scholarly journals Requirements for sulfate transport and the diastrophic dysplasia sulfate transporter in fibronectin matrix assembly

2007 ◽  
Vol 179 (5) ◽  
pp. 999-1009 ◽  
Author(s):  
Leontine L. Galante ◽  
Jean E. Schwarzbauer
Keyword(s):  
Cell Surface ◽  
Sulfate Transporter ◽  
Matrix Assembly ◽  
Diastrophic Dysplasia ◽  
Matrix Deposition ◽  
Fibrosarcoma Cells ◽  
Extracellular Matrix Deposition ◽  
Necessary And Sufficient ◽  
Rna Knockdown ◽  
Interfering Rna

Diastrophic dysplasia sulfate transporter (DTDST) is a sulfate/chloride antiporter whose function is impaired in several human chondrodysplasias. We show that DTDST is upregulated by dexamethasone stimulation of HT1080 fibrosarcoma cells and is required for fibronectin (FN) extracellular matrix deposition by these cells. DTDST imports sulfate for the modification of glycosaminoglycans. We find that N-sulfation of these chains is important for FN matrix assembly and that sulfation of cell surface proteoglycans is reduced in the absence of DTDST. Of the candidate HT1080 cell surface proteoglycans, only loss of syndecan-2 compromises FN assembly, as shown by syndecan-2 small interfering RNA knockdown. DTDST is both necessary and sufficient to induce FN matrix assembly in HT1080 cells. Knockdown of DTDST ablates FN matrix, whereas its overexpression increases assembly without dexamethasone stimulation. These results identify a previously unrecognized regulatory pathway for matrix assembly via modulation of a sulfate transporter and proteoglycan sulfation. These data raise the possibility that FN assembly defects contribute to chondrodysplasias.

scholarly journals Giantin is required for intracellular N-terminal processing of type I procollagen

2020 ◽  
Author(s):  
Nicola L. Stevenson ◽  
J. M. Bergen Dylan ◽  
Chrissy L. Hammond ◽  
David J. Stephens
Keyword(s):  
Extracellular Matrix ◽  
Type I Collagen ◽  
Fish Analysis ◽  
Type I ◽  
Matrix Assembly ◽  
Matrix Deposition ◽  
Type I Procollagen ◽  
Main Protein ◽  
Extracellular Matrix Deposition ◽  
Craniofacial Defects

AbstractKnockout of the golgin giantin leads to skeletal and craniofacial defects driven by poorly studied changes in glycosylation and extracellular matrix deposition. Here, we sought to determine how giantin impacts the production of healthy bone tissue by focussing on the main protein component of the osteoid, type I collagen. Giantin mutant zebrafish accumulate multiple spontaneous fractures in their caudal fin, suggesting their bones may be more brittle. Inducing new experimental fractures revealed defects in the mineralisation of newly deposited collagen as well as diminished procollagen reporter expression in mutant fish. Analysis of giantin knockout cells expressing a GFP-tagged procollagen showed that procollagen trafficking is independent of giantin. However, our data show that intracellular N-propeptide processing of pro-α1(I) is defective in the absence of giantin. These data demonstrate a conserved role for giantin in collagen biosynthesis and extracellular matrix assembly. Our work also provides evidence of a giantin-dependent pathway for intracellular procollagen processing.

Localization of urokinase to focal adhesions by human fibrosarcoma cells synthesizing recombinant vitronectin

1996 ◽  
Vol 74 (6) ◽  
pp. 899-910 ◽  
Author(s):  
Sarah A. Wilcox ◽  
Thomas Reho ◽  
Eiman Tominna-Sebald ◽  
Paula J. Mckeown-Longo ◽  
Paul J. Higgins
Keyword(s):  
Cell Migration ◽  
Cell Surface ◽  
Intracellular Signaling ◽  
De Novo ◽  
Focal Adhesions ◽  
Migration And Invasion ◽  
Cdna Insert ◽  
Transfected Cells ◽  
Matrix Deposition ◽  
Fibrosarcoma Cells

