epidermal-growth-factor and arginyl-glycyl-aspartic-acid

Recovery from ischemic and nephrotoxic acute renal failure (ARF) requires the replacement of damaged tubular cells with new ones that restore the continuity of the renal epithelium. The repair process involves a number of growth factors produced in renal tissue that participate as autocrine or paracrine regulators in the repair process. The aim of this review is threefold: (1) to focus on the role of local growth factors such as EGF, TGF-alpha, IGF-1, HGF and TGF-beta in renal regeneration immediately after an acute renal insult. Receptors for these growth factors have been found in renal epithelial cells, medullary interstitial cells and glomeruli. These mediators play an important role in renal repair by promoting tubular cell proliferation. (2) A review the data supporting the administration of these growth factors in animal models of ARF, and the possibility of using these mediators in humans for the purpose of accelerating renal recovery and decreasing the morbidity and mortality rates, and the costs of multidisciplinary medical care, is presented. (3) Finally, the possibilities of introducing supportive therapy aimed at specific targets such as RGD peptides to reduce intratubular obstruction, atrial natriuretic factor to improve altered glomerular hemodynamics, and cell therapy such as bioartificial renal tubule in association with dialysis are discussed.

Topics: Acute Kidney Injury; Animals; Cell Division; Epidermal Growth Factor; Growth Substances; Hepatocyte Growth Factor; Humans; Insulin-Like Growth Factor I; Kidney Tubules; Oligopeptides; Regeneration; Transforming Growth Factor alpha; Transforming Growth Factor beta

Controlled presentation of biomolecules on synthetic substrates is an important aspect for biomaterials development. If the immobilization of multiple biomolecules is required, highly efficient orthogonal surface chemistries are needed to ensure the precision of the immobilization. In this communication, chemical vapor deposition (CVD) copolymerization is used to fabricate polymer coatings with controlled ratio of alkyne and pentafluorophenyl ester (Pfp-ester) groups. Cyclic argine-glycine-aspartic acid (cRGD) adhesion peptide and epidermal growth factor (EGF) are immobilized through alkyne-azide cycloaddtion ("click" chemistry) and active ester-amine reaction, respectively. Cell studies with human umbilical vein endothelial cells (HUVEC) and A431 cell lines demonstrate the biological activity of the coimmobilized biomolecules.

Topics: Alkynes; Amines; Azides; Cell Line, Tumor; Click Chemistry; Epidermal Growth Factor; Esters; Gases; Human Umbilical Vein Endothelial Cells; Humans; Immobilized Proteins; Oligopeptides; Polymers

Developmental endothelial cell locus-1 (Del-1) glycoprotein is secreted by endothelial cells and a subset of macrophages. Del-1 plays a regulatory role in vascular remodeling and functions in innate immunity through interaction with integrin α(V)β(3). Del-1 contains 3 epidermal growth factor (EGF)-like repeats and 2 discoidin-like domains. An Arg-Gly-Asp (RGD) motif in the second EGF domain (EGF2) mediates adhesion by endothelial cells and phagocytes. We report the crystal structure of its 3 EGF domains. The RGD motif of EGF2 forms a type II' β turn at the tip of a long protruding loop, dubbed the RGD finger. Whereas EGF2 and EGF3 constitute a rigid rod via an interdomain calcium ion binding site, the long linker between EGF1 and EGF2 lends considerable flexibility to EGF1. Two unique O-linked glycans and 1 N-linked glycan locate to the opposite side of EGF2 from the RGD motif. These structural features favor integrin binding of the RGD finger. Mutagenesis data confirm the importance of having the RGD motif at the tip of the RGD finger. A database search for EGF domain sequences shows that this RGD finger is likely an evolutionary insertion and unique to the EGF domain of Del-1 and its homologue milk fat globule-EGF 8.

Topics: Amino Acid Motifs; Calcium-Binding Proteins; Carrier Proteins; Cell Adhesion Molecules; Epidermal Growth Factor; Fucose; Glycosylation; HEK293 Cells; Humans; Integrins; Models, Molecular; Oligopeptides; Protein Structure, Tertiary

The development of different techniques based on natural and polymeric scaffolds are useful for the design of different biomimetic materials. These approaches, however, require supplementary steps for the chemical or physical modification of the biomaterial. To avoid such steps, in the present study, we constructed a new multifunctional protein that can be easily immobilized onto hydrophobic surfaces, and at the same time helps enhance specific cell adhesion and proliferation onto collagen substrates. A collagen binding domain was fused to a previously constructed protein, which had an epidermal growth factor fused to a hydrophobic peptide that allows for cell adhesion. The new fusion protein, designated fnCBD-ERE-EGF is produced in Escherichia coli, and its abilities to bind to collagen and promote cell proliferation were investigated. fnCBD-ERE-EGF was shown to keep both collagen binding and cell growth-promoting activities comparable to those of the corresponding unfused proteins. The results obtained in this study also suggest the use of a fnCBD-ERE-EGF as an alternative for the design of multifunctional ECM-bound growth factor based materials.

Topics: Biomimetic Materials; Cell Adhesion; Cell Line; Cell Proliferation; Collagen; Elastin; Epidermal Growth Factor; Escherichia coli; Humans; Hydrophobic and Hydrophilic Interactions; Immobilized Proteins; Models, Molecular; Oligopeptides; Recombinant Fusion Proteins; Tissue Engineering

Chitosan scaffolds were prepared by freeze-drying method and modified with Arg-Gly-Asp (RGD) sequence of fibronectin or epidermal growth factor (EGF) by covalent immobilization. The results obtained from FTIR-ATