Heparan sulfate chains of syndecan-1 regulate ectodomain shedding

Matrix metalloproteinases release intact syndecan-1 ectodomains from the cell surface giving rise to a soluble, shed form of the proteoglycan. Although it is known that shed syndecan-1 controls diverse pathophysiological responses in cancer, wound healing, inflammation, infection, and immunity, the mechanisms regulating shedding remain unclear. We have discovered that the heparan sulfate chains present on syndecan core proteins suppress shedding of the proteoglycan. Syndecan shedding is dramatically enhanced when the heparan sulfate chains are enzymatically degraded or absent from the core protein. Exogenous heparan sulfate or heparin does not inhibit shedding, indicating that heparan sulfate must be attached to the core protein to suppress shedding. Regulation of shedding by heparan sulfate occurs in multiple cell types, for both syndecan-1 and syndecan-4 and in murine and human syndecans. Mechanistically, the loss of heparan sulfate enhances the susceptibility of the core protein to proteolytic cleavage by matrix metalloproteinases. Enhanced shedding of syndecan-1 following loss of heparan sulfate is accompanied by a dramatic increase in core protein synthesis. This suggests that in response to an increase in the rate of shedding, cells attempt to maintain a significant level of syndecan-1 on the cell surface. Together these data indicate that the amount of heparan sulfate present on syndecan core proteins regulates both the rate of syndecan shedding and core protein synthesis. These findings assign new functions to heparan sulfate chains, thereby broadening our understanding of their physiological importance and implying that therapeutic inhibition of heparan sulfate degradation could impact the progression of some diseases.     

Lipid peroxides induce expression of catalase in cultured vascular cells

Various forms of oxidized low-density lipoproteins (Ox-LDL) are thought to play a major role in the development of atherosclerosis. The lipid components of Ox-LDL present a plethora of proatherogenic effects in in vitro cell culture systems, suggesting that oxidative stress could be an important risk factor for coronary artery disease. However, buried among these effects are those that could be interpreted as antiatherogenic. The present study demonstrates that various oxidants, including oxidized fatty acids and mildly oxidized forms of LDL (MO-LDL), are able to induce catalase (an antioxidant enzyme) expression in rabbit femoral arterial smooth muscle cells (RFASMC), RAW cells (macrophages), and human umbilical vein endothelial cells (HUVEC). In RFASMC, catalase protein, mRNA, and the enzyme activity are increased in response to oxidized linoleic acid (13-hydroperoxy-9,11-octadecadienoic acid [13-HPODE] and 13-hydroxy-9,11-octadecadienoic acid [13-HODE]), MO-LDL, or hydrogen peroxide (H(2)O(2)). Such an increase in catalase gene expression cannot totally be attributed to the cellular response to an intracellular generation of H(2)O(2) after the addition of 13-HPODE or 13-HODE because these agents induce a further increase of catalase as seen in catalase-transfected RFASMC. Taken together with the induction of heme oxygenase, NO synthase, manganese superoxide dismutase (Mn-SOD), and glutathione synthesis by oxidative stress, our results provide yet more evidence suggesting that a moderate oxidative stress can induce cellular antioxidant response in vascular cells, and thereby could be beneficial for preventing further oxidative stress.     

EJ-ras oncogene transfection of endothelial cells upregulates the expression of syndecan-4 and downregulates heparan sulfate sulfotransferases and epimerase

The EC rabbit endothelial cell line was transfected with the EJ-ras oncogene (EJ-ras EC). EJ-ras EC cells display over expression of the Ras oncogene, morphological changes and deregulation of the cell cycle, becoming more densely populated and serum-independent. In addition, EJ-ras-transfectant cells show higher levels of the syndecan-4 mRNA. In addition to the increase in the core protein, a parallel increase in the glycosylation of the syndecan-4 protein, a proteoglycan that bears heparan sulfate chains, also occurs. This increase is observed both for the heparan sulfate proteoglycan synthesized by the cells and for that secreted to the culture medium. This enhancement in heparan sulfate synthesis was observed through metabolic labeling of the cells, immunoprecipitation of syndecan-4 and heparitinases treatment. Furthermore, the EJ-ras-transfectant cells do not exhibit decreased synthesis of heparan sulfate during the G(1)-S phase transition, as observed for the parental cell line. Also, heparan sulfate synthesis is not stimulated by PMA as displayed by parental endothelial cells. Significant structural changes of heparan sulfate, such as decreased O-sulfation, were observed in the EJ-ras-transfected cells. Decreases in the mRNA levels of some enzymes (glucuronosyl C-5 epimerase, iduronosyl-2-O-sulfotransferase, glucosaminyl-6-O-sulfotransferase-1 and N-deacetylase/N-sulfotransferase-1), involved in the biosynthetic pathway of heparan sulfate, were also observed. The results suggest that overexpression of the EJ-ras oncogene alters the cell cycle, through signal transduction cascades, upregulates the expression of syndecan-4, and downregulates enzymes involved in the heparan sulfate biosynthesis related to chain modification, leading to the structural changes of the heparan sulfate syndecan-4 proteoglycan in endothelial cells.     

