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1.
Exp Cell Res ; 300(1): 94-108, 2004 Oct 15.
Article in English | MEDLINE | ID: mdl-15383318

ABSTRACT

The presence of many laminin receptors of the beta1 integrin family on most cells makes it difficult to define the biological functions of other major laminin receptors such as integrin alpha6beta4 and dystroglycan. We therefore tested the binding of a beta1 integrin-null cell line GD25 to four different laminin variants. The cells were shown to produce dystroglycan, which based on affinity chromatography bound to laminin-1, -2/4, and -10/11, but not to laminin-5. The cells also expressed the integrin alpha6Abeta4A variant. GD25 beta1 integrin-null cells are known to bind poorly to laminin-1, but we demonstrate here that these cells bind avidly to laminin-2/4, -5, and -10/11. The initial binding at 20 min to each of these laminins could be inhibited by an integrin alpha6 antibody, but not by a dystroglycan antibody. Hence, integrin alpha6Abeta4A of GD25 cells was identified as a major receptor for initial GD25 cell adhesion to three out of four tested laminin isoforms. Remarkably, cell adhesion to laminin-5 failed to promote cell spreading, proliferation, and extracellular signal-regulated kinase (ERK) activation, whereas all these responses occurred in response to adhesion to laminin-2/4 or -10/11. The data establish GD25 cells as useful tools to define the role integrin alpha6Abeta4A and suggest that laminin isoforms have distinctly different capacities to promote cell adhesion and signaling via integrin alpha6Abeta4A.


Subject(s)
Cell Movement/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Integrin beta1/genetics , Laminin/metabolism , Animals , Binding Sites/drug effects , Binding Sites/genetics , Cell Adhesion/drug effects , Cell Adhesion/genetics , Cell Adhesion Molecules/metabolism , Cell Adhesion Molecules/pharmacology , Cell Division/drug effects , Cell Division/genetics , Cell Movement/drug effects , Cells, Cultured , Dystroglycans/drug effects , Dystroglycans/metabolism , Enzyme Activation/genetics , Extracellular Signal-Regulated MAP Kinases/drug effects , Integrin alpha6beta4/drug effects , Integrin alpha6beta4/genetics , Integrin alpha6beta4/metabolism , Laminin/pharmacology , Mice , Protein Binding/drug effects , Protein Binding/genetics , Protein Isoforms/metabolism , Protein Isoforms/pharmacology , Signal Transduction/drug effects , Signal Transduction/genetics , Kalinin
2.
J Cell Sci ; 117(Pt 12): 2611-22, 2004 May 15.
Article in English | MEDLINE | ID: mdl-15159456

ABSTRACT

Basement membranes generally determine different tissue compartments in complex organs, such as skin, playing not only an important structural but also a regulatory role. We have previously demonstrated the formation of a regular basement membrane in organotypic three-dimensional (3D)-cocultures of human skin keratinocytes and fibroblasts by indirect immunofluorescence and transmission electron microscopy. In this assembly process, cross-linking of type IV collagen and the laminin gamma1 chain by nidogen is considered a crucial step. For a functional proof, we have now competitively inhibited nidogen binding to laminin in 3D-cocultures with a recombinant laminin gamma1 fragment (gamma1III3-5 module) spanning this binding site. Repeated treatment abolished the deposition of nidogen at the epithelial-matrix interface but also greatly perturbed the presence of other matrix constituents such as laminin and perlecan. This effect persisted over the entire observation period of 10 to 21 days. In contrast, some components of the basement membrane zone were only moderately affected, with the laminin-5 isoform (gamma2 chain), type IV collagen and integrin alpha6ss4 still showing a distinct staining at their regular position, when seen by light microscopy. Furthermore, epidermal morphology and differentiation remained largely normal as indicated by the regular location of keratins K1/K10 and also of late differentiation markers. Ultrastructural examination demonstrated that the gamma1 fragment completely suppressed any formation of basement membrane structures (lamina densa) and also of hemidesmosomal adhesion complexes. As a consequence of hemidesmosome deficiency, keratin filament bundles were not attached to the ventral basal cell aspect. These findings were further substantiated by immuno-electron microscopy, revealing either loss or drastic reduction and dislocation of basement membrane and hemidesmosomal components. Taken together, in this simplified human skin model (representing a 'closed system') a functional link has been demonstrated between compound structures of the extra- and intracellular space at the junctional zone providing a basis to interfere at distinct points and in a controlled fashion.


