Specific syndecan-1 domains regulate mesenchymal tumor cell adhesion, motility and migration

Background

Syndecans are proteoglycans whose core proteins have a short cytoplasmic domain, a transmembrane domain and a large N-terminal extracellular domain possessing glycosaminoglycan chains. Syndecans are involved in many important cellular processes. Our recent publications have demonstrated that syndecan-1 translocates into the nucleus and hampers tumor cell proliferation. In the present study, we aimed to investigate the role of syndecan-1 in tumor cell adhesion and migration, with special focus on the importance of its distinct protein domains, to better understand the structure-function relationship of syndecan-1 in tumor progression.

Methodology/Principal Findings

We utilized two mesenchymal tumor cell lines which were transfected to stably overexpress full-length syndecan-1 or truncated variants: the 78 which lacks the extracellular domain except the DRKE sequence proposed to be essential for oligomerization, the 77 which lacks the whole extracellular domain, and the RMKKK which serves as a nuclear localization signal. The deletion of the RMKKK motif from full-length syndecan-1 abolished the nuclear translocation of this proteoglycan. Various bioassays for cell adhesion, chemotaxis, random movement and wound healing were studied. Furthermore, we performed gene microarray to analyze the global gene expression pattern influenced by syndecan-1. Both full-length and truncated syndecan-1 constructs decrease tumor cell migration and motility, and affect cell adhesion. Distinct protein domains have differential effects, the extracellular domain is more important for promoting cell adhesion, while the transmembrane and cytoplasmic domains are sufficient for inhibition of cell migration. Cell behavior seems to depend also on the nuclear translocation of syndecan-1. Many genes are differentially regulated by syndecan-1 and a number of genes are actually involved in cell adhesion and migration.

Conclusions/Significance

Our results demonstrate that syndecan-1 regulates mesenchymal tumor cell adhesion and migration, and different domains have differential effects. Our study provides new insights into better understanding of the role of syndecans in tumor progression.     

Trans-regulation of Syndecan Functions by Hetero-oligomerization

Syndecans, a family of transmembrane heparansulfate proteoglycans, are known to interact through their transmembrane domains to form non-covalently linked homodimers, a process essential for their individual functions. Because all syndecan transmembrane domains are highly conserved and thus might mediate interactions between different members of the syndecan family, we investigated syndecan interactions in detail. All recombinant syndecan-2 and -4 protein variants containing the transmembrane domain formed not only sodium dodecyl sulfate (SDS)-resistant homodimers but also SDS-resistant heterodimers. Biochemical and structural data revealed that recombinant syndecan-2 and -4 formed intermolecular interactions in vitro, and the GXXXG motif in transmembrane domain mediated this interaction. When exogenously expressed in rat embryonic fibroblasts, syndecan-2 interacted with syndecan-4 and vice versa. Furthermore, bimolecular fluorescence complementation-based assay demonstrated specific hetero-molecular interactions between syndecan-2 and -4, supporting hetero-oligomer formation of syndecans in vivo. Interestingly, hetero-oligomerization significantly reduced syndecan-4-mediated cellular processes such as protein kinase Cα activation and protein kinase Cα-mediated cell adhesion as well as syndecan-2-mediated tumorigenic activities in colon cancer cells such as migration and anchorage-independent growth. Taken together, these data provide evidence that hetero-oligomerization produces distinct syndecan functions and offer insights into the underlying signaling mechanisms of syndecans.     

Syndecan-4 cytoplasmic domain regulation of turkey satellite cell focal adhesions and apoptosis

