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1.
Proc Natl Acad Sci U S A ; 98(17): 9642-7, 2001 Aug 14.
Article in English | MEDLINE | ID: mdl-11493690

ABSTRACT

Redistribution of specialized molecules in migrating cells develops asymmetry between two opposite cell poles, the leading edge and the uropod. We show that acquisition of a motile phenotype in T lymphocytes results in the asymmetric redistribution of ganglioside GM3- and GM1-enriched raft domains to the leading edge and to the uropod, respectively. This segregation to each cell pole parallels the specific redistribution of membrane proteins associated to each raft subfraction. Our data suggest that raft partitioning is a major determinant for protein redistribution in polarized T cells, as ectopic expression of raft-associated proteins results in their asymmetric redistribution, whereas non-raft-partitioned mutants of these proteins are distributed homogeneously in the polarized cell membrane. Both acquisition of a migratory phenotype and SDF-1alpha-induced chemotaxis are cholesterol depletion-sensitive. Finally, GM3 and GM1 raft redistribution requires an intact actin cytoskeleton, but is insensitive to microtubule disruption. We propose that membrane protein segregation not only between raft and nonraft domains but also between distinct raft subdomains may be an organizational principle that mediates redistribution of specialized molecules needed for T cell migration.


Subject(s)
Chemotaxis, Leukocyte/physiology , Membrane Microdomains/metabolism , T-Lymphocytes/metabolism , Actins/physiology , Animals , Cell Line , Cell Polarity , Cholesterol/physiology , Culture Media, Serum-Free , Cytoskeleton/physiology , G(M1) Ganglioside/metabolism , G(M3) Ganglioside/metabolism , Humans , Jurkat Cells , Mice , Models, Biological , T-Lymphocytes/ultrastructure
2.
Semin Immunol ; 13(2): 147-57, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11308298

ABSTRACT

Cell chemotaxis requires the acquisition and maintenance of both spatial and functional asymmetry between initially equivalent cell parts. In leukocytes one becomes the leading edge and the other, the rear edge or uropod. The acquisition of this cell polarity is controlled by an array of chemoattractants, including those of the chemokine family. We propose that chemokine receptor activation in highly organized lipid raft domains is a major determinant for the correct localization of the signaling pathways leading to the cell asymmetries required for migration. The lateral organization imposed by membrane raft microdomains is discussed in the context of other chemokine receptor activities, such as its role as a human immunodeficiency virus (HIV) coreceptor.


Subject(s)
Membrane Microdomains/immunology , Receptors, Chemokine/immunology , Signal Transduction/immunology , Animals , Cell Movement , Cell Polarity , Chemotaxis/immunology , Humans , Proteins/metabolism
3.
EMBO Rep ; 2(2): 151-6, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11258708

ABSTRACT

Complex cell responses require the integration of signals delivered through different pathways. We show that insulin-like growth factor (IGF)-I induces specific transactivation of the Gi-coupled chemokine receptor CCR5, triggering its tyrosine phosphorylation and Galpha recruitment. This transactivation occurs via a mechanism involving transcriptional upregulation and secretion of RANTES, the natural CCR5 ligand. CCR5 transactivation is an essential downstream signal in IGF-I-induced cell chemotaxis, as abrogation of CCR5 function with a transdominant-negative KDELccr5A32 mutant abolishes IGF-I-induced migration. The relevance of this transactivation pathway was shown in vivo, as KDELccr5A32 overexpression prevents invasion by highly metastatic tumor cells; conversely, RANTES overexpression confers built-in invasive capacity on a non-invasive tumor cell line. Our results suggest that this extracellular growth factor-chemokine network represents a general mechanism connecting tumorigenesis and inflammation.


