RESUMEN
Skeletal muscle demonstrates a high degree of regenerative capacity repeating the embryonic myogenic program under strict control. Rhabdomyosarcoma is the most common sarcoma in childhood and is characterized by impaired muscle differentiation. In this study, we observed that silencing the expression of syndecan-4, the ubiquitously expressed transmembrane heparan sulfate proteoglycan, significantly enhanced myoblast differentiation, and fusion. During muscle differentiation, the gradually decreasing expression of syndecan-4 allows the activation of Rac1, thereby mediating myoblast fusion. Single-molecule localized superresolution direct stochastic optical reconstruction microscopy (dSTORM) imaging revealed nanoscale changes in actin cytoskeletal architecture, and atomic force microscopy showed reduced elasticity of syndecan-4-knockdown cells during fusion. Syndecan-4 copy-number amplification was observed in 28% of human fusion-negative rhabdomyosarcoma tumors and was accompanied by increased syndecan-4 expression based on RNA sequencing data. Our study suggests that syndecan-4 can serve as a tumor driver gene in promoting rabdomyosarcoma tumor development. Our results contribute to the understanding of the role of syndecan-4 in skeletal muscle development, regeneration, and tumorigenesis.
Asunto(s)
Actinas/metabolismo , Rabdomiosarcoma/patología , Sindecano-4/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Citoesqueleto de Actina , Animales , Diferenciación Celular , Línea Celular , Variaciones en el Número de Copia de ADN , Humanos , Masculino , Ratones , Desarrollo de Músculos , Músculo Esquelético/metabolismo , Mioblastos/citología , Mioblastos/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Wistar , Rabdomiosarcoma/metabolismo , Sindecano-4/antagonistas & inhibidores , Sindecano-4/genética , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T/metabolismoRESUMEN
Background: YKL-40, a secreted glycoprotein, has a role in promoting tumor angiogenesis through syndecan-1 receptor. Syndecan-4 is a member of syndecan family. However, the effects of YKL-40 on migration and tube formation of human umbilical vein cells (HUVECs) mediated by syndecan-4 receptor are unknown. Materials and methods: HUVECs were transfected with lentivirus encoding syndecan-4 short hairpin (sh) RNAs (lenti-synd4 shRNAs) and the efficiency of transfection was measured using qRT-PCR and western blotting. The effects of recombinant protein of YKL-40 on migration and angiogenesis of HUVECs adjusted by syndecan-4 were determined by wound healing and tube formation assay. The expressions of protein kinase Cα (PKCα) and extracellular signal regulated kinases (ERKs) 1 and 2 (ERK1/2) in HUVECs were measured using western blotting. Results: The mRNA and protein expression of syndecan-4 were significantly decreased in HUVECs successfully transfected with lenti-synd4 shRNAs. Lenti-synd4 shRNAs remarkably inhibited the migration and tube formation of HUVECs stimulated by recombinant protein of YKL-40. The levels of PKCα and ratio of p-ERK1/2 to ERK1/2 in HUVECs were also decreased by down-regulating syndecan-4. Conclusion: The effects of YKL-40 on migration and tube formation of HUVECs are partly inhibited by knock-downing syndecan-4 through suppressing PKCα and ERK1/2 signaling pathways.