Syndecan-4 is a cell membrane proteoglycan composed of a transmembrane core protein and substituted glycosaminoglycan (GAG) and N-linked glycosylated (N-glycosylated) chains. The core protein has three domains: extracellular, transmembrane and cytoplasmic domains. The GAG and N-glycosylated chains and the cytoplasmic domain of syndecan-4, especially the amino acids: Ser(178) and Tyr(187) are critical in regulation of turkey satellite cell growth and development. How these processes are regulated is still unknown. The objective of the current study was to determine whether the syndecan-4 GAG and N-glycosylated chains and the cytoplasmic domain functions through modulating focal adhesion formation and apoptosis. Twelve mutant clones were generated: a truncated syndecan-4 without the cytoplasmic domain with or without GAG and N-glycosylated chains, and Ser(178) and Tyr(187) mutants with or without GAG and N-glycosylated chains. The wild type syndecan-4 and all of the syndecan-4 mutants were transfected into turkey myogenic satellite cells after which cell apoptosis and focal adhesion formation were measured. Syndecan-4 increased cell membrane localization of β1 integrin and the activity of focal adhesion kinase (FAK) whereas the cytoplasmic domain mutation decreased the phosphorylation of FAK. However, syndecan-4 and syndecan-4 mutants did not influence cell apoptosis. They also had no effect on vinculin or paxillin-containing focal adhesion formation. These results suggested that the syndecan-4 cytoplasmic domain plays an important role in regulating FAK activity and β1 integrin cell membrane localization but not cell apoptosis and vinculin or paxillin-containing focal adhesion formation.     

Syndecan-2 is a novel ligand for the protein tyrosine phosphatase receptor CD148

Syndecan-2 is a heparan sulfate proteoglycan that has a cell adhesion regulatory domain contained within its extracellular core protein. Cell adhesion to the syndecan-2 extracellular domain (S2ED) is β1 integrin dependent; however, syndecan-2 is not an integrin ligand. Here the protein tyrosine phosphatase receptor CD148 is shown to be a key intermediary in cell adhesion to S2ED, with downstream β1 integrin-mediated adhesion and cytoskeletal organization. We show that S2ED is a novel ligand for CD148 and identify the region proximal to the transmembrane domain of syndecan-2 as the site of interaction with CD148. A mechanism for the transduction of the signal from CD148 to β1 integrins is elucidated requiring Src kinase and potential implication of the C2β isoform of phosphatidylinositol 3 kinase. Our data uncover a novel pathway for β1 integrin-mediated adhesion of importance in cellular processes such as angiogenesis and inflammation.     

Hierarchy between the transmembrane and cytoplasmic domains in the regulation of syndecan-4 functions

Syndecan-4, a transmembrane heparan sulfate proteoglycan, plays a critical role in cell adhesion. Both the transmembrane and cytoplasmic domains of syndecan-4 are known to contribute to its functions, but the regulatory mechanisms underlying the functional interplay between the two domains were previously unclear. Here, we examined the functional relationship between these two domains. Fluorescence resonance energy transfer (FRET)-based assays showed that syndecan-4 expression enhanced RhoA activation. Furthermore, rat embryonic fibroblasts (REFs) plated on fibronectin fragments lacking the heparin-binding domain that interacts with syndecan-4 showed much lower RhoA activation than that in cells plated on full-length fibronectin, indicating that RhoA is involved in syndecan-4-mediated cell adhesion signaling. Syndecan-4 mutants defective in transmembrane domain-induced oligomerization and syndecan-4 phosphorylation-mimicking cytoplasmic domain mutants showed decreases in RhoA activation and RhoA-related functions, such as adhesion, spreading and focal adhesion formation, and subsequent increase in cell migration, but the inhibitory effect was much higher in cells expressing the transmembrane domain mutants. The cytoplasmic domain mutants (but not the transmembrane domain mutants) retained the capacity to form SDS-resistant dimers, and the cytoplasmic mutants showed less inhibition of syndecan-4-mediated protein kinase C activation compared to the transmembrane domain mutants. Finally, cytoplasmic domain activation failed to overcome the inhibition conferred by mutation of the transmembrane domain. Taken together, these data suggest that the transmembrane domain plays a major role in regulating syndecan-4 functions, and further show that a domain hierarchy exists in the regulation of syndecan-4.     

