RESUMO
Multiple myeloma arises when malignant plasma cells invade and form multiple tumors in the bone marrow. High levels of heparanase (HPSE) correlate with poor prognosis in myeloma patients. A likely target of the enzyme is the heparan sulfate (HS) proteoglycan syndecan-1 (Sdc1, CD138), which is highly expressed on myeloma cells and contributes to poor prognosis in this disease. We find that HPSE promotes an invasive phenotype mediated by the very late antigen-4 (VLA-4, or α4ß1 integrin) in myeloma cells plated on either fibronectin (FN) or vascular endothelial cell adhesion molecule-1 (VCAM-1), ligands that are prevalent in the bone marrow. The phenotype depends on vascular endothelial cell growth factor receptor-2 (VEGFR2), which is aberrantly expressed in myeloma, and is characterized by a highly protrusive lamellipodium and cell invasion. HPSE-mediated trimming of the HS on Sdc1 and subsequent matrix metalloproteinase-9-mediated shedding of the syndecan exposes a juxtamembrane site in Sdc1 that binds VEGFR2 and VLA-4, thereby coupling VEGFR2 to the integrin. Shed Sdc1 can be mimicked by recombinant Sdc1 ectodomain or by a peptide based on its binding motif, which causes VLA-4 to re-orient from the lagging edge (uropod) to the leading edge of migrating cells, couple with and activate VEGFR2. Peptides (called 'synstatins') containing only the VLA-4 or VEGFR2 binding sites competitively inhibit invasion, as they block coupling of the receptors. This mechanism is also utilized by vascular endothelial cells, in which it is also activated by HPSE, during endothelial cell tube formation. Collectively, our findings reveal for the first time the mechanism through which HPSE modulates Sdc1 function to promote both tumor cell invasion and angiogenesis, thereby driving multiple myeloma progression. The inhibitory synstatins, or inhibitors of HPSE enzyme activity, are likely to show promise as therapeutics against myeloma extravasation and spread.
RESUMO
FGF signaling uses receptor tyrosine kinases that form high-affinity complexes with FGFs and heparan sulfate (HS) proteoglycans at the cell surface. It is hypothesized that assembly of these complexes requires simultaneous recognition of distinct sulfation patterns within the HS chain by FGF and the FGF receptor (FR), suggesting that tissue-specific HS synthesis may regulate FGF signaling. To address this, FGF-2 and FGF-4, and extracellular domain constructs of FR1-IIIc (FR1c) and FR2-IIIc (FR2c), were used to probe for tissue-specific HS in embryonic day 18 mouse embryos. Whereas FGF-2 binds HS ubiquitously, FGF-4 exhibits a restricted pattern, failing to bind HS in the heart and blood vessels and failing to activate signaling in mouse aortic endothelial cells. This suggests that FGF-4 seeks a specific HS sulfation pattern, distinct from that of FGF-2, which is not expressed in most vascular tissues. Additionally, whereas FR2c binds all FGF-4-HS complexes, FR1c fails to bind FGF-4-HS in most tissues, as well as in Raji-S1 cells expressing syndecan-1. Proliferation assays using BaF3 cells expressing either FR1c or FR2c support these results. This suggests that FGF and FR recognition of specific HS sulfation patterns is critical for the activation of FGF signaling, and that synthesis of these patterns is regulated during embryonic development.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Heparitina Sulfato/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Fosfatase Alcalina , Animais , Encéfalo/irrigação sanguínea , Encéfalo/embriologia , Células Cultivadas , Embrião de Mamíferos/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 4 de Crescimento de Fibroblastos , Proteínas Ligadas por GPI , Heparina/farmacologia , Heparitina Sulfato/química , Imuno-Histoquímica , Isoenzimas/genética , Isoenzimas/metabolismo , Rim/citologia , Rim/embriologia , Rim/metabolismo , Fígado/citologia , Fígado/embriologia , Fígado/metabolismo , Pulmão/citologia , Pulmão/embriologia , Pulmão/metabolismo , Camundongos , Estrutura Molecular , Miocárdio/química , Miocárdio/metabolismo , Ligação Proteica , Receptores Proteína Tirosina Quinases/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Pele/química , Pele/citologia , Pele/embriologiaAssuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Heparitina Sulfato/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Biotina , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Técnicas In Vitro , Ligação Proteica , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Distribuição TecidualRESUMO
The syndecans, cell surface heparan sulfate proteoglycans (HSPGs), bind numerous ligands via their HS glycosaminoglycan chains. The response to this binding is flavored by the identity of the core protein that bears the HS chains. Each of the syndecan core proteins has a short cytoplasmic domain that binds cytosolic regulatory factors. The syndecans also contain highly conserved transmembrane domain and extracellular domains for which important activities are slowly emerging. These protein domains, which will be the focus of this review, localize the syndecan to sites at the cell surface during development where they collaborate with other receptors to regulate signaling and cytoskeletal organization.