Membrane-anchored proteoglycans of mouse macrophages: P388D1 cells express a syndecan-4–like heparan sulfate proteoglycan and a distinct chondroitin sulfate form

Proteoglycan accumulation by thioglycollate-elicited mouse peritoneal macrophages and a panel of murine monocyte-macrophage cell lines has been examined to determine whether these cells express plasma membrane-anchored heparan sulfate proteoglycans. Initially, cells were screened for heparan sulfate and chondroitin sulfate glycosaminoglycans after metabolic labeling with radiosulfate. Chondroitin sulfate is secreted to a variable extent by every cell type examined. In contrast, heparan sulfate is all but absent from immature pre-monocytes and is associated predominantly with the cell layer of mature macrophage-like cells. In the P388D1 cell line, the cell-associated chondroitin sulfate is largely present as a plasma membrane-anchored proteoglycan containing a 55 kD core protein moiety, which appears to be unique. In contrast, the cell-associated heparan sulfate is composed of a proteoglycan fraction and protein-free glycosaminoglycan chains, which accumulate intracellularly. A fraction of the heparan sulfate proteoglycan contains a lipophilic domain and can be released from cells following mild treatment with trypsin, suggesting that it is anchored in the plasma membrane. Isolation of this proteoglycan indicates that it is likely syndecan-4: it is expressed as a heparan sulfate proteoglycan at the cell surface, it is cleaved from the plasma membrane by low concentrations of trypsin, and it consists of a single 37 kD core protein moiety that co-migrates with syndecan-4 isolated from NMuMG mouse mammary epithelial cells. Northern analysis reveals that a panel of macrophage-like cell lines accumulate similar amounts of syndecan-4 mRNA, demonstrating that this proteoglycan is expressed by a variety of mature macrophage-like cells. Syndecan-1 mRNA is present only in a subset of these cells, suggesting that the expression of this heparan sulfate proteoglycan may be more highly regulated by these cells. © 1993 Wiley-Liss, Inc.     

Heparanase enhances syndecan-1 shedding: a novel mechanism for stimulation of tumor growth and metastasis

When shed from the cell surface, the heparan sulfate proteoglycan syndecan-1 can facilitate the growth, angiogenesis, and metastasis of tumors. Here we report that tumor cell expression of heparanase, an enzyme known to be a potent promoter of tumor progression and metastasis, regulates both the level and location of syndecan-1 within the tumor microenvironment by enhancing its synthesis and subsequent shedding from the tumor cell surface. Heparanase regulation of syndecan-1 is detected in both human myeloma and breast cancer cell lines. This regulation requires the presence of active enzyme, because mutated forms of heparanase lacking heparan sulfate-degrading activity failed to influence syndecan-1 expression or shedding. Removal of heparan sulfate from the cell surface using bacterial heparitinase dramatically accelerated syndecan-1 shedding, suggesting that the effects of heparanase on syndecan-1 expression by tumor cells may be due, at least in part, to enzymatic removal or reduction in the size of heparan sulfate chains. Animals bearing tumors formed from cells expressing high levels of heparanase or animals transgenic for heparanase expression exhibited elevated levels of serum syndecan-1 as compared with controls, indicating that heparanase regulation of syndecan-1 expression and shedding can occur in vivo and impact cancer progression and perhaps other pathological states. These results reveal a new mechanism by which heparanase promotes an aggressive tumor phenotype and suggests that heparanase and syndecan-1 act synergistically to fine tune the tumor microenvironment and ensure robust tumor growth.     