Strain-induced Differentiation of Fetal Type II Epithelial Cells Is Mediated via the Integrin α6β1-ADAM17/Tumor Necrosis Factor-α-converting Enzyme (TACE) Signaling Pathway

Mechanical forces are critical for normal fetal lung development. However, the mechanisms regulating this process are not well-characterized. We hypothesized that strain-induced release of HB-EGF and TGF-α is mediated via integrin-ADAM17/TACE interactions. Employing an in vitro system to simulate mechanical forces in fetal lung development, we showed that mechanical strain of fetal epithelial cells actives TACE, releases HB-EGF and TGF-α, and promotes differentiation. In contrast, in samples incubated with the TACE inhibitor IC-3 or in cells isolated from TACE knock-out mice, mechanical strain did not release ligands or promote cell differentiation, which were both rescued after transfection of ADAM17. Cell adhesion assay and co-immunoprecipitation experiments in wild-type and TACE knock-out cells using several TACE constructs demonstrated not only that integrins α6 and β1 bind to TACE via the disintegrin domain but also that mechanical strain enhances these interactions. Furthermore, force applied to these integrin receptors by magnetic beads activated TACE and shed HB-EGF and TGF-α. The contribution of integrins α₆ and β₁ to differentiation of fetal epithelial cells by strain was demonstrated by blocking their binding site with specific antibodies and by culturing the cells on membranes coated with anti-integrin α₆ and β₁ antibodies. In conclusion, mechanical strain releases HB-EGF and TGF-α and promotes fetal type II cell differentiation via α₆β₁ integrin-ADAM17/TACE signaling pathway. These investigations provide novel mechanistic information on how mechanical forces promote fetal lung development and specifically differentiation of epithelial cells. This information could be also relevant to other tissues exposed to mechanical forces.     

Syndecan-1 mediates cell spreading in transfected human lymphoblastoid (Raji) cells

Syndecan-1 is a cell surface proteoglycan containing a highly conserved transmembrane and cytoplasmic domain, and an extracellular domain bearing heparan sulfate glycosaminoglycans. Through these domains, syndecan-1 is proposed to have roles in growth factor action, extracellular matrix adhesion, and cytoskeletal organization that controls cell morphology. To study the role of syndecan-1 in cell adhesion and cytoskeleton reorganization, mouse syndecan-1 cDNA was transfected into human Raji cells, a lymphoblastoid cell line that grows as suspended cells and exhibits little or no endogenous cell surface heparan sulfate. High expressing transfectants (Raji-Sl cells) bind to and spread on immobilized thrombospondin or fibronectin, which are ligands for the heparan sulfate chains of the proteoglycan. This binding and spreading as not dependent on the cytoplasmic domain of the core protein, is mutants expressing core proteins with cytoplasmic deletions maintain the ability to spread. The spreading is mediated through engagement of the syndecan-1 core protein, as the Raji-S 1 cells also bind to and spread on immobilized mAb 281.2, an antibody specific for the ectodomain of the syndecan-1 core protein. Spreading on the antibody is independent of the heparan sulfate glycosaminoglycan chains and can be inhibited by competition with soluble mAb 281.2. The spreading can be inhibited by treatment with cytochalasin D or colchicine. These data suggest that the core protein of syndecan-1 mediates spreading through the formation of a multimolecular signaling complex at the cell surface that signals cytoskeleton reorganization. This complex may form via intramembrane or extracellular interactions with the syndecan core protein.     

Clustering of Syndecan-4 and Integrin β1 by Laminin α3 Chain–derived Peptide Promotes Keratinocyte Migration

Syndecans function as receptors for extracellular matrix (ECM) with integrins in cell spreading. However, the molecular mechanism of their specific involvement in cell migration or in wound healing has not been elucidated yet. Here, we report that a synthetic peptide, PEP75, which contains the syndecan-binding sequence of the laminin α3LG4 module, induces keratinocyte migration in in vitro and in vivo. Soluble PEP75 induced the clustering of syndecan-4 and conformation-modified integrin β1 colocalized with syndecan-4 in soluble PEP75-induced clusters. Treatment of cells in solution with PEP75 resulted in the exposure of the P4G11 antibody epitope of integrin β1 in immunostaining as well as in flow cytometry and augmented integrin β1–dependent cell adhesion to ECM. Pulldown assays demonstrated that PEP75 bound to syndecan-4, but not to integrin β1. A siRNA study revealed a role for syndecan-4 in PEP75-induced up-regulation of P4G11 antibody binding and migration of HaCaT cells. We conclude that binding of soluble PEP75 to syndecan-4 induces the coupling of integrin β1, which is associated with integrin β1-conformational changes and activation, and leads to keratinocyte migration. To activate integrin function through syndecans could be a novel therapeutic approach for chronic wound.     

