Cell surface heparan sulfates mediate internalization of the PWWP/HATH domain of HDGF via macropinocytosis to fine-tune cell signalling processes involved in fibroblast cell migration

2010 ◽  
Vol 433 (1) ◽  
pp. 127-138 ◽  
Author(s):  
Chia-Hui Wang ◽  
Fabian Davamani ◽  
Shih-Che Sue ◽  
Shao-Chen Lee ◽  
Po-long Wu ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Surface ◽  
Receptor Binding ◽  
Cell Signalling ◽  
Biological Significance ◽  
Matrix Metalloproteases ◽  
Membrane Receptor ◽  
Mutant Form ◽  
Heparin Binding ◽  
Nih 3T3

HDGF (hepatoma-derived growth factor) stimulates cell proliferation by functioning on both sides of the plasma membrane as a ligand for membrane receptor binding to trigger cell signalling and as a stimulator for DNA synthesis in the nucleus. Although HDGF was initially identified as a secretory heparin-binding protein, the biological significance of its heparin-binding ability remains to be determined. In the present study we demonstrate that cells devoid of surface HS (heparan sulfate) were unable to internalize HDGF, HATH (N-terminal domain of HDGF consisting of amino acid residues 1–100, including the PWWP motif) and HATH(K96A) (single-site mutant form of HATH devoid of receptor binding activity), suggesting that the binding of HATH to surface HS is important for HDGF internalization. We further demonstrate that both HATH and HATH(K96A) could be internalized through macropinocytosis after binding to the cell surface HS. Interestingly, HS-mediated HATH(K96A) internalization is found to exhibit an inhibitory effect on cell migration and proliferation in contrast with that observed for HATH action on NIH 3T3 cells, suggesting that HDGF exploits the innate properties of both cell surface HS and membrane receptor via the HATH domain to affect related cell signalling processes. The present study indicates that MAPK (mitogen-activated protein kinase) signalling pathways could be affected by the HS-mediated HATH internalization to regulate cell migration in NIH 3T3 fibroblasts, as judged from the differential effect of HATH and HATH(K96A) treatment on the expression level of matrix metalloproteases.

scholarly journals The membrane protein CD9/DRAP 27 potentiates the juxtacrine growth factor activity of the membrane-anchored heparin-binding EGF-like growth factor.

1995 ◽  
Vol 128 (5) ◽  
pp. 929-938 ◽  
Author(s):  
S Higashiyama ◽  
R Iwamoto ◽  
K Goishi ◽  
G Raab ◽  
N Taniguchi ◽  
...  
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Cell Surface ◽  
Biological Activities ◽  
Transmembrane Protein ◽  
Biological Significance ◽  
Mitogenic Activity ◽  
Heparin Binding ◽  
Factor Activity ◽  
Growth Activity

The membrane-anchored heparin-binding EGF-like growth factor precursor (proHB-EGF)/diphtheria toxin receptor (DTR) belongs to a class of transmembrane growth factors and physically associates with CD9/DRAP27 which is also a transmembrane protein. To evaluate the biological activities of proHB-EGF/DTR as a juxtacrine growth factor and the biological significance of its association with CD9/DRAP27, the mitogenic activity of proHB-EGF/DTR was analyzed using stable transfectants of mouse L cells expressing both human proHB-EGF/DTR and monkey CD9/DRAP27, or either one alone. Juxtacrine activity was assayed by measuring the ability of cells in co-culture to stimulate DNA synthesis in an EGF receptor ligand dependent cell line, EP170.7. LH-2 cells expressing human proHB-EGF/DTR stimulated EP170.7 cell growth moderately. However, LCH-1 cells, a stable co-transfectant expressing both human proHB-EGF/DTR and monkey CD9/DRAP27 cDNAs, dramatically unregulated the juxtacrine growth factor activity of proHB-EGF/DTR approximately 25 times over that of LH-2 cells even though both cell types expressed similar levels of proHB-EGF/DTR on the cell surface. Anti-CD9/DRAP27 antibodies which were not able to neutralize the mitogenic activity of soluble HB-EGF suppressed LCH-1 cell juxtacrine growth activity to the same extent as did anti-HB-EGF neutralizing antibodies and CRM 197, specific inhibitors of human HG-EGF. These findings suggest that optimal expression of the juxtacrine growth activity of proHB-EGF/DTR requires co-expression of CD9/DRAP27. These studies also indicate that growth factor potentiation effects which have been observed previously for soluble growth factors also occurs at the level of cell surface associated growth factors.