Assuntos
Diferenciação Celular/fisiologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiologia , Proteoglicanas/química , Proteoglicanas/fisiologia , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , SindecanasRESUMO
Myogenesis in the embryo and the adult mammal consists of a highly organized and regulated sequence of cellular processes to form or repair muscle tissue that include cell proliferation, migration, and differentiation. Data from cell culture and in vivo experiments implicate both FGFs and HGF as critical regulators of these processes. Both factors require heparan sulfate glycosaminoglycans for signaling from their respective receptors. Since syndecans, a family of cell-surface transmembrane heparan sulfate proteoglycans (HSPGs) are implicated in FGF signaling and skeletal muscle differentiation, we examined the expression of syndecans 1-4 in embryonic, fetal, postnatal, and adult muscle tissue, as well as on primary adult muscle fiber cultures. We show that syndecan-1, -3, and -4 are expressed in developing skeletal muscle tissue and that syndecan-3 and -4 expression is highly restricted in adult skeletal muscle to cells retaining myogenic capacity. These two HSPGs appear to be expressed exclusively and universally on quiescent adult satellite cells in adult skeletal muscle tissue, suggesting a role for HSPGs in satellite cell maintenance or activation. Once activated, all satellite cells maintain expression of syndecan-3 and syndecan-4 for at least 96 h, also implicating these HSPGs in muscle regeneration. Inhibition of HSPG sulfation by treatment of intact myofibers with chlorate results in delayed proliferation and altered MyoD expression, demonstrating that heparan sulfate is required for proper progression of the early satellite cell myogenic program. These data suggest that, in addition to providing potentially useful new markers for satellite cells, syndecan-3 and syndecan-4 may play important regulatory roles in satellite cell maintenance, activation, proliferation, and differentiation during skeletal muscle regeneration.
Assuntos
Glicoproteínas de Membrana/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Proteoglicanas/metabolismo , Regeneração , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores/análise , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Cloratos/farmacologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Membro Anterior , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Heparitina Sulfato/farmacologia , Laminina/análise , Camundongos , Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/embriologia , Músculo Esquelético/crescimento & desenvolvimento , Proteína MyoD/análise , Proteínas Proto-Oncogênicas c-met/análise , Receptores Proteína Tirosina Quinases/análise , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/análise , Regeneração/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sindecana-3 , Sindecana-4RESUMO
Syndecan-1-expressing Raji lymphoid cells (Raji-S1 cells) bind and spread rapidly when attaching to matrix ligands that contain heparan sulfate-binding domains. However, these ligands also contain binding sites for integrins, which are widely known to signal, raising the question of whether the proteoglycan core protein participates in generation of the signal for spreading. To address this question, the spreading of the Raji-S1 cells is examined on ligands specific for either beta1 integrins, known to be present on the Raji cells, or the syndecan-1 core protein. The cells adhere and spread on invasin, a ligand that activates beta1 integrins, the IIICS fragment of fibronectin, which is a specific ligand for the alpha4beta1 integrin, or mAb281.2, an antibody specific for the syndecan-1 core protein. The signaling resulting from adhesion to the syndecan-specific antibody appears integrin independent as (i) the morphology of the cells spreading on the antibody is distinct from spreading initiated by the integrins alone; (ii) spreading on the syndecan or integrin ligands is affected differently by the kinase inhibitors tyrphostin 25, genistein, and staurosporine; and (iii) spreading on the syndecan-specific antibody is not disrupted by blocking beta1 integrin activation with mAb13, a beta1 inhibitory antibody. These data demonstrate that ligation of syndecan-1 initiates intracellular signaling and suggest that this signaling occurs when cells expressing syndecan-1 adhere to matrix ligands containing heparan sulfate-binding domains.
