Heparan Sulfate Proteoglycan Syndecan-1 Research Articles

Growth factor–induced shedding of syndecan-1 confers glypican-1 dependence on mitogenic responses of cancer cells

The cell surface heparan sulfate proteoglycan (HSPG) glypican-1 is up-regulated by pancreatic and breast cancer cells, and its removal renders such cells insensitive to many growth factors. We sought to explain why the cell surface HSPG syndecan-1, which is also up-regulated by these cells and is a known growth factor coreceptor, does not compensate for glypican-1 loss. We show that the initial responses of these cells to the growth factor FGF2 are not glypican dependent, but they become so over time as FGF2 induces shedding of syndecan-1. Manipulations that retain syndecan-1 on the cell surface make long-term FGF2 responses glypican independent, whereas those that trigger syndecan-1 shedding make initial FGF2 responses glypican dependent. We further show that syndecan-1 shedding is mediated by matrix metalloproteinase-7 (MMP7), which, being anchored to cells by HSPGs, also causes its own release in a complex with syndecan-1 ectodomains. These results support a specific role for shed syndecan-1 or MMP7–syndecan-1 complexes in tumor progression and add to accumulating evidence that syndecans and glypicans have nonequivalent functions in vivo.

The matrix protein CCN1 (CYR61) induces apoptosis in fibroblasts

Integrin-mediated cell adhesion to extracellular matrix proteins is known to promote cell survival, whereas detachment from the matrix can cause rapid apoptotic death in some cell types. Contrary to this paradigm, we show that fibroblast adhesion to the angiogenic matrix protein CCN1 (CYR61) induces apoptosis, whereas endothelial cell adhesion to CCN1 promotes cell survival. CCN1 induces fibroblast apoptosis through its adhesion receptors, integrin α6β1 and the heparan sulfate proteoglycan (HSPG) syndecan-4, triggering the transcription-independent p53 activation of Bax to render cytochrome c release and activation of caspase-9 and -3. Neither caspase-8 activity nor de novo transcription or translation is required for this process. These results show that cellular interaction with a specific matrix protein can either induce or suppress apoptosis in a cell type–specific manner and that integrin α6β1-HSPGs can function as receptors to induce p53-dependent apoptosis.

Syndecan-1 expression by stromal fibroblasts promotes breast carcinoma growth in vivo and stimulates tumor angiogenesis

The induction of the cell surface heparan sulfate proteoglycan syndecan-1 (Sdc1) in stromal fibroblasts is observed in more than 70% of human breast carcinomas. Using a coculture model, we have recently shown that stromal cell-derived Sdc1 stimulates carcinoma cell proliferation in vitro, and that this activity requires Sdc1 glycanation. In the present study, we investigated the effect of stromal cell Sdc1 on breast carcinoma growth in vivo. MDA-MB-231 human breast carcinoma cells were inoculated into the flanks of athymic nude mice either alone, or as mixed suspensions with Sdc1-transfected or mock-transfected 3T3 mouse fibroblasts. The mixed tumors showed an intimate association between carcinoma cells and stromal fibroblasts and histologically closely resembled poorly differentiated human breast carcinomas. The presence of fibroblasts led to significantly accelerated tumor growth, which was further augmented (88% increase) by forced expression of stromal Sdc1. The hyperemic macroscopic appearance of tumors containing Sdc1-positive stromal cells contrasted with pale tumors developing in the presence of mock-transfected fibroblasts, which prompted us to examine tumor microvessels. Stromal Sdc1 expression was associated with a significantly elevated microvessel density (36% increase) and a larger vessel area (153% increase). To evaluate the relevance of this finding in human breast cancer, the relationship between stromal Sdc1 and tumor vascularity was also examined in a tissue array containing 207 human breast carcinoma samples. Similar to the xenografts, stromal Sdc1 expression correlated with both vessel density (P=0.013) and total vessel area (P=0.0026). In conclusion, stromal fibroblast-derived Sdc1 stimulates breast carcinoma growth and angiogenesis in vivo.

