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J Immunol. 2018 Nov 15;201(10):3106-3118. doi: 10.4049/jimmunol.1701556. Epub 2018 Oct 24.

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

Author information

1
Institute of Biochemistry, Christian-Albrechts-University of Kiel, 24118 Kiel, Germany.
2
Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Eppendorf, 20246 Hamburg, Germany.
3
Institute of Anatomy, Christian-Albrechts-University of Kiel, 24118 Kiel, Germany.
4
Division of Infection and Immunity, Systems Immunity Research Institute, Cardiff University School of Medicine, Cardiff CF10 3AT, United Kingdom.
5
Department of Molecular Medicine and Surgery, Karolinska Institutet and University Hospital, Solna, SE-171 76 Stockholm, Sweden; and.
6
Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.
7
Institute of Biochemistry, Christian-Albrechts-University of Kiel, 24118 Kiel, Germany; rosejohn@biochem.uni-kiel.de fzunke@biochem.uni-kiel.de.

Abstract

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 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.

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