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E Ruoslahti

Publications and source records attributed to E Ruoslahti.

At least 19 recordsLinked to original sources
PubMed1992

Natural inhibitor of transforming growth factor-beta protects against scarring in experimental kidney disease.

The central pathological feature of human kidney disease that leads to kidney failure is the accumulation of extracellular matrix in glomeruli. Overexpression of transforming growth factor-beta (TGF-beta) underlies the accumulation of pathological matrix in experimental glomerulonephritis. Administration of an antibody raised against TGF-beta to glomerulonephritic rats suppresses glomerular matrix production and prevents matrix accumulation in the injured glomeruli. One of the matrix components induced by TGF-beta, the proteoglycan decorin, can bind TGF-beta and neutralize its biological activity, so decorin may be a natural regulator of TGF-beta (refs 3, 4). We tested whether decorin could antagonize the action of TGF-beta in vivo using the experimental glomerulonephritis model. We report here that administration of decorin inhibits the increased production of extracellular matrix and attenuates manifestations of disease, confirming our hypothesis. On the basis of our results, decorin may eventually prove to be clinically useful in diseases associated with overproduction of TGF-beta.

Animals↗
PubMed1992

Hyaluronate binding properties of versican.

We have previously cloned a large chondroitin sulfate proteoglycan (versican) from human fibroblasts. The primary sequence shows that the N terminus contains sequence homology with known hyaluronate-binding molecule, suggesting that versican can bind hyaluronate. To test this hypothesis we have reconstructed a full-length versican cDNA and a versican cDNA fragment encoding the N terminus and have transfected Chinese hamster ovary cells and mouse 3T3 fibroblasts, respectively, with these constructs. The transfected Chinese hamster ovary cells make a proteoglycan shown to be versican by enzymatic and immunologic analysis. No corresponding proteoglycan was seen in the control cells. Using hyaluronate affinity chromatography, we show that recombinant versican specifically binds hyaluronate and does not bind to heparin or chondroitin sulfate. The transfected fibroblasts make a 78-kDa truncated form of versican that also binds hyaluronate and does not bind the related polysaccharides, showing that the hyaluronate binding activity resides at the N terminus of versican. The binding of versican to hyaluronate is substrate-concentration dependent and time dependent and can be competed with unlabeled versican. The dissociation constant for versican binding to hyaluronate was determined to be 4 x 10(-9) M.

3T3 Cells↗
PubMed1992

An alternative form of the integrin beta 1 subunit with a variant cytoplasmic domain.

We have identified an alternatively spliced form of the human beta 1 integrin subunit, beta 1S. The beta 1S mRNA is expressed in human platelets, HEL and K562 erythroleukemia cell lines, and THP1 monocytic and HL60 promyelocytic cell lines. It is undetectable in peripheral blood lymphocytes and cultured umbilical vein endothelial cells at early passages. The beta 1S cDNA encodes a new cytoplasmic domain distinct from the previously reported alternative cytoplasmic domain of the beta 1 subunit. The sequence reveals the presence of an insert of 116 nucleotides which produces a frame shift in the previously reported 3' end of the beta 1 integrin subunit and codes for a unique 48-amino acid COOH-terminal sequence. An antiserum prepared against a synthetic peptide generated from the deduced sequence of the beta 1S cytoplasmic domain immunoprecipitated an HEL cell surface molecule that comigrated with the usual beta 1 subunit in sodium dodecyl sulfate electrophoresis. The immunoprecipitation indicated that beta 1S constitutes a minor portion of total beta 1 subunit in these cells. This variant beta 1 cytoplasmic tail may modulate integrin affinity and may provide additional modes for the transduction of extracellular signals and modulation of cytoskeletal organization by beta 1 integrins.

Amino Acid Sequence↗
PubMed1992

Role of the alpha v beta 3 integrin in human melanoma cell invasion.