Subject(s)
Insulin-Like Growth Factor I/pharmacology , Receptors, CCR5/metabolism , Signal Transduction/drug effects , Transcriptional Activation/drug effects , Breast Neoplasms/pathology , Cell Polarity/drug effects , Chemokine CCL5/genetics , Chemokine CCL5/metabolism , Chemotaxis/drug effects , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Humans , Ligands , Mutation/genetics , Neoplasm Invasiveness/pathology , Oligopeptides/genetics , Phosphorylation/drug effects , Protein Sorting Signals/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, CCR5/chemistry , Receptors, CCR5/genetics , Tumor Cells, Cultured
4.
IUBMB Life ; 49(2): 89-96, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10776589

ABSTRACT

Throughout evolution, both prokaryotic and eukaryotic cells have developed a variety of biochemical mechanisms to define the direction and proximity of extracellular stimuli. This process is essential for the cell to reply properly to the environmental cues that determine cell migration, proliferation, and differentiation. Chemotaxis is the cellular response to chemical attractants that direct cell migration, a process that plays a central role in many physiological situations, such as host immune responses, angiogenesis, wound healing, embryogenesis, and neuronal patterning, among others. In addition, cell migration takes part in pathological states, including inflammation and tumor metastasis. Indeed, tumor progression to invasion and metastasis depends on the active motility of the invading cancer cells and the endothelial cell bed during tumor neovascularization. Cell migration switches "off" and "on," based on quantitative differences in molecular components such as adhesion receptors, cytoskeletal linking proteins, and extracellular matrix ligands, and by regulating the affinity of membrane-bound chemoattractant receptors. A clear understanding of how cells sense chemoattractants is, therefore, of pivotal importance in the biology of the normal cell as well as in prevention of malignant cell invasion. Here we offer a perspective on cell migration that emphasizes the relationship between cell polarization and cell movement and the importance of the equilibrium between the signals that drive each process for the control of tumor cell invasion.


Subject(s)
Cell Movement/physiology , Cell Polarity/physiology , Animals , Cell Adhesion , Cell Differentiation , Cell Division , Chemotaxis , Humans , Neoplasm Metastasis
5.
EMBO Rep ; 1(2): 190-6, 2000 Aug.
Article in English | MEDLINE | ID: mdl-11265761

ABSTRACT

HIV-1 infection triggers lateral membrane diffusion following interaction of the viral envelope with cell surface receptors. We show that these membrane changes are necessary for infection, as initial gp120-CD4 engagement leads to redistribution and clustering of membrane microdomains, enabling subsequent interaction of this complex with HIV-1 co-receptors. Disruption of cell membrane rafts by cholesterol depletion before viral exposure inhibits entry by both X4 and R5 strains of HIV-1, although viral replication in infected cells is unaffected by this treatment. This inhibitory effect is fully reversed by cholesterol replenishment of the cell membrane. These results indicate a general mechanism for HIV-1 envelope glycoprotein-mediated fusion by reorganization of membrane microdomains in the target cell, and offer new strategies for preventing HIV-1 infection.


Subject(s)
CD4 Antigens/metabolism , HIV Envelope Protein gp120/metabolism , HIV-1/metabolism , Membrane Fusion/physiology , Membrane Microdomains/metabolism , Receptors, CXCR4/metabolism , beta-Cyclodextrins , Animals , Cell Line , Cholesterol/metabolism , Cyclodextrins/metabolism , Cyclodextrins/pharmacology , Genes, Reporter , Humans , Membrane Microdomains/chemistry , Membrane Microdomains/drug effects , Microscopy, Confocal , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
6.
EMBO J ; 18(22): 6211-20, 1999 Nov 15.
Article in English | MEDLINE | ID: mdl-10562533

ABSTRACT

The acquisition of spatial and functional asymmetry between the rear and the front of the cell is a necessary step for cell chemotaxis. Insulin-like growth factor-I (IGF-I) stimulation of the human adenocarcinoma MCF-7 induces a polarized phenotype characterized by asymmetrical CCR5 chemokine receptor redistribution to the leading cell edge. CCR5 associates with membrane raft microdomains, and its polarization parallels redistribution of raft molecules, including the raft-associated ganglioside GM1, glycosylphosphatidylinositol-anchored green fluorescent protein and ephrinB1, to the leading edge. The non-raft proteins transferrin receptor and a mutant ephrinB1 are distributed homogeneously in migrating MCF-7 cells, supporting the raft localization requirement for polarization. IGF-I stimulation of cholesterol-depleted cells induces projection of multiple pseudopodia over the entire cell periphery, indicating that raft disruption specifically affects the acquisition of cell polarity, but not IGF-I-induced protrusion activity. Cholesterol depletion inhibits MCF-7 chemotaxis, which is restored by replenishing cholesterol. Our results indicate that initial segregation between raft and non-raft membrane proteins mediates the necessary redistribution of specialized molecules for cell migration.