Asunto(s)
Proteína 1 Similar a Quitinasa-3/fisiología , Células Endoteliales de la Vena Umbilical Humana/fisiología , Neovascularización Fisiológica/genética , Sindecano-4/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Células Cultivadas , Proteína 1 Similar a Quitinasa-3/antagonistas & inhibidores , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Neovascularización Fisiológica/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sindecano-4/antagonistas & inhibidoresRESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel emerging pathogen causing an unprecedented pandemic in 21st century medicine. Due to the significant health and economic burden of the current SARS-CoV-2 outbreak, there is a huge unmet medical need for novel interventions effectively blocking SARS-CoV-2 infection. Unknown details of SARS-CoV-2 cellular biology hamper the development of potent and highly specific SARS-CoV-2 therapeutics. Angiotensin-converting enzyme-2 (ACE2) has been reported to be the primary receptor for SARS-CoV-2 cellular entry. However, emerging scientific evidence suggests the involvement of additional membrane proteins, such as heparan sulfate proteoglycans, in SARS-CoV-2 internalization. Here, we report that syndecans, the evolutionarily conserved family of transmembrane proteoglycans, facilitate the cellular entry of SARS-CoV-2. Among syndecans, the lung abundant syndecan-4 was the most efficient in mediating SARS-CoV-2 uptake. The S1 subunit of the SARS-CoV-2 spike protein plays a dominant role in the virus's interactions with syndecans. Besides the polyanionic heparan sulfate chains, other parts of the syndecan ectodomain, such as the cell-binding domain, also contribute to the interaction with SARS-CoV-2. During virus internalization, syndecans colocalize with ACE2, suggesting a jointly shared internalization pathway. Both ACE2 and syndecan inhibitors exhibited significant efficacy in reducing the cellular entry of SARS-CoV-2, thus supporting the complex nature of internalization. Data obtained on syndecan specific in vitro assays present syndecans as novel cellular targets of SARS-CoV-2 and offer molecularly precise yet simple strategies to overcome the complex nature of SARS-CoV-2 infection.
Asunto(s)
COVID-19/metabolismo , Receptores de Coronavirus/metabolismo , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/metabolismo , Sindecanos/metabolismo , Internalización del Virus , Amilorida/farmacología , Enzima Convertidora de Angiotensina 2/antagonistas & inhibidores , Enzima Convertidora de Angiotensina 2/metabolismo , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , COVID-19/virología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Bloqueadores del Canal de Sodio Epitelial/farmacología , Humanos , Péptidos/farmacología , Dominios Proteicos , SARS-CoV-2/metabolismo , Sindecano-4/antagonistas & inhibidores , Sindecano-4/metabolismo , Sindecanos/antagonistas & inhibidoresRESUMEN
The membranous receptor syndecan-4 (SDC-4) and the nuclear transcription factor hypoxia-induced factor-2α (HIF-2α) play critical roles in the pathogenesis of osteoarthritis (OA). The aim of this study was to determine whether SDC-4 inhibition downregulates HIF-2a expression by microRNA-96-5p (miR-96-5p) in murine chondrocyte and cartilage tissue. The OA model was induced surgically in mice, and SDC-4 polyclonal antibody, HIF-2α small interfering RNA (siRNA) and its control, miR-96-5p mimics and its scrambled controls or anti-miR-96-5p and its control were then injected into the knee joints. At 2 and 4 weeks after surgery, OA progression was evaluated microscopically, histologically, radiographically and immunohistochemically in these mice. Real-time polymerase chain reaction (RT-PCR) and western blotting were performed after treating with antibody and transfecting with miRNA mimic or siRNA to determine their effects on OA-related mediators. The potential miRNAs related to OA development were identified by using miRNA microarray analysis. Whether miRNAs play a pivotal role in OA development in vivo or in vitro was also investigated. MiR-96-5p expression was upregulated by SDC-4-specific antibodies in chondrocytes and cartilage tissue, and miR-96-5p directly targeted the 3'-UTR of HIF-2α to inhibit HIF-2α signaling in murine chondrocytes. Moreover, we demonstrated that anti-SDC-4-attenuated IL-1ß-induced chondrocyte hypertrophy and cartilage degradation by inhibiting HIF-2α signaling by a miR-96-5p-dependent mechanism. Our study revealed that the inhibition of SDC-4 exerts its effects on both cartilage homeostasis and the chondrocyte hypertrophy phenotype by inducing miR-96-5p expression, which results in targeting HIF-2α 3'-UTR sequences and inhibiting HIF-2α in murine cartilage tissue and chondrocytes.