Epithelial Syndecan-1 Expression Is Associated with Stage and Grade in Colorectal Cancer

Objectives: Loss of epithelial heparan sulfate proteoglycan syndecan-1 has been associated with a more aggressive behavior in various cancer forms, but the prognostic significance of syndecan-1 expression in colorectal cancer is unclear. The aim of this study was to evaluate the prognostic value of immunohistochemical syndecan-1 expression in a series of 237 patients with colorectal cancer. Methods: Paraffin-embedded formalin-fixed specimens were stained with a syndecan-1-specific monoclonal antibody, and both the epithelial and stromal expression were analyzed. Results: Epithelial expression of syndecan-1 was seen in 222 tumors (94%), and it was associated with low stage of disease (p = 0.002) and low histological differentiation grade (p = 0.048). The cumulative 5-year survival of patients with weak and strong syndecan-1 expression was 49 and 54 %, respectively (p = 0.234). Syndecan-1 stromal immunoreactivity was observed in 138 tumors (58%), but lacked prognostic significance. Staining pattern and distribution can be viewed from digitized representative microscope slides (virtual slides) at http://www.webmicroscope.net/supplements/syndecan. Conclusions: The results are in line with previous reports in that low epithelial syndecan-1 expression was associated with a higher histological grade and a more advanced clinical stage of the patients. This study shows that syndecan-1 is expressed also in stromal tissue of colorectal cancer, but it does not support the proposed role of stromal syndecan-1 expression as a marker of poor clinical outcome.

Effects of posthatch feed deprivation on heparan sulfate proteoglycan, syndecan-1, and glypican expression: implications for muscle growth potential in chickens

The heparan sulfate proteoglycans, syndecan-1 and glypican-1 (glypican), are low affinity receptors for fibroblast growth factor 2 (FGF2). Because FGF2 stimulates skeletal muscle cell proliferation but inhibits differentiation, changes in FGF2 signaling due to early posthatch feed deprivation may play a significant role in modulating muscle growth. To study the effect of early posthatch feed deprivation in chickens on heparan sulfate proteoglycan relative protein concentration, syndecan-1 expression, and glypican mRNA expression, pectoralis major muscle tissue was isolated from pretreatment d 0 chicks and chicks fed or feed deprived for 3 d, and after d 3 feeding was resumed in the feed-deprived birds until d 7. Heparan sulfate proteoglycan protein concentration was measured by ELISA analysis and was significantly decreased in the feed-deprived birds beginning at d 2 (P < 0.05). The expression of syndecan-1 and glypican was measured by semi-quantitative reverse transcription PCR. Syndecan-1 expression was unaffected by feed withdrawal and refeeding (P > 0.05). Glypican mRNA expression was decreased in the muscle tissue from feed-deprived birds at d 3 (P < 0.05), but by d 7, after initiating feeding on d 4, it was significantly elevated compared with in muscle tissue from chicks maintained on feed (P < 0.05). The results from the present study demonstrate that the heparan sulfate proteoglycan protein concentration and syndecan-1 and glypican mRNA expressions are differentially affected by early posthatch feed deprivation, which may alter signaling events associated with muscle growth.

Syndecan-4 Is Required for Thrombin-induced Migration and Proliferation in Human Vascular Smooth Muscle Cells

Thrombin is a mitogen and chemoattractant for vascular smooth muscle cells (SMCs) and may contribute to vascular lesion formation. We have previously shown that human SMCs, when stimulated with thrombin, release basic fibroblast growth factor (bFGF), causing phosphorylation of FGF receptor-1 (FGFR-1). Treatment with bFGF-neutralizing antibodies (anti-bFGF) or heparin inhibits thrombin-induced DNA synthesis. We concluded that thrombin may stimulate entry into the cell cycle via bFGF release and FGFR-1 activation. In the present study, we demonstrate a requirement for not only FGFR-1 but also syndecan-4, a transmembrane heparan-sulfate proteoglycan. Inhibition of syndecan-4 expression using small interfering RNA (siRNA) resulted in reduced DNA synthesis by human SMCs after stimulation with thrombin (10 nmol/liter). Anti-bFGF antibody, which inhibits DNA synthesis in control cells, had no inhibitory effect when syndecan-4 expression was reduced by siRNA. Thrombin- or bFGF-induced SMC migration, determined in Boyden chamber assays, was reduced in cells treated with syndecan-4 or FGFR-1 siRNA or by anti-bFGF. Thrombin induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in a biphasic pattern. Although thrombin-mediated ERK phosphorylation at 5 min was not affected by syndecan-4 or FGFR-1 siRNA, ERK phosphorylation at later time points was reduced. We conclude that thrombin-released bFGF binds to syndecan-4 and FGFR-1, which is required for thrombin-induced mitogenesis and migration.