Assuntos
Linfócitos B/citologia , Integrina beta1/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteoglicanas/metabolismo , Transdução de Sinais/fisiologia , Anticorpos Monoclonais/farmacologia , Linfócitos B/fisiologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Inibidores Enzimáticos/farmacologia , Proteínas da Matriz Extracelular/fisiologia , Genisteína/farmacologia , Proteoglicanas de Heparan Sulfato/fisiologia , Humanos , Integrina beta1/imunologia , Linfoma de Células B , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Testes de Neutralização , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteoglicanas/genética , Proteoglicanas/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Estaurosporina/farmacologia , Sindecana-1 , Sindecanas , Células Tumorais Cultivadas , Tirfostinas/farmacologiaRESUMO
Fibroblast growth factors (FGFs) require heparan sulfate proteoglycans (HSPGs) as cofactors for signaling. The heparan sulfate chains (HS) mediate stable high affinity binding of FGFs to their receptor tyrosine kinases (FR) and may specifically regulate FGF activity. A novel in situ binding assay was developed to examine the ability of HSPGs to promote FGF/FR binding using a soluble FR fusion construct (FR1-AP). This fusion protein probe forms a dimer in solution, simulating the dimerization or oligomerization that is thought to occur at the cell surface physiologically. In frozen sections of human skin, FGF-2 binds to keratinocytes and basement membranes of epidermis and dermal blood vessels. In contrast, in skin preincubated with FGF-2, FR1-AP binds avidly to FGF-2 immobilized on keratinocyte cell surfaces, but fails to bind to basement membranes at the dermo-epidermal junction or dermal microvessels despite the fact that these structures bind large amounts of FGF-2. Apparently, basement membrane and cell surface HSPGs differ in their ability to mediate the assembly of a FGF/FR signaling complex presumably due to structural differences of the heparan sulfate chains.
Assuntos
Proteoglicanas de Heparan Sulfato/fisiologia , Processamento de Proteína Pós-Traducional , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Células CHO , Células COS , Cricetinae , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Transdução de SinaisRESUMO
The syndecans, a family of cell surface proteoglycans, have highly conserved cytoplasmic domains that bind proteins containing PDZ domains and co-localize with the actin cytoskeleton. The syndecan cytoplasmic domains contain four conserved tyrosine residues, two of which are located within favorable sequences for phosphorylation. Endogenous tyrosine phosphorylation of syndecans-1 and -4 is detected in adherent B82 fibroblasts. Approximately 1.5% of total syndecan is endogenously phosphorylated, while most, if not all, cell surface syndecan is phosphorylated following treatment with the tyrosine phosphatase inhibitor pervanadate. Syndecan phosphorylation is also detected in Raji-S1 and NMuMG cells, but only following treatment with vanadate or pervanadate, suggesting that endogenous phosphorylation is maintained in an "off" state in these cells. Endogenous syndecan phosphorylation in B82 cells is rapidly blocked by genistein (IC50 < 10 microM) confirming the presence of a constitutively active kinase and a corresponding tyrosine phosphatase. Phosphorylation is also inhibited by herbimycin A (IC50 < 1.0 microM) and staurosporine (IC50 < 1.0 nM), suggesting a role for Src family kinases in regulating syndecan phosphorylation. Together, these data suggest an important role for tyrosine phosphorylation of the syndecan cytoplasmic domains in regulating downstream signaling events in response to cell adhesion and/or growth factor activity.