Cell surface plasminogen activators have been proposed to participate in cell migration and invasion by activating both intracellular signaling pathways and extracellular proteolysis. Urokinase-type plasminogen activator (uPA) is secreted from many cell types and localizes to focal contact areas when cells are seeded onto the plasma protein vitronectin. Induction of vitronectin synthesis during migration of neural crest cells and growth of certain tumors suggests that the de novo synthesis and deposition of vitronectin into the tissue matrix may remodel the matrix to provide an environment suitable for cell migration and (or) tumor invasion. To investigate the effects of vitronectin secretion and matrix deposition on the localization and activity of cell-associated uPA, HT-1080 fibrosarcoma cells were transfected with the Rc/CMV expression vector containing a vitronectin cDNA insert and stable cell lines expressing vitronectin were selected. Vitronectin-secreting cells were allowed to attach and spread on collagen- and fibronectin-coated substrates. Within 6 h, vitronectin was detected on the substrate; vitronectin synthesis was accompanied by the clustering of both the αvβ5 vitronectin receptor and uPA into vinculin-containing focal adhesions. Although mock transfected cells formed small focal adhesions on both collagen and fibronectin, no co-localization of uPA or αvβ5 to focal adhesions was evident in these cells. Vitronectin-secreting cells also exhibited decreased levels of plasminogen activation and increased levels of cell adhesion as compared with the mock transfected cells. These data demonstrate that the synthesis of vitronectin and its matrix association by transfected HT-1080 fibrosarcoma cells results in localization of uPA to oαvβ5 containing focal adhesions, decreased cell surface uPA activity, and an increase in cell adhesion.Key words: urokinase, vitronectin, focal adhesions, αvβ5 integrin.

scholarly journals Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

2011 ◽  
Vol 301 (4) ◽  
pp. C780-C791 ◽  
Author(s):  
Jane E. Murphy ◽  
Dirk Roosterman ◽  
Graeme S. Cottrell ◽  
Benjamin E. Padilla ◽  
Micha Feld ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein Phosphatase ◽  
Binding Sites ◽  
Protein Phosphatase 2A ◽  
Surface Binding ◽  
Neurokinin 1 Receptor ◽  
Phosphatase 2A ◽  
Neurokinin 1 ◽  
Rna Knockdown ◽  
Interfering Rna

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK1R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK1R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca2+ signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK1R. SP induced association of β-arrestin1 and PP2A with noninternalized NK1R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK1R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK1R with PP2A. Resensitization of NK1R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK1R and mediates resensitization. PP2A interaction with NK1R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK1R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs.

scholarly journals Three-dimensional Culture Regulates Raf-1 Expression to Modulate Fibronectin Matrix Assembly

2006 ◽  
Vol 17 (8) ◽  
pp. 3386-3396 ◽  
Author(s):  
B. S. Winters ◽  
B. K. Mohan Raj ◽  
E. E. Robinson ◽  
R. A. Foty ◽  
S. A. Corbett
Keyword(s):  
Monolayer Culture ◽  
Three Dimensional ◽  
3D Culture ◽  
Mrna Levels ◽  
Matrix Assembly ◽  
3 Dimensional ◽  
2D System ◽  
Rna Knockdown ◽  
Interfering Rna ◽  
Insight Into