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Cartílago Articular , MicroARNs , Osteoartritis , Sindecano-4 , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cartílago Articular/metabolismo , Cartílago Articular/patología , Condrocitos/metabolismo , Regulación hacia Abajo/genética , Masculino , Ratones , Ratones Endogámicos ICR , MicroARNs/genética , MicroARNs/metabolismo , Osteoartritis/genética , Osteoartritis/metabolismo , Osteoartritis/patología , Sindecano-4/antagonistas & inhibidores , Sindecano-4/genética , Sindecano-4/metabolismoRESUMEN
OBJECTIVE: Syndecan-4 (sdc4) is a cell-anchored proteoglycan that consists of a transmembrane core protein and glucosaminoglycan (GAG) side chains. Binding of soluble factors to the GAG chains of sdc4 may result in the dimerisation of sdc4 and the initiation of downstream signalling cascades. However, the question of how sdc4 dimerisation and signalling affects the response of cells to inflammatory stimuli is unknown. METHODS: Sdc4 immunostaining was performed on rheumatoid arthritis (RA) tissue sections. Interleukin (IL)-1 induced extracellular signal-regulated kinases (ERK) phosphorylation and matrix metalloproteinase-3 production was investigated. Il-1 binding to sdc4 was investigated using immunoprecipitation. IL-1 receptor (IL1R1) staining on wild-type, sdc4 and IL1R1 knockout fibroblasts was performed in fluorescence-activated cell sorting analyses. A blocking sdc4 antibody was used to investigate sdc4 dimerisation, IL1R1 expression and the histological paw destruction in the human tumour necrosis factor-alpha transgenic mouse. RESULTS: We show that in fibroblasts, the loss of sdc4 or the antibody-mediated inhibition of sdc4 dimerisation reduces the cell surface expression of the IL-1R and regulates the sensitivity of fibroblasts to IL-1. We demonstrate that IL-1 directly binds to sdc4 and in an IL-1R-independent manner leads to its dimerisation. IL-1-induced dimerisation of sdc4 regulates caveolin vesicle-mediated trafficking of the IL1R1, which in turn determines the responsiveness to IL-1. Administration of antibodies (Ab) against the dimerisation domain of sdc4, thus, strongly reduces the expression IL1R1 on arthritic fibroblasts both in vitro and an animal model of human RA. CONCLUSION: Collectively, our data suggest that Ab that specifically inhibit sdc4 dimerisation may support anti-IL-1 strategies in diseases such as inflammatory arthritis.
Asunto(s)
Anticuerpos Bloqueadores/farmacología , Artritis Reumatoide/metabolismo , Receptores Tipo I de Interleucina-1/efectos de los fármacos , Sindecano-4/antagonistas & inhibidores , Animales , Artritis Reumatoide/genética , Artritis Reumatoide/patología , Dimerización , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Heparitina Sulfato , Miembro Posterior , Humanos , Interleucina-1/metabolismo , Interleucina-1beta/metabolismo , Sistema de Señalización de MAP Quinasas , Metaloproteinasa 3 de la Matriz/metabolismo , Ratones , Ratones Transgénicos , Células 3T3 NIH , Osteoartritis/genética , Osteoartritis/metabolismo , Osteoartritis/patología , Fosforilación/efectos de los fármacos , Transporte de Proteínas , Receptores Tipo I de Interleucina-1/metabolismo , Transducción de Señal , Sindecano-4/genética , Sindecano-4/metabolismo , Membrana Sinovial/metabolismo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Dysregulation of Wnt signaling has been implicated in developmental defects and in the pathogenesis of many diseases such as osteoarthritis; however, the underlying mechanisms are poorly understood. Here, we report that non-canonical Wnt signaling induced loss of chondrocyte phenotype through activation of Fz-6/DVL-2/SYND4/CaMKIIα/B-raf/ERK1/2 cascade. We show that in response to Wnt-3a, Frizzled 6 (Fz-6) triggers the docking of CaMKIIα to syndecan 4 (SYND4) and that of B-raf to DVL-2, leading to the phosphorylation of B-raf by CaMKIIα and activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling, which leads to chondrocyte de-differentiation. We demonstrate that CaMKIIα associates and phosphorylates B-raf in vitro and in vivo. Our study reveals the mechanism by which non-canonical Wnt activates ERK1/2 signaling that induces loss of chondrocyte phenotype, and demonstrates a direct functional relationship between CaMKIIα and B-raf during chondrocyte de-differentiation. The identification of Fz-6, SYND4, and B-raf as novel physiological regulators of chondrocyte phenotype may provide new potential anti-osteoarthritic targets.