Syndecan-1 is a novel regulator of angiogenesis

The cell surface heparan sulfate proteoglycan syndecan-1 (Sdc1) binds to several proangiogenic factors, including bFGF and two VEGF isoforms, under physiological conditions. We used Sdc1 knockout (KO)- and overexpressing mice to study its role in angiogenesis. The interactions of leukocytes and ECs of WT and Sdc1 KO mice were studied in vivo, using a perfusion technique with FITC-coupled ConA and intravital microscopy. Static in vitro adhesion assays were used to study the role of HS in the adhesion process. Angiogenesis was studied in vivo using a cornea angiogenesis assay. Sdc1 KO mice showed dramatically enhanced leukocyte-EC adhesion in the perfusion model (5-fold, p<0.005) and in intravital microscopy (80-fold, TNF-α-stimulation, p<0.001). This effect was leukocyte-dependent, as shown in hematopoietic chimaeras and by in vitro assays measuring adhesion of Sdc1 KO monocytes and PMN's to HUVEC cells. Addition of heparin (10µg/ml) reverted increased adhesion of Sdc1 KO cells to WT levels, suggesting an involvement of the Sdc1 HS chains. However, a HS fingerprint analysis revealed no qualitative differences in the organ-specific disaccharide patterns of HS isolated from Sdc1 KO and wild-type mice. Increased angiogenesis (1.8-fold, p<0.0001) and abnormal blood vessel morphology in the Sdc1 KO mice were demonstrated in the cornea angiogenesis assay. In Syndecan-1-overexpressing mice, we observed abnormally dilated blood vessels in the granulation tissue of healing dermal wounds, accompanied by a reduction of cell proliferation and an increase in the elastolytic activity of wound fluids, which could be attributed to Sdc-1. Using siRNA knockdown technology, we could demonstrate that FGF-2 mediated MAPK signalling was reduced in MCF-7 cells upon treatment with Sdc-1 siRNA. Our data indicate that syndecan-1 acts as a novel regulator of angiogenesis, having implications for several pathologic conditions that involve abnormal angiogenesis, including breast cancer and endometriosis.

Dynamin II interacts with syndecan-4, a regulator of focal adhesion and stress-fiber formation

Dynamin is a large mechanochemical GTPase that has been implicated in vesicle formation in multiple cellular compartments. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. To identify potential intracellular proteins that interact with the PH domain of dynamin II, we carried out a yeast two-hybrid screen in which the PH domain of dynamin II was used as bait. The cell surface heparan sulfate proteoglycan syndecan-4 that acts in conjunction with integrins to promote the formation of actin stress fibers and focal adhesions was isolated as a binding partner for the PH domain of dynamin II. In vitro binding assays, immunoprecipitation, and confocal microscopy analysis confirmed the association of dynamin II with syndecan-4. Most dramatic finding of our study is that the cytoplasmic distribution of dynamin II and syndecan-4 changes in fibroblasts that have been stimulated to form the focal adhesions and stress fibers with LPA. In quiescent cells, dynamin II is evenly distributed in the cytoplasm and colocalizes with syndecan-4 near the nucleus. Upon treatment with LPA to induce focal adhesions and stress-fiber formation, dynamin II becomes markedly associated with syndecan-4 at focal adhesion sites. We further established the colocalization of syndecan-4 and dynamin with paxillin and actin as marker proteins for focal adhesions and stress fibers, respectively. All of these results suggest that the interaction between dynamin II and syndecan-4 is important in mediating focal adhesion and stress-fiber formation.