Oncogenic transformation has been associated with decreased fibronectin (FN) matrix assembly. For example, both the HT-1080 fibrosarcoma and MAT-LyLu cell lines fail to assemble a FN matrix when grown in monolayer culture (2-dimensional [2D] system). In this study, we show that these cells regain the ability to assemble a FN matrix when they are grown as aggregates (3-dimensional [3D] system). FN matrix assembly in 3D correlates with decreased Raf-1 protein expression compared with cells grown in monolayer culture. This effect is associated with reduced Raf-1 mRNA levels as determined by quantitative RT-PCR and not proteasome-mediated degradation of endogenous Raf-1. Interestingly, transient expression of a Raf-1 promoter-reporter construct demonstrates increased Raf-1 promoter activity in 3D, suggesting that the transition to 3D culture may modulate Raf-1 mRNA stability. Finally, to confirm that decreased Raf-1 expression results in increased FN matrix assembly, we used both pharmacological and small interfering RNA knockdown of Raf-1. This restored the ability of cells in 2D culture to assemble a FN matrix. Moreover, overexpression of Raf-1 prevented FN matrix assembly by cells cultured in 3D, resulting in decreased aggregate compaction. This work provides new insight into how the cell microenvironment may influence Raf-1 expression to modulate cell–FN interactions in 3D.

Cell-surface transglutaminase promotes fibronectin assembly via interaction with the gelatin-binding domain of fibronectin

2001 ◽  
Vol 114 (16) ◽  
pp. 2989-3000
Author(s):  
Sergey S. Akimov ◽  
Alexey M. Belkin
Keyword(s):  
Wound Healing ◽  
Cell Surface ◽  
Transforming Growth Factor ◽  
Tissue Transglutaminase ◽  
Binding Domain ◽  
Matrix Assembly ◽  
Cell Growth And Migration ◽  
Matrix Deposition ◽  
Fibronectin Assembly ◽  
Α5β1 Integrin

Assembly of fibronectin into a fibrillar matrix is critical for regulation of cell growth and migration, embryogenesis and wound healing. We have previously shown that cell-surface tissue transglutaminase serves as an integrin-binding adhesion coreceptor for fibronectin. Here we report that transglutaminase strongly promotes fibronectin assembly mediated byα5β1 integrin. This effect is independent from transglutaminase-mediated enzymatic crosslinking of fibronectin and separate from the ability of transglutaminase to stimulate cell spreading. Surface transglutaminase increases the binding of fibronectin to cells via interaction with its gelatin-binding domain that contains modules I6II1,2I7-9 and lacks integrin-binding motifs. The gelatin-binding fragment of fibronectin binds to surface transglutaminase on cells in suspension but does not interact with cell monolayers where surface transglutaminase is occupied by fibronectin. Surface transglutaminase colocalizes with growing fibronectin fibrils at early timepoints of matrix formation and remains codistributed with fibronectin matrices thereafter. The observed stimulation of matrix assembly by transglutaminase is blocked by the gelatin-binding fragment of fibronectin,but is not strongly perturbed by its N-terminal fragment consisting of modules I1-5. These results implicate an interaction between transglutaminase and the gelatin-binding domain of fibronectin in matrix assembly and suggest its role in initiation of fibrillogenesis. However,blocking antibodies against α5β1 integrin or the cell-binding fragment of fibronectin that contains modules III2-11 most strongly suppress matrix formation and abolish the effects of transglutaminase. Hence,transglutaminase cooperates with but can not substitute for α5β1 integrin in fibronectin assembly. Treatment of fibroblasts with transforming growth factor β (TGFβ) significantly increases surface expression of transglutaminase and its association with β1 integrins, but not withαVβ3 integrin. TGFβ enhances the binding of fibronectin to the cell surface and elevates matrix formation, whereas antibody against transglutaminase or the gelatin-binding fragment of fibronectin suppresses these effects, indicating an involvement of transglutaminase in TGFβ-dependent fibronectin assembly. Therefore, TGFβ-induced fibronectin matrix deposition during normal wound healing or fibrotic disorders may depend on upregulation of integrin-associated surface transglutaminase.

scholarly journals Giantin is required for intracellular N-terminal processing of type I procollagen

2021 ◽  
Vol 220 (6) ◽  
Author(s):  
Nicola L. Stevenson ◽  
Dylan J.M. Bergen ◽  
Yinhui Lu ◽  
M. Esther Prada-Sanchez ◽  
Karl E. Kadler ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Type I Collagen ◽  
Fish Analysis ◽  
Type I ◽  
Matrix Assembly ◽  
Matrix Deposition ◽  
Type I Procollagen ◽  
Main Protein ◽  
Extracellular Matrix Deposition ◽  
Craniofacial Defects