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Desdiferenciación Celular , Proteínas Dishevelled/metabolismo , Receptores Frizzled/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Sindecano-4/metabolismo , Proteínas Wnt/metabolismo , Células Cultivadas , Condrocitos/citología , Condrocitos/metabolismo , Proteínas Dishevelled/antagonistas & inhibidores , Proteínas Dishevelled/genética , Receptores Frizzled/antagonistas & inhibidores , Receptores Frizzled/genética , Humanos , Sistema de Señalización de MAP Quinasas , Osteoartritis/metabolismo , Osteoartritis/patología , Fenotipo , Fosforilación , Proteínas Proto-Oncogénicas B-raf/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Sindecano-4/antagonistas & inhibidores , Sindecano-4/genética , beta Catenina/antagonistas & inhibidores , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
The most common complication after cataract surgery is postoperative capsular opacification, which includes anterior capsular opacification (ACO) and posterior capsular opacification (PCO). Increased adhesion of lens epithelial cells (LECs) to the intraocular lens material surface promotes ACO formation, whereas proliferation and migration of LECs to the posterior capsule lead to the development of PCO. Cell adhesion is mainly mediated by the binding of integrin to extracellular matrix proteins, while cell proliferation and migration are regulated by fibroblast growth factor (FGF). Syndecan-4 (SDC-4) is a co-receptor for both integrin and FGF signaling pathways. Therefore, SDC-4 may be an ideal therapeutic target for the prevention and treatment of postoperative capsular opacification. However, how SDC-4 contributes to FGF-mediated proliferation, migration, and integrin-mediated adhesion of LECs is unclear. Here, we found that downregulation of SDC-4 inhibited FGF signaling through the blockade of ERK1/2 and PI3K/Akt/mTOR activation, thus suppressing cell proliferation and migration. In addition, downregulation of SDC-4 suppressed integrin-mediated cell adhesion through inhibiting focal adhesion kinase (FAK) phosphorylation. Moreover, SDC-4 knockout mice exhibited normal lens morphology, but had significantly reduced capsular opacification after injury. Finally, SDC-4 expression level was increased in the anterior capsule LECs of age-related cataract patients. Taken together, we for the first time characterized the key regulatory role of SDC-4 in FGF and integrin signaling in human LECs, and provided the basis for future pharmacological interventions of capsular opacification.
Asunto(s)
Opacificación Capsular/patología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Integrinas/metabolismo , Transducción de Señal/efectos de los fármacos , Sindecano-4/metabolismo , Animales , Cámara Anterior/metabolismo , Cámara Anterior/patología , Opacificación Capsular/metabolismo , Opacificación Capsular/cirugía , Adhesión Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Células Epiteliales/citología , Células Epiteliales/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Cristalino/citología , Ratones , Ratones Noqueados , Proteínas Quinasas/metabolismo , Interferencia de ARN , Retina/metabolismo , Retina/patología , Sindecano-4/antagonistas & inhibidores , Sindecano-4/genéticaRESUMEN