Cys Palmitoylation of the β Subunit Modulates Gating of the Epithelial Sodium Channel

The epithelial Na⁺ channel (ENaC) is comprised of three homologous subunits (α, β, and γ) that have a similar topology with two transmembrane domains, a large extracellular region, and cytoplasmic N and C termini. Although ENaC activity is regulated by a number of factors, palmitoylation of its cytoplasmic Cys residues has not been previously described. Fatty acid-exchange chemistry was used to determine whether channel subunits were Cys-palmitoylated. We observed that only the β and γ subunits were modified by Cys palmitoylation. Analyses of ENaCs with mutant β subunits revealed that Cys-43 and Cys-557 were palmitoylated. Xenopus oocytes expressing ENaC with a β C43A,C557A mutant had significantly reduced amiloride-sensitive whole cell currents, enhanced Na⁺ self-inhibition, and reduced single channel P_o when compared with wild-type ENaC, while membrane trafficking and levels of surface expression were unchanged. Computer modeling of cytoplasmic domains indicated that β Cys-43 is in proximity to the first transmembrane α helix, whereas β Cys-557 is within an amphipathic α-helix contiguous with the second transmembrane domain. We propose that β subunit palmitoylation modulates channel gating by facilitating interactions between cytoplasmic domains and the plasma membrane.     

MMP7 Shedding of Syndecan-1 Facilitates Re-Epithelialization by Affecting α2β1 Integrin Activation

Background

Lung injury promotes the expression of matrix metalloproteinase-7 (MMP7, matrilysin), which is required for neutrophil recruitment and re-epithelialization. MMP7 governs the lung inflammatory response through the shedding of syndecan-1. Because inflammation and repair are related events, we evaluated the role of syndecan-1 shedding in lung re-epithelialization.

Methodology/Principal Finding

Epithelial injury induced syndecan-1 shedding from wild-type epithelium but not from Mmp7^−/− mice in vitro and in vivo. Moreover, cell migration and wound closure was enhanced by MMP7 shedding of syndecan-1. Additionally, we found that syndecan-1 augmented cell adhesion to collagen by controlling the affinity state of the α₂β₁ integrin.

Conclusion/Significance

MMP7 shedding of syndecan-1 facilitates wound closure by causing the α₂β₁ integrin to assume a less active conformation thereby removing restrictions to migration. MMP7 acts in the lungs to regulate inflammation and repair, and our data now show that both these functions are controlled through the shedding of syndecan-1.     

N-Ethylmaleimide-sensitive Factor Attachment Protein α (αSNAP) Regulates Matrix Adhesion and Integrin Processing in Human Epithelial Cells

Integrin-based adhesion to the extracellular matrix (ECM) plays critical roles in controlling differentiation, survival, and motility of epithelial cells. Cells attach to the ECM via dynamic structures called focal adhesions (FA). FA undergo constant remodeling mediated by vesicle trafficking and fusion. A soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein α (αSNAP) is an essential mediator of membrane fusion; however, its roles in regulating ECM adhesion and cell motility remain unexplored. In this study, we found that siRNA-mediated knockdown of αSNAP induced detachment of intestinal epithelial cells, whereas overexpression of αSNAP increased ECM adhesion and inhibited cell invasion. Loss of αSNAP impaired Golgi-dependent glycosylation and trafficking of β1 integrin and decreased phosphorylation of focal adhesion kinase (FAK) and paxillin resulting in FA disassembly. These effects of αSNAP depletion on ECM adhesion were independent of apoptosis and NSF. In agreement with our previous reports that Golgi fragmentation mediates cellular effects of αSNAP knockdown, we found that either pharmacologic or genetic disruption of the Golgi recapitulated all the effects of αSNAP depletion on ECM adhesion. Furthermore, our data implicates β1 integrin, FAK, and paxillin in mediating the observed pro-adhesive effects of αSNAP. These results reveal novel roles for αSNAP in regulating ECM adhesion and motility of epithelial cells.     