Assuntos
Glicoproteínas de Membrana/metabolismo , Proteoglicanas/metabolismo , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Benzoquinonas , Adesão Celular , Polaridade Celular , Citoplasma , Fibroblastos/metabolismo , Genisteína/farmacologia , Lactamas Macrocíclicas , Dados de Sequência Molecular , Fosforilação , Inibidores de Proteínas Quinases , Quinonas/farmacologia , Rifabutina/análogos & derivados , Estaurosporina/farmacologia , Sindecana-2 , Sindecana-4 , Sindecanas , Tirosina/metabolismo , Quinases da Família src/antagonistas & inibidoresRESUMO
The syndecan family of cell-surface heparan sulfate proteoglycans participate in multiple cell behaviors ranging from growth factor signaling to cell adhesion. Participation in these activities is dependent on specific binding interactions of their heparan sulfate chains and molecular interactions of their core proteins with cytoskeletal and signaling molecules. The highly conserved features of the core proteins have long suggested important functions, which are only now beginning to be understood. Recent advances point to important roles for the extracellular, transmembrane and cytoplasmic domains of the syndecan core proteins in the assembly of these proteoglycans into an intracellular cytoskeletal and signaling apparatus. The proteins display interactions that may be common among the different family members, as well as interactions that provide signaling capabilities that are specific to individual members.
Assuntos
Proteoglicanas de Heparan Sulfato/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteoglicanas/metabolismo , Sequência de Aminoácidos , Animais , Polaridade Celular , Humanos , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , SindecanasRESUMO
The syndecan family of cell surface proteoglycans regulates cell adhesion via their glycosaminoglycan chains and discrete domains of their core proteins. Core protein domains that are variable between syndecan family members may regulate syndecan-specific associations, thereby endowing individual syndecans with unique functions. A syndecan-4-specific domain has been identified in the extracellular syndecan-4 protein. This region mediates cell adhesion when provided as an artificial substratum and is localized within amino acids 56-109 of the recombinant extracellular protein domain of mouse syndecan-4 (mS4ED) (McFall, A. J., and Rapraeger, A. C. (1997) J. Biol. Chem. 272, 12901-12904). To characterize its interaction with the cell surface, radiolabeled ligand binding studies were performed. A single high affinity interaction, with a dissociation constant of 2 x 10(-9) M, was observed between mS4ED and both human and mouse cells. Both chicken S4ED and mS4ED compete for this interaction, although they are only 34% identical within the cell-binding domain sequence. The extracellular protein domains of syndecan-1, -2, and -3, however, fail to compete. The interaction is also observed with native syndecan-4 shed from cell surfaces. Interestingly, the extracellular protein domain of syndecan-1 also mediates cell adhesion, suggesting a similar but discrete interaction for this family member.
Assuntos
Glicoproteínas de Membrana/metabolismo , Proteoglicanas/metabolismo , Animais , Sítios de Ligação , Cátions Bivalentes , Adesão Celular , Galinhas , Endotélio Vascular , Fibroblastos , Humanos , Camundongos , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Ensaio Radioligante , Sindecana-1 , Sindecana-4 , SindecanasRESUMO
Binding of fibroblast growth factors (FGFs) to receptor tyrosine kinases (FGFRs) and signaling is facilitated by binding of FGF to heparan sulfate proteoglycans (HSPGs). There are multiple families of HSPGs, including extracellular and cell surface forms. An important and potentially controversial question is whether cell surface forms of HSPGs act as positive or negative regulators of FGF signaling. This study examines the ability of the cell surface HSPG syndecan-1 to regulate FGF binding and signaling. HSPG-deficient Raji lymphoma cells, expressing a transfected syndecan-1 cDNA (Raji S1 cells), were used as HSPG "donor" cells. BaF3 cells, expressing an FGFR1 cDNA (FR1C-11 cells), were used as FGFR "reporter" cells. Using Raji S1 cells preincubated with FGF, it was found that they formed heterotypic aggregates with FR1C-11 cells in the presence of FGF-2, but not FGF-1. In addition, the FR1C-11 cells demonstrated FGF-2, but not FGF-1, dependent survival when cultured on fixed Raji S1 cells. Thus, Raji syndecan-1 1) differentially regulates the binding and signaling of FGFs 1 and 2 and 2) acts as a positive regulator of FGF-2 signaling.