The human melanoma cell line A375M expresses the vitronectin receptor (alpha v beta 3 integrin) on its cell surface. Treatment of A375M cells with either polyclonal or monoclonal anti-alpha v beta 3 antibodies resulted in stimulation of invasion through basement membrane matrices in vitro. Similar treatment of these cells with a monoclonal anti-alpha v antibody, which does not inhibit the adhesive function of the alpha v beta 3 antigen, also stimulated invasion; however, anti-beta 3 antibody treatment had no effect. Furthermore, pretreatment of the cells with vitronectin or addition of vitronectin to the basement membrane matrix also resulted in stimulation of invasion. Similar treatments with fibronectin receptor antibody or fibronectin had no effect on invasion. Analysis of type IV collagenase expression in cells treated with anti-alpha v beta 3 antibody showed higher levels of both the secreted 72-kDa enzyme and its mRNA. Signal transduction through the alpha v beta 3 integrin could underlie the elevated expression of metalloproteinase and the enhanced invasion of A375M cells through basement membrane matrices.

Cell Adhesion↗
PubMed1992

Immunolocalization of ciliary neuronotrophic factor in adult rat sciatic nerve.

Two rabbit polyclonal antibodies were raised against synthetic peptides corresponding to residue numbers 45-59 and 181-200 of rat ciliary neuronotrophic factor (CNTF). The resulting antibodies were purified by affinity chromatography and both purified antibodies reacted by enzyme-linked immunoassay (ELISA) and immunoblotting with rat sciatic nerve CNTF. The anti-CNTF peptide antibodies were used to immunostain sections of adult rat sciatic nerve, previously known as the richest tissue source of CNTF. By light microscopy both antibodies appeared to stain exclusively Schwann cells and axons and both did so with the same pattern of specific staining. Immunostaining was eliminated by absorption of the anti-peptide antibodies with either their corresponding peptide or with purified rat nerve CNTF or by using purified nonspecific IgG. Schwann cells were stained and in semi-thin sections this staining appeared to be in the Schwann cell cytoplasm. Axons could be stained in addition to Schwann cells providing higher concentrations of antibodies were used. Epineurial, endoneurial and endothelial cells appeared unstained. Since all Schwann cells and axons appear to contain CNTF and since CNTF is known to act in vitro to support sensory and sympathetic ganglionic and motor neurons, we suggest that Schwann cells may normally provide CNTF to those neurons contributing axons to the peripheral nerve.

Amino Acid Sequence↗
PubMed1992

The Walter Herbert Lecture. Control of cell motility and tumour invasion by extracellular matrix interactions.

Integrins are heterodimeric transmembrane proteins with large ectodomains and a short cytoplasmic tail inside the cell. They mediate cell adhesion to extracellular matrix proteins and to the surfaces of other cells. In many cases the sequence recognised by the integrins in the extracellular matrix proteins is the tripeptide Arg-Gly-Asp (RGD). Short synthetic peptides containing this sequence can inhibit invasion in vitro and tumour dissemination in vivo. Thus, the alpha 5 beta 1 fibronectin binding integrin appears to be the key integrin in the invasion of at least melanoma, osteosarcoma and glioblastoma cells. Modulation of the level and activities of this integrin can suppress invasion, whereas the alpha v beta 3 vitronectin binding integrin appears to be associated with increased invasiveness. There is increasing evidence that some of these effects are mediated through signals elicited by the binding of integrins to their target proteins. This possibility has generated a great deal of interest in the cytoplasmic molecules that might mediate the integrin-associated signalling.

Amino Acid Sequence↗
PubMed1992

Production of extracellular matrix by glomerular epithelial cells is regulated by transforming growth factor-beta 1.