Domain 1 of Mucosal Addressin Cell Adhesion Molecule Has an I1-set Fold and a Flexible Integrin-binding Loop

Mucosal addressin cell adhesion molecule (MAdCAM) binds integrin α₄β₇. Their interaction directs lymphocyte homing to mucosa-associated lymphoid tissues. The interaction between the two immunoglobulin superfamily (IgSF) domains of MAdCAM and integrin α₄β₇ is unusual in its ability to mediate either rolling adhesion or firm adhesion of lymphocytes on vascular surfaces. We determined four crystal structures of the IgSF domains of MAdCAM to test for unusual structural features that might correlate with this functional diversity. Higher resolution 1.7- and 1.4-Å structures of the IgSF domains of MAdCAM in a previously described crystal lattice revealed two alternative conformations of the integrin-binding loop, which were deformed by large lattice contacts. New crystal forms in the presence of two different Fabs to MAdCAM demonstrate a shift in IgSF domain topology from the I2- to I1-set, with a switch of integrin-binding loop from CC′ to CD. The I1-set fold and CD loop appear biologically relevant. The different conformations seen in crystal structures suggest that the integrin-binding loop of MAdCAM is inherently flexible. This contrasts with rigidity of the corresponding loops in vascular cell adhesion molecule, intercellular adhesion molecule (ICAM)-1, ICAM-2, ICAM-3, and ICAM-5 and may reflect a specialization of MAdCAM to mediate both rolling and firm adhesion by binding to different α₄β₇ integrin conformations.     

Syndecan-2 overexpression regulates adhesion and migration through cooperation with integrin α2

Syndecan-2, a transmembrane heparan sulfate proteoglycan, is known to serve as an adhesion receptor, but details of the regulatory mechanism governing syndecan-2 cell adhesion and migration remain unclear. Here, we examined this regulatory mechanism, showing that overexpression of syndecan-2 enhanced collagen adhesion, cell migration and invasion of normal rat intestinal epithelial cells (RIE1), and increased integrin α2 expression levels. Interestingly, RIE1 cells transfected with either syndecan-2 or integrin α2 showed similar adhesion and migration patterns, and a function-blocking anti-integrin α2 antibody abolished syndecan-2-mediated adhesion and migration. Consistent with these findings, transfection of integrin α2 siRNA diminished syndecan-2-induced cell migration in HCT116 human colon cancer cells. Taken together, these results demonstrate a novel cooperation between syndecan-2 and integrin α2β1 in adhesion-mediated cell migration and invasion. This interactive dynamic might be a possible mechanism underlying the tumorigenic activities of colon cancer cells.     

Syndecans promote integrin-mediated adhesion of mesenchymal cells in two distinct pathways

Syndecans are transmembrane proteoglycans that support integrin-mediated adhesion. Well documented is the contribution of syndecan-4 that interacts through its heparan sulphate chains to promote focal adhesion formation in response to fibronectin domains. This process has requirements for integrin and signaling through the cytoplasmic domain of syndecan-4. Here an alternate pathway mediated by the extracellular domains of syndecans-2 and -4 is characterized that is independent of both heparan sulphate and syndecan signaling. This pathway is restricted to mesenchymal cells and was not seen in any epithelial cell line tested, apart from vascular endothelia. The syndecan ectodomains coated as substrates promoted integrin-dependent attachment, spreading and focal adhesion formation. Syndecan-4 null cells were competent, as were fibroblasts compromised in heparan sulphate synthesis that were unable to form focal adhesions in response to fibronectin. Consistent with actin cytoskeleton organization, the process required Rho-GTP and Rho kinase. While syndecan-2 and -4 ectodomains could both promote integrin-mediated adhesion, their pathways were distinct, as shown by competition assays. Evidence for an indirect interaction of beta1 integrin with both syndecan ectodomains was obtained, all of which suggests a distinct mechanism of integrin-mediated adhesion.     

