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Osteocytes Control Myeloid Cell Proliferation and Differentiation Through Gsα-Dependent and -Independent Mechanisms
Başlık:
Osteocytes Control Myeloid Cell Proliferation and Differentiation Through Gsα-Dependent and -Independent Mechanisms
Yazar:
Azab, Ehab, author.
ISBN:
9780438128668
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (140 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 79-11(E), Section: B.
Advisors: Paola Divieti Pajevic Committee members: Manish Bais; Paola Divieti Pajevic; Makoto Senoo; Philip Trackman.
Özet:
Introduction: Previous studies have shown that osteocytes, the matrix-embedded cells in bone, control bone modeling and remodeling through direct contact with adjacent cells and via secreted factors that can reach cells in the bone marrow microenvironment (BMM). Osteocytes express several receptors including G protein-coupled receptors (GPCRs) and mice lacking the stimulatory subunit of G-proteins (Gsalpha) in osteocytes have abnormal myelopoiesis, skeletal abnormalities and reduced adipose tissue. This study aimed at evaluating the effects of osteocyte-secreted factors on myeloid cell proliferation and differentiation in vitro. To investigate cross-talk between osteocytes and the BMM, we established osteocytic cell lines lacking Gsalpha expression to study the molecular mechanisms by which osteocytes control myeloid cell proliferation and differentiation.
Methods: CRISPR/Cas9 was used to knockout Gsalpha in the osteocytic cell line Ocy454. Conditioned media (CM) from differentiated Ocy-Gsalpha Ctrl and Ocy-GsalphaKO cells were used to treat myeloid cells and bone marrow mononuclear cells (BMNCs) isolated from long bones of 6-8-week-old C57/BL6 mice. BMNCs were cultured with Macrophage Colony Stimulating Factor (M-CSF), Receptor Activator of Nuclear Factor Kappa beta Ligand (RANKL) to induce osteoclast differentiation. Proliferation, TRAP staining, TRAP activity, resorption pit assay, F-actin ring formation and mRNA expression were used to evaluate cell proliferation, differentiation and function of the induced osteoclasts. Proteomics analysis of CM was performed to identify osteocyte-secreted factors capable of controlling myelopoiesis and osteoclastogenesis.
Results: Myeloid cells treated with CM from Ocy-Gsalpha KO showed a significant increase in cell proliferation compared to Ocy-GsalphaCtrl CM and non-treated control. BMNCs treated with CM from Ocy-GsalphaCtrl and Ocy-GsalphaKO showed a significant increase in cell proliferation as compared to non-treated control. Osteoclast differentiation was significantly suppressed by CM from Ocy-GsalphaCtrl and further suppressed by CM from Ocy-Gsalpha KO compared to non-treated control. Osteoclasts exposed to CM from Ocy-GsalphaKO showed a significant defect in activity and function as compared to cells exposed to CM from Ocy-GsalphaCtrl and non-treated cells. Osteoclast apoptosis was significantly enhanced by Ocy-GsalphaCtrl and Ocy-GsalphaKO CM compared to non-treated control. Among osteocyte secreted factors, we identified neuropilin-1 (NRP-1) as a Gsalpha-dependent osteocytic factor capable of suppressing osteoclastogenesis. CM from Ocy-GsalphaKO in which M-CSF was reduced by shRNA demonstrated decrease in BMNCs proliferation, demonstrating that osteocytes are also important sources of this cytokine.
Conclusions: Osteocytes produce several Gsalpha-dependent and -independent secreted factors capable of supporting myelopoiesis, promoting macrophage proliferation and suppressing osteoclast formation. We identified osteocyte-derived NRP-1 as a novel factor capable of decreasing osteoclastogenesis. In addition, we found that M-CSF secreted by osteocytes is responsible in part for BMNC proliferation. Future studies should focus on determining the role of osteocyte-mediated NRP-1 and other secreted factor(s) in control of myelopoiesis and osteoclastogenesis.
Notlar:
School code: 0017
Tüzel Kişi Ek Girişi:
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(696017.1) | 696017-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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