Subject(s)
Basement Membrane/metabolism , Laminin/metabolism , Membrane Glycoproteins/metabolism , Skin/cytology , Basement Membrane/ultrastructure , Binding Sites , Carrier Proteins/drug effects , Carrier Proteins/metabolism , Carrier Proteins/ultrastructure , Cell Differentiation , Cells, Cultured , Coculture Techniques , Collagen Type IV/drug effects , Collagen Type IV/metabolism , Collagen Type IV/ultrastructure , Cross-Linking Reagents/metabolism , Dose-Response Relationship, Drug , Eukaryotic Initiation Factors , Extracellular Matrix/chemistry , Fluorescent Antibody Technique, Indirect , Hemidesmosomes/ultrastructure , Heparan Sulfate Proteoglycans/metabolism , Humans , Integrin alpha6beta4/drug effects , Integrin alpha6beta4/metabolism , Intermediate Filament Proteins/drug effects , Intermediate Filament Proteins/metabolism , Intermediate Filament Proteins/ultrastructure , Keratin-1 , Keratin-10 , Keratinocytes/metabolism , Keratins/metabolism , Keratins/ultrastructure , Laminin/drug effects , Laminin/genetics , Laminin/pharmacology , Organ Culture Techniques , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Time Factors
3.
Cell Motil Cytoskeleton ; 54(1): 64-80, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12451596

ABSTRACT

Transforming growth factor-beta1 (TGF-beta1) has the ability to induce epithelial cell migration while stopping proliferation. In this study, we show that, concomitant to promoting migration of normal human keratinocytes in vitro, TGF-beta1 induced a marked decrease in their adhesion capacity to processed alpha3-containing laminin 5-coated surfaces. Indeed, the expression levels of alpha3 and alpha6 integrin subunit mRNA and protein, as well as the cell surface alpha3beta1 and alpha6beta4 integrins, were down-regulated. Recent studies showed that keratinocytes over express and deposit laminin 5 during migration and we have shown that laminin 5 found in the matrix of TGF-beta1 induced migrating keratinocytes is present in its unprocessed form [Décline and Rousselle, 2001: J. Cell Sci. 114:811-823]. We show here that TGF-beta1 treatment of the cells promoted a significant increase in their adhesion to the alpha3 chain carboxy-terminal LG4/5 subdomain and that this interaction is likely to be mediated by a heparan sulfate proteoglycan type of receptor. Our results indicate that alpha6beta4 and alpha3beta1 integrin interactions with laminin 5 are diminished during migration while a specific interaction occurs between an additional cellular receptor and the alpha3 LG4/5 module present on unprocessed laminin 5.


Subject(s)
Cell Adhesion/drug effects , Cell Communication/physiology , Cell Movement/physiology , Keratinocytes/physiology , Laminin/metabolism , Transforming Growth Factor beta/pharmacology , Cell Communication/drug effects , Cell Movement/drug effects , Cells, Cultured , Cytoskeleton/drug effects , Down-Regulation , Extracellular Matrix/metabolism , Humans , Integrin alpha3beta1/biosynthesis , Integrin alpha3beta1/drug effects , Integrin alpha3beta1/genetics , Integrin alpha6beta4/biosynthesis , Integrin alpha6beta4/drug effects , Integrin alpha6beta4/genetics , Keratinocytes/cytology , Keratinocytes/drug effects , Male , Phenotype , Receptors, Laminin/drug effects , Receptors, Laminin/metabolism , Transforming Growth Factor beta1 , Wound Healing
4.
Int J Dermatol ; 41(12): 836-40, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12492965

ABSTRACT

BACKGROUND: Interferon-alpha and -gamma are glycoproteins with antiviral and immunoregulatory properties. In vitro studies have shown a role for these cytokines in the regulation of epidermal keratinocyte growth and differentiation. In the same way, integrins are adhesion molecules which regulate keratinocyte proliferation and differentiation. AIM: To determine whether the regulatory activity of interferons on keratinocyte proliferation and differentiation is related to a modulation of keratinocyte integrins. METHODS: Two different methods were used: monolayers and reconstituted skin, incubated either with 1,200 U/mL interferon-alpha or 500 U/mL interferon-gamma or control medium for 48 h. The integrin expression was assessed by flow cytometry and immunohistochemistry. RESULTS: In monolayers, only the alpha3 subunit was significantly inhibited by interferon-gamma. In reconstituted skin, where keratinocytes are differentiated, both interferons had an inductive effect on beta1 expression and interferon-alpha had an inhibitory effect on alpha6 expression. CONCLUSION: Interferon-alpha and -gamma induce a modulatory effect on alpha3, alpha6 and beta1 which appears to be related to the state of differentiation. Moreover, the decreased expression of alpha6 and alpha3 could be one of the mechanisms involved in the formation of bullous lesions during long-term interferon therapy.


Subject(s)
Antigenic Modulation/drug effects , Immunologic Factors/pharmacology , Integrins/analysis , Integrins/drug effects , Interferon-alpha/pharmacology , Keratinocytes/drug effects , Cell Differentiation/drug effects , Humans , In Vitro Techniques , Integrin alpha2beta1/analysis , Integrin alpha2beta1/drug effects , Integrin alpha3beta1/analysis , Integrin alpha3beta1/drug effects , Integrin alpha5beta1/analysis , Integrin alpha5beta1/drug effects , Integrin alpha6beta4/analysis , Integrin alpha6beta4/drug effects , Keratinocytes/pathology , Receptors, Vitronectin/analysis , Receptors, Vitronectin/drug effects , Time Factors
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