Proteoglycans are macromolecules that consist of a core protein and one or more glycosaminoglycan side chains. Previously, we reported that transforming growth factor-ß1 (TGF-ß1 ) regulates the synthesis of a large heparan sulfate proteoglycan, perlecan, and a small leucine-rich dermatan sulfate proteoglycan, biglycan, in vascular endothelial cells depending on cell density. Recently, we found that TGF-ß1 first upregulates and then downregulates the expression of syndecan-4, a transmembrane heparan sulfate proteoglycan, via the TGF-ß receptor ALK5 in the cells. In order to identify the intracellular signal transduction pathway that mediates this modulation, bovine aortic endothelial cells were cultured and treated with TGF-ß1 . Involvement of the downstream signaling pathways of ALK5-the Smad and MAPK pathways-in syndecan-4 expression was examined using specific siRNAs and inhibitors. The data indicate that the Smad3-p38 MAPK pathway mediates the early upregulation of syndecan-4 by TGF-ß1 , whereas the late downregulation is mediated by the Smad2/3 pathway. Multiple modulations of proteoglycan synthesis may be involved in the regulation of vascular endothelial cell functions by TGF-ß1 . J. Cell. Biochem. 118: 2009-2017,2017. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Proteína Smad2/genética , Proteína smad3/genética , Sindecano-4/genética , Factor de Crecimiento Transformador beta1/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/metabolismo , Bovinos , Células Endoteliales/citología , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Proteína Smad2/antagonistas & inhibidores , Proteína Smad2/metabolismo , Proteína smad3/antagonistas & inhibidores , Proteína smad3/metabolismo , Sindecano-4/antagonistas & inhibidores , Sindecano-4/metabolismo , Factores de Tiempo , Factor de Crecimiento Transformador beta1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Pulmonary tuberculosis (TB) is an airborne disease caused by the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb). Alveolar epithelial cells and macrophages are the first point of contact for Mtb in the respiratory tract. However, the mechanisms of mycobacterial attachment to, and internalization by, nonprofessional phagocytes, such as epithelial cells, remain incompletely understood. We identified syndecan 4 (Sdc4) as mycobacterial attachment receptor on alveolar epithelial cells. Sdc4 mRNA expression was increased in human and mouse alveolar epithelial cells after mycobacterial infection. Sdc4 knockdown in alveolar epithelial cells or blocking with anti-Sdc4 antibody reduced mycobacterial attachment and internalization. At the molecular level, interactions between epithelial cells and mycobacteria involved host Sdc and the mycobacterial heparin-binding hemagglutinin adhesin. In vivo, Sdc1/Sdc4 double-knockout mice were more resistant to Mtb colonization of the lung. Our work reveals a role for distinct Sdcs in promoting mycobacterial entry into alveolar epithelial cells with impact on outcome of TB disease.
Asunto(s)
Células Epiteliales/microbiología , Interacciones Huésped-Patógeno , Pulmón/microbiología , Sindecano-4/inmunología , Tuberculosis Pulmonar/inmunología , Células A549 , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Animales , Anticuerpos Neutralizantes/farmacología , Adhesión Bacteriana/efectos de los fármacos , Células Epiteliales/inmunología , Regulación de la Expresión Génica , Humanos , Pulmón/inmunología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/microbiología , Ratones , Ratones Noqueados , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , ARN Mensajero/genética , ARN Mensajero/inmunología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Sindecano-1/deficiencia , Sindecano-1/genética , Sindecano-1/inmunología , Sindecano-4/antagonistas & inhibidores , Sindecano-4/deficiencia , Sindecano-4/genética , Tuberculosis Pulmonar/microbiología , Tuberculosis Pulmonar/patologíaRESUMEN
In this study, we examined the role(s) of syndecan-4 in regulating the formation of an actin geodesic dome structure called a cross-linked actin network (CLAN) in which syndecan-4 has previously been localized. CLANs have been described in several different cell types, but they have been most widely studied in human trabecular meshwork (HTM) cells where they may play a key role in controlling intraocular pressure by regulating aqueous humor outflow from the eye. In this study we show that a loss of cell surface synedcan-4 significantly reduces CLAN formation in HTM cells. Analysis of HTM cultures treated with or without dexamethasone shows that laminin 5 deposition within the extracellular matrix is increased by glucocorticoid treatment and that a laminin 5-derived, syndecan-4-binding peptide (PEP75), induces CLAN formation in TM cells. This PEP75-induced CLAN formation was inhibited by heparin and the broad spectrum PKC inhibitor Ro-31-7549. In contrast, the more specific PKCα inhibitor Gö 6976 had no effect, thus excluding PKCα as a downstream effector of syndecan-4 signaling. Analysis of PKC isozyme expression showed that HTM cells also expressed both PKCγ and PKCε. Cells treated with a PKCε agonist formed CLANs while a PKCα/γ agonist had no effect. These data suggest that syndecan-4 is essential for CLAN formation in HTM cells and that a novel PKCε-mediated signaling pathway can regulate formation of this unique actin structure.