Mechanochemical Regulation of Cell Adhesion by Incorporation of Synthetic Polymers to Plasma Membranes.

Chemical control of cell-cell interactions using synthetic materials is useful for a wide range of biomedical applications. Herein, we report a method to regulate cell adhesion and dispersion by introducing repulsive forces to live cell membranes. To induce repulsion, we tethered amphiphilic polymers, such as cholesterol-modified poly(ethylene glycol) (PEG-CLS), to cell membranes. We found that the repulsive forces introduced by these tethered polymers induced cell detachment from a substrate and allowed cell dispersion in a suspension, modulated the speed of cell migration, and improved the separation of cells from tissues. Our analyses showed that coating the cells with tethered polymers most likely generated two distinct repulsive forces, lateral tension and steric repulsion, on the surface, which were tuned by altering the polymer size and density. We modeled how these two forces are generated in kinetically distinctive manners to explain the various responses of cells to the coating. Collectively, our observations demonstrate mechanochemical regulation of cell adhesion and dispersion by simply adding polymers to cells without genetic manipulation or chemical synthesis in the cells, which may contribute to the optimization of chemical coating strategies to regulate various types of cell-cell interacting systems.

A plasma protein indistinguishable from ribosomal protein S19: conversion to a monocyte chemotactic factor by a factor XIIIa-catalyzed reaction on activated platelet membrane phosphatidylserine in association with blood coagulation.

A monocyte-chemoattracting factor is generated during blood coagulation and during clotting of platelet-rich plasma. This chemotactic factor attracts monocytes as a ligand of the C5a receptor; however, it inhibits C5a-induced neutrophil chemotaxis as an apparent receptor antagonist. The curious dual function of the serum monocyte chemotactic factor resembles that of the cross-linked homodimer of ribosomal protein S19 (RP S19). Indeed, the inactive precursor of the monocyte chemotactic factor was present in plasma, and the precursor molecule and RP S19, as well as the active form and the RP S19 dimer, were indistinguishable in terms of immunological reactivity and molecular size. Coagulation factor XIIIa, plasma transglutaminase, and membrane phosphatidylserine on the activated platelets were required for conversion of the precursor to the active form. In addition, the precursor molecule in plasma could be replaced by wild-type recombinant RP S19 but not by mutant forms of it. These results indicate that a molecule indistinguishable from RP S19 was present in plasma, and that the RP S19-like molecule was converted to the active form by a transglutaminase-catalyzed reaction on a scaffold that included the phosphatidylserine-exposed platelet membrane.

Role of ribosomal protein S19-like plasma protein in blood coagulum resorption.

Western blot analyses and monocyte chemoattraction analyses of guinea pig plasma and serum indicated the presence of a plasma protein indistinguishable from ribosomal protein S19 and the cross-linked dimerization of it gaining monocyte chemotactic capacity in association with blood coagulation as in the case of human. When coagula preformed in vitro were intraperitoneally inserted into guinea pigs, they were rapidly covered by macrophages within 24h concomitant with an intra-coagulum macrophage infiltration. Differences were observed between the surface macrophages and the penetrating macrophages in ultrastructural, histochemical and immunohistochemical analyses. The inserted coagula were resorbed by day 7. When either anti-RP S19 antibodies or Gln137Asn-RP S19, a competitive inhibitor against RP S19, was premixed into the inserted coagulum, the attachment and penetration by macrophages decreased and the coagulum resorption retarded. These results indicate the role of the plasma RP S19-like molecule in coagulum resorption via macrophage recruitment.

Pro- and anti-apoptotic dual functions of the C5a receptor: involvement of regulator of G protein signaling 3 and extracellular signal-regulated kinase.

When apoptosis is initiated by manganese (II) loading, hyperthermia or thapsigargin treatment, human HL-60 and AsPC-1 cells initiate de novo synthesis of the C5a receptor (C5aR) and generation of its ligand, the ribosomal protein S19 (RP S19) homodimer. The ligand-receptor interaction, in an autocrine/paracrine fashion, promotes apoptosis, which can be bypassed by exogenous administration of C5a, another ligand. The proapoptotic function of the RP S19 dimer is reproduced by a C5a/RPS19 chimera that contains the body of C5a and the C-terminal region (Ile134-His145) of RP S19. The RP S19 dimer or C5a/RPS19 and C5a inversely regulate the expression of Regulator of G protein Signaling 3 (RGS3) gene in the apoptosis-initiated cells. Namely, the RP S19-type proteins upregulate RGS3 expression, whereas the C5a reduce it. Transformation of HL-60 cells to overexpress RGS3 promotes apoptosis in association with the downregulation of the Extracellular signal-Regulated Kinase (ERK) signal, and vice versa in the RGS3 knocked-down cells. Consistent with this result, an inhibitor of ERK phosphorylation effectively enhances the apoptotic rate in wild-type HL-60 cells. Moreover, a dominant negative effect on the RP S19 dimer production encourages apoptosis-initiated HL-60 cells with a longer lifespan in mouse than the natural effect. Our data indicate that, in apoptosis-initiated cells, the ligand-dependent C5aR-mediated dual signal affects the fate of cells, either apoptosis execution or survival, through regulation of RGS3 gene expression and subsequent modulation of ERK signal.