Subject(s)
Cell Polarity/physiology , Chemotaxis/physiology , Receptors, CCR5/physiology , Adenocarcinoma , Breast Neoplasms , Cell Membrane/drug effects , Cell Membrane/physiology , Cell Membrane/ultrastructure , Cell Polarity/drug effects , Chemotaxis/drug effects , Cholesterol/pharmacology , Cholesterol/physiology , Female , Humans , Insulin-Like Growth Factor I/pharmacology , Membrane Lipids/physiology , Receptor, IGF Type 1/physiology , Signal Transduction , Tumor Cells, Cultured
7.
J Biol Chem ; 274(11): 6935-45, 1999 Mar 12.
Article in English | MEDLINE | ID: mdl-10066747

ABSTRACT

The androgen-independent human prostate adenocarcinoma cell line DU-145 proliferates in serum-free medium and produces insulin-like growth factors (IGF)-I, IGF-II, and the IGF type-1 receptor (IGF-1R). They also secrete three IGF-binding proteins (IGFBP), IGFBP-2, -3, and -4. Of these, immunoblot analysis revealed selective proteolysis of IGFBP-3, yielding fragments of 31 and 19 kDa. By using an anti-IGF-I-specific monoclonal antibody (mAb), we detect surface receptor-bound IGF-I on serum-starved DU-145 cells, which activates IGF-1R and triggers a mitogenic signal. Incubation of DU-145 cells with blocking anti-IGF-I, anti-IGF-II, or anti-IGF-I plus anti-IGF-II mAb does not, however, inhibit serum-free growth of DU-145. Conversely, anti-IGF-1R mAb and IGFBP-3 inhibit DNA synthesis. IGFBP-3 also modifies the DU-145 cell cycle, decreases p34(cdc2) levels, and IGF-1R autophosphorylation. The antiproliferative IGFBP-3 activity is not IGF-independent, since des-(1-3)IGF-I, which does not bind to IGFBP-3, reverses its inhibitory effect. DU-145 also secretes the matrix metalloproteinase (MMP)-9, which can be detected in both a soluble and a membrane-bound form. Matrix metalloproteinase inhibitors, but not serpins, abrogate DNA synthesis in DU-145 associated with the blocking of IGFBP-3 proteolysis. Overexpression of an antisense cDNA for MMP-9 inhibits 80% of DU-145 cell proliferation that can be reversed by IGF-I in a dose-dependent manner. Inhibition of MMP-9 expression is also associated with a decrease in IGFBP-3 proteolysis and with reduced signaling through the IGF-1R. Our data indicate an IGF autocrine loop operating in DU-145 cells, specifically modulated by IGFBP-3, whose activity may in turn be regulated by IGFBP-3 proteases such as MMP-9.


Subject(s)
Cell Division/physiology , Collagenases/physiology , Somatomedins/physiology , 3T3 Cells , Animals , Antibodies, Monoclonal/immunology , Base Sequence , DNA Primers , Humans , Insulin-Like Growth Factor Binding Proteins/immunology , Insulin-Like Growth Factor Binding Proteins/metabolism , Matrix Metalloproteinase 9 , Mice , Mice, Inbred BALB C , Receptor, IGF Type 1/immunology , Receptor, IGF Type 1/metabolism , Somatomedins/metabolism , Tumor Cells, Cultured
8.
Mol Cell Biol ; 19(4): 3125-35, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10082579