Increased Leukocyte-Endothelial Interactions in Syndecan-1–Deficient Mice Involve Heparan Sulfate–Dependent and –Independent Steps

Purpose: Increased leukocyte-endothelial interactions and angiogenesis are observed in the ocular vasculature of mice lacking the cell surface heparan sulfate proteoglycan syndecan-1. Here we investigate the interaction of defined leukocyte populations of syndecan-1 knockout (KO) and wild-type mice with endothelial cells in vitro. Heparin is used to substitute for the lack of syndecan-1 heparan sulfate. Methods: The adhesion of polymorphonuclear cells and monocytes purified from syndecan-1 KO and wild-type mice to unstimulated and TNF-α–treated human umbilical vein endothelial cells (HUVECs) was measured in a static adhesion assay. Results: Adhesion of syndecan-1 KO leukocytes to HUVECs is increased relative to wild-type leukocytes, being more pronounced in TNF-α –stimulated HUVECs. Heparin reverted this adhesion to wild-type levels in unstimulated endothelium. Conclusions: Syndecan-1 acts as a negative regulator of polymorphonuclear leukocytes (PMNs) and monocyte adhesion to endothelial cells. Its heparan sulfate chains play different roles in this process in unstimulated endothelia compared to TNF-α –stimulated endothelia.

Heparanase Promotes the Spontaneous Metastasis of Myeloma Cells to Bone.

Although widespread skeletal dissemination is a critical step in the progression of myeloma, little is known regarding mechanisms that control this process. High levels of the syndecan-1 heparan sulfate proteoglycan are present in the myeloma microenvironment where they bind numerous growth factors (e.g., HGF, FGF-2) that control myeloma growth, angiogenesis and dissemination. Heparanase-1 (HPSE1) is an enzyme that cleaves heparan sulfate chains of proteoglycans and thus may regulate heparan-binding growth factor activity. We have previously demonstrated that human myeloma cells express heparanase and that active enzyme is present in the plasma harvested from the marrow of myeloma patients (Cancer Res. 63: 8749–56). In the present study, experiments were performed to determine the effects of enhanced expression of heparanase on myeloma tumor growth and dissemination in vivo. The human myeloma cell line, CAG, was transfected with human HPSE1 cDNA (CAGHPSE1) or with corresponding vector only (CAGNeo). The transfected cells were injected either directly into the tibia or subcutaneously into the flank of severe combined immune deficient (SCID) mice and tumor growth rate, microvessel density and metastasis to bone were analyzed. We discovered that expression of heparanase: (i) accelerates the initial growth of the primary tumor, (ii) increases whole body tumor burden as compared to controls, and (iii) enhances both the number and size of microvessels within the primary tumor. In addition, enhanced expression of heparanase dramatically upregulates spontaneous metastasis of subcutaneously-injected myeloma cells to bone (95% in mice bearing CAGHPSE1 tumors, n = 21; as compared to 6% in mice bearing CAGNeo tumors, n=16). When tumor was injected directly into the tibia, 100% of the mice bearing CAGHPSE1 tumor cells had metastases within the contralateral femur (n=10) while only 29% of the mice bearing CAGNeo tumor had metastases (n=7). These studies describe a novel experimental animal model for examining the spontaneous metastasis of bone-homing tumors and indicate that heparanase is a critical determinant of myeloma dissemination and growth in vivo. Thus, therapeutic modulation of heparanase expression or function may be of value in the treatment of myeloma and other cancers that metastasize to bone.

Inhibition by the Soluble Syndecan-1 Ectodomains Delays Wound Repair in Mice Overexpressing Syndecan-1

Wound repair is a tightly regulated process stimulated by proteases, growth factors, and chemokines, which are modulated by heparan sulfate. To characterize further the role of the heparan sulfate proteoglycan syndecan-1 in wound repair, we generated mice overexpressing syndecan-1 (Snd/Snd) and studied dermal wound repair. Wound closure, reepithelialization, granulation tissue formation, and remodeling were delayed in Snd/Snd mice. Soluble syndecan-1 was increased, and shedding was prolonged in wounds from Snd/Snd mice. Excess syndecan-1 increased the elastolytic activity of wound fluids. Additionally, cells in the granulation tissue and keratinocytes at wound edges showed markedly reduced proliferation rates in Snd/Snd mice. Skin grafting experiments between Snd/Snd and control mice indicated that the slower growth rate was mainly due to a soluble factor in the Snd/Snd mouse skin. Syndecan-1 immunodepletion and further degradation experiments identified syndecan-1 ectodomain as a dominant negative inhibitor of cell proliferation. These studies indicate that shed syndecan-1 ectodomain may enhance proteolytic activity and inhibit cell proliferation during wound repair.