Transforming growth factor-beta (TGF-beta) has widespread effects on extracellular matrix production by many cultured cell lines and appears to play a role in the pathological accumulation of extracellular matrix that accompanies inflammatory and fibrotic diseases such as glomerulonephritis. Earlier experiments have shown that mesangial cells respond to TGF-beta 1 with a marked increase in the production of two chondroitin/dermatan sulfate proteoglycans, decorin and biglycan, but their production of other matrix components elevated in glomerulonephritis is not substantially affected by TGF-beta 1. Since the glomerular epithelial cells are also thought to contribute to matrix production in the glomerulus, we examined the ability of these cells to produce some of the nonproteoglycan matrix components in response to TGF-beta 1. Exposure of glomerular epithelial cells to TGF-beta 1 increased the production of fibronectin and type IV collagen, in addition to biglycan. Enhancement of the cell layer accumulation of laminin was also observed. These results show that TGF-beta 1 has a differential effect on extracellular matrix production by epithelial and mesangial cells from glomeruli. TGF-beta 1 released in the glomerulus secondary to injury could thus affect both cell types and lead to increased intraglomerular production of proteoglycans, whereas the increased fibronectin, type IV collagen, and laminin may primarily originate from the epithelial cells.

Animals↗
PubMed1992

Motility of fibronectin receptor-deficient cells on fibronectin and vitronectin: collaborative interactions among integrins.

Cells are capable of adhering to and migrating on protein components of the extracellular matrix. These cell-matrix interactions are thought to be mediated largely through a family of cell surface receptors termed integrins. However, the manner in which individual integrins are involved in cell adhesion and motility has not been fully determined. To explore this issue, we previously selected a series of CHO variants that are deficient in expression of the integrin alpha 5 beta 1, the "classical" fibronectin receptor. Two sets of subclones of these variants were defined which respectively express approximately 20% or 2% of fibronectin receptor on the cell surface when compared to wild-type cells (Schreiner, C. L., J. S. Bauer, Y. N. Danilov, S. Hussein, M. M. Sczekan, and R. L. Juliano. 1989. J. Cell Biol. 109:3157-3167). In the current study, the variant clones were tested for haptotactic motility on substrata coated with fibronectin or vitronectin. Data from assays using fibronectin show that cellular motility of the 20% variants was substantially decreased (30-75% of wild type), while the motility of the 2% variants was nearly abolished (2-20% of wild type). Surprisingly, a similar pattern was seen for haptotactic motility of both 2% and 20% variants when vitronectin was used (approximately 20-30% of wild type). The reduced haptotactic motility of the fibronectin receptor-deficient variant clones on vitronectin was shown not to be due to reduced vitronectin receptor (alpha v beta 3) expression nor to a failure of these variants to adhere to vitronectin substrata. Transfection of the deficient variants with a cDNA for the human alpha 5 subunit resulted in normal levels of fibronectin receptor expression (as a human alpha 5/hamster beta 1 chimera) and restored the motility of the CHO variants on fibronectin and vitronectin. This indicates that expression of the alpha 5 subunit is required for normal haptotactic motility on vitronectin substrata and suggests that the fibronectin receptor (alpha 5 beta 1) plays a cooperative role with vitronectin receptors in cell motility.

Animals↗
PubMed1992

A fibronectin self-assembly site involved in fibronectin matrix assembly: reconstruction in a synthetic peptide.

The active form of fibronectin is its extracellular matrix form, which allows for the attachment of cells and influences both the growth and migration of cells. The matrix form is assembled by cells; however, many cells are defective in this regard. Several regions within fibronectin have been shown to play a role in matrix assembly by cells. One such region has been localized into the first type III repeat of fibronectin (Chernousov, M. A., F. J. Fogerty, V. E. Koteliansky, and D. F. Mosher. J. Biol. Chem. 266:10851-10858). We have identified this site as a fibronectin-fibronectin binding site and reproduced it as a synthetic peptide. This site is contained in a 14-kD fragment that corresponds to portions of the first two type III repeats. The 14-kD fragment was found to bind to cell monolayers and to inhibit fibronectin matrix assembly. The 14-kD fragment only slightly reduced the binding of fibronectin to cell surfaces but it significantly inhibited the subsequent incorporation of fibronectin into the extracellular matrix. The 14-kD fragment also bound to purified fibronectin and inhibited fibronectin-fibronectin binding. A synthetic 31-amino acid peptide (P1) representing a segment of the 14-kD fragment retained the ability to inhibit fibronectin-fibronectin binding. Peptide P1 specifically bound fibronectin from plasma in affinity chromatography, whereas a column containing another peptide from the 14-kD fragment did not. These results define a fibronectin-fibronectin binding site that appears to promote matrix assembly by allowing the assembly of fibronectin molecules into nascent fibrils. The 14-kD fragment and the P1 peptide that contain this site inhibit matrix assembly by competing for the fibronectin-fibronectin binding.

