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M Aumailley

Publications and source records attributed to M Aumailley.

At least 55 records · Page 3Linked to original sources
PubMed1992

Increased adhesion of fibroblasts from patients with scleroderma to extracellular matrix components: in vitro modulation by IFN-gamma but not by TGF-beta.

A characteristic feature of systemic scleroderma is fibrosis of the skin and eventually of internal organs resulting from an overproduction of collagen and other connective tissue components by the resident fibroblasts. The balance between the cells and the amount of the surrounding extracellular matrix is then altered. Because cellular metabolism depends to a large extent on cellular contacts and communications with connective tissue molecules, we have therefore investigated the interactions with extracellular matrix components of fibroblasts obtained from skin of patients affected with scleroderma. In comparison to fibroblasts from healthy skin, all fibroblasts from scleroderma patients had an increased adhesion capacity to collagens I, IV, VI, fibronectin, and laminin. In addition, whereas adhesion of control fibroblasts was stimulated by a pre-treatment with transforming growth factor-beta, adhesion patterns of scleroderma fibroblasts remained unchanged. However, pre-incubation of the cells with interferon-gamma decreased the adhesion of both scleroderma and control fibroblasts.

Cell Adhesion↗
PubMed1991

Localization of a major nidogen-binding site to domain III of laminin B2 chain.

The large pepsin fragments P1 and P1X, which comprise most of the rod-like domains III of the three short arms of laminin from the mouse Engelbreth-Holm-Swarm tumor, possess full binding activity for nidogen in radioligand assays. Partial reduction (70-80%) of disulfide bonds in P1 did not reduce binding activity and allowed the separation of domain III segments originating from the A, B1 and B2 chains of laminin as demonstrated by sequence analysis. Only the B2 chain segment consisting of seven cysteine-rich repeats with similarity to epidermal growth factor showed substantial nidogen-binding activity. Further degradation of this component to an active 28-kDa fragment was achieved by a second pepsin digestion of partially reduced P1. This indicates that a major binding structure for nidogen is located within three or four cysteine-rich repeats occupying sequence positions 755 to about 920 in the B2 chain. The data also show that fragments P1 and P1X differ by the absence or presence of a large portion, domain IIIb, of the laminin A chain but are indistinguishable in nidogen binding.

Amino Acid Sequence↗
PubMed1991

Arg-Gly-Asp constrained within cyclic pentapeptides. Strong and selective inhibitors of cell adhesion to vitronectin and laminin fragment P1.

Cyclic Arg-Gly-Asp-Phe-Val peptides with either D-Phe or D-Val residues were 20- to more than 100-fold better inhibitors of cell adhesion to vitronectin and/or laminin fragment P1 when compared to a linear variant or Gly-Arg-Gly-Asp-Ser. No or only little increase in inhibitory capacity was observed for fibronectin adhesion and for the binding of platelet receptor alpha IIb beta 3 to fibrinogen. NMR studies of the two most active cyclic peptides showed for both an all-trans conformation with a beta II' and gamma turn. Subtle conformational differences, however, exist between both peptides and may contribute to selectivity of inhibition.

Amino Acid Sequence↗
PubMed1991

Recombinant expression and properties of the human calcium-binding extracellular matrix protein BM-40.

A cDNA construct (approximately 1 kb) of human BM-40 in a plasmid with the cytomegalovirus promoter and enhancer was used to produce several stable clones by transfecting two human cell lines (293, HT 1080). These clones showed a high expression of exogenous 1-kb BM-40 mRNA and no or only little endogenous 2.2-kb mRNA. These clones also secreted BM-40 at high rates (5-50 micrograms ml-1 day-1) into serum-free culture medium as shown by electrophoresis, radioimmunoassay and metabolic labelling. Transfection with the plasmid and overexpression of BM-40 had no effect on cell spreading, proliferation rate and adhesion patterns to extracellular matrix substrates. Recombinant human BM-40 was purified by anion-exchange chromatography and showed the expected N-terminal sequence and amino acid composition. The protein was also identical or similar to authentic BM-40 purified from the mouse Engelbreth-Holm-Swarm tumor in hexosamine content, electrophoretic mobility, circular dichroism and binding activity for calcium and collagen IV. Reduction of both authentic and recombinant BM-40 decreased binding activity which indicates correct formation of disulfide bonds in the recombinant protein. A specific and sensitive radioimmunoassay for human BM-40 was shown to be useful for detecting small quantities of the protein in human cell culture medium and blood. No significant cross-reaction was, however, detected between human and mouse BM-40.