Syndecan-1 transmembrane and extracellular domains have unique and distinct roles in cell spreading

Raji cells expressing syndecan-1 (Raji-S1) adhere and spread when plated on heparan sulfate-binding extracellular matrix ligands or monoclonal antibody 281.2, an antibody directed against the syndecan-1 extracellular domain. Cells plated on monoclonal antibody 281.2 initially extend a broad lamellipodium, a response accompanied by membrane ruffling at the cell margin. Membrane ruffling then becomes polarized, leading to an elongated cell morphology. Previous work demonstrated that the syndecan-1 cytoplasmic domain is not required for these activities, suggesting important roles for the syndecan-1 transmembrane and/or extracellular domains in the assembly of a signaling complex necessary for spreading. Work described here demonstrates that truncation of the syndecan-1 extracellular domain does not affect the initial lamellipodial extension in the Raji-S1 cells but does inhibit the active membrane ruffling that is necessary for cell polarization. Replacement of the entire syndecan-1 transmembrane domain with leucine residues completely blocks the cell spreading. These data demonstrate that the syndecan-1 transmembrane and extracellular domains have important but distinct roles in Raji-S1 cell spreading; the extracellular domain mediates an interaction that is necessary for dynamic cytoskeletal rearrangements whereas an interaction of the transmembrane domain is required for the initial spreading response.     

Transmembrane domain-induced oligomerization is crucial for the functions of syndecan-2 and syndecan-4

The syndecans are known to form homologous oligomers that may be important for their functions. We have therefore determined the role of oligomerization of syndecan-2 and syndecan-4. A series of glutathione S-transferase-syndecan-2 and syndecan-4 chimeric proteins showed that all syndecan constructs containing the transmembrane domain formed SDS-resistant dimers, but not those lacking it. SDS-resistant dimer formation was hardly seen in the syndecan chimeras where each transmembrane domain was substituted with that of platelet-derived growth factor receptor (PDGFR). Increased MAPK activity was detected in HEK293T cells transfected with syndecan/PDGFR chimeras in a syndecan transmembrane domain-dependent fashion. The chimera-induced MAPK activation was independent of both ligand and extracellular domain, implying that the transmembrane domain is sufficient to induce dimerization/oligomerization in vivo. Furthermore, the syndecan chimeras were defective in syndecan-4-mediated focal adhesion formation and protein kinase Calpha activation or in syndecan-2-mediated cell migration. Taken together, these data suggest that the transmembrane domains are sufficient for inducing dimerization and that transmembrane domain-induced oligomerization is crucial for syndecan-2 and syndecan-4 functions.     

Functional role of syndecan-1 cytoplasmic V region in lamellipodial spreading, actin bundling, and cell migration

Cell protrusions contribute to cell motility and migration by mediating the outward extension and initial adhesion of cell edges. In many cells, these extensions are supported by actin bundles assembled by the actin cross-linking protein, fascin. Multiple extracellular cues regulate fascin and here we focus on the mechanism by which the transmembrane proteoglycan, syndecan-1, specifically activates lamellipodial cell spreading and fascin-and-actin bundling when clustered either by thrombospondin-1, laminin, or antibody to the syndecan-1 extracellular domain. There is almost no knowledge of the signaling mechanisms of syndecan-1 cytoplasmic domain and we have tested the hypothesis that the unique V region of syndecan-1 cytoplasmic domain has a crucial role in these processes. By four criteria--the activities of N-cadherin/V region chimeras, syndecan-1 deletion mutants, or syndecan-1 point mutants, and specific inhibition by a membrane-permeable TAT-V peptide--we demonstrate that the V region is necessary and sufficient for these cell behaviors and map the molecular basis for its activity to multiple residues located across the V region. These activities correlate with a V-region-dependent incorporation of cell-surface syndecan-1 into a detergent-insoluble form. We also demonstrate functional roles of syndecan-1 V region in laminin-dependent C2C12 cell adhesion and three-dimensional cell migration. These data identify for the first time specific cell behaviors that depend on signaling through the V region of syndecan-1.     