Interleukin-1α promotes extracellular shedding of syndecan-2 via induction of matrix metalloproteinase-7 expression

The cell surface heparan sulfate proteoglycan, syndecan-2, is known to play an important role in the tumorigenic activity of colon cancer cells. In addition, the extracellular domain of syndecan-2 is cleaved by matrix metalloproteinase-7 (MMP-7) in various colon cancer cells, but factors involved in regulating this process remain unknown. Here, we demonstrate a role for interleukin-1α (IL-1α) in syndecan-2 shedding in colon cancer cells. Treatment of low metastatic (HT-29) and highly metastatic (HCT-116) colon cancer cells with various soluble growth factors and cytokines revealed that IL-1α specifically increased extracellular shedding of syndecan-2 in a concentration- and time-dependent manner. IL-1α did not affect the expression of syndecan-2, but did significantly reduce its cell surface levels. Notably, IL-1α increased the mRNA expression and subsequent secreted levels of MMP-7 protein and enhanced the phosphorylation of p38 and ERK mitogen-activated protein kinases. Furthermore, increased syndecan-2 shedding was dependent on the mitogen-activated protein kinase-mediated MMP-7 expression. Taken together, these data suggest that IL-1α regulates extracellular domain shedding of syndecan-2 through regulation of the MAP kinase-mediated MMP-7 expression in colon cancer cells.     

Constitutive and accelerated shedding of murine syndecan-1 is mediated by cleavage of its core protein at a specific juxtamembrane site

Syndecan-1 is a developmentally regulated cell surface heparan sulfate proteoglycan (HSPG). It functions as a coreceptor for a variety of soluble and insoluble ligands and is implicated in several biological processes, including differentiation, cell migration, morphogenesis, and recently feeding behavior. The extracellular domain of syndecan-1 is proteolytically cleaved at a juxtamembrane site by tissue inhibitor of metalloprotease-3 (TIMP-3)-sensitive metalloproteinases in response to a variety of physiological stimulators and stress in a process known as shedding. Shedding converts syndecan-1 from a membrane-bound coreceptor into a soluble effector capable of binding the same ligands. We found that replacing syndecan-1 juxtamembrane amino acid residues A243-S-Q-S-L247 with human CD4 amino acid residues can completely block PMA-induced syndecan-1 ectodomain shedding. Furthermore, using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS), we identified the proteolytic cleavage site of syndecan-1 as amino acids A243 and S244, generated by constitutive and PMA-induced shedding from murine NMuMG cells. Finally, we show that basal cleavage of syndecan-1 utilizes the same in vivo site as the in vitro site. Indeed, as predicted, transgenic mice expressing the syndecan-1/CD4 cDNA do not shed the syndecan-1 ectodomain in vivo. These results suggest that the same cleavage site is utilized for basal syndecan-1 ectodomain shedding both in vitro from NMuMG and CHO cells and in vivo.     

PR-39 coordinates changes in vascular smooth muscle cell adhesive strength and locomotion by modulating cell surface heparan sulfate-matrix interactions

PR-39 is proline-rich peptide produced at sites of tissue injury. While the functional properties of this peptide have not been fully defined, PR-39 may be an important regulator of processes related to cell-matrix adhesion since it reportedly upregulates syndecan-4, which is a critical determinant of focal adhesion formation. The ability of PR-39 to modulate the adhesion and chemokinetic migration behavior of arterial smooth muscle cells (SMCs) in a fashion coordinated with syndecan-4 expression was investigated. Treatment of SMCs with PR-39 did not alter syndecan-1 mRNA, but did induce a two-fold increase in syndecan-4 mRNA (P < 0.0001) and significantly enhanced cell surface expression of both syndecan-4 (P < 0.01) and heparan sulfate (HS) (P < 0.05). These observations were consistent with an observed increase in cell-matrix adhesive strength (P < 0.05) and a reduction in cell speed (P < 0.01) on fibronectin-coated substrates. Incubation of PR-39 treated cells with a soluble fibronectin derived heparin-binding peptide, as a competitive inhibitor of heparan sulfate/matrix interactions, abolished these effects. These data suggest that PR-39 mediated alterations of cell adhesion and motility may be related, in part, to the increased expression of heparan sulfate glycosaminoglycans (GAGs) that accompany the upregulation of cell surface syndecan-4. Furthermore, this investigation supports the notion that factors which control syndecan-4 expression may play an important role in regulating adhesion related cell processes.     