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Glicoproteínas de Membrana/fisiologia , Proteoglicanas/fisiologia , Receptores Proteína Tirosina Quinases , Animais , Linfoma de Burkitt , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/fisiologia , Heparina/metabolismo , Heparitina Sulfato/farmacologia , Humanos , Camundongos , Microesferas , Ligação Proteica/efeitos dos fármacos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/biossíntese , Receptores de Fatores de Crescimento de Fibroblastos/efeitos dos fármacos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Sefarose/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sindecana-1 , Sindecanas , Células Tumorais CultivadasRESUMO
The syndecan family of cell surface proteoglycans regulates cell adhesion and growth factor signaling by binding components of the extracellular matrix and growth factors. To date, all known ligand interactions are via the covalently attached glycosaminoglycan chains. To assay for potential extracellular interactions via the core proteins directly, the recombinant extracellular domain of syndecan-4 (S4ED), one of the four syndecan family members, was tested as a substratum for the attachment of mammalian cells. Human foreskin fibroblasts bind to mouse S4ED, and both mouse and chicken S4ED can block this binding, with 50% inhibition observed between 0.1 and 1 x 10(-7) M. The extracellular domain of another syndecan family member, syndecan-1, fails to compete for cell binding to mouse S4ED. Amino acids 56-109 of the 120-amino acid mouse S4ED compete fully, suggesting that the cell binding domain is within this region. The ability of syndecan-4 to interact with molecules at the cell surface via its core protein as well as its glycosaminoglycan chains may uniquely regulate the formation of cell surface signaling complexes following engagement of this proteoglycan with its extracellular ligands.
Assuntos
Moléculas de Adesão Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteoglicanas/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Ligantes , Glicoproteínas de Membrana/química , Camundongos , Dados de Sequência Molecular , Proteoglicanas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sindecana-4RESUMO
Fibroblast growth factors (FGFs) play multiple roles during development and in adult tissues as paracrine regulators of growth and differentiation. FGFs signal through transmembrane receptor tyrosine kinases, but heparan sulfate is also required for signaling by members of the FGF family. In addition, heparan sulfate may be involved in determining tissue distribution of FGFs. Using biotinylated FGF-2 and FGF-7 (KGF) as probes, we have identified specific interactions between FGFs and heparan sulfates in human tissues. Both FGF species bind to tissue mast cells and to epithelial cell membranes. Binding to basement membrane heparan sulfate is tissue source dependent and specific. Although FGF-2 strongly binds to basement membrane heparan sulfate in skin and most other tissue sites examined, FGF-7 fails to bind to basement membrane heparan sulfate in most locations. However, in subendothelial matrix in blood vessels and in the basement membrane of a papillary renal cell carcinoma, strong FGF-7 binding is seen. In summary, distinct and specific affinities of heparan sulfates for different FGFs were identified that may affect growth factor activation and local distribution. Heparan sulfate may have a gatekeeper function to either restrict or permit diffusion of heparin-binding growth factors across the basement membrane.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos , Substâncias de Crescimento/metabolismo , Heparitina Sulfato/metabolismo , Células 3T3 , Animais , Membrana Basal/metabolismo , Membrana Basal/patologia , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Epiderme/metabolismo , Epiderme/patologia , Fator 10 de Crescimento de Fibroblastos , Fator 7 de Crescimento de Fibroblastos , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Camundongos , Ligação Proteica , Pele/metabolismo , Pele/patologiaRESUMO