ABSTRACT

The coordinated interplay of substrate adhesion and deadhesion is necessary for cell motility. Using MCF-7 cells, we found that insulin-like growth factor I (IGF-I) induces the adhesion of MCF-7 to vitronectin and collagen in a dose- and time-dependent manner, suggesting that IGF-I triggers the activation of different integrins. On the other hand, IGF-I promotes the association of insulin receptor substrate 1 with the focal adhesion kinase (FAK), paxillin, and the tyrosine phosphatase SHP-2, resulting in FAK and paxillin dephosphorylation. Abrogation of SHP-2 catalytic activity with a dominant-negative mutant (SHP2-C>S) abolishes IGF-I-induced FAK dephosphorylation, and cells expressing SHP2-C>S show reduced IGF-I-stimulated chemotaxis compared with either mock- or SHP-2 wild-type-transfected cells. This impairment of cell migration is recovered by reintroduction of a catalytically active SHP-2. Interestingly, SHP-2-C>S cells show a larger number of focal adhesion contacts than wild-type cells, suggesting that SHP-2 activity participates in the integrin deactivation process. Although SHP-2 regulates mitogen-activated protein kinase activity, the mitogen-activated protein kinase kinase inhibitor PD-98059 has only a marginal effect on MCF-7 cell migration. The role of SHP-2 as a general regulator of cell chemotaxis induced by other chemotactic agents and integrins is discussed.


Subject(s)
Cell Adhesion Molecules/metabolism , Cell Movement/physiology , Insulin-Like Growth Factor I/pharmacology , Protein Tyrosine Phosphatases/metabolism , Protein-Tyrosine Kinases/metabolism , Receptor Cross-Talk/physiology , Cell Adhesion/physiology , Chemokine CCL5/pharmacology , Chemotactic Factors/metabolism , Chemotaxis/physiology , Cytoskeletal Proteins/metabolism , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Humans , Insulin Receptor Substrate Proteins , Integrins/metabolism , Intracellular Signaling Peptides and Proteins , Models, Biological , Neoplasm Invasiveness , Paxillin , Phosphoproteins/metabolism , Phosphorylation , Protein Binding , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Protein Tyrosine Phosphatase, Non-Receptor Type 6 , Protein Tyrosine Phosphatases/genetics , Receptor, IGF Type 1/metabolism , Signal Transduction , Tumor Cells, Cultured
9.
J Endocrinol ; 154(2): 293-302, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9291840

ABSTRACT

The primary structure of recombinant human (h) insulin-like growth factor-I (IGF-I) epitopes recognized by a panel of 28 monoclonal antibodies (mAbs) is characterized. Pairwise mAb epitope mapping defines eight 'epitopic clusters' (I-VIII) which cover nearly the entire solvent-exposed IGF-I surface. Monoclonal antibody reactivity with 32 overlapping synthetic peptides and with IGF-I mutants is used to associate these epitopic clusters with the probable primary IGF-I sequences recognized. Epitopic cluster I involves residues in the C-domain and the first alpha-helix of the A-domain; clusters II, V and VII involve principally the B-domain; clusters III and IV map to amino acid sequences (55-70) and (1-13) respectively; cluster VI includes the A- and B-domains; and cluster VIII involves mainly the C-terminal part of the B-domain. Data indicate that this mAb panel defines 14 distinct IGF-I epitopes. The specific inhibition of HEL 92.1.7 IGF-I-promoted proliferation by these mAbs was explored. Direct correlation between mAb affinity and inhibitory activity was observed except in the case of clusters III- and VIII-specific mAbs. Finally, the combination of epitopic cluster I and II mAbs detect 0.5-10 ng/ml hIGF-I in a sandwich immunoassay, with no IGF-II crossreactivity. These anti-IGF-I mAbs are, therefore, useful for both the inhibition of IGF-I mitogenic activity and for the quantification of this growth factor. The potential use of this mAb panel in tumor cell growth control is discussed.


Subject(s)
Antibodies, Monoclonal , Epitope Mapping , Insulin-Like Growth Factor I/immunology , Humans
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