Agonistic and antagonistic effects of C5a-chimera bearing S19 ribosomal protein tail portion on the C5a receptor of monocytes and neutrophils, respectively.

C-terminus of S19 ribosomal protein (RP S19) endows the cross-linked homodimer with a dual effect on the C5a receptor in leucocyte chemoattraction; agonistic effect on the monocyte receptor, and antagonistic effect on the neutrophil receptor. C5a exhibits the uniform agonistic effect on this receptor of both cell types. We have currently prepared a recombinant C5a-chimeric protein bearing the C-terminus of RP S19 (C5a/RP S19 chimera) to be used as a substitute of the RP S19 dimer. In vitro, this chimera similarly inhibited the intracellular Ca(2+) mobilization of neutrophils induced by C5a to the RP S19 dimer did. In the guinea pig skin, 10(-7) M C5a/RP S19 chimera exhibited an inhibitory capacity to the neutrophil infiltration induced by 3 x 10(-7) M C5a without enhancing monocyte infiltration. In reverse passive Arthus reaction, the neutrophil infiltration associated with plasma extravasation was significantly reduced by the simultaneous administration of 10(-7) M C5a/RP S19 chimera with antibodies. The C5a/RP S19 chimera is a useful tool not only to examine the molecular mechanism that underlies the functional difference of the C5a receptor between monocytes and neutrophils, but also to prevent C5a-mediated hyper-response of neutrophils in acute inflammation.

Roles of the ribosomal protein S19 dimer and chemically induced apoptotic cells as a tumor vaccine in syngeneic mouse transplantation models.

We examined the roles of a chemotherapeutic reagent in both inducing apoptosis and conferring acquired tumor immunity using mouse syngeneic tumor transplantation models. A chemotherapeutic reagent, cis-diamminedichloroplatinum (II), effectively induced apoptosis in Colon-26 cells originating from a BALB/c mouse. Three intradermal inoculations of cis-diamminedichloroplatinum (II)-treated Colon-26 cells conferred tumor immunity against viable Colon-26 cells in BALB/c mice but not in athymic BALB/c mice. The acquired immunity was Colon-26 cell specific and was adoptively transferable with splenic cells. We next examined the involvement of the cross-linked ribosomal protein S19 dimer (RP S19), which is released during apoptosis, in the acquisition of tumor immunity. A Colon-26 transformant that produces a Gln137Asn-mutant RP S19 prevents the formation of a functional RP S19 dimer. Acquired tumor immunity was significantly reduced when the transformant was used in combination with anti-RP S19 antibodies as the vaccination source. These data suggest the importance of the RP S19 dimer in chemotherapeutic agent-induced acquired immunity.

Base of molecular mimicry between human ribosomal protein S19 dimer and human C5a anaphylatoxin.

The crosslinked homodimer of human ribosomal protein S19 (hRP S19) but not hRP S19 monomer shares the hC5a receptor ligation capacity with anaphylatoxin hC5a. The hRP S19 dimer engages hC5a receptor-bearing monocytes in chemotactic movement and secretion as does hC5a. Two submolecular regions essential for the receptor ligation were already identified in hRP S19 as well as in hC5a. Using the tertiary structure data base of an archaeobacterial RP S19 as template, we made a tertiary structure model of hRP S19. The obtained structure was almost entirely α-helical with two short ß-sheet regions, and folds a five α-helix bundle organized around a central amphipathic α-helix. While the secondary structure components were similar to those of hC5a, the gross tertiary structure of hRP S19 was loose and the distance between the two receptor binding regions was rather big in comparison to that of hC5a. Anti-recombinant hC5a rabbit antibodies cross-recognized not only the crosslinked hRP S19 dimer but also the guinea pig (gp) RP S19 dimer, however, these antibodies reacted hRP S19 monomer and crosslinked Gln137Asn-hRP S19 mutant dimer at significantly less extents. These antibodies neutralized the monocyte attracting capacity of the hRP S19 dimer in vitro and that of the gpRP S19 dimer in vivo. We assume that the crosslinkage between Lys122 of one hRP S19 molecule and Gln137 of the other one would assemble the hC5a-like structure probably providing one of two receptor binding regions by each hRP S19 subunit.