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Molecular Mechanisms underlying the link between the vitamin K-dependent proteins, Gas6 and protein-S, and phagocytosis of apoptotic cells during cell differentiation processes

Final Report Summary - PHAGOCYTOSIS (Molecular mechanisms underlying the link between the vitamin K-dependent proteins, Gas6 and protein-S, and phagocytosis of apoptotic cells ...)

Vitamin K-dependent factors are a family of mainly secreted proteins whose secretion and activity depends on vitamin K. Warfarin is a vitamin K antagonist which inhibits the activity of vitamin K-dependent proteins. Since most of vitamin K-dependent proteins belong to the blood coagulation system this property of Warfarin has led to its wide use in anticoagulant therapy known also as AVK. Among the vitamin K-dependent proteins, the anti-coagulant factor protein-S and its structural homolog Gas6 have several unique features. Protein-S and Gas6, are produced in the liver but also in the retina, brain, and testis. The anti-coagulant factor protein-S and its structural homologue Gas6 are ligands for the Tyro3/Axl/Mer family of related tyrosine kinase receptors (also termed TAM for Tyro3, Axl and Mer).

Vitamin K-dependent proteins have been mainly associated with blood coagulation disorders, however, published reports from several laboratories including ours had led us to hypothesise that protein-S and Gas6 as well as other vitamin K-dependent proteins regulate apoptotic cell phagocytosis. Defects in phagocytosis lead to cell differentiation-related pathologies which are to date hardly characterised and poorly understood. The major aims of the research project PHAGOCYTOSIS was to assess in what extent substrate binding, ingestion and degradation stages during non-professional phagocytosis may be tissue-specific, how the phagocytosis process may be regulated by vitamin K-dependent proteins and which are the signalling pathways that regulate this process.

During the Marie Curie Fellowship, Dr Marina Yefimova established and standardised a chimerical phagocytosis system where testicular Sertoli cell primary cultures were exposed to either tissue specific substrates (testis: spermatid residual bodies) or tissue none-specific substrates (retina: shed photoreceptor tips) and analysed under these conditions the course of main phagocytosis stages. Our study establishes that substrate binding and ingestion phagocytosis stages occur similarly whether Sertoli cell cultures are exposed to apoptotic substrates derived from the testis or the retina. However, for the degradation of ingested retina-derived substrates, in contrast to that of ingested testis-derived substrates, Sertoli cell cultures mobilise their autophagic machinery, which is known as an autonomous pathway designed primarily for intracellular protein and organelle degradation.

We demonstrate that testicular Sertoli cells protrude pseudopods to ingest both testis and retina-derived substrates implying thereby the implication of phagocytosis type I towards both substrate types. Phagocytosis by Sertoli cells of both testis- and retina- derived substrates is activated by the vitamin K-dependent factor, protein S, is associated with the phosphorylation of its tyrosine kinase receptor (Mertk) and results in the redistribution of myosin II which co-localises with phosphorylated Mertk on the site of phagocytes-substrates contact suggesting thereby an association between phagocytosis substrates, activated phagocytosis-mediated receptor (Mertk) and a motor (non-muscle myosin II) to drive substrate ingestion by Sertoli cells.

Although the physiological relevance of the vitamin K-dependent proteins Gas6- versus protein S-mediated phagocytosis by Sertoli cells is not clear, the production of both Gas6 and protein S by the testis and their seminiferous epithelium cycle stage-dependent expression suggests that Gas6 and protein S could both regulate Mertk-dependent phagocytosis by Sertoli cell. Our study establishes that on morphological and molecular levels, the recognition and the ingestion phagocytosis steps of phosphatidylserine-exposing substrates by Sertoli cells follow a similar pattern for both tissue specific and tissue non-specific substrates. However, retina but not testis ingested by Sertoli cells, were found inside double-membrane limited vacuoles, which further mature into single-membrane-limited autolysosome. In addition, we found a co-localisation of the autophagic membrane marker LC3 and rhodopsin in these vacuoles.

Altogether, our study underlines that the nature of phagocytosis substrates is a major factor that should be taken into account for the study of phagocytosis machinery by non-professional phagocytes. Therefore, the conclusions drawn from previous studies should be reconsidered on the light of our findings. Moreover, our electron microscopy observations revealed that activation of autophagy following Sertoli cell cultures exposure to ROS did not lead to Sertoli cell death, suggesting thereby that it rather represents a cell survival mechanism similar to that triggered following Sertoli cell exposure to toxic substrates.

The overlap between phagocytosis and autophagy we discovered is a novel property that may be of major importance for a better understanding of fundamentals of apoptotic substrates clearance in healthy and diseased states. The research that was conducted during the Marie Curie fellowship is of major importance in elucidating the role of the vitamin K-dependent factor, protein S, in regulation phagocytosis of apoptotic cells. Dr Marina Yefimova research work on phagocytosis will hopefully lead, in few years, to the development of novel therapies aiming at an effective targeting of pathologies associated with a defect of phagocytosis. A defect in phagocytosis is associated with retina degeneration and blindness, neurodegenerative diseases and infertility which are major health problem in Europe. These health problems are worsening as the European population is ageing. Additionally, as the population is ageing, the use of anti-coagulant (anti-vitamin K also termed AVK) is rapidly increasing.

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