Pyong Woo Park

Matrilysin shedding of syndecan-1 regulates chemokine mobilization and transepithelial efflux of neutrophils in acute lung injury

The influx of inflammatory cells to sites of injury is largely directed by signals from the epithelium, but how these cells form chemotactic gradients is not known. In matrilysin null mice, neutrophils remained confined in the interstitium of injured lungs and did not advance into the alveolar space. Impaired transepithelial migration was accompanied by a lack of both shed syndecan-1, a heparan sulfate proteoglycan, and KC, a CXC chemokine, in the alveolar fluid. KC was bound to shed syndecan-1, and it was not detected in the lavage of syndecan-1 null mice. In vitro, matrilysin cleaved syndecan-1 from the surface of cells. Thus, matrilysin-mediated shedding of syndecan-1/KC complexes from the mucosal surface directs and confines neutrophil influx to sites of injury.

Cell Surface Heparan Sulfate Proteoglycans: Selective Regulators of Ligand-Receptor Encounters

Cell surface heparan sulfate proteoglycans (HSPGs), substantially more abundant than most receptors, modulate encounters of extracellular protein ligands with their receptors by forming HSprotein complexes. Two gene families account for most cell surface HSPGs. Both consist of discrete core proteins covalently attached to two or three chains of HS, an Nand O-sulfated linear polysaccharide of repeating disaccharides containing N-acetylglucosamine (GlcNAc) and uronic acid (glucuronic acid (GlcA) or iduronic acid (IdoUA)). The syndecan family was the first discovered, which in mammals contains four gene products with distinctive extracellular domains (ectodomains) and highly conserved short cytoplasmic domains. These apparently extended proteins place the HS chains distal from the plasma membrane (1, 2). The syndecan family contrasts with the glypican family, which in mammals contains six gene products that are covalently linked to plasma membrane lipid by glycosylphosphatidylinositol anchor (1, 3). The glypican core proteins contain six invariant disulfide bonds, are likely to be globular, and place HS chains adjacent to the plasma membrane. Expression of both the syndecans and glypicans is extensively regulated during mouse embryogenesis and results in discrete adult expression patterns for each HSPG such that every adherent cell exhibits a distinct repertoire of cell surface HSPGs. Binding to HS chains is remarkably widespread among extracellular proteins, especially matrix proteins, proteases and their inhibitors, lipases, lipoproteins, growth factors and their binding proteins, cytokines, chemokines, collectins, and antimicrobial peptides. These proteins are involved in morphogenesis, tissue repair, energy balance, and host defense (Fig. 1). Additionally, numerous pathogens (e.g. herpes simplex virus, Neisseria, Plasmodium) bind to the cell surface via HS (4). Importantly, many of these ligand-HS interactions are essentially identical from Drosophila to the mouse, including those involved in generation of the basic metazoan body plan, e.g. dpp (bone morphogenetic proteins 2–4), wg (Wnt-1), and sog (chordin). Formation of the complexes can enhance or reduce receptor activation, often depending on the concentrations of ligand, receptor, and HSPG. The HS chains catalyze encounters between ligand and signaling receptor by bringing them together. Because binding to the HS chain reduces the dimensionality of this interaction from three (when the ligand is soluble) to two (when the ligand is bound to the HS chain), interaction could result from a localized increase in ligand concentration at optimal HS concentrations (5). However, at HS levels lower or higher than optimal, the effective ligand concentration for engaging the receptor will fall, potentially accounting for the bell-shaped activity curve typically seen experimentally when HSPG (or heparin) concentrations are varied. The curve may be concave or convex depending on whether ligand binding to the HSPG is inhibitory or stimulatory (6). Furthermore, the cytoplasmic domains of the syndecans also form complexes with cytoplasmic enzymes and scaffolding proteins, adding to the modulating influence of these proteoglycans (7). The HS chains are structurally diverse by virtue of their biosynthesis. A non-sulfated repeating disaccharide precursor is generated while attached to a core protein and is then sequentially modified by a variety of enzymes in reactions that do not go to completion. The details of this biosynthetic scheme have been recently reviewed in this series (8). Three major characteristics of this scheme produce HS chains with selectivity for protein binding. First, the process yields an extraordinary variety of saccharide sequences. Second, clustering of the modifications along the HS chain yields highly N-sulfated domains (NS domains) of approximately 12–20 residues that alternate with typically larger sized, relatively unmodified N-acetyl-rich domains (NA domains). The NS domains are rich in IdoUA, which can assume several different conformations and thus influence the orientation of the sulfate residues in space. This domain organization places relatively flexible NA domains adjacent to relatively rigid NS domains, thus facilitating protein interactions with the sulfate residues. Finally, this microsequence diversity and macro-organization are cell typespecific and do not appear to be core protein-specific (e.g. HS chains of syndecan-1 and -4 from mammary epithelia are indistinguishable), presumably the result of cell type-specific repertoires of the HS chain-modifying enzymes. Distinct oligosaccharide sequences in HS chains are recognized by the various proteins whose function depends on this interaction. The best characterized of these interactions is the recognition of a specific pentasaccharide sequence by antithrombin III (9). FGF-2 binds most tightly to a specific hexasaccharide sequence, but an additional 4–6 sugar residues are required to activate the receptor (10). Specific oligosaccharide binding sequences are known for multiple ligands (11–14); however, there is no universal consensus amino acid sequence for protein binding to HS chains. Most studies suggest that multiple arginine and/or lysine residues aligned on the protein surface accommodate a distinctive array of anionic sites on the HS chain (14).