A Disintegrin and Metalloproteinase 17 (ADAM17) Mediates Inflammation-induced Shedding of Syndecan-1 and -4 by Lung Epithelial Cells

Syndecans are cell surface proteoglycans that bind and modulate various proinflammatory mediators and can be proteolytically shed from the cell surface. Within the lung, syndecan-1 and -4 are expressed as transmembrane proteins on epithelial cells and released in the bronchoalveolar fluid during inflammation. We here characterize the mechanism leading to the generation of soluble syndecan-1 and -4 in cultured epithelial cells and murine lung tissue. We show that the bladder carcinoma epithelial cell line ECV304, the lung epithelial cell line A459 and primary alveolar epithelial cells express and constitutively release syndecan-1 and -4. This release involves the activity of the disintegrin-like metalloproteinase ADAM17 as demonstrated by use of specific inhibitors and lentivirally transduced shRNA. Stimulation of epithelial cells with PMA, thrombin, or proinflammatory cytokines (TNFα/IFNγ) led to the down-regulation of surface-expressed syndecan-1 and -4, which was associated with a significant increase of soluble syndecans and cell-associated cleavage fragments. The enhanced syndecan release was not related to gene induction of syndecans or ADAM17, but rather due to increased ADAM17 activity. Soluble syndecan-1 and -4 were also released into the bronchoalveolar fluid of mice. Treatment with TNFα/IFNγ increased ADAM17 activity and syndecan release in murine lungs. Both constitutive and induced syndecan shedding was prevented by the ADAM17 inhibitor. ADAM17 may therefore be an important regulator of syndecan functions on inflamed lung epithelium.     

Heparan Sulfates Mediate the Interaction between Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) and the Gαq/11 Subunits of Heterotrimeric G Proteins

The endothelial cell-cell junction has emerged as a major cell signaling structure that responds to shear stress by eliciting the activation of signaling pathways. Platelet endothelial cell adhesion molecule-1 (PECAM-1) and heterotrimeric G protein subunits Gα_q and 11 (Gα_q/11) are junctional proteins that have been independently proposed as mechanosensors. Our previous findings suggest that they form a mechanosensitive junctional complex that discriminates between different flow profiles. The nature of the PECAM-1·Gα_q/11 interaction is still unclear although it is likely an indirect association. Here, we investigated the role of heparan sulfates (HS) in mediating this interaction and in regulating downstream signaling in response to flow. Co-immunoprecipitation studies show that PECAM-1·Gα_q/11 binding is dramatically decreased by competitive inhibition with heparin, pharmacological inhibition with the HS antagonist surfen, and enzymatic removal of HS chains with heparinase III treatment as well as by site-directed mutagenesis of basic residues within the extracellular domain of PECAM-1. Using an in situ proximity ligation assay, we show that endogenous PECAM-1·Gα_q/11 interactions in endothelial cells are disrupted by both competitive inhibition and HS degradation. Furthermore, we identified the heparan sulfate proteoglycan syndecan-1 in complexes with PECAM-1 that are rapidly decreased in response to flow. Finally, we demonstrate that flow-induced Akt activation is attenuated in endothelial cells in which PECAM-1 was knocked down and reconstituted with a binding mutant. Taken together, our results indicate that the PECAM-1·Gα_q/11 mechanosensitive complex contains an endogenous heparan sulfate proteoglycan with HS chains that is critical for junctional complex assembly and regulating the flow response.     