The heparan sulfate proteoglycan syndecan-1 is a novel regulator of leukocyte-endothelial cell adhesion and leukocyte transmigration

The heparan sulfate proteoglycan syndecan-1 is a novel regulator of leukocyte-endothelial cell adhesion and leukocyte transmigration

Eosinophil trafficking: new answers to old questions

Eosinophil trafficking: new answers to old questions

Cleavage of Syndecan-1 by Membrane Type Matrix Metalloproteinase-1 Stimulates Cell Migration

The transmembrane heparan sulfate proteoglycan syndecan-1 was identified from a human placenta cDNA library by the expression cloning method as a gene product that interacts with membrane type matrix metalloproteinase-1 (MT1-MMP). Co-expression of MT1-MMP with syndecan-1 in HEK293T cells promoted syndecan-1 shedding, and concentration of cell-associated syndecan-1 was reduced. Treatment of cells with MMP inhibitor BB-94 or tissue inhibitor of MMP (TIMP)-2 but not TIMP-1 interfered with the syndecan-1 shedding promoted by MT1-MMP expression. In contrast, syndecan-1 shedding induced by 12-O-tetradecanoylphorbol-13-acetate treatment was inhibited by BB-94 but not by either TIMP-1 or TIMP-2. Shedding of syndecan-1 was also induced by MT3-MMP but not by other MT-MMPs. Recombinant syndecan-1 core protein was shown to be cleaved by recombinant MT1-MMP or MT3-MMP preferentially at the Gly245-Leu246 peptide bond. HT1080 fibrosarcoma cells stably transfected with the syndecan-1 cDNA (HT1080/SDC), which express endogenous MT1-MMP, spontaneously shed syndecan-1. Migration of HT1080/SDC cells on collagen-coated dishes was significantly slower than that of control HT1080 cells. Treatment of HT1080/SDC cells with BB-94 or TIMP-2 induced accumulation of syndecan-1 on the cell surface, concomitant with further retardation of cell migration. Substitution of Gly245 of syndecan-1 with Leu significantly reduced shedding from HT1080/SDC cells and cell migration. These results suggest that the shedding of syndecan-1 promoted by MT1-MMP through the preferential cleavage of Gly245-Leu246 peptide bond stimulates cell migration.

Heparan Sulfate Targeting of Syndecan-1

Cell-surface heparan sulfate proteoglycans bind to various extracellular proteins, including growth factors and proteases, and can thereby affect their function, increase their local concentration, and modify their interactions with receptors. In migrating plasma and myeloma cells, which become polarized, the transmembrane heparan sulfate proteoglycan syndecan-1 localizes to a membrane region called the uropod at the trailing edge of the cell. Syndecan-1 in the uropod plays a role in cell-cell and cell-extracellular matrix adhesion and may regulate the activity of heparin-binding cytokines and the bone destruction seen in myeloma. Yang et al. investigated the mechanism of syndecan-1 targeting to the uropod of myeloma cells and discovered that this was mediated by its heparan sulfate side chains and was regulated at the cell surface. Adding exogenous heparin (which structurally and functionally resembles heparan sulfate) to cultured myeloma cells or treating the cells with heparitinase to remove cell-surface heparan sulfate side chains triggered redistribution of immunofluorescently labeled syndecan-1 over the cell surface. Heparin-mediated redistribution was reversible; recovery was accelerated by antibody binding to syndecan-1 and was abolished by disruption of the actin cytoskeleton. Other heparan sulfate proteoglycans expressed in myeloma cells did not localize to the uropod. The authors expressed syndecan-1 mutants to determine that the heparan sulfate side chains were required for targeted localization, whereas the cytoplasmic and transmembrane regions were not. Thus, heparan sulfate side chains are not only important in mediating proteoglycan interactions with extracellular proteins, but may also play a role in cell-surface localization of proteoglycans.Y. Yang, M. Børset, J. K. Langford, R. D. Sanderson, Heparan sulfate regulates targeting of syndecan-1 to a functional domain on the cell surface. J. Biol. Chem. 278, 12888-12893 (2003). [Abstract] [Full Text]