Knockout of the golgin giantin leads to skeletal and craniofacial defects driven by poorly studied changes in glycosylation and extracellular matrix deposition. Here, we sought to determine how giantin impacts the production of healthy bone tissue by focusing on the main protein component of the osteoid, type I collagen. Giantin mutant zebrafish accumulate multiple spontaneous fractures in their caudal fin, suggesting their bones may be more brittle. Inducing new experimental fractures revealed defects in the mineralization of newly deposited collagen as well as diminished procollagen reporter expression in mutant fish. Analysis of a human giantin knockout cell line expressing a GFP-tagged procollagen showed that procollagen trafficking is independent of giantin. However, our data show that intracellular N-propeptide processing of pro-α1(I) is defective in the absence of giantin. These data demonstrate a conserved role for giantin in collagen biosynthesis and extracellular matrix assembly. Our work also provides evidence of a giantin-dependent pathway for intracellular procollagen processing.

scholarly journals GCN2 drives macrophage and MDSC function and immunosuppression in the tumor microenvironment

2019 ◽  
Vol 4 (42) ◽  
pp. eaax8189 ◽  
Author(s):  
Marie Jo Halaby ◽  
Kebria Hezaveh ◽  
Sara Lamorte ◽  
M. Teresa Ciudad ◽  
Andreas Kloetgen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Macrophage Polarization ◽  
Suppressor Cells ◽  
Interferon Γ ◽  
Mass Cytometry ◽  
General Control ◽  
Immune Attack ◽  
Environmental Sensor ◽  
Rna Knockdown ◽  
Interfering Rna

General control nonderepressible 2 (GCN2) is an environmental sensor controlling transcription and translation in response to nutrient availability. Although GCN2 is a putative therapeutic target for immuno-oncology, its role in shaping the immune response to tumors is poorly understood. Here, we used mass cytometry, transcriptomics, and transcription factor–binding analysis to determine the functional impact of GCN2 on the myeloid phenotype and immune responses in melanoma. We found that myeloid-lineage deletion of GCN2 drives a shift in the phenotype of tumor-associated macrophages and myeloid-derived suppressor cells (MDSCs) that promotes antitumor immunity. Time-of-flight mass cytometry (CyTOF) and single-cell RNA sequencing showed that this was due to changes in the immune microenvironment with increased proinflammatory activation of macrophages and MDSCs and interferon-γ expression in intratumoral CD8+ T cells. Mechanistically, GCN2 altered myeloid function by promoting increased translation of the transcription factor CREB-2/ATF4, which was required for maturation and polarization of macrophages and MDSCs in both mice and humans, whereas targeting Atf4 by small interfering RNA knockdown reduced tumor growth. Last, analysis of patients with cutaneous melanoma showed that GCN2-dependent transcriptional signatures correlated with macrophage polarization, T cell infiltrates, and overall survival. Thus, these data reveal a previously unknown dependence of tumors on myeloid GCN2 signals for protection from immune attack.

A multifunctional substance P-conjugated chitosan hydrochloride hydrogel accelerates full-thickness wound healing by enhancing synchronized vascularization, extracellular matrix deposition, and nerve regeneration

2021 ◽  
Author(s):  
Hao Li ◽  
Mengna Li ◽  
Pei Liu ◽  
Kai-Yang Wang ◽  
Haoyu Fang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Wound Healing ◽  
Full Thickness ◽  
Matrix Deposition ◽  
Chitosan Hydrochloride ◽  
Reconstructive Microsurgery ◽  
Extracellular Matrix Deposition ◽  
Advanced Biomaterials ◽  
Native Skin ◽  
Full Thickness Wound

Due to the native skin limitations and the complexity of reconstructive microsurgery, advanced biomaterials are urgently required to promote wound healing for severe skin defects caused by accidents and disasters....

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