Phospholipid Scramblase 1 Modulates FcR-Mediated Phagocytosis in Differentiated Macrophages. Cecile Herate, Ghania Ramdani, Nancy Grant, Sabrina Marion, Stephane Gasman, Florence Niedergang, Serge Benichou, Jerome Bouchet To cite this version: Cecile Herate, Ghania Ramdani, Nancy Grant, Sabrina Marion, Stephane Gasman, et al.. Phospho- lipid Scramblase 1 Modulates FcR-Mediated Phagocytosis in Differentiated Macrophages.. PLoS ONE, Public Library of Science, 2016, 11 (1), pp.e0145617. 10.1371/journal.pone.0145617. hal-02922246 HAL Id: hal-02922246 https://hal.archives-ouvertes.fr/hal-02922246 Submitted on 25 Aug 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. RESEARCH ARTICLE Phospholipid Scramblase 1 Modulates FcR- Mediated Phagocytosis in Differentiated Macrophages Cecile Herate1,2,3, Ghania Ramdani1,2,3, Nancy J. Grant4, Sabrina Marion1,2,3, Stephane Gasman4, Florence Niedergang1,2,3, Serge Benichou1,2,3☯*, Jerome Bouchet1,2,3☯ 1 Inserm U1016, Institut Cochin, Paris, France, 2 CNRS, UMR8104, Paris, France, 3 Université Paris- Descartes, Sorbonne Paris-Cité, Paris, France, 4 Institut des Neurosciences Cellulaires et Intégratives, CNRS UPR3212, and Université de Strasbourg, Strasbourg, France ☯ These authors contributed equally to this work. * [email protected] Abstract Phospholipid Scramblase 1 (PLSCR1) was initially characterized as a type II transmem- brane protein involved in bilayer movements of phospholipids across the plasma membrane leading to the cell surface exposure of phosphatidylserine, but other cellular functions have been ascribed to this protein in signaling processes and in the nucleus. In the present study, OPEN ACCESS expression and functions of PLSCR1 were explored in specialized phagocytic cells of the monocyte/macrophage lineage. The expression of PLSCR1 was found to be markedly Citation: Herate C, Ramdani G, Grant NJ, Marion S, Gasman S, Niedergang F, et al. (2016) Phospholipid increased in monocyte-derived macrophages compared to undifferentiated primary mono- Scramblase 1 Modulates FcR-Mediated cytes. Surprisingly, this 3-fold increase in PLSCR1 expression correlated with an apparent Phagocytosis in Differentiated Macrophages. PLoS modification in the membrane topology of the protein at the cell surface of differentiated ONE 11(1): e0145617. doi:10.1371/journal. pone.0145617 macrophages. While depletion of PLSCR1 in the monocytic THP-1 cell-line with specific shRNA did not inhibit the constitutive cell surface exposure of phosphatidylserine observed Editor: Christophe Lamaze, Institut Curie, FRANCE in differentiated macrophages, a net increase in the FcR-mediated phagocytic activity was Received: October 16, 2015 measured in PLSCR1-depleted THP-1 cells and in bone marrow-derived macrophages Accepted: November 4, 2015 from PLSCR1 knock-out mice. Reciprocally, phagocytosis was down-regulated in cells Published: January 8, 2016 overexpressing PLSCR1. Since endogenous PLSCR1 was recruited both in phagocytic cups and in phagosomes, our results reveal a specific role for induced PLSCR1 expression Copyright: © 2016 Herate et al. This is an open access article distributed under the terms of the in the modulation of the phagocytic process in differentiated macrophages. Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction Data Availability Statement: All relevant data are Phospholipid scramblase 1 (PLSCR1) is a member of a protein family referenced as phospho- within the paper. lipid scramblases that are conserved in all eukaryotic organisms. In human, the scramblase Funding: This work was supported by Inserm, CNRS family is constituted of four known homologues named PLSCR1, 2, 3 and 4 [1]. As the most and Universite Paris-Descartes. studied member of the scramblase family, the 37 kD ubiquitous PLSCR1 protein has been Competing Interests: The authors have declared described as a type-II transmembrane protein comprised of a short 9 amino acid (aa)-long C- that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0145617 January 8, 2016 1/19 Phospholipid Scramblase 1 Function in Macrophages terminal extracellular domain (aa 310–318), a single transmembrane helix (aa 291–309) and a long intracytoplasmic N-terminal domain of 290 aa (aa 1–290), containing a cysteine-rich pal- mitoylation motif (C184CCPCC189) that could stabilize PLSCR1 anchoring in biological mem- branes [2–4]. PLSCR1 mutants with substitutions in this palmitoylation motif have been shown to localize