Syndecan-1 is a transmembrane haparan sulphate proteoglycan that binds extracellular matrices and growth factors, making it a candidate to act between these regulatory molecules and intracellular signalling pathways. It has a highly conserved transmembrane/cytoplasmic domain that contains four conserved tyrosines. One of these is in a consensus sequence for tyrosine kinase phosphorylation. As an initial step to investigating whether or not phosphorylation of these tyrosines is part of a signal-transduction pathway, we have monitored the tyrosine phosphorylation of syndecan-1 by cytoplasmic tyrosine kinases in intact cells. Tyrosine phosphorylation of syndecan-1 is observed when NMuMG cells are treated with sodium orthovanadate or pervanadate, which have been shown to activate intracellular tyrosine kinases. Initial studies with sodium orthovanadate demonstrate a slow accumulation of phosphotyrosine on syndecan-1 over the course of several hours. Pervanadate, a more effective inhibitor of phosphatases, allows detection of phosphotyrosine on syndecan-1 within 5 min, with peak phosphorylation seen by 15 min. Concurrently, in a second process activated by pervanadate, syndecan-1 ectodomain is cleaved and released into the culture medium. Two phosphorylated fragments of syndecan-1 of apparent sizes 6 and 8 kDa remain with the cell after shedding of the ectodomain. The 8 kDa size class appears to be a highly phosphorylated form of the 6 kDa product, as it disappears if samples are dephosphorylated. These fragments contain the C-terminus of syndecan-1 and also retain at least a portion of the transmembrane domain, suggesting that they are produced by a cell surface cleavage event. Thus pervanadate treatment of cells results in two effects of syndecan-1: (i) phosphorylation of one or more of its tyrosines via the action of a cytoplasmic kinase(s) and (ii) cleavage and release of the ectodomain into the medium, producing a C-terminal fragment containing the transmembrane/cytoplasmic domain.
Assuntos
Glicoproteínas de Membrana/metabolismo , Proteínas Quinases/metabolismo , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteoglicanas/metabolismo , Tirosina/metabolismo , Vanadatos/farmacologia , Animais , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Peróxido de Hidrogênio/farmacologia , Camundongos , Fosforilação , Sindecana-1 , Sindecanas , TransfecçãoRESUMO
FGF-2 activates multiple signaling pathways by a cell surface signaling complex assembled with FGF, its receptor tyrosine kinase, and heparan sulfate proteoglycan. Heparan sulfate binds to a site on the receptor and at least one site on the growth factor. Several models propose an important role for heparan sulfate not only in facilitating FGF-2 binding to its receptor tyrosine kinase but also in promoting signaling via formation of receptor dimers. Such dimers are capable of transphosphorylation of the cytoplasmic domain of the receptor, leading to the generation of phosphotyrosines that are important initiators of intracellular signaling pathways. To explore the participation of heparan sulfates in the formation of a signaling complex that activates these pathways, the binding and activity of FGF-2 on Swiss 3T3 fibroblasts and F32 lymphoid cells is examined with either native or modified forms of heparin. As shown previously, fibroblasts treated with chlorate, which inhibits the sulfation of heparan sulfate and its subsequent binding to FGF-2, display a dramatically reduced response to picomolar concentrations of FGF-2, but binding to receptors and a mitogenic response is restored by heparin. However, the restoration of high-affinity binding is seen only at an optimal concentration of heparin. Excess heparin competes for binding sites within the signaling complex such that high-affinity binding and receptor transphosphorylation are reduced. Despite this, mitogenic signaling is not diminished. A similar result is observed using heparin fragments that promote mitogenesis but not high-affinity binding. These results suggest that the high-affinity signaling complex that is necessary for stable receptor transphosphorylation differs from the signaling complex sufficient for triggering mitogenesis. We speculate that heparan sulfate in vivo participates in two hierarchies of receptor activation. In one, heparan sulfate participates in FGF-2 binding to its receptor tyrosine kinase and activation of mitogenic signaling, perhaps through monomeric receptors or the transient formation of receptor dimers. In the second hierarchy, heparan sulfate participates in the stabilization of a signaling complex that is likely to be comprised of receptor multimers that carry out effective receptor transphosphorylation. A further description of this mechanism may lead to an understanding of how heparan sulfate or its homologues can regulate specific signaling pathways within the cell.