Amino Acid Sequence↗
PubMed1992

Proteolytic processing of endogenous and recombinant beta 4 integrin subunit.

The alpha 6 beta 4 integrin is a receptor involved in the interaction of epithelial cells with basement membranes. This integrin is unique among the known integrins in that its beta 4 subunit has a large cytoplasmic domain. The function of this cytoplasmic domain is not known. In this paper we show that the beta 4 subunit undergoes proteolytic processing in cultured cells and provide evidence that this also happens in tissues. Immunoprecipitation experiments indicated that the cytoplasmic domain of beta 4 is susceptible to a calcium-dependent protease present in cellular extracts. In vitro assays with purified calpain showed that this enzyme can cleave beta 4 at two distinct sites in the cytoplasmic domain, generating truncated molecules of 165 and 130 kD. Immunoblotting experiments performed on cultured epithelial cells using an antibody to a peptide modeled after the COOH-terminus of the beta 4 subunit showed 70-kD fragments and several fragments of molecular masses between 185 and 115 kD. Similar fragments were detected in CHO cells transfected with the full-length beta 4 cDNA, but not in control transfected cells or in cells transfected with a mutant cDNA lacking the epitope of the cytoplasmic peptide antibody. The sizes of the fragments indicated that both the intracellular and extracellular domains of beta 4 are proteolytically processed. To examine the processing of the beta 4 subunit in epithelial tissues in vivo, human skin frozen sections were stained with antibodies to the ectodomain or the cytoplasmic domain of beta 4. The distinct staining patterns obtained with the two types of antibodies provided evidence that beta 4 is proteolytically processed in vivo in skin. Analogous experiments performed on sections of the cornea suggested that beta 4 is not proteolytically processed at a detectable level in this tissue. Thus, cleavage of the beta 4 subunit occurs in a tissue-specific fashion. These results suggest a potential mechanism of modulating the activities of the alpha 6 beta 4 integrin.

Amino Acid Sequence↗
PubMed1991

Dietary protein restriction rapidly reduces transforming growth factor beta 1 expression in experimental glomerulonephritis.

Dietary protein restriction has been shown to slow the rate of loss of kidney function in humans with progressive glomerulosclerosis due to glomerulonephritis or diabetes mellitus. A central feature of glomerulosclerosis is the pathological accumulation of extracellular matrix within the diseased glomeruli. Transforming growth factor beta 1 (TGF-beta 1) is known to have widespread regulatory effects on extracellular matrix and has been implicated as a major cause of increased extracellular matrix synthesis and buildup of pathological matrix within glomeruli in experimental glomerulonephritis. In the present study, it is shown that administration of a low protein diet to rats rapidly reduces the elevated expression of TGF-beta 1 mRNA and TGF-beta 1 protein that is known to occur within glomeruli after induction of glomerulonephritis. Compared to a normal protein diet, glomerulonephritic rats receiving the low protein diet did not develop an increase in glomerular extracellular matrix and showed significantly less proteinuria. Glomeruli isolated from glomerulonephritic rats fed the normal protein diet showed a marked increase in proteoglycan synthesis on day 7 of disease and were demonstrated to be secreting increased amounts of TGF-beta 1 into the medium, whereas glomeruli at the same point in time isolated from rats on a low protein diet showed no increase in proteoglycan production or TGF-beta 1 secretion. These results suggest that a mechanism of the rapid therapeutic effect of a low protein diet on experimental glomerulonephritis is through suppression of TGF-beta 1 expression and prevention of the induction of extracellular matrix synthesis within the injured glomeruli.

Animals↗
PubMed1991

The alpha 5 beta 1 fibronectin receptor. Characterization of the alpha 5 gene promoter.