Amino Acid Sequence↗
PubMed1991

Calcium-dependent binding of basement membrane protein BM-40 (osteonectin, SPARC) to basement membrane collagen type IV.

Basement membrane protein BM-40, prepared from the mouse Engelbreth-Holm-Swarm tumor, was used in native, denatured and proteolytically processed form for binding to various extracellular matrix proteins. BM-40 and its derivatives were also characterized by CD spectroscopy, calcium binding and epitope analysis. Of several basement membrane proteins tested only collagen IV showed a distinct and calcium-dependent binding of BM-40 in an immobilized ligand assay. This interaction was specific as shown by a low activity of other collagen types (I, III, V, VI) in direct binding and competition assays. The binding was reduced or abolished by metal-ion-chelating or chaotropic agents, high salt and reduction of disulfide bonds in BM-40. Fragment studies indicated that domains III (alpha-helix) and/or IV (EF hand) of BM-40 possess the binding site(s) for collagen IV, while the N-terminal domains I and II provide the major antigenic determinants. A major BM-40-binding site on collagen IV was dependent on a triple-helical conformation and could be localized to a pepsin fragment from the central portion of the triple-helical domain, in agreement with electron microscopic visualization of BM-40--collagen-IV complexes.

Amino Acid Sequence↗
PubMed1991

Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV.

Recombinant mouse nidogen and two fragments were produced in mammalian cells and purified from culture medium without resorting to denaturing conditions. The truncated products were fragments Nd-I (positions 1-905) comprising the N-terminal globule and rod-like domain and Nd-II corresponding mainly to the C-terminal globule (position 906-1217). Recombinant nidogen was indistinguishable from authentic nidogen obtained by guanidine dissociation from tumor tissue with respect to size, N-terminal sequence, CD spectra and immunochemical properties. They differed in protease stability and shape indicating that the N-terminal domain of the more native, recombinant protein consists of two globules connected by a flexible segment. This established a new model for the shape of nidogen consisting of three globes of variable mass (31-56 kDa) connected by either a rod-like or a thin segment. Recombinant nidogen formed stable complexes (Kd less than or equal to 1 nM) with laminin and collagen IV in binding assays with soluble and immobilized ligands and as shown by electron microscopy. Inhibition assays demonstrated different binding sites on nidogen for both ligands with different specificities. This was confirmed in studies with fragment Nd-I binding to collagen IV and fragment Nd-II binding to laminin fragment P1. In addition, recombinant nidogen but not Nd-I was able to bridge between laminin or P1 and collagen IV. Formation of such ternary complexes implicates a similar role for nidogen in the supramolecular organization of basement membranes.

Animals↗
PubMed1991

Differences in laminin fragment interactions of normal and transformed endothelial cells.

Bovine aortic and microvascular endothelial cells showed good adhesion with spreading on fibronectin or collagen IV and to a lower extent on laminin. Recognition of native laminin was due to its long arm fragment E8 and was mediated by alpha 6 integrins as demonstrated by antibody inhibition. A considerably stronger, RGD-dependent interaction was observed with the isolated laminin short arm fragment P1 previously shown to represent a cryptic cell-binding site. No adhesion was observed with the heparin-binding fragment E3. In contrast, murine microvascular endothelial cells transformed by the polyoma middle T oncogene showed preferential adherence and spreading on laminin via its E8 cell-binding site and also showed adhesion to fragment E3. Attachment to laminin fragment P1 and to collagen IV was low or negative and was never followed by spreading. These data show that the transformation of microvascular endothelial cells, which give them the property to form hemangiomas, also leads to changes in cell adhesion to extracellular matrix proteins, particularly to laminin fragments.

Amino Acid Sequence↗
PubMed1991

Isolation of alpha 6 beta 1 integrins from platelets and adherent cells by affinity chromatography on mouse laminin fragment E8 and human laminin pepsin fragment.