scholarly journals Gold Nanoparticles Inhibit VEGF165-Induced Migration and Tube Formation of Endothelial Cells via the Akt Pathway

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Yunlong Pan ◽  
Qing Wu ◽  
Li Qin ◽  
Jiye Cai ◽  
Bin Du
Keyword(s):  
Gold Nanoparticles ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Umbilical Vein ◽  
Critical Role ◽  
Chorioallantoic Membrane ◽  
Tube Formation ◽  
Endothelial Cell Proliferation ◽  
Akt Pathway

The early stages of angiogenesis can be divided into three steps: endothelial cell proliferation, migration, and tube formation. Vascular endothelial growth factor (VEGF) is considered the most important proangiogenic factor; in particular, VEGF165plays a critical role in angiogenesis. Here, we evaluated whether gold nanoparticles (AuNPs) could inhibit the VEGF165-induced human umbilical vein endothelial cell (HUVEC) migration and tube formation. AuNPs and VEGF165were coincubated overnight at 4°C, after which the effects on cell migration and tube formation were assessed. Cell migration was assessed using a modified wound-healing assay and a transwell chamber assay; tube formation was assessed using a capillary-like tube formation assay and a chick chorioallantoic membrane (CAM) assay. We additionally detected the cell surface morphology and ultrastructure using atomic force microscopy (AFM). Furthermore, Akt phosphorylation downstream of VEGFR-2/PI3K in HUVECs was determined in a Western blot analysis. Our study demonstrated that AuNPs significantly inhibited VEGF165-induced HUVEC migration and tube formation by affecting the cell surface ultrastructure, cytoskeleton and might have inhibited angiogenesis via the Akt pathway.

scholarly journals Induction of β3-Integrin Gene Expression by Sustained Activation of the Ras-Regulated Raf–MEK–Extracellular Signal-Regulated Kinase Signaling Pathway

2001 ◽  
Vol 21 (9) ◽  
pp. 3192-3205 ◽  
Author(s):  
Douglas Woods ◽  
Holly Cherwinski ◽  
Eleni Venetsanakos ◽  
Arun Bhat ◽  
Stephan Gysin ◽  
...  
Keyword(s):  
Cell Surface ◽  
Signaling Pathway ◽  
Erk Signaling ◽  
Integrin Expression ◽  
3T3 Cells ◽  
Extracellular Signal ◽  
Integrin Gene ◽  
Β3 Integrin ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT Alterations in the expression of integrin receptors for extracellular matrix (ECM) proteins are strongly associated with the acquisition of invasive and/or metastatic properties by human cancer cells. Despite this, comparatively little is known of the biochemical mechanisms that regulate the expression of integrin genes in cells. Here we demonstrate that the Ras-activated Raf–MEK–extracellular signal-regulated kinase (ERK) signaling pathway can specifically control the expression of individual integrin subunits in a variety of human and mouse cell lines. Pharmacological inhibition of MEK1 in a number of human melanoma and pancreatic carcinoma cell lines led to reduced cell surface expression of α6- and β3-integrin. Consistent with this, conditional activation of the Raf-MEK-ERK pathway in NIH 3T3 cells led to a 5 to 20-fold induction of cell surface α6- and β3-integrin expression. Induced β3-integrin was expressed on the cell surface as a heterodimer with αv-integrin; however, the overall level of αv-integrin expression was not altered by Ras or Raf. Raf-induced β3-integrin was observed in primary and established mouse fibroblast lines and in mouse and human endothelial cells. Consistent with previous reports of the ability of the Raf-MEK-ERK signaling pathway to induce β3-integrin gene transcription in human K-562 erythroleukemia cells, Raf activation in NIH 3T3 cells led to elevated β3-integrin mRNA. However, unlike immediate-early Raf targets such as heparin binding epidermal growth factor and Mdm2, β3-integrin mRNA was induced by Raf in a manner that was cycloheximide sensitive. Surprisingly, activation of the Raf-MEK-ERK signaling pathway by growth factors and mitogens had little or no effect on β3-integrin expression, suggesting that the expression of this gene requires sustained activation of this signaling pathway. In addition, despite the robust induction of cell surface αvβ3-integrin expression by Raf in NIH 3T3 cells, such cells display decreased spreading and adhesion, with a loss of focal adhesions and actin stress fibers. These data suggest that oncogene-induced alterations in integrin gene expression may participate in the changes in cell adhesion and migration that accompany the process of oncogenic transformation.