A genomic clone containing the gene for the alpha 5 subunit of the human alpha 5 beta 1 integrin complex was isolated by screening a human genomic library with the previously described alpha 5 cDNA (Argraves, W. S., Suzuki, S., Arai, H., Thompson, K., Pierschbacher, M. D., and Ruoslahti, E. (1987) J. Cell Biol 105, 1183-1190). The alpha 5 gene 5'-flanking region lacks both TATA and CCAAT boxes, and it is located in a CpG island. This region was an active promoter in transfection assays using the HT-1080 cell line (fibrosarcoma), which expresses alpha 5, but was inactive in the Raji cell line (B cell), which does not express alpha 5. These results indicate that the alpha 5 gene 5'-flanking region acts as a promoter that exhibits the expected cell-type specificity. Deletion of alpha 5 promoter sequences from position -657 to -178 caused transcription stimulation, suggesting that a silencer is located between these sites. Successive 5' deletions from position -178 decreased promoter activity until activity was essentially eliminated on deletion to position -27. Isolation of a functional promoter for the alpha 5 gene is a first step in understanding how expression of this gene is controlled at the molecular level.

Amino Acid Sequence↗
PubMed1991

The human leukocyte-adhesion ligand, intercellular-adhesion molecule 2. Expression and characterization of the protein.

The leukocyte cell-adhesion receptors, complexes of the cluster of differentiation antigen 11a with cluster of differentiation antigen 18 (CD11a/CD18), cluster of differentiation antigen 11b with cluster of differentiation antigen 18 (CD11b CD18) and cluster of differentiation antigen 11c with cluster of differentiation antigen 18 (CD11c CD18), are of major importance in several leukocyte functions. Previously a cellular ligand named intercellular-adhesion molecule 1 (ICAM-1) was identified, isolated and extensively characterized. Recently a second similar molecule, intercellular-adhesion molecule 2 (ICAM-2), was found by a functional DNA-cloning method. We have now synthesized the ICAM-2 DNA by the polymerase chain reaction (PCR), sequenced it, and transferred it into mammalian and bacterial expression vectors. A functional leukocyte-binding glycoprotein was obtained by transfection of COS-1 cells. A soluble protein-A - ICAM-2 fusion protein was made in Escherichia coli, purified and used for antiserum production. The antiserum precipitated a cell-surface protein with an apparent molecular mass of 55 kDa from ICAM-2 transfected COS-1 cells, leukocytes and endothelial cells, and inhibited leukocyte binding to transfected COS-1 cells. The bacterial fusion protein, lacking carbohydrate, specifically bound to leukocyte receptors.

Animals↗
PubMed1991

Molecular cloning of the cDNA encoding human laminin A chain.

Laminin is a large basement membrane glycoprotein composed of three subunits designated the A, B1, and B2. We report here the isolation and nucleotide sequence of human laminin A chain cDNA. The nucleotide sequence spans 9505 bases and has an open reading frame encoding 3075-amino acids. The sequence covers a 77-nucleotide long 5' untranslated region and a 190-nucleotide long 3' sequence in front of the poly (A)+ tail. In analogy with the mouse A chain sequence, the deduced human amino acid sequence contains eight-distinct domains of four-globular regions, three-cysteine-rich domains and an alpha-helical region, which is though to interact with the B chains of laminin. The deduced amino acid sequence is 14-amino acids shorter than the mouse A chain sequence. Seven of these amino acids are located in the putative signal sequence. The overall identity between the sequences from the two species is 78%. The carboxylterminal globular (G) domain contains five homologous subdomains characterized by a conserved seven-amino acid repeat within each subdomain. Both human and mouse A chain are about 39% identical to the G domain of merosin, a recently discovered A chain homologue. Unlike the mouse A chain, the human A chain contains a potential cell binding sequence (RGD) in this domain. The RGD sequence that is thought to be a cryptic cell attachment site in the amino-terminal domain IIIb of mouse laminin is not conserved in the human sequence.

Amino Acid Sequence↗
PubMed1991

Integrins.

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Animals↗