Ligand affinity chromatography was used to identify receptors on platelets and two adherent cell lines, OV-CAR-4 and HBL-100, for the E8 fragment of murine laminin. A complex of two polypeptides (140 and 110 kDa nonreduced) was bound by the E8 affinity columns from all three cell types and was eluted with EDTA. This heterodimeric complex was identified as the alpha 6 beta 1 integrin by immunoprecipitation with specific antibodies against either the alpha 6 or the beta 1 subunit. The alpha 6 beta 1 integrin did not bind to an affinity column containing fragment P1 originating from a different part of murine laminin which, however, bound the alpha IIb beta 3 integrin from platelets. Furthermore, in immunofluorescence staining, the alpha 6 beta 1 integrin localizes in focal contacts of OVCAR-4 cells attached to laminin and E8 but not to fibronectin substrates. These results, combined with previous antibody inhibition studies, unequivocally identify the alpha 6 beta 1 integrin as a specific receptor for fragment E8. Affinity chromatography of OVCAR-4 and HBL-100 cells on a large pepsin fragment of laminin from human placenta yielded integrin alpha 3 beta 1. When alpha 3 beta 1 was removed from lysates of OVCAR-4 cells by preclearing with an alpha 3-specific monoclonal antibody, alpha 6 beta 1 was able to bind to human laminin as well. Integrin alpha 6 beta 1 on platelets which do not express alpha 3 beta 1 binds directly to human laminin. These results indicate that both alpha 3 beta 1 and alpha 6 beta 1 can act as receptors for human laminin and may interfere by steric hindrance. The alpha 6 beta 4 complex, which is strongly expressed on HBL-100 cells, did not bind to either mouse laminin fragment E8 or human laminin affinity columns.

Amino Acid Sequence↗
PubMed1991

Multiple cell surface receptors for the short arms of laminin: alpha 1 beta 1 integrin and RGD-dependent proteins mediate cell attachment only to domains III in murine tumor laminin.

Cell surface molecules that interact with the cross formed by the three short arms of murine tumor laminin were studied using thermal perturbation, antibody and peptide blocking, and affinity chromatography. Several potential receptors for the laminin short arms were revealed that differed from those mediating cell attachment to the E8 (long arm) fragment. Two cell lines, Rugli and L8 attached well to E1-X (short arm) fragments of laminin. This attachment was blocked by antibodies against alpha 1 integrin chains. Other cells were unable to attach strongly to E1-X, but attached to P1. This attachment was unaffected by anti-beta 1 integrin antibodies, but specifically blocked by the peptide GRGDS. By contrast, binding of Rugli cells was RGD independent and blocked by anti-beta 1 integrin antibodies. G7 and C2C12 myoblasts were very sensitive to GRGDS (ID50 approximately 2 micrograms.ml-1) for attachment to P1 which implied that a non-beta 1 series integrin, possibly alpha v beta 3, was involved. On heat denaturation of P1(3) attachment remained sensitive to RGDS and ID50 was unchanged. On heat denaturation of E1-X, attachment remained sensitive to RGDS but the ID50 increased to approximately 200 micrograms.ml-1. Cellular beta 1 integrins were retained on laminin affinity columns. A beta 1 integrin with an approximately 190 kD alpha-chain could be isolated from Rugli cells whose attachment could be blocked by anti-alpha 1 antibodies and not from cells blocked by RGDS peptides. Anti-alpha 1 antibodies blocked Rugli attachment to native laminin, but only when the E8 cell binding sites on laminin were also blocked. Thus, a receptor related to alpha 1 beta 1 integrin can function simultaneously with a receptor for E8. Anti-alpha 1 also blocked attachment to heated laminin, suggesting that the heat-stable attachment activity in laminin involved the E1-X binding site. Thus, at least two putative receptors mediate attachment to the short arms of laminin. One, related to alpha 1 beta 1 integrin, recognizes RGDS-independent sites in E1-X defined by P1 (within domains III, IIIa, IIIb), and one is an RGD-dependent molecule recognizing sites in P1, and is not a beta 1 integrin.

Amino Acid Sequence↗
PubMed1991

Activation of collagen gene expression in keloids: co-localization of type I and VI collagen and transforming growth factor-beta 1 mRNA.