Heparan Sulfate Targeting of Syndecan-1

Cell-surface heparan sulfate proteoglycans bind to various extracellular proteins, including growth factors and proteases, and can thereby affect their function, increase their local concentration, and modify their interactions with receptors. In migrating plasma and myeloma cells, which become polarized, the transmembrane heparan sulfate proteoglycan syndecan-1 localizes to a membrane region called the uropod at the trailing edge of the cell. Syndecan-1 in the uropod plays a role in cell-cell and cell-extracellular matrix adhesion and may regulate the activity of heparin-binding cytokines and the bone destruction seen in myeloma. Yang et al. investigated the mechanism of syndecan-1 targeting to the uropod of myeloma cells and discovered that this was mediated by its heparan sulfate side chains and was regulated at the cell surface. Adding exogenous heparin (which structurally and functionally resembles heparan sulfate) to cultured myeloma cells or treating the cells with heparitinase to remove cell-surface heparan sulfate side chains triggered redistribution of immunofluorescently labeled syndecan-1 over the cell surface. Heparin-mediated redistribution was reversible; recovery was accelerated by antibody binding to syndecan-1 and was abolished by disruption of the actin cytoskeleton. Other heparan sulfate proteoglycans expressed in myeloma cells did not localize to the uropod. The authors expressed syndecan-1 mutants to determine that the heparan sulfate side chains were required for targeted localization, whereas the cytoplasmic and transmembrane regions were not. Thus, heparan sulfate side chains are not only important in mediating proteoglycan interactions with extracellular proteins, but may also play a role in cell-surface localization of proteoglycans. Y. Yang, M. Børset, J. K. Langford, R. D. Sanderson, Heparan sulfate regulates targeting of syndecan-1 to a functional domain on the cell surface. J. Biol. Chem . 278 , 12888-12893 (2003). [Abstract] [Full Text]

Role of heparan sulfate in interactions of Listeria monocytogenes with enterocytes.

Heparan sulfate is known to participate in binding a wide variety of microbes to mammalian cells, but few studies have focused on the enterocyte. Normal human colonic and small intestinal enterocytes, and cultured HT-29 (but not Caco-2) enterocytes, reacted prominently with antibodies specific for heparan sulfate and for the core protein of syndecan-1 (a heparan sulfate proteoglycan). The heparan sulfate analog, heparin, inhibited interactions of Listeria monocytogenes (adherence and internalization) with HT-29, but not Caco-2, enterocytes. Internalization of L. monocytogenes by HT-29 enterocytes was inhibited by heparan sulfate and to a lesser extent by chondroitin sulfate, but not by the non-sulfated glycosaminoglycan hyaluronic acid. Compared to plasmid control ARH-77 cells, adherence of L. monocytogenes, was increased using ARH-77 cells transfected with syndecan-1 cDNA. Heparin binding protein(s) on L. monocytogenes were confirmed using biotinylated heparin. To determine if these in vitro observations might have in vivo relevance, L. monocytogenes was preincubated with heparin and then orally inoculated into mice. Compared to L. monocytogenes not pretreated with heparin, L. monocytogenes pretreated with heparin was associated with decreased extraintestinal dissemination to the mesenteric lymph nodes and liver of orally inoculated mice. Thus, heparan sulfate (possibly as the heparan sulfate proteoglycan syndecan-1) appears to participate in interactions of L. monocytogenes with enterocytes.

Syndecan-1 as a regulator of chemokine function.

Chemokines are a family of chemotactic cytokines that play critical roles in leukocyte recruitment to sites of inflammation. Characterized by the presence of conserved aminoterminal cysteine residues, these lowmolecular weight proteins signal through G-protein–coupled receptors with seven transmembrane domains[1,2]. Apart from their function in leukocyte recruitment, chemokines and their receptors have been ascribed roles in angiogenesis and tumor growth, infections, Th1 and Th2 responses, as well as the maturation and development of several leukocyte subpopulations[1]. Considering their fundamental role in orchestrating the inflammatory response, it is no surprise that a dysregulation of chemokine function is observed in a variety of pathological conditions, such as pulmonary fibrosis, hypersensitivity responses, chronic inflammation, and cancer[1,2,3,4]. For this reason, it is important to know how chemokine action is regulated in molecular detail, in order to develop more efficient therapies for diseases characterized by chemokine malfunction. Two recent papers shed more light on the regulation of chemokine function, uncovering a crucial role for the cell surface heparan sulfate (HS) proteoglycan syndecan-1 in the generation of chemokine gradients[5,6]. Using mice deficient in the matrix-metalloprotease matrilysin in an experimental model of lung fibrosis, Li et al.[5] observed an impaired transepithelial migration of neutrophils, which resulted in protection against the lethal effect of bleomycin-induced lung injury. They found that matrilysin sheds a complex of syndecan-1 and the CXC chemokine KC from the mucosal surface, which serves as a chemotactic gradient for neutrophils. Marshall et al.[6] used an in vitro model of transendothelial migration to demonstrate that syndecan-1 forms a chemotactic IL-8 gradient at the endothelial cell surface. An increase of constitutive syndecan-1/IL-8 complex shedding in the presence of a neutralizing antibody to plasminogen activator inhibitor-1 (PAI-1) resulted in an inhibition of transendothelial neutrophil migration. The syndecans are a family of cell surface heparan sulfate proteoglycans (HSPG), which act as adhesion molecules, modulators of growth factor function, and coreceptors in processes as diverse as morphogenesis, tissue repair, host defense, tumor development, and energy metabolism[7,8]. Protease-mediated cleavage of the intact syndecan ectodomains (“shedding”) converts the cell-surface molecules into soluble effectors[9]. Increasing evidence suggests an important role for the syndecans in the