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Fragmentos de Péptidos/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Sindecano-4/metabolismo , Malla Trabecular/citología , Actinas/genética , Adolescente , Adulto , Antiinflamatorios/farmacología , Western Blotting , Moléculas de Adhesión Celular/genética , Células Cultivadas , Reactivos de Enlaces Cruzados/farmacología , Dexametasona/farmacología , Oftalmopatías/metabolismo , Oftalmopatías/patología , Técnica del Anticuerpo Fluorescente , Humanos , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Sindecano-4/antagonistas & inhibidores , Sindecano-4/genética , Malla Trabecular/efectos de los fármacos , Malla Trabecular/metabolismo , KalininaRESUMEN
The phosphatidylinositol 3 kinase (Pi3K)/Akt pathway is a major regulator of cell growth, proliferation, metabolism, survival, and angiogenesis. Despite extensive study, a thorough understanding of the modulation and regulation of this pathway has remained elusive. We have previously demonstrated that syndecan 4 (S4) regulates the intracellular localization of mTORC2, thus altering phosphorylation of Akt at serine473 (Ser473), one of two critical phosphorylation sites essential for the full activation of Akt [1]. Here we report that S4 also regulates the phosphorylation of Akt at threonine308 (Thr308), the second phosphorylation site required for the full Akt activation. A deletion of S4 resulted in lower levels of Thr308 phosphorylation both in vitro and in vivo. Furthermore, a deletion or knockdown of the S4 effector molecule PKCα led to a similar reduction in phosphorylation of Thr308 while overexpression of myristoylated PKCα rescued AktThr308 phosphorylation in endothelial cells lacking S4. Finally, PAK1/2 is also recruited to the rafts by the S4-PKCα complex and is required for AKT activation.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sindecano-4/metabolismo , Animales , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/metabolismo , Activación Enzimática , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteína Quinasa C-alfa/antagonistas & inhibidores , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Sindecano-4/antagonistas & inhibidores , Sindecano-4/genética , Quinasas p21 Activadas/metabolismoRESUMEN
Folliculostellate (FS) cells in the anterior pituitary gland appear to have multifunctional properties. FS cells connect to each other at gap junctions and thereby form a histological and functional network. We have performed a series of studies on network formation in FS cells and recently reported that FS cells markedly prolong their cytoplasmic processes and form numerous interconnections with neighboring FS cells in the presence of laminin, an extracellular matrix (ECM) component of the basement membrane. In this study, we investigated the mechanism of this extension of FS cell cytoplasmic processes under the influence of laminin and found that laminin promoted stress fiber formation within FS cells. Next, we noted that formation of stress fibers in FS cells was mediated by syndecan-4, a transmembrane proteoglycan that binds ECM and soluble factors via their extracellular glycosaminoglycan chain. We then observed that expressions of syndecan-4 and α-actinin (a microfilament bundling protein that cross-links actin stress fibers in FS cells) were upregulated by laminin. Using specific siRNA of syndecan-4, actin polymerization of FS cells was inhibited. Our findings suggest that FS cells received a signal from laminin-syndecan-4 interaction, which resulted in morphological changes, and that the formation of a morphological and functional network in FS cells was transduced by a syndecan-4-dependent mechanism in the presence of ECM.