Untreated, clinically active keloids were examined as model system to study the spatial expression of extracellular matrix and transforming growth factor-beta 1 (TGF-beta 1) genes in fibrotic skin diseases. In situ hybridizations localized active expression of type I and VI collagen genes to the areas containing an abundance of fibroblasts and apparently representing the expanding border of the lesions. Within this zone, microvascular endothelial cells also expressed the type I collagen genes, as evaluated by simultaneous use of in situ hybridization for collagen gene expression and immunolocalization for factor VIII-related antigen, a marker for endothelial cell differentiation. Slot-blot hybridizations of RNA isolated from this zone suggested that the expression of type I and IV collagen genes was selectively enhanced, as compared to type III collagen gene expression. TGF-beta 1 protein and mRNA were also detected in areas active in type I and type VI collagen gene expression, indicating that TGF-beta 1 gene is transcribed and the corresponding protein is deposited in areas of elevated collagen gene expression, including microvascular endothelial cells. We conclude that the initial step in the development of fibrotic reaction in keloids involves the expression of the TGF-beta 1 gene by the neovascular endothelial cells, thus activating the adjacent fibroblasts to express markedly elevated levels of TGF-beta 1, as well as type I and VI collagen genes.

Adolescent↗
PubMed1990

Cell adhesion, spreading and neurite stimulation by laminin fragment E8 depends on maintenance of secondary and tertiary structure in its rod and globular domain.

The cell adhesion, spreading and neurite-promoting properties of mouse tumor laminin fragment E8, which contains major site(s) responsible for laminin-cell interactions, were probed by proteolytic degradation, denaturation, synthetic peptides and antibody inhibition. Removal of more than half of the N-terminal portion contributing to the rod-like domain did not effect cell attachment or spreading although neurite-promoting activity was reduced. More extensive degradation of the rod or of the globular domains of E8, or separation of the globule from the rod, also resulted in loss of cell spreading activity although weak attachment was found to an A chain subfragment comprising the globular domain and a short piece of the rod. Exposure of E8 to increasing concentrations of dissociating agents produce an apparently reversible denaturation but an irreversible loss of both attachment and neurite-promoting activities, as did reduction and alkylation of disulfide bonds in the globular domain. Although cell adhesion and spreading were blocked by antibodies to an alpha 6 integrin subunit, neurite outgrowth was unaffected, indicating two distinct receptors for these two activities. Furthermore, a synthetic peptide, the sequence of which is found in the vicinity of adhesion and neurite-promoting sites and previously implicated in neurite growth and cell attachment activities, was found to be inactive. These results indicate that the major cell attachment and neurite-promoting sites of laminin are distinct although both require the native conformation of parts of the rod and the terminal globular domain of the long arm of laminin.

Amino Acid Sequence↗
PubMed1990

Identification of the Arg-Gly-Asp sequence in laminin A chain as a latent cell-binding site being exposed in fragment P1.

A single RGD-containing sequence present within an epidermal growth factor-like repeat of the short arms of laminin is shown by peptide inhibition to block integrin receptors recognizing a latent cell-binding site of laminin. Based on proteolysis data it is proposed that masking occurs by folding of the globular domain IVa over the cell-binding site in the adjacent rod-like structures of laminin A chain.

Binding Sites↗
PubMed1990

Nerve growth factor induces increased expression of a laminin-binding integrin in rat pheochromocytoma PC12 cells.

Rat pheochromocytoma PC12 cells exposed to nerve growth factor differentiate as sympathetic neurons and extend neurites on laminin and to a much lesser extent on fibronectin. Analysis of laminin fragments indicated that neurite outgrowth occurs mainly on fragment P1, corresponding to the center of the cross, and only poorly on fragment E8, a long arm structure that is active with other neuronal cells. Integrin antibodies prevented adhesion and neurite sprouting of these cells on laminin, fragment P1, and fibronectin. By affinity chromatography we isolated an integrin-type receptor for laminin consisting of two subunits with molecular massess of 180 and 135 kDa. The latter is recognized by an antiserum to integrin beta 1 subunit. The bound laminin receptor could be displaced by EDTA, but not by Arg-Gly-Asp or Tyr-Ile-Gly-Ser-Arg peptides. Affinity chromatography on laminin fragments showed that the 180/135 kDa receptor binds to P1. The expression of the 180-kDa alpha subunit of the laminin receptor at the cell surface was increased 10-fold after NGF treatment. The effect of NGF is specific since the amount of a 150-kDa fibronectin-binding integrin alpha subunit remained unchanged. Moreover, the increased expression of the 180/135 kDa receptor at the cell surface corresponded to a selective increase in cell adhesion to laminin and to fragment P1. The 180/135-kDa complex is thus an integrin-type receptor for laminin whose expression and binding specificity correlates with the capacity of NGF-induced PC12 cells to extend neurites on laminin.

Adrenal Gland Neoplasms↗
PubMed1990

Antibody to integrin alpha 6 subunit specifically inhibits cell-binding to laminin fragment 8.