835 P21CIP1 siRNA enhanced TGF-B-mediated apoptosis in human HCC cells

Temporally and spatially dynamic Wnt and BMP signals are essential to pattern foregut endoderm progenitors that give rise to the liver, pancreas and lungs, but how these two signaling pathways are coordinated in the extracellular space is unknown. Here we identify the transmembrane heparan sulphate proteoglycan Syndecan-4 (Sdc4), as a key regulator of both non-canonical Wnt and BMP signaling in the Xenopus foregut. Foregut-specific Sdc4 depletion results in a disrupted Fibronectin (Fn1) matrix, reduced cell adhesion, and failure to maintain foregut gene expression ultimately leading to foregut organ hypoplasia. Sdc4 is required to maintain robust Wnt/JNK and BMP/Smad1 signaling in the hhex+ foregut progenitors. Pathway analysis suggests that Sdc4 functionally interacts with Fzd7 to promote Wnt/JNK signaling, which maintains foregut identity and cell adhesion. In addition, the Sdc4 ectodomain is required to support Fn1 matrix assembly, which is essential for the robust BMP signaling that promotes foregut gene expression. This work sheds lights on how the extracellular matrix can coordinate different signaling pathways during organogenesis.

Syndecan-4 Modulates Focal Adhesion Kinase Phosphorylation

The cell-surface heparan sulfate proteoglycan syndecan-4 acts in conjunction with the alpha(5)beta(1) integrin to promote the formation of actin stress fibers and focal adhesions in fibronectin (FN)-adherent cells. Fibroblasts seeded onto the cell-binding domain (CBD) fragment of FN attach but do not fully spread or form focal adhesions. Activation of Rho, with lysophosphatidic acid (LPA), or protein kinase C, using the phorbol ester phorbol 12-myristate 13-acetate, or clustering of syndecan-4 with antibodies directed against its extracellular domain will stimulate formation of focal adhesions and stress fibers in CBD-adherent fibroblasts. The distinct morphological differences between the cells adherent to the CBD and to full-length FN suggest that syndecan-4 may influence the organization of the focal adhesion or the activation state of the proteins that comprise it. FN-null fibroblasts (which express syndecan-4) exhibit reduced phosphorylation of focal adhesion kinase (FAK) tyrosine 397 (Tyr(397)) when adherent to CBD compared with FN-adherent cells. Treating the CBD-adherent fibroblasts with LPA, to activate Rho, or the tyrosine phosphatase inhibitor sodium vanadate increased the level of phosphorylation of Tyr(397) to match that of cells plated on FN. Treatment of the fibroblasts with PMA did not elicit such an effect. To confirm that this regulatory pathway includes syndecan-4 specifically, we examined fibroblasts derived from syndecan-4-null mice. The phosphorylation levels of FAK Tyr(397) were lower in FN-adherent syndecan-4-null fibroblasts compared with syndecan-4-wild type and these levels were rescued by the addition of LPA or re-expression of syndecan-4. These data indicate that syndecan-4 ligation regulates the phosphorylation of FAK Tyr(397) and that this mechanism is dependent on Rho but not protein kinase C activation. In addition, the data suggest that this pathway includes the negative regulation of a protein-tyrosine phosphatase. Our results implicate syndecan-4 activation in a direct role in focal adhesion regulation.