Reciprocal regulation of syndecan-2 and Notch signaling in vascular smooth muscle cells

Vascular cell interactions mediated through cell surface receptors play a critical role in the assembly and maintenance of blood vessels. These signaling interactions transmit important information that alters cell function through changes in protein dynamics and gene expression. Here, we identify syndecan-2 (SDC2) as a gene whose expression is induced in smooth muscle cells upon physical contact with endothelial cells. Syndecan-2 is a heparan sulfate proteoglycan that is known to be important for developmental processes, including angiogenesis. Our results show that endothelial cells induce mRNA expression of syndecan-2 in smooth muscle cells by activating Notch receptor signaling. Both NOTCH2 and NOTCH3 contribute to the increased expression of syndecan-2 and are themselves sufficient to promote its expression independent of endothelial cells. Syndecan family members serve as coreceptors for signaling molecules, and interestingly, our data show that syndecan-2 regulates Notch signaling and physically interacts with NOTCH3. Notch activity is attenuated in smooth muscle cells made deficient in syndecan-2, and this specifically prevents expression of the differentiation marker smooth muscle α-actin. These results show a novel mechanism in which Notch receptors control their own activity by inducing the expression of syndecan-2, which then acts to propagate Notch signaling by direct receptor interaction.     

Characterization of syndecan-4 expression in 3T3-F442A mouse adipocytes: link between syndecan-4 induction and cell proliferation.

Heparan sulfate proteoglycans are found on the surface of most cells. Syndecan-4 is a widely expressed transmembrane heparan sulfate proteoglycan. Using quantitative RNase protection assays and immunoblotting, syndecan-4 expression was characterized in 3T3-F442A mouse adipoblasts. These cells exhibit dramatic changes in their biological and morphological characteristics during differentiation to adipocytes. During this process, the levels of syndecan-4 protein and mRNA expression changed dramatically. They peaked at the time when quiescent cells reentered the cell cycle before differentiation. Serum depletion-repletion also replicated the syndecan-4 mRNA induction when the cells were released back into proliferation, and a cycloheximide treatment abolished the peak of induction. In addition, inhibiting syndecan-4 induction with antisense oligonucleotides inhibited the proliferation of 3T3-F442A cells. In the terminally differentiated adipocytes characterized by the loss of proliferation capability, the serum inducibility of syndecan-4 is repressed, emphasizing the link between syndecan-4 induction in 3T3-F442A cells and cell proliferation.     

Putative role of heparan sulfate proteoglycan expression and shedding on the proliferation and survival of cells after photodynamic therapy

INTRODUCTION: Photodynamic therapy is based on the selective retention of a photosensitizer by highly proliferating cells and its activation with light at the appropriate wavelength. This combination generates reactive oxygen species that ultimately kill the cells. Some cells, however, may survive photodynamic therapy and the interaction of these cells with the extracellular matrix has profound effect in tumor biology. The knowledge of photodynamic therapy action on the extracellular matrix has not been fully explored. It has been focused mainly on integrins, matrix metalloproteinases and on growth factors and immunological mediators. Other important molecules involved in the regulation of many cell processes are the glycosaminoglycans, polymers of disaccharide units, present on the cell surface and in the extracellular matrix. In most cases, the glycosaminoglycans occur as proteoglycans. AIMS: The purpose of the present investigation is to evaluate heparan sulfate proteoglycan expression and shedding, and its relation to the survival of the remaining cells, after a liposomal-AlClPc based photodynamic treatment. MATERIALS: A wild-type endothelial cell derived from rabbit aorta and its counterpart transfected with EJ-ras oncogene were used. RESULTS: Both cell lines presented augmented heparan sulfate proteoglycan syndecan-4 mRNA expression, augmented synthesis of heparan sulfate chains and increased shedding. Also, the formation of stress fibers on the border of the cells and the arrest in G(1) phase of the cell cycle was observed. CONCLUSIONS: These results show that surviving cells after photodynamic therapy exhibit changes in their morphology and cell processes that differ from that of non-treated cells, and these changes are probably hindering the cells from resuming normal proliferation.     

Cleavage of syndecan-4 by ADAMTS1 provokes defects in adhesion

Syndecan-4 is a membrane-bound heparan sulfate proteoglycan that participates in cell-cell and cell-matrix interactions and modulates adhesion and migration of many cell types. Through its extracellular domain, syndecan-4 cooperates with adhesion molecules and binds matrix components relevant for cell migration. Importantly, syndecan-4 is a substrate of extracellular proteases, however the biological significance of this cleavage has not been elucidated. Here, we show that the secreted metalloprotease ADAMTS1, involved in angiogenesis and inflammatory processes, cleaves the ectodomain of syndecan-4. We further showed that this cleavage results in altered distribution of cytoskeleton components, functional loss of adhesion, and gain of migratory capacities. Using syndecan-4 null cells, we observed that ADAMTS1 proteolytic action mimics the outcome of genetic deletion of this proteoglycan with regards to focal adhesion. Our findings suggest that the shedding of syndecan-4 by ADAMTS1 disrupts cell adhesion and promotes cell migration.     