scholarly journals Mechanical Control of Cell Migration by the Metastasis Suppressor Tetraspanin CD82/KAI1

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1545
Author(s):  
Laura Ordas ◽  
Luca Costa ◽  
Anthony Lozano ◽  
Christopher Chevillard ◽  
Alexia Calovoulos ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Surface ◽  
Focal Adhesion ◽  
Nuclear Translocation ◽  
Single Cells ◽  
Integral Membrane Protein ◽  
Metastasis Suppressor ◽  
Dependent Manner ◽  
Strong Inhibitor ◽  
Caveolin 1

The plasma membrane is a key actor of cell migration. For instance, its tension controls persistent cell migration and cell surface caveolae integrity. Then, caveolae constituents such as caveolin-1 can initiate a mechanotransduction loop that involves actin- and focal adhesion-dependent control of the mechanosensor YAP to finely tune cell migration. Tetraspanin CD82 (also named KAI-1) is an integral membrane protein and a metastasis suppressor. Its expression is lost in many cancers including breast cancer. It is a strong inhibitor of cell migration by a little-known mechanism. We demonstrated here that CD82 controls persistent 2D migration of EGF-induced single cells, stress fibers and focal adhesion sizes and dynamics. Mechanistically, we found that CD82 regulates membrane tension, cell surface caveolae abundance and YAP nuclear translocation in a caveolin-1-dependent manner. Altogether, our data show that CD82 controls 2D cell migration using membrane-driven mechanics involving caveolin and the YAP pathway.

scholarly journals The binding of NCAM to FGFR1 induces a specific cellular response mediated by receptor trafficking

2009 ◽  
Vol 187 (7) ◽  
pp. 1101-1116 ◽  
Author(s):  
Chiara Francavilla ◽  
Paola Cattaneo ◽  
Vladimir Berezin ◽  
Elisabeth Bock ◽  
Diletta Ami ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cellular Response ◽  
Critical Role ◽  
Biological Significance ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Fgf Receptor ◽  
Neural Cell Adhesion ◽  
Sustained Activation

Neural cell adhesion molecule (NCAM) associates with fibroblast growth factor (FGF) receptor-1 (FGFR1). However, the biological significance of this interaction remains largely elusive. In this study, we show that NCAM induces a specific, FGFR1-mediated cellular response that is remarkably different from that elicited by FGF-2. In contrast to FGF-induced degradation of endocytic FGFR1, NCAM promotes the stabilization of the receptor, which is recycled to the cell surface in a Rab11- and Src-dependent manner. In turn, FGFR1 recycling is required for NCAM-induced sustained activation of various effectors. Furthermore, NCAM, but not FGF-2, promotes cell migration, and this response depends on FGFR1 recycling and sustained Src activation. Our results implicate NCAM as a nonconventional ligand for FGFR1 that exerts a peculiar control on the intracellular trafficking of the receptor, resulting in a specific cellular response. Besides introducing a further level of complexity in the regulation of FGFR1 function, our findings highlight the link of FGFR recycling with sustained signaling and cell migration and the critical role of these events in dictating the cellular response evoked by receptor activation.

scholarly journals HHV-8 reduces dendritic cell migration through down-regulation of cell-surface CCR6 and CCR7 and cytoskeleton reorganization

2012 ◽  
Vol 9 (1) ◽  
Author(s):  
Mara Cirone ◽  
Valeria Conte ◽  
Antonella Farina ◽  
Sandro Valia ◽  
Pankaj Trivedi ◽  
...  
Keyword(s):  
Cell Migration ◽  
Dendritic Cell ◽  
Cell Surface ◽  
Down Regulation ◽  
Cytoskeleton Reorganization ◽  
Dendritic Cell Migration

scholarly journals Constitutive and Regulated Shedding of Soluble FGF Receptors Releases Biologically Active Inhibitors of FGF-2

2021 ◽  
Vol 22 (5) ◽  
pp. 2712
Author(s):  
Anne Hanneken ◽  
Maluz Mercado ◽  
Pamela Maher
Keyword(s):  
Cell Surface ◽  
Ligand Binding ◽  
Cellular Proliferation ◽  
Biological Activities ◽  
Biological Properties ◽  
Matrix Metalloproteases ◽  
Biologically Active ◽  
Ectodomain Shedding ◽  
High Affinity