Plasma restoration of endothelial glycocalyx in a rodent model of hemorrhagic shock

BACKGROUND: The use of plasma-based resuscitation for trauma patients in hemorrhagic shock has been associated with a decrease in mortality. Although some have proposed a beneficial effect through replacement of coagulation proteins, the putative mechanisms of protection afforded by plasma are unknown. We have previously shown in a cell culture model that plasma decreases endothelial cell permeability in comparison with crystalloid. The endothelial glycocalyx consists of proteoglycans and glycoproteins attached to a syndecan backbone, which together protect the underlying endothelium. We hypothesize that endothelial cell protection by plasma is due, in part, to its restoration of the endothelial glycocalyx and preservation of syndecan-1 after hemorrhagic shock. METHODS: Rats were subjected to hemorrhagic shock to a mean arterial blood pressure of 30 mm Hg for 90 minutes followed by resuscitation with either lactated Ringers (LR) solution or fresh plasma to a mean arterial blood pressure of 80 mm Hg and compared with shams or shock alone. After 2 hours, lungs were harvested for syndecan mRNA, immunostained with antisyndecan-1, or stained with hematoxylin and eosin. To specifically examine the effect of plasma on the endothelium, we infused small bowel mesentery with a lanthanum-based solution, identified venules, and visualized the glycocalyx by electron microscopy. All data are presented as mean ± SEM. Results were analyzed by 1-way analysis of variance with Tukey post hoc tests. RESULTS: Electron microscopy revealed degradation of the glycocalyx after hemorrhagic shock, which was partially restored by plasma but not LR. Pulmonary syndecan-1 mRNA expression was higher in animals resuscitated with plasma (2.76 ± 0.03) in comparison with shock alone (1.39 ± 0.22) or LR (0.82 ± 0.03) and correlated with cell surface syndecan-1 immunostaining. Shock also resulted in significant lung injury by histopathology scoring (1.63 ± 0.26), which was mitigated by resuscitation with plasma (0.67 ± 0.17) but not LR (2.0 ± 0.25). CONCLUSION: The protective effects of plasma may be due in part to its ability to restore the endothelial glycocalyx and preserve syndecan-1 after hemorrhagic shock.

Molecular functions of syndecan-1 in disease.

Syndecan-1 is a cell surface heparan sulfate proteoglycan that binds to many mediators of disease pathogenesis. Through these molecular interactions, syndecan-1 can modulate leukocyte recruitment, cancer cell proliferation and invasion, angiogenesis, microbial attachment and entry, host defense mechanisms, and matrix remodeling. The significance of syndecan-1 interactions in disease is underscored by the striking pathological phenotypes seen in the syndecan-1 null mice when they are challenged with disease-instigating agents or conditions. This review discusses the key molecular functions of syndecan-1 in modulating the onset, progression, and resolution of inflammatory diseases, cancer, and infection.

Heparan sulfate and syndecan-1 are essential in maintaining murine and human intestinal epithelial barrier function

Patients with protein-losing enteropathy (PLE) fail to maintain intestinal epithelial barrier function and develop an excessive and potentially fatal efflux of plasma proteins. PLE occurs in ostensibly unrelated diseases, but emerging commonalities in clinical observations recently led us to identify key players in PLE pathogenesis. These include elevated IFN-gamma, TNF-alpha, venous hypertension, and the specific loss of heparan sulfate proteoglycans from the basolateral surface of intestinal epithelial cells during PLE episodes. Here we show that heparan sulfate and syndecan-1, the predominant intestinal epithelial heparan sulfate proteoglycan, are essential in maintaining intestinal epithelial barrier function. Heparan sulfate- or syndecan-1-deficient mice and mice with intestinal-specific loss of heparan sulfate had increased basal protein leakage and were far more susceptible to protein loss induced by combinations of IFN-gamma, TNF-alpha, and increased venous pressure. Similarly, knockdown of syndecan-1 in human epithelial cells resulted in increased basal and cytokine-induced protein leakage. Clinical application of heparin has been known to alleviate PLE in some patients but its unknown mechanism and severe side effects due to its anticoagulant activity limit its usefulness. We demonstrate here that non-anticoagulant 2,3-de-O-sulfated heparin could prevent intestinal protein leakage in syndecan-deficient mice, suggesting that this may be a safe and effective therapy for PLE patients.

Molecular and Cellular Mechanisms of Ectodomain Shedding

The extracellular domain of several membrane‐anchored proteins is released from the cell surface as soluble proteins through a regulated proteolytic mechanism called ectodomain shedding. Cells use ectodomain shedding to actively regulate the expression and function of surface molecules, and modulate a wide variety of cellular and physiological processes. Ectodomain shedding rapidly converts membrane‐associated proteins into soluble effectors and, at the same time, rapidly reduces the level of cell surface expression. For some proteins, ectodomain shedding is also a prerequisite for intramembrane proteolysis, which liberates the cytoplasmic domain of the affected molecule and associated signaling factors to regulate transcription. Ectodomain shedding is a process that is highly regulated by specific agonists, antagonists, and intracellular signaling pathways. Moreover, only about 2% of cell surface proteins are released from the surface by ectodomain shedding, indicating that cells selectively shed their protein ectodomains. This review will describe the molecular and cellular mechanisms of ectodomain shedding, and discuss its major functions in lung development and disease. Anat Rec, 293:925–937, 2010.

Activation of syndecan-1 ectodomain shedding by Staphylococcus aureus α-toxin and β-toxin

Exploitation of host components by microbes to promote their survival in the hostile host environment has been a recurring theme in recent years. Available data indicate that bacterial pathogens activate ectodomain shedding of host cell surface molecules to enhance their virulence. We reported previously that several major bacterial pathogens activate ectodomain shedding of syndecan-1, the major heparan sulfate proteoglycan of epithelial cells. Here we define the molecular basis of how Staphylococcus aureus activates syndecan-1 shedding. We screened mutant S. aureus strains devoid of various toxin and protease genes and found that only strains lacking both α-toxin and β-toxin genes do not stimulate shedding. Mutations in the agr global regulatory locus, which positively regulates expression of α- and β-toxins and other exoproteins, also abrogated the capacity to stimulate syndecan-1 shedding. Furthermore, purified S. aureus α- and β-toxins, but not enterotoxin A and toxic shock syndrome toxin-1, rapidly potentiated shedding in a concentration-dependent manner. These results establish that S. aureus activates syndecan-1 ectodomain shedding via its two virulence factors, α- and β-toxins. Toxin-activated shedding was also selectively inhibited by antagonists of the host cell shedding mechanism, indicating that α- and β-toxins shed syndecan-1 ectodomains through stimulation of the host cells shedding machinery. Interestingly, β-toxin, but not α-toxin, also enhanced ectodomain shedding of syndecan-4 and heparin-binding epidermal growth factor. Because shedding of these ectodomains has been implicated in promoting bacterial pathogenesis, activation of ectodomain shedding by α-toxin and β-toxin may be a previously unknown virulence mechanism of S. aureus.

Syndecan-1 shedding facilitates the resolution of neutrophilic inflammation by removing sequestered CXC chemokines

Heparan sulfate binds to and regulates many inflammatory mediators in vitro, suggesting that it serves an important role in directing the progression and outcome of inflammatory responses in vivo. Here, we evaluated the role of syndecan-1, a major heparan sulfate proteoglycan, in modulating multiorgan host injury responses in murine endotoxemia. The extent of systemic inflammation was similar between endotoxemic syndecan-1-null and wild-type mice. However, high levels of CXC chemokines (KC and MIP-2), particularly at later times after LPS, were specifically sustained in multiple organs in syndecan-1-null mice and associated with exaggerated neutrophilic inflammation, organ damage, and lethality. Syndecan-1 shedding was activated in several organs of endotoxemic wild-type mice, and this associated closely with the removal of tissue-bound CXC chemokines and resolution of accumulated neutrophils. Moreover, administration of a shedding inhibitor exacerbated disease by impeding the removal of CXC chemokines and neutrophils, whereas administration of heparan sulfate inhibited the accumulation of CXC chemokines and neutrophils in tissues and attenuated multiorgan injury and lethality. These data show that syndecan-1 shedding is a critical endogenous mechanism that facilitates the resolution of neutrophilic inflammation by aiding the clearance of proinflammatory chemokines in a heparan sulfate-dependent manner.

α-Toxin Facilitates the Generation of CXC Chemokine Gradients and Stimulates Neutrophil Homing in Staphylococcus aureus Pneumonia

BACKGROUND Staphylococcus aureus alpha-toxin is a major virulence factor, but its mechanism of action in vivo is incompletely understood. METHODS We examined the role of alpha-toxin in S. aureus pneumonia using the mouse model of intranasal lung infection with S. aureus strain 8325-4 (hla(+) S. aureus) and an alpha-toxin-deficient mutant strain made on the 8325-4 background (hla(-) S. aureus). RESULTS Intranasal infection of mice with hla(-) S. aureus resulted in substantially less lung injury and inflammation, pulmonary edema, and tissue bacterial burden than did infection with hla(+) S. aureus. Furthermore, fewer mice infected with hla(-) S. aureus died of the infection, compared with those infected with hla(+) S. aureus. Levels of the CXC chemokines keratinocyte-derived chemokine and macrophage inflammatory protein-2 were significantly lower in the airways of mice infected with hla(-) S. aureus, and this difference was the result of reduced secretion of newly synthesized chemokines into the airway. Consistent with these data, significantly fewer neutrophils were present in the airways and lungs of mice infected with hla(-) S. aureus, compared with those infected with hla(+) S. aureus. CONCLUSIONS These data suggest that alpha-toxin enhances virulence by facilitating the generation of CXC chemokine gradients and stimulating chemokine-induced neutrophil influx in S. aureus pneumonia.

The mitogenic effects of the B beta chain of fibrinogen are mediated through cell surface calreticulin.

We have previously shown that soluble partially degraded fibrin(ogen) remains in solution after fibrin clot formation and is a potent fibroblast mitogen (Gray, A. J., Bishop, J. E., Reeves, J. T., Mecham, R. P., and Laurent, G. J.(1995) Am. J. Cell Mol. Biol. 12, 684-690). Mitogenic sites within the fibrin(ogen) molecule are located on the Aα and Bβ chains of the protein (Gray, A. J., Bishop, J. E., Reeves, J. T., and Laurent, G. J.(1993) J. Cell Sci. 104, 409-413). However, receptor pathways [Abstract] through which mitogenic effects are mediated are unknown. The present study sought to determine the nature of fibrin (ogen) receptors expressed on human fibroblasts which interact with the fibrinogen Bβ chain. Receptor complexes were isolated from 125I-surface-labeled fibroblasts and purified on a fibrinogen Bβ chain affinity column. Subsequent high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis analysis indicated fibrinogen Bβ chain bound specifically to a 60-kDa surface protein. Sequence analysis of the amino terminus of this protein indicated 100% homology to human calreticulin. Immunoprecipitation experiments employing a polyclonal anti-calreticulin antibody provided further evidence that the 60-kDa protein isolated in this study was calreticulin. Further, polyclonal antibodies to human calreticulin significantly inhibited the mitogenic activity of fibrinogen Bβ chain on human fibroblasts. The present study has shown that cell surface calreticulin binds to the Bβ chain of fibrinogen mediating its mitogenic activity.