Asunto(s)
Adenohipófisis/citología , Adenohipófisis/metabolismo , Fibras de Estrés/metabolismo , Sindecano-4/genética , Animales , Células Cultivadas , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Laminina/metabolismo , Masculino , Factores de Crecimiento Nervioso/genética , Adenohipófisis/efectos de los fármacos , Adenohipófisis/fisiología , Unión Proteica/efectos de los fármacos , Proteoglicanos/genética , Proteoglicanos/metabolismo , ARN Interferente Pequeño/farmacología , Ratas , Ratas Transgénicas , Subunidad beta de la Proteína de Unión al Calcio S100 , Proteínas S100/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Fibras de Estrés/genética , Sindecano-4/antagonistas & inhibidores , Sindecano-4/metabolismoRESUMEN
Aggrecan cleavage by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 5 (ADAMTS-5) is crucial for the breakdown of cartilage matrix during osteoarthritis, a degenerative joint disease that leads to the progressive destruction of articular structures. The mechanisms of ADAMTS-5 activation and their links to the pathogenesis of osteoarthritis remain poorly understood, but syndecans have been shown to be involved in the activation of ADAMTS-4 (ref. 3). Here we show that syndecan-4 is specifically induced in type X collagen-producing chondrocytes both in human osteoarthritis and in murine models of the disease. The loss of syndecan-4 in genetically modified mice and intra-articular injections of syndecan-4-specific antibodies into wild-type mice protect from proteoglycan loss and thereby prevent osteoarthritic cartilage damage in a surgically induced model of osteoarthritis. The occurrence of less severe osteoarthritis-like cartilage destruction in both syndecan-4-deficient mice and syndecan-4-specific antibody-treated wild-type mice results from a marked decrease in ADAMTS-5 activity. Syndecan-4 controls the activation of ADAMTS-5 through direct interaction with the protease and through regulating mitogen-activated protein kinase (MAPK)-dependent synthesis of matrix metalloproteinase-3 (MMP-3). Our data suggest that strategies aimed at the inhibition of syndecan-4 will be of great value for the treatment of cartilage damage in osteoarthritis.
Asunto(s)
Proteínas ADAM/fisiología , Osteoartritis/etiología , Osteoartritis/fisiopatología , Sindecano-4/fisiología , Proteínas ADAM/genética , Proteína ADAMTS5 , Animales , Cartílago/patología , Condrocitos/patología , Condrocitos/fisiología , Colágeno Tipo X/biosíntesis , Modelos Animales de Enfermedad , Humanos , Sistema de Señalización de MAP Quinasas , Metaloproteinasa 3 de la Matriz/deficiencia , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/fisiología , Ratones , Ratones Noqueados , Osteoartritis/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Sindecano-4/antagonistas & inhibidores , Sindecano-4/deficiencia , Sindecano-4/genéticaRESUMEN
Glycosaminoglycan (GAG) sugars are largely responsible for the bioactivity of the proteoglycan proteins they decorate, and are particularly important for mediating the processes of cell attachment and growth factor signaling. Here, we show that chlorate-induced de-sulfation of GAGs expressed by MG-63 osteosarcoma cells results in delayed cell proliferation when the cells are exposed to chlorate for short or medium periods, but a disrupted mineralization without altered cell proliferation in response to long-term chlorate exposure. Analysis of GAG-binding growth factor activity indicated that chlorate disrupted BMP2/noggin signaling, but not FGF2 activity. Microarray analyses, which were confirmed by subsequent cell-based assays, indicated that chlorate predominantly disrupted the cell cycle and actin cytoskeleton and upregulated cholesterol synthesis, without affecting cell migration or attachment. Furthermore, we observed that disruption of the functions of the proteoglycan syndecan-4 replicated phenotypes induced by chlorate, implicating a primary role for this proteoglycan in providing bioactivity for these cells. J. Cell. Physiol. 219: 572-583, 2009. (c) 2009 Wiley-Liss, Inc.
