow. Both Grk3-/- BmMSC proliferation and osteogenic differentiation were reduced to WT level upon reduction of sphingosine-1-phosphate (S1P), and Grk3-/- BmMSCs have sustained ERK1/2 signaling upon stimulation of sphingosine-1-phosphate receptor (S1PR) with S1P in comparison to WT BmMSCs. In addition, we report GRK3 recruits β-arrestin, a protein necessary for receptor internalization, to the C-terminus of S1PR1, and we demonstrate BmMSCs lacking GRK3 regulation have impaired S1PR1 internalization. Our findings suggest GRK3 regulates GPCR S1PR on BmMSCs. Grk3-/- mice have increased bone marrow lineage negative (Lin-) Sca1+ c-Kit+ (LSK) HSPC and oligopotent progenitor numbers, as well as increased total leukocytes in the peripheral blood. Since increased stem cell numbers and function potentiate cellular engraftment and hematopoiesis, we tested whether GRK3 deficiency enhances hematopoietic cell function in vivo after short-term transplantation, termed colony forming unit-spleen (CFU-S) assay. Transplanted Grk3-/- LSK HSPCs or Grk3-/- whole bone marrow increases colony counts on the explanted spleen in comparison to WT controls, suggesting hematopoiesis of Grk3-/- HSPC is enhanced. Further, both Grk3-/- hematopoietic myeloid granulocytic and monocytic (CFU-GM) and lymphoid (CFU-Pre-B) colony counts increased ex vivo upon CXCR4 ligand stimulation (CXCL12), and Grk3-/- myeloid colony counts reduced to WT levels with CXCR4 antagonist treatment (AMD3100). Taken together, in vivo and ex vivo CFU data suggest GRK3 regulates bone marrow HSPC numbers, and this is, at least in part, mediated through CXCL12/CXCR4 stimulation. Herein, we describe a newly elucidated pathway of regulation on two niche cells, BmMSCs and HSPCs. Specifically, our data suggest GRK3 functions as a negative regulator of GPCRs on both BmMSCs and HSPCs and can modulate stem cell function.
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  • Whitmire, Jason
  • Matsushima, Glenn
  • Caron, Kathleen
  • Tisch, Roland
  • Tarrant, Teresa
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2018
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