| Human MRP8 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),one of which, located in the MRP8 C-terminal half with a rather high affinity. Calcium binding results in an exposure of hydrophobic domains to the surface of the protein, which may alleviate interaction with target proteins, plays a role in biological activities. MRP8 is originally discovered as immunogenic protein expressed and secreted by neutrophils. Subsequently, it has emerged as important pro-inflammatory mediator in acute and chronic inflammation, thus contributing substantially to the recruitment of monocytes to inflammatory sites.Altogether, elevated S100A8 and S100A9 protein levels are a hallmark of numerous pathological conditions associated with inflammation.Endotoxin, or lipopolysaccharide (LPS), is an effective trigger of the inflammatory response during infection with Gram-negative bacteria. This bacterial product induces specific responses in cells of the immune system, especially in phagocytes. Uncontrolled activation of LPS-induced mechanisms results in sepsis or systemic inflammatory response syndrome, the latter of which may also have a noninfectious etiology. Despite enormous efforts in the development of antimicrobial therapies,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 MRP8 is actively secreted from monocytes and macrophages following stimulation with LPS. MRP8 can also be passively released from necrotic cells. Once released, MRP8 is able to activate many other cells including monocytes, macrophages, endothelial cells and epithelium to produce proinflammatory cytokine and adhesion molecules. MRP8 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 MRP8 plays a key role in inflammatory reactions.Although, many possible functions have been proposed for MRP8, its biological role still remains to be defined. Altogether, its expression and potential cytokine-like function in inflammation and in cancer suggests that MRP8 may play a key role in inflammation-associated cancer. In vitro studies demonstrated interaction of MRP8 with heparan sulfate proteoglycans and carboxylated glycans. Moreover,the molecules CD36 and RAGE (receptor of advanced glycation end products) are two other putative receptors for MRP8. However, blocking studies performed with all the receptor-blocking antibodies never completely prevented cellular activation by MRP8. It is thus likely that there are alternative MRP8-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 MRP8 may internalize into mammalian cells and reduce cell responses.Endocytosis is a complex and efficient process that cells utilize to take up nutrients and communicate with other cells. Endocytic vesicle formation is a complex process that couples sequential protein recruitment and lipid modifications with dramatic shape transformations of the plasma membrane. Endocytosis is required not only for uptake of nutrients, but also to regulate the amount of cell surface receptors including signalling receptors, adhesion molecules and GPI-linked receptors. Cargoes use different procedures to enter the cell. In clathrin-mediated endocytosis, various receptors, such as those for transferrin (TfnR), epidermal growth factor (EGFR), and low-density lipoprotein (LDLR), are enriched through interactions with AP-2 and other adaptors. In caveolae, certain receptors are accumulated in cholesterol-enriched microdomains into which caveolin is inserted. 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. Clathrin-independent carriers, which among other molecules incorporate GPI-linked receptors and glycolipids, may be of several kinds and are characterised by their pleiomorphic structures and by their absence of a rigid coat. Heparan sulfate proteoglycan is a major component of the extracellular matrix, and is essential for the self-assembly, insolubility and barrier properties of basement membranes. 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.For observation, the recombinant MRP8 protein fused with enhanced green fluorescent protein (EGFP) was expressed and purified. For electron microscopy analyses, the cells were treated with protein to examine the internalization process of MRP8. Then to study the internalization mechanism of extracellular MRP8, a series of co-localization experiments using molecular fluorescent probes and specific drugs were performed. To futher characterize the internalization domain, we construct different domains of MRP8 to determine which part can internalize independently. At last, we proposed the relationship between internalization and proinflammationary effect of MRP8 by detecting the production of cytokines stimulated by MRP8.In this study, we draw the following conclusions. Firstly, the internalization of MRP8 by mammalian cells is a time-and energy-dependent process, which begins at 15 min after treatment with the protein, stabilization at 1 h and degradation at 9h. Secondly, MRP8 is internalized through caveolae-mediated endocytosis by interacting with cell membrane surface HSPG firstly. The caveosomes transport in cytoplasm is related to cytoskeleton. Thirdly, the internalization of MRP8 was accomplished by synergy of two calcium binding motifs at the present of calcium. Fourthly, MRP8 ubiqutinated proteins are targeted to the proteasome for degradation. Moreover, polyubiquitination of at least four ubiqutin moieties via K92 linkage mainly leads to degradation of MRP8 in proteasome. Finally, the internalization and proinflammatory effect of MRP8 may be accomplished by two absolutely different pathways, internalization of MRP8 is in order to degradation, accordingly inhibits inflammatory reaction to regulate internal environment homeostasis.MRP8 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 MRP8 may widen the therapeutic window for endotoxemia, septic shock and sepsis if this newly recognized cytokine proves to be a clinically accessible target. MRP8 is already useful in a clinical setting and we suggest that other members of the S100 family will emerge as important biomarkers in the future and may provide new therapeutic opportunities. |
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