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Structural and Functional Analyses of the Shedding Protease ADAM17 in HoxB8-Immortalized Macrophages and Dendritic-like Cells.

ABSTRACT: A disintegrin and metalloproteinase (ADAM) 17 has been implicated in many shedding processes. Major substrates of ADAM17 are TNF-?, IL-6R, and ligands of the epidermal growth factor receptor. The essential role of the protease is emphasized by the fact that ADAM17 deficiency is lethal in mice. To study ADAM17 function in vivo, we generated viable hypomorphic ADAM17 mice called ADAM17ex/ex mice. Recent studies indicated regulation of proteolytic ADAM17 activity by cellular processes such as cytoplasmic phosphorylation and removal of the prodomain by furin cleavage. Maturation and thus activation of ADAM17 is not fully understood. So far, studies of ADAM17 maturation have been mainly limited to mouse embryonic fibroblasts or transfected cell lines relying on nonphysiologic stimuli such as phorbol esters, thus making interpretation of the results difficult in a physiologic context. In this article, we present a robust cell system to study ADAM17 maturation and function in primary cells of the immune system. To this end, HoxB8 conditionally immortalized macrophage precursor cell lines were derived from bone marrow of wild-type and hypomorphic ADAM17ex/ex mice, which are devoid of measurable ADAM17 activity. ADAM17 mutants were stably expressed in macrophage precursor cells, differentiated to macrophages under different growth factor conditions (M-CSF versus GM-CSF), and analyzed for cellular localization, proteolytic activity, and podosome disassembly. Our study reveals maturation and activity of ADAM17 in a more physiological-immune cell system. We show that this cell system can be further exploited for genetic modifications of ADAM17 and for studying its function in immune cells.

SUBMITTER: Cabron AS

PROVIDER: S-EPMC6215251 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Structural and Functional Analyses of the Shedding Protease ADAM17 in HoxB8-Immortalized Macrophages and Dendritic-like Cells.

Cabron Anne-Sophie AS El Azzouzi Karim K Boss Melanie M Arnold Philipp P Schwarz Jeanette J Rosas Marcela M Dobert Jan Philipp JP Pavlenko Egor E Schumacher Neele N Renné Thomas T Taylor Philip R PR Linder Stefan S Rose-John Stefan S Zunke Friederike F

Journal of immunology (Baltimore, Md. : 1950) 20181024 10

A disintegrin and metalloproteinase (ADAM) 17 has been implicated in many shedding processes. Major substrates of ADAM17 are TNF-α, IL-6R, and ligands of the epidermal growth factor receptor. The essential role of the protease is emphasized by the fact that ADAM17 deficiency is lethal in mice. To study ADAM17 function in vivo, we generated viable hypomorphic ADAM17 mice called ADAM17<sup>ex/ex</sup> mice. Recent studies indicated regulation of proteolytic ADAM17 activity by cellular processes su ...[more]

PMID: 30355783

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Project description:In vitro differentiation into functional osteoclasts is routinely achieved by incubation of embryonic stem cells, induced pluripotent stem cells, or primary as well as cryopreserved spleen and bone marrow-derived cells with soluble receptor activator of nuclear factor kappa-B ligand and macrophage colony-stimulating factor. Additionally, osteoclasts can be derived from co-cultures with osteoblasts or by direct administration of soluble receptor activator of nuclear factor kappa-B ligand to RAW 264.7 macrophage lineage cells. However, despite their benefits for osteoclast-associated research, these different methods have several drawbacks with respect to differentiation yields, time and animal consumption, storage life of progenitor cells or the limited potential for genetic manipulation of osteoclast precursors. In the present study, we therefore established a novel protocol for the differentiation of osteoclasts from murine ER-Hoxb8-immortalized myeloid stem cells. We isolated and immortalized bone marrow cells from wild type and genetically manipulated mouse lines, optimized protocols for osteoclast differentiation and compared these cells to osteoclasts derived from conventional sources. In vitro generated ER-Hoxb8 osteoclasts displayed typical osteoclast characteristics such as multi-nucleation, tartrate-resistant acid phosphatase staining of supernatants and cells, F-actin ring formation and bone resorption activity. Furthermore, the osteoclast differentiation time course was traced on a gene expression level. Increased expression of osteoclast-specific genes and decreased expression of stem cell marker genes during differentiation of osteoclasts from ER-Hoxb8-immortalized myeloid progenitor cells were detected by gene array and confirmed by semi-quantitative and quantitative RT-PCR approaches. In summary, we established a novel method for the quantitative production of murine bona fide osteoclasts from ER-Hoxb8 stem cells generated from wild type or genetically manipulated mouse lines. These cells represent a standardized and theoretically unlimited source for osteoclast-associated research projects.

Dataset Information

Structural and Functional Analyses of the Shedding Protease ADAM17 in HoxB8-Immortalized Macrophages and Dendritic-like Cells.

Publications

Structural and Functional Analyses of the Shedding Protease ADAM17 in HoxB8-Immortalized Macrophages and Dendritic-like Cells.

Similar Datasets

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