Inter-��-trypsin Inhibitor Promotes Bronchial Epithelial Repair after Injury through Vitronectin Binding

Pulmonary epithelial injury is central to the pathogenesis of many lung diseases, such as asthma, pulmonary fibrosis, and the acute respiratory distress syndrome. Regulated epithelial repair is crucial for lung homeostasis and prevents scar formation and inflammation that accompany dysregulated healing. The extracellular matrix (ECM) plays an important role in epithelial repair after injury. Vitronectin is a major ECM component that promotes epithelial repair. However, the factors that modify cell-vitronectin interactions after injury and help promote epithelial repair are not well studied. Inter-α-trypsin inhibitor (IaI) is an abundant serum protein. IaI heavy chains contain von Willebrand A domains that can bind the arginine-glycine-aspartate domain of vitronectin. We therefore hypothesized that IaI can bind vitronectin and promote vitronectin-induced epithelial repair after injury. We show that IaI binds vitronectin at the arginine-glycine-aspartate site, thereby promoting epithelial adhesion and migration in vitro. Furthermore, we show that IaI-deficient mice have a dysregulated response to epithelial injury in vivo, consisting of decreased proliferation and epithelial metaplasia. We conclude that IaI interacts not only with hyaluronan, as previously reported, but also other ECM components like vitronectin and is an important regulator of cellular repair after injury.Epithelial injury is a crucial component in the pathogenesis of many lung diseases. Bronchial epithelial injury occurs chronically in asthmatic patients (1, 2). Furthermore, alveolar and bronchiolar epithelial injury are early triggers in idiopathic pulmonary fibrosis (3) and in lung transplant rejection (4), respectively. In acute respiratory distress syndrome, diffuse alveolar epithelial injury initiates the inflammatory and fibrotic response that leads to lung dysfunction (5). It is now believed that a dysregulated response to epithelial injury ultimately causes fibroproliferation, scar formation, and respiratory failure in acute as well as chronic lung injury (3). It is therefore important to understand the epithelial repair process after pulmonary epithelial injury, if we are to develop causal treatments for these diseases.The mechanisms governing epithelial repair are incompletely understood. Epithelial repair encompasses cell proliferation, migration, and differentiation. All of these processes require cell interactions with the extracellular matrix (ECM).² ECM components like tenascin C, fibronectin, and vitronectin promote epithelial regeneration through integrin binding. Vitronectin (Vn) is a pluripotent 75-kDa plasma and ECM glycoprotein that regulates a number of biological processes such as coagulation, complement activation, and wound healing. Vn promotes cell adhesion and migration via binding primarily to integrins α_vβ₁, α_vβ₃, α_vβ₅, and α_vβ₆. After cell binding, Vn can protect bronchial epithelial cells from apoptosis (6) by inducing Akt phosphorylation and preventing caspase and Fas-associated with death domain (FADD) activation (7). Vn also binds to non-integrin cell receptors such as urokinase-type plasminogen activator receptor to promote changes in cell morphology, migration, and signal transduction (8). Consequently, Vn deficiency impairs bronchial (6) and alveolar (9) epithelial repair.Cell-Vn interactions are modulated by other extracellular factors. For example, plasminogen activator inhibitor 1 (PAI-1) is bound to circulating Vn and forms multimers with Vn upon extravasation to the extracellular space (10), thus possibly activating Vn into an adhesive form (11). Furthermore, Vn possesses several domains that can function as possible ligands, such as a somatomedin B domain, an arginine-glycine-aspartate (RGD) domain, and a heparin-binding domain. However, the extent to which other serum or ECM factors may interact with Vn and influence cell-Vn interactions is unclear.In this report, we investigated possible interactions between Vn and the serum and ECM protein inter-α-trypsin inhibitor. Inter-α-trypsin inhibitor (IaI) is a complex protein found in relatively high concentrations in mammalian plasma. It is made up of a light chain (called bikunin for its two Kunitz domains), which confers the protease inhibitory activity, as well as two heavy chains (12). The precise functions of the heavy chains are unknown. Heavy chains contain a von Willebrand Type A (vWA) domain, and they have been shown to bind to hyaluronan and thereby stabilize the extracellular matrix. However, vWA domains are fairly promiscuous and can bind to a large array of proteins, including RGD domains. Furthermore, IaI is expressed by epithelial cells under stress conditions and is incorporated into de novo ECM structures produced by stressed epithelia (13). We therefore hypothesized that IaI may interact with Vn and thus promote epithelial survival after injury.