The identification of soluble fibroblast growth factor (FGF) receptors in blood and the extracellular matrix has led to the prediction that these proteins modulate the diverse biological activities of the FGF family of ligands in vivo. A recent structural characterization of the soluble FGF receptors revealed that they are primarily generated by proteolytic cleavage of the FGFR-1 ectodomain. Efforts to examine their biological properties are now focused on understanding the functional consequences of FGFR-1 ectodomain shedding and how the shedding event is regulated. We have purified an FGFR-1 ectodomain that is constitutively cleaved from the full-length FGFR-1(IIIc) receptor and released into conditioned media. This shed receptor binds FGF-2; inhibits FGF-2-induced cellular proliferation; and competes with high affinity, cell surface FGF receptors for ligand binding. FGFR-1 ectodomain shedding downregulates the number of high affinity receptors from the cell surface. The shedding mechanism is regulated by ligand binding and by activators of PKC, and the two signaling pathways appear to be independent of each other. Deletions and substitutions at the proposed cleavage site of FGFR-1 do not prevent ectodomain shedding. Broad spectrum inhibitors of matrix metalloproteases decrease FGFR-1 ectodomain shedding, suggesting that the enzyme responsible for constitutive, ligand-activated, and protein kinase C-activated shedding is a matrix metalloprotease. In summary, shedding of the FGFR-1 ectodomain is a highly regulated event, sharing many features with a common system that governs the release of diverse membrane proteins from the cell surface. Most importantly, the FGFR ectodomains are biologically active after shedding and are capable of functioning as inhibitors of FGF-2.

Induction of acetylcholine receptor clustering by native polystyrene beads. Implication of an endogenous muscle-derived signalling system

1992 ◽  
Vol 102 (3) ◽  
pp. 543-555 ◽  
Author(s):  
L.P. Baker ◽  
Q. Chen ◽  
H.B. Peng
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Acetylcholine Receptor ◽  
Hot Spots ◽  
Hot Spot ◽  
Muscle Cells ◽  
Membrane Receptors ◽  
Heparin Binding ◽  
Polystyrene Beads ◽  
Myotomal Muscle

Aneural muscle cells in culture often form acetylcholine receptor (AChR) clusters, termed hot spots, which are similar to those found at the postsynaptic membrane both in structure and in molecular composition. Although hot spots form on both dorsal and ventral surfaces of the cell, the ventral ones are better characterized because of their association with sites of cell-substratum contact. To understand the stimuli and mechanisms involved in ventral hot spot formation, native, uncoated polystyrene beads were applied to cultured Xenopus myotomal muscle cells to create local membrane-substratum contacts. These beads were able to induce a postsynaptic-type development as evidenced by the clustering of AChRs and the development of a set of ultrastructural specializations, including membrane infoldings and a basement membrane. Whereas these native beads were effective in inducing clustering, beads coated with bovine serum albumin or treated with serum-containing medium were ineffective. Native beads were also capable of inducing clusters in serum-free medium, indicating that their effect was mediated by endogenous molecules that were locally presented by the beads, rather than by bead adsorption of components in the medium. Heparan sulfate proteoglycan (HSPG) is a major component of the muscle extracellular matrix and our previous study has shown that basic fibroblast growth factor (bFGF), a member of the heparin-binding growth factor (HBGF) family, and its receptor are present in Xenopus myotomal muscle during the period of synaptogenesis. Therefore, we tested the involvement of HBGF in bead induction. The results of this study show the following: (1) preincubation of cultures in heparin, which solubilizes matrix-bound HBGFs, suppressed the bead-induced AChR clustering. (2) Suramin, which interferes with the interaction between several growth factors and their receptors, also inhibited bead-induced clustering. (3) Tyrphostin, which blocks tyrosine kinase activity associated with a number of growth factor receptors, was also inhibitory to the bead effect. (4) The percentage of bead-induced AChR clusters was significantly enhanced by pretreating the cultures with bFGF prior to bead application. This exogenously applied bFGF could be largely removed by treatment of cultures with heparin, suggesting its association with HSPG at the cell surface. (5) An anti-bFGF neutralizing antiserum significantly reduced the efficacy of the bead stimulation. These data suggest that uncoated beads, which adhere to the cell surface and can mimic the cell-substratum interaction, effect a local presentation of HBGFs, such as bFGF, residing with the HSPG to their membrane receptors, thereby locally activating receptor-associated tyrosine kinases.(ABSTRACT TRUNCATED AT 400 WORDS)

Clathrin-mediated endocytosis and recycling of autocrine motility factor receptor to fibronectin fibrils is a limiting factor for NIH-3T3 cell motility