in the nucleus where PLSCR1 can also carry out biological functions, such as transcriptional activity [5]. The main function ascribed to PLSCR1 has been related to its potential involvement in bidi- rectional and nonspecific movements of phospholipids between the inner and outer leaflets of the plasma membrane in response to intracellular calcium mobilization [6–8]. Scrambling of membrane phospholipids then leads to the cell surface exposure of phosphatidylserine (PS), a critical signal for biological processes such as cell activation, coagulation, apoptosis and secre- tion [9,10]. However, this specific role of PLSCR1 in regulating phospholipid movements within the plasma membrane has been recently challenged in several experimental systems (for reviews, [2,9]). While the exact involvement of PLSCR1 in the translocation of membrane phospholipids remains controversial, increasing evidence now indicates that this transmembrane protein could also be involved in cell signaling processes at the plasma membrane. Indeed, PLSCR1 is found in lipid rafts where it has been shown to interact directly with several plasma membrane receptors, including the epidermal growth factor receptor, the high-affinity IgE receptor FcεRI and the CD4 T-cell receptor [11–14]. In T lymphocytes, we have shown that both PLSCR1 and PLSCR4 are cellular receptors for the secretory leucocyte protease inhibitor (SLPI) and interact with CD4 at the plasma membrane [14]. In addition, PLSCR1 can also associate with cellular tyrosine kinases containing Src-homology 3 (SH3) domains, such as c-Abl [15] and Syk [16], and Src family kinases including Src and Lyn [13,16]. Association of PLSCR1 with these kinases is probably related to the multiple SH3-binding proline-rich motifs found in the long cytoplasmic domain of PLSCR1 (for review, [2]). However, the exact contributions of these interactions to specific functions of PLSCR1 are still poorly understood. To further characterize these functions, PLSCR1 expression was first examined in CD4-po- sitive myeloid and lymphoid cells, and PLSCR1 levels were found to be higher in monocytic cells than in T lymphocytes. We next analyzed the expression and potential functions of PLSCR1 in the professional phagocytic myeloid cells, monocytes and macrophages. We found that the level of PLSCR1 was markedly increased during differentiation of primary monocytes to macrophages, and more interestingly, PLSCR1 specifically modulated phagocytosis in differ- entiated macrophages. Materials and Methods Cell culture and differentiation Adherent HeLa cells were grown in Dulbecco minimal essential medium supplemented with 10% fetal calf serum (FCS), 100 IU of penicillin/ml, and 100 μg of streptomycin/ml (Invitro- gen). Human THP-1 monocytic and HPB-ALL T lymphoid cells have been already described [17]. THP-1 and HPB-ALL non-adherent cells were cultured in RPMI 1640 medium with Glu- tamax-1 (Invitrogen) supplemented with 10 mM HEPES, 10% FCS, 100 IU of penicillin/ml, and 0.1 mg streptomycin/ml (complete medium). For differentiation in macrophages, THP-1 cells were treated in complete medium, supplemented with 1 μM phorbol 12-myristate 13-ace- tate (PMA) (Sigma) alone or in combination with ionomycin where indicated, for the indicated time periods. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gra- dient centrifugation from whole blood donated by healthy volunteers (Etablissement Français du Sang, Hôpital Saint Vincent de Paul, Paris, France). The study has been approved by the PLOS ONE | DOI:10.1371/journal.pone.0145617 January 8, 2016 2/19 Phospholipid Scramblase 1 Function in Macrophages Ethic Committee from Inserm (Institut National de la Santé et de la Recherche Médicale, Paris, France), and subjects gave written, informed consent. PBMCs were incubated for 1 hour in a humidified atmosphere at 37°C with 5% CO2, and then washed twice in RPMI to keep only adherent cells. Monocytes were then differentiated into macrophages for 7 days in RPMI 1640 medium supplemented with 10% of human serum from the same donor. PLSCR-1 -/- mice were purchased from CDTA (Cryo-preservation, Distribution, Typage et Archivage animal), housed and raised at Chronobiotron UMS 3415. All mice were bred, handled, and maintained in agreement with European council directive 86/609/EEC and resulting French regulations. Mouse management and sample collection were conducted by trained personnel under the supervision of a veterinarian,