Assuntos
Divisão Celular , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Heparitina Sulfato/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Células 3T3 , Animais , Sítios de Ligação , Cloratos/farmacologia , Reagentes de Ligações Cruzadas/metabolismo , Glicosaminoglicanos/metabolismo , Proteoglicanas de Heparan Sulfato , Heparina/análogos & derivados , Heparina/metabolismo , Heparina/farmacologia , Humanos , Linfócitos/metabolismo , Camundongos , Oligossacarídeos , Fosforilação , Ligação Proteica , Proteoglicanas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Tirosina/metabolismoRESUMO
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.
Assuntos
Linfócitos B/ultraestrutura , Adesão Celular/fisiologia , Glicoproteínas de Membrana/fisiologia , Proteoglicanas/fisiologia , Transdução de Sinais/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Linfoma de Burkitt/patologia , Tamanho Celular , Colchicina/farmacologia , Cicloeximida/farmacologia , Citocalasina D/farmacologia , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , DNA Complementar/genética , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Inibidores da Síntese de Proteínas/farmacologia , Proteoglicanas/química , Proteoglicanas/genética , Proteoglicanas/imunologia , Proteínas Recombinantes/metabolismo , Sindecana-1 , Sindecanas , Transfecção , Células Tumorais CultivadasRESUMO
Fibroblast growth factors and their receptors bind to heparan sulfate glycosaminoglycans. This is thought to promote ligand-receptor binding and enhance signaling by promoting receptor multimerization. Synthetic mimetics designed to occupy these binding sites may provide the means to understand and to regulate FGF signaling.
Assuntos
Proteoglicanas de Heparan Sulfato/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Humanos , Oligossacarídeos/química , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/efeitos dos fármacosRESUMO
Fibroblast growth factors (FGFs) are a family of nine proteins that bind to three distinct types of cell surface molecules: (i) FGF receptor tyrosine kinases (FGFR-1 through FGFR-4); (ii) a cysteine-rich FGF receptor (CFR); and (iii) heparan sulfate proteoglycans (HSPGs). Signaling by FGFs requires participation of at least two of these receptors: the FGFRs and HSPGs form a signaling complex. The length and sulfation pattern of the heparan sulfate chain determines both the activity of the signaling complex and, in part, the ligand specificity for FGFR-1. Thus, the heparan sulfate proteoglycans are likely to play an essential role in signaling. We have recently identified a role for FGF in limb bud development in vivo. In the chick limb bud, ectopic expression of the 18 kDa form of FGF-2 or FGF-2 fused to an artificial signal peptide at its amino terminus causes skeletal duplications. These data, and the observations that FGF-2 is localized to the subjacent mesoderm and the apical ectodermal ridge in the early developing limb, suggest that FGF-2 plays an important role in limb outgrowth. We propose that FGF-2 is an apical ectodermal ridge-derived factor that participates in limb outgrowth and patterning.
Assuntos
Fatores de Crescimento de Fibroblastos/fisiologia , Mesoderma/fisiologia , Músculos/embriologia , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Asas de Animais/embriologia , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Diferenciação Celular , Divisão Celular , Embrião de Galinha , Fatores de Crescimento de Fibroblastos/metabolismo , Proteoglicanas de Heparan Sulfato , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Dados de Sequência Molecular , Músculos/citologia , Osteogênese , Proteoglicanas/química , Proteoglicanas/metabolismo , Transdução de SinaisRESUMO
The integral role of heparan sulfate proteoglycans in FGF signaling provides a potential means of regulating FGF activity. This regulation may be used by the cell, where the modification of heparan sulfate glycosaminoglycans during their synthesis in the Golgi can produce cell type- and potentially ligand-specific sulfation sequences. The description of these sequences will not only provide information on how this regulation is achieved, perhaps lending insight into other heparan sulfate-ligand interactions, but may also discern sulfated mimetics that can be used to disrupt or alter FGF signaling. These mimetics may be useful in the treatment disrupt or alter FGF signaling. These mimetics may be useful in the treatment of disease, or in understanding how FGF signaling via discrete pathways within the cell leads to specific cellular responses, such as activation of mitogenic signaling pathways, calcium fluxes, and cellular differentiation.