2000 ◽  
Vol 113 (18) ◽  
pp. 3227-3240 ◽  
Author(s):  
P.U. Le ◽  
N. Benlimame ◽  
A. Lagana ◽  
A. Raz ◽  
I.R. Nabi
Keyword(s):  
Cell Surface ◽  
Cell Motility ◽  
Endocytic Pathway ◽  
Limiting Factor ◽  
Autocrine Motility Factor ◽  
Endocytic Compartment ◽  
Motility Factor ◽  
Microtubule Disruption ◽  
Nih 3T3 ◽  
Fibronectin Fibrils

Autocrine motility factor receptor (AMF-R) is internalized via a clathrin-independent pathway to smooth endoplasmic reticulum tubules. This endocytic pathway is shown here to be inhibited by methyl-(beta)-cyclodextrin (m(beta)CD) implicating caveolae or caveolae-like structures in AMF internalization to smooth ER. AMF-R is also internalized via a clathrin-dependent pathway to a transferrin receptor-negative, LAMP-1/lgpA-negative endocytic compartment identified by electron microscopy as a multivesicular body (MVB). Endocytosed AMF recycles to cell surface fibrillar structures which colocalize with fibronectin; AMF-R recycling is inhibited at 20 degrees C, which blocks endocytosis past the early endosome, but not by m(beta)CD demonstrating that AMF-R recycling to fibronectin fibrils is mediated by clathrin-dependent endocytosis to MVBs. Microtubule disruption with nocodazole did not affect delivery of bAMF to cell surface fibrils indicating that recycling bAMF traverses the MVB but not a later endocytic compartment. Plating NIH-3T3 cells on an AMF coated substrate did not specifically affect cell adhesion but prevented bAMF delivery to cell surface fibronectin fibrils and reduced cell motility. AMF-R internalization and recycling via the clathrin-mediated pathway are therefore rate-limiting for cell motility. This recycling pathway to the site of deposition of fibronectin may be implicated in the de novo formation of cellular attachments or the remodeling of the extracellular matrix during cell movement.

scholarly journals Modulation of cardiac Na+,K+-ATPase cell surface abundance by simulated ischemia-reperfusion and ouabain preconditioning

2013 ◽  
Vol 304 (1) ◽  
pp. H94-H103 ◽  
Author(s):  
Aude Belliard ◽  
Yoann Sottejeau ◽  
Qiming Duan ◽  
Jessa L. Karabin ◽  
Sandrine V. Pierre
Keyword(s):  
Cell Surface ◽  
Atpase Activity ◽  
Ion Transport ◽  
Cell Viability ◽  
Ischemia Reperfusion ◽  
Resistant Mutant ◽  
Surface Expression ◽  
Live Cells ◽  
Mutant Form ◽  
Dependent Manner

Na+,K+-ATPase and cell survival were investigated in a cellular model of ischemia-reperfusion (I/R)-induced injury and protection by ouabain-induced preconditioning (OPC). Rat neonatal cardiac myocytes were subjected to 30 min of substrate and coverslip-induced ischemia followed by 30 min of simulated reperfusion. This significantly compromised cell viability as documented by lactate dehydrogenase release and Annexin V/propidium iodide staining. Total Na+,K+-ATPase α1- and α3-polypeptide expression remained unchanged, but cell surface biotinylation and immunostaining studies revealed that α1-cell surface abundance was significantly decreased. Na+,K+-ATPase-activity in crude homogenates and 86Rb+ transport in live cells were both significantly decreased by about 30% after I/R. OPC, induced by a 4-min exposure to 10 μM ouabain that ended 8 min before the beginning of ischemia, increased cell viability in a PKCε-dependent manner. This was comparable with the protective effect of OPC previously reported in intact heart preparations. OPC prevented I/R-induced decrease of Na+,K+-ATPase activity and surface expression. This model also revealed that Na+,K+-ATPase-mediated 86Rb+ uptake was not restored to control levels in the OPC group, suggesting that the increased viability was not conferred by an increased Na+,K+-ATPase-mediated ion transport capacity at the cell membrane. Consistent with this observation, transient expression of an internalization-resistant mutant form of Na+,K+-ATPase α1 known to have increased surface abundance without increased ion transport activity successfully reduced I/R-induced cell death. These results suggest that maintenance of Na+,K+-ATPase cell surface abundance is critical to myocyte survival after an ischemic attack and plays a role in OPC-induced protection. They further suggest that the protection conferred by increased surface expression of Na+,K+-ATPase may be independent of ion transport.

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