Asunto(s)
Actinas/metabolismo , Ciclo Celular/fisiología , Colesterol/biosíntesis , Glicosaminoglicanos/metabolismo , Osteogénesis/fisiología , Proteína Morfogenética Ósea 2/metabolismo , Proteínas Portadoras/metabolismo , Adhesión Celular/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cloratos/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Glicosaminoglicanos/química , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Osteosarcoma/metabolismo , Osteosarcoma/patología , Transducción de Señal/efectos de los fármacos , Sulfatos/química , Sindecano-4/antagonistas & inhibidores , Sindecano-4/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The ability of cells to respond to external mechanical stimulation is a complex and robust process involving a diversity of molecular interactions. Although mechanotransduction has been heavily studied, many questions remain regarding the link between physical stimulation and biochemical response. Of significant interest has been the contribution of the transmembrane proteins involved, and integrins in particular, because of their connectivity to both the extracellular matrix and the cytoskeleton. Here, we demonstrate the existence of a mechanically based initiation molecule, syndecan-4. We first demonstrate the ability of syndecan-4 molecules to support cell attachment and spreading without the direct extracellular binding of integrins. We also examine the distribution of focal adhesion-associated proteins through controlling surface interactions of beads with molecular specificity in binding to living cells. Furthermore, after adhering cells to elastomeric membranes via syndecan-4-specific attachments we mechanically strained the cells via our mechanical stimulation and polymer surface chemical modification approach. We found ERK phosphorylation similar to that shown for mechanotransductive response for integrin-based cell attachments through our elastomeric membrane-based approach and optical magnetic twisting cytometry for syndecan-4. Finally, through the use of cytoskeletal disruption agents, this mechanical signaling was shown to be actin cytoskeleton dependent. We believe that these results will be of interest to a wide range of fields, including mechanotransduction, syndecan biology, and cell-material interactions.
Asunto(s)
Mecanotransducción Celular/fisiología , Sindecano-4/fisiología , Animales , Anticuerpos , Bioingeniería , Fenómenos Biomecánicos , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Fibronectinas/fisiología , Integrinas/fisiología , Sistema de Señalización de MAP Quinasas , Mecanotransducción Celular/efectos de los fármacos , Ratones , Modelos Biológicos , Células 3T3 NIH , Unión Proteica , Propiedades de Superficie , Sindecano-4/antagonistas & inhibidores , Sindecano-4/inmunología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
In addition to their physiologic effects in inflammation and angiogenesis, chemokines are involved in cancer pathology. The aim of this study was to determine whether the chemokine stromal cell-derived factor 1 (SDF-1) induces the growth, migration, and invasion of human hepatoma cells. We show that SDF-1 G protein-coupled receptor, chemokine (C-X-C motif) receptor 4 (CXCR4), and SDF-1 mRNA are expressed in human hepatoma Huh7 cells, which secrete and bind SDF-1. This binding depends on CXCR4 and glycosaminoglycans. SDF-1 associates with CXCR4, and syndecan-4 (SDC-4), a heparan sulfate proteoglycan at the plasma membrane of Huh7 cells, induces the growth of Huh7 cells by promoting their entry into the cell cycle, and inhibits the tumor necrosis factor-alpha-mediated apoptosis of the cells. SDF-1 also reorganizes Huh7 cytoskeleton and induces tyrosine phosphorylation of focal adhesion kinase. Finally, SDF-1 activates matrix metalloproteinase-9, resulting in increased migration and invasion of Huh7 cells. These biological effects of SDF-1 were strongly inhibited by the CXCR4 antagonist AMD3100, by a glycosaminoglycan, heparin, as well as by beta-D-xyloside treatment of the cells, or by c-jun NH(2)-terminal kinase/stress-activated protein kinase inhibitor. Therefore, the CXCR4, glycosaminoglycans, and the mitogen-activated protein kinase signaling pathways are involved in these events. The fact that reducing SDC-4 expression by RNA interference decreased SDF-1-induced Huh7 hepatoma cell migration and invasion strongly indicates that SDC-4 may be an auxiliary receptor for SDF-1. Finally, the fact that CXCR4 is expressed in hepatocellular carcinoma cells from liver biopsies indicates that the in vitro results reported here could be extended to in vivo conditions.