A large number of cell lines which attach and spread on laminin show a comparable binding either to both laminin fragments P1 and E8 or exclusively to E8. Adhesion to fragment E8 was with one exception completely inhibited by a monoclonal antibody to the alpha 6 integrin subunit, indicating that VLA-6 or a related structure is the major cellular receptor for laminin. It is not involved in fragment P1 adhesion. Synthetic peptides possessing RGD or YIGSR sequences were without inhibitory activity for alpha 6-mediated adhesion to fragment E8.

Amino Acid Sequence↗
PubMed1990

Integrin recognition of different cell-binding fragments of laminin (P1, E3, E8) and evidence that alpha 6 beta 1 but not alpha 6 beta 4 functions as a major receptor for fragment E8.

The involvement of integrins in mediating interaction of cells to well-characterized proteolytic fragments (P1, E3, and E8) of laminin was assessed by antibody blocking studies. Cell adhesion to fragment P1 was affected by mAbs against the integrin beta 1 and beta 3 subunits and furthermore could be prevented completely by a synthetic peptide containing the Arg-Gly-Asp sequence. Because the beta 3 antibody-sensitive cell lines expressed the vitronectin receptor (alpha v beta 3) at high levels, the involvement of this receptor in cell adhesion to P1 is strongly suggested. Integrin-mediated cell adhesion to E3 is of low affinity and was inhibited by antibodies against the integrin beta 1 subunit. In contrast, adhesion of some cell types to E3 was not or only partially sensitive to inhibition by anti-integrin subunit antibodies. Cell adhesion to E8 was blocked completed by integrin alpha 6 or beta 1 antibodies. The alpha 6-specific antibody did not inhibit cell adhesion to E3 or P1. Furthermore, the antibody only blocked adhesion to laminin of those cells that adhered exclusively to the E8 fragment. In addition, expression of alpha 6 beta 1 was closely correlated with the ability of cells to bind to the E8 fragment of laminin. These results indicate that the alpha 6 beta 1 integrin is a specific receptor for the E8 fragment of laminin. Many cell types expressed, instead of or in addition to alpha 6 beta 1 the recently described integrin alpha 6 beta 4. Although the ligand of alpha 6 beta 4 was not identified, it must be different from that of alpha 6 beta 1, because cells that express alpha 6 beta 4, but not alpha 6 beta 1, do not adhere to E8, and cell adhesion to E8 was specifically blocked by beta 1 specific antibodies. In conclusion, the data indicate that distinct integrin receptors belonging to the beta 1 or beta 3 subfamily are involved in adhesion of cells to the various laminin fragments. Adhesion to E3 may also be brought about by other receptor molecules, possibly proteoglycans, not belonging to the integrin family.

Animals↗
PubMed1990

Recognition of the laminin E8 cell-binding site by an integrin possessing the alpha 6 subunit is essential for epithelial polarization in developing kidney tubules.

It has been previously shown that A-chain and domain(E8)-specific antibodies to laminin that inhibit cell adhesion also interfere with the establishment of epithelial cell polarity during kidney tubule development (Klein, G., M. Langegger, R. Timpl, and P. Ekblom. 1988. Cell. 55:331-341). A monoclonal antibody specific for the integrin alpha 6 subunit, which selectively blocks cell binding to E8, was used to study the receptors involved. Immunofluorescence staining of embryonic kidneys and of organ cultures of metanephric mesenchyme demonstrated coappearance of the integrin alpha 6 subunit and the laminin A-chain in regions where nonpolarized mesenchymal cells convert into polarized epithelial cells. Both epitopes showed marked colocalization in basal areas of tubules, while an exclusive immunostaining for alpha 6 was observed in lateral and apical cell surfaces of the tubular epithelial cells. Organ culture studies demonstrated a consistent inhibition of kidney epithelium development by antibodies against the alpha 6 subunit. The data suggest that the recognition of E8 cell-binding site of laminin by a specific integrin is crucial for the formation of kidney tubule epithelium from undifferentiated mesenchymal stem cells. In some other cell types (endothelium, some ureter cells) an exclusive expression of alpha 6 with no apparent colocalization of laminin A-chain in the corresponding basement membrane was seen. Thus, in these cells, integrins possessing the alpha 6 subunit may bind to laminin isoforms that differ from those synthesized by developing tubules.

Animals↗