Studies On The Molecular Mechanisms And Biological Functions Of Cellular Internalization Of MRP14

Human MRP14 is a Ca²⁺-binding protein from the S100 family of proteins. Members of this family are characterized by a relatively low molecular weight and the presence of two calcium-binding sites of EF-hand type（helix-loop-helix calcium-binding domains）.Because of such structure which has calcium-binding sites,MRP14 are implicated in Ca²⁺-dependent regulation of a variety of intracellular activities such as protein phosphorylation,cell proliferation and differentiation,the dynamics of cytoskeleton constituents,and intracellular Ca²⁺ homeostasis.MRP14 is predominantly expressed in cells of the myelomonocytic lineage,used to form heterodimer with human MRP8 which another member of S100 family of proteins.The myeloid expression and its association with a number of inflammatory diseases led to the assumption that this molecule is involved in the body’s defense against inflammation.Indeed,the first cells,which infiltrate and dominate acute inflammatory lesions,have been shown to express S100A8 and S100A9.Phagocytes expressing S100A8 and S100A9 are found in a variety of inflammatory conditions,including rheumatoid arthritis,allograft rejections,and inflammatory bowel and lung diseases.In addition,inflammatory disorders,such as chronic bronchitis,cystic fibrosis,and rheumatoid arthritis,are associated with elevated plasma levels of S100A8/A9.These findings stimulated the research community to focus the efforts on the role of this protein in inflammation.Although MRP14 is generally co-expressed with MRP8,several studies demonstrated that in inflammatory diseases,MRP14 and MRP8 are independently expressed and exert different functions.Accordingly,we just focus on the roles of MRP14 in inflammation.Sepsis remains to be one of the chief causes of death in intensive care units at present,with the mortality is between 30～70%.Several therapeutic agents that target tumor necrosis factor（TNF） and interleukin-1（IL-1） have been tested in clinical trials of sepsis,and a significant survival advantage has not been observed.It has been found that MRP14 is actively secreted from monocytes and macrophages following stimulation with LPS.MRP14 can also be passively released from necrotic cells.Once released,MRP14 is able to activate many other cells including monocytes,macrophages,endothelial cells and epithelium to produce proinflammatory cytokine and adhesion molecules.MRP14 causes inflammatory responses in various systems in vivo,including brain,lung,gastrointestinal tract and heart,leading to the systemic inflammatory response even death.This implicates MRP14 plays a key role in inflammatory reactions.The signalling mechanisms by which MRP14 activates the cells are only partially understood.Some MRP14 receptors on mammalian cell membrane have been found,including receptor for advanced glycation end products（RAGE） and toll-like receptor-2/4（TLR-2/4）.However,blocking studies performed with all the receptor-blocking antibodies never completely prevented cellular activation by MRP14.It is thus likely that there are alternative MRP14-binding receptors or other pathways.Meanwhile,researchers found that more and more cytokines,growth factors or their receptors can be internalized by mammalian cells and this take part in signaling pathways of the cells.All these observations implicate that MRP14 may internalize into mammalian cells and reduce cell responses.Mammalian cells have evolved a variety of mechanisms to internalize small molecules,macromolecules and particles and target them to specific sealed organelles within the cytoplasm.Internalization serves many important cellular functions including the uptake of extracellular nutrients,regulation of cell-surface receptor expression and other cell functions.Endocytosis is one of the most important way by which macromolecules internalize.Endocytosis is a complex process that involves different pathways and a large network of protein-protein, protein-lipid and protein-glycan interactions.The first and best-characterized pathway is clathrin-dependent endocytosis,which starts on the plasma membrane with the formation of clathrin-coated invaginations that pinch off to make up clathrin-coated vesicles.Some molecules,including low density lipoproteins（LDL）, transferrin,epidermal growth factor（EGF） and insulin,make use of this endocytosis pathway.Less defined are the non-classical,clathrin-independent pathways,among which is caveolae-mediated endocytosis.Caveolae are flask-shaped,small invaginations with caveolin-1 in the plasma membrane that constitute a subclass of domains enriched in cholesterol and sphingolipids that are called lipid rafts. Caveolae are involved in signal transduction and the intracellular transportation. Several bacterial toxins,including cholera toxin（CTx）,and some viruses,including simian virus 40（SV40）,make use of caveolae-mediated endocytosis to enter the cells.Heparan sulfate proteoglycan（HSPG） is a particular class of proteoglycans expressed by most of the mammalian cells and is found on cell surfaces and in the extracellular matrix.It is formed of the core protein and the heparan sulfate（HS） side chains.The unique structural feature endows it with properties that may influence a wide range of biological processes such as embryonic development and tissue repair.These years it was also found to play an important role in the internalization of macromolecules.In this research,the recombinant MRP14 protein fused with enhanced green fluorescent protein（EGFP） was expressed and purified,and the internalization by microscopic analysis of cells treated with the protein was assessed firstly.Then to study the molecular pathway involved in the internalization of extracellular MRP14, a series of co-localization experiments using molecular fluorescent probes and specific drugs were performed.After this,EGFP fusion proteins with the different domains of MRP14 were constructed to study which part of MRP14 can internalize independently.At last,we revealed the relationship of internalization and proinflammationary effect of MRP14 by detecting the production of cytokines stimulated by MRP14.Through these studies,we draw the following conclusions.Firstly,the internalization of MRP14 by mammalian cells is a time- and energy-dependent process,which begins at 15 min after treatment with the protein and stabilizes at 1 h. Secondly,MRP14 is internalized through caveolae-mediated endocytosis by interact with cell surface HSPG firstly.The caveosomes transport in cytoplasm is related to cell cytoskeleton and enter into proteinases to degradate finally.Thirdly,the internalization of MRP14 were accomplished by synergy of two calcium binding motifs with the existence of calcium.Finally,the internalization and proinflammatory reaction of MRP14 may be accomplished by two absolutely different pathways,internalization of MRP14 is in order to degradation,accordingly inhibits inflammatory reaction to regulate internal environment homeostasis.MRP14 is an important proinflammatory cytokine released into the extracellular milieu while its signalling mechanisms are incompletely understood. The insights from our and others’ studies on MRP14 may widen the therapeutic window for endotoxemia,septic shock and sepsis if this newly recognized cytokine proves to be a clinically accessible target.