The matrix metalloproteinase-7 regulates the extracellular shedding of syndecan-2 from colon cancer cells

The cell surface heparan sulfate proteoglycan syndecan-2 regulates the activation of matrix metalloproteinase-7 (MMP-7) as a docking receptor. Here, we demonstrate the role of MMP-7 on syndecan-2 shedding in colon cancer cells. Western blot analysis showed that shed syndecan-2 was found in the culture media from various colon cancer cells. Overexpression of MMP-7 enhanced syndecan-2 shedding, whereas the opposite was true when MMP-7 levels were knocked-down using small inhibitory RNAs. Consistently, HT29 cells treated with MMP-7, but neither MMP-2 nor MMP-9, showed increased shed syndecan-2 in a time- and concentration-dependent manner. Furthermore, MALDI-TOF MS analysis and N-terminal amino acid sequencing revealed that MMP-7 cleaved both recombinant syndecan-2 and an endogenously glycosylated syndecan-2 ectodomain in the N-terminus at Leu(149) residue in vitro. Taken together, the data suggest that MMP-7 directly mediates shedding of syndecan-2 from colon cancer cells.     

Synergistic effect of tumor necrosis factor-alpha and interferon-gamma on enterocyte shedding of syndecan-1 and associated decreases in internalization of Listeria monocytogenes and Staphylococcus aureus

Syndecan-1 is a heparan sulfate proteoglycan expressed on epithelia, and its ectodomain can be shed into the extracellular milieu, affecting a variety of cellular functions. Using two bacteria known to react with heparan sulfate, Listeria monocytogenes and Staphylococcus aureus, experiments were designed to clarify the effect of syndecan-1 shedding on bacterial internalization by human HT-29 enterocytes. Mature enterocytes were incubated with tumor necrosis factor (TNF)-alpha and/or interferon (IFN)-gamma for 16h prior to addition of bacteria. These cytokines acted synergistically to decrease syndecan-1 expression, assessed by visual observations of syndecan-1 expression on enterocytes using immunohistochemistry and a monoclonal antibody to the syndecan-1 core protein, by quantifying this fluorescent intensity, and by quantifying the concentration of shed syndecan-1 using an enzyme-linked immunoabsorbent assay. Neither IFN-gamma nor TNF-alpha alone had a noticeable effect on L. monocytogenes internalization, but a mixture of both cytokines resulted in decreased (P<0.01) internalization. Enterocyte preincubation with TNF-alpha alone, and with both cytokines, was associated with decreased S. aureus internalization, at P<0.05 and P<0.01, respectively. Thus, TNF-alpha and IFN-gamma acted synergistically to shed syndecan-1 ectodomains from HT-29 enterocytes, and shedding was associated with decreased internalization of two pathogenic bacteria, suggesting that syndecan-1 shedding may modulate the pathogenesis of specific microbes.     

Syndecan-1 downregulates syndecan-4 expression by suppressing the ERK1/2 and p38 MAPK signaling pathways in cultured vascular endothelial cells

Syndecan-1 and syndecan-4 are members of the syndecan family of transmembrane heparan sulfate proteoglycans. Vascular endothelial cells synthesize both species of proteoglycans and use them to regulate the blood coagulation-fibrinolytic system and their proliferation via their heparin-like activity and FGF-2 binding activity, respectively. However, little is known about the crosstalk between the expressions of the proteoglycan species. Previously, we reported that biglycan, a small leucine-rich dermatan sulfate proteoglycan, intensifies ALK5–Smad2/3 signaling by TGF-β₁ and downregulates syndecan-4 expression in vascular endothelial cells. In the present study, we investigated the crosstalk between the expressions of syndecan-1 and other proteoglycan species (syndecan-4, perlecan, glypican-1, and biglycan) in bovine aortic endothelial cells in a culture system. These data suggested that syndecan-1 downregulated syndecan-4 expression by suppressing the endogenous FGF-2-dependent ERK1/2 pathway and FGF-2-independent p38 MAPK pathway in the cells. Moreover, this crosstalk was a one-way communication from syndecan-1 to syndecan-4, suggesting that syndecan-4 compensated for the reduced activity in the regulation of vascular endothelial cell functions caused by the decreased expression of syndecan-1 under certain conditions.     

Th1 cytokine-induced syndecan-4 shedding by airway smooth muscle cells is dependent on mitogen-activated protein kinases

In asthma, airway smooth muscle (ASM) chemokine secretion can induce mast cell recruitment into the airways. The functions of the mast cell chemoattractant CXCL10, and other chemokines, are regulated by binding to heparan sulphates such as syndecan-4. This study is the first demonstration that airway smooth muscle cells (ASMC) from people with and without asthma express and shed syndecan-4 under basal conditions. Syndecan-4 shedding was enhanced by stimulation for 24 h with the Th1 cytokines interleukin-1β (IL-1β) or tumor necrosis factor-α (TNF-α), but not interferon-γ (IFNγ), nor the Th2 cytokines IL-4 and IL-13. ASMC stimulation with IL-1β, TNF-α, and IFNγ (cytomix) induced the highest level of syndecan-4 shedding. Nonasthmatic and asthmatic ASM cell-associated syndecan-4 protein expression was also increased by TNF-α or cytomix at 4-8 h, with the highest levels detected in cytomix-stimulated asthmatic cells. Cell-associated syndecan-4 levels were decreased by 24 h, whereas shedding remained elevated at 24 h, consistent with newly synthesized syndecan-4 being shed. Inhibition of ASMC matrix metalloproteinase-2 did not prevent syndecan-4 shedding, whereas inhibition of ERK MAPK activation reduced shedding from cytomix-stimulated ASMC. Although ERK inhibition had no effect on syndecan-4 mRNA levels stimulated by cytomix, it did cause an increase in cell-associated syndecan-4 levels, consistent with the shedding being inhibited. In conclusion, ASMC produce and shed syndecan-4 and although this is increased by the Th1 cytokines, the MAPK ERK only regulates shedding. ASMC syndecan-4 production during Th1 inflammatory conditions may regulate chemokine activity and mast cell recruitment to the ASM in asthma.     

Syndecan-1 ectodomain shedding is regulated by the small GTPase Rab5

The ectodomain shedding of syndecan-1, a major cell surface heparan sulfate proteoglycan, modulates molecular and cellular processes central to the pathogenesis of inflammatory diseases. Syndecan-1 shedding is a highly regulated process in which outside-in signaling accelerates the proteolytic cleavage of syndecan-1 ectodomains at the cell surface. Several extracellular agonists that induce syndecan-1 shedding and metalloproteinases that cleave syndecan-1 ectodomains have been identified, but the intracellular mechanisms that regulate syndecan-1 shedding are largely unknown. Here we examined the role of the syndecan-1 cytoplasmic domain in the regulation of agonist-induced syndecan-1 shedding. Our results showed that the syndecan-1 cytoplasmic domain is essential because mutation of invariant cytoplasmic Tyr residues abrogates ectodomain shedding, but not because it is Tyr phosphorylated upon shedding stimulation. Instead, our data showed that the syndecan-1 cytoplasmic domain binds to Rab5, a small GTPase that regulates intracellular trafficking and signaling events, and this interaction controls the onset of syndecan-1 shedding. Syndecan-1 cytoplasmic domain bound specifically to Rab5 and preferentially to inactive GDP-Rab5 over active GTP-Rab5, and shedding stimulation induced the dissociation of Rab5 from the syndecan-1 cytoplasmic domain. Moreover, the expression of dominant-negative Rab5, unable to exchange GDP for GTP, interfered with the agonist-induced dissociation of Rab5 from the syndecan-1 cytoplasmic domain and significantly inhibited syndecan-1 shedding induced by several distinct agonists. Based on these data, we propose that Rab5 is a critical regulator of syndecan-1 shedding that serves as an on-off molecular switch through its alternation between the GDP-bound and GTP-bound forms.     

Activation of syndecan-1 ectodomain shedding by Staphylococcus aureus alpha-toxin and beta-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 alpha-toxin and beta-toxin genes do not stimulate shedding. Mutations in the agr global regulatory locus, which positively regulates expression of alpha- and beta-toxins and other exoproteins, also abrogated the capacity to stimulate syndecan-1 shedding. Furthermore, purified S. aureus alpha- and beta-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, alpha- and beta-toxins. Toxin-activated shedding was also selectively inhibited by antagonists of the host cell shedding mechanism, indicating that alpha- and beta-toxins shed syndecan-1 ectodomains through stimulation of the host cell's shedding machinery. Interestingly, beta-toxin, but not alpha-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 alpha-toxin and beta-toxin may be a previously unknown virulence mechanism of S. aureus.