Structural and functional analyses of the shedding protease ADAM17 in HoxB8-Immortalized macrophages and dendritic-like cells

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

A disordered protein domain involved in morphogenesis and cell cycle progression.

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Podosomes in space

International audienceMigration of macrophages is a key process for a variety of physiological functions, such as pathogen clearance or tissue homeostasis. However, it can also be part of pathological scenarios, as in the case of tumor-associated macrophages. This review presents an overview of the different migration modes macrophages can adopt, depending on the physical and chemical properties of specific environments, and the constraints they impose upon cells. We discuss the importance of these environmental and also of cellular parameters, as well as their relative impact on macrophage migration and on the formation of matrix-lytic podosomes in 2D and 3D. Moreover, we present an overview of routinely used and also newly developed assays for the study of macrophage migration in both 2D and 3D contexts, their respective advantages and limitations, and also their potential to reliably mimic in vivo situations

Knr4 N-terminal domain controls its localization and function during sexual differentiation and vegetative growth

International audienceThe Saccharomyces cerevisiae protein Knr4 is composed of a globular central core flanked by two natively disordered regions. Although the central part of the protein holds most of its biological function, the N-terminal domain (amino acids 1-80) is essential in the absence of a functional CWI pathway. We show that this specific protein domain is required for the proper cellular localization of Knr4 at sites of polarized growth during vegetative growth and sexual differentiation (bud tip and 'shmoo' tip). Moreover, Knr4 N-terminal domain is also necessary for cell cycle arrest and shmoo formation in response to pheromone to occur at the correct speed. Thus, the presence of Knr4 at the incipient mating projection site seems important for the establishment of the following polarized growth. Cell wall integrity (CWI) and calcineurin pathways are known to share a common essential function, for which they can substitute for one another. Searching for Knr4 partners responsible for survival in a CWI-defective background, we found that the catalytic subunit of calcineurin Cna1 physically interacts with Knr4 in the yeast two-hybrid assay, in a manner dependent on the presence of the Knr4 N-terminal domain. In addition, we present evidence that Knr4 protein participates in the morphogenesis checkpoint, a safety mechanism that holds the cell cycle in response to bud formation defects or insults in cytoskeleton organization, and in which both the CWI pathway and calcineurin are involved

The subclassification of papillary renal cell carcinoma does not affect oncological outcomes after nephron sparing surgery

International audienceObjectives To evaluate the oncological outcomes of papillary renal cell carcinoma (pRCC) following nephron sparing surgery (NSS) and to determine whether the subclassification type of pRCC could be a prognostic factor for recurrence, progression, and specific death. Materials and methods An international multicentre retrospective study involving 19 institutions and the French network for research on kidney cancer was conducted after IRB approval. We analyzed data of all patients with pRCC who were treated by NSS between 2004 and 2014. Results We included 486 patients. Tumors were type 1 pRCC in 369 (76 %) cases and type 2 pRCC in 117 (24 %) cases. After a mean follow-up of 35 (1–120) months, 8 (1.6 %) patients experienced a local recurrence, 12 (1.5 %) had a metastatic progression, 24 (4.9 %) died, and 7 (1.4 %) died from cancer. Patients with type I pRCC had more grade II (66.3 vs. 46.1 %; p \textless 0.001) and less grade III (20 vs. 41 %; p \textless 0.001) tumors. Three-year estimated cancer-free survival (CFS) rate for type 1 pRCC was 96.5 % and for type 2 pRCC was 95.1 % (p = 0.894), respectively. Three-year estimated cancer-specific survival rate for type 1 pRCC was 98.4 % and for type 2 pRCC was 97.3 % (p = 0.947), respectively. Tumor stage superior to pT1 was the only prognostic factor for CFS (HR 3.5; p = 0.03). Conclusion Histological subtyping of pRCC has no impact on oncologic outcomes after nephron sparing surgery. In this selected population of pRCC tumors, we found that tumor stage is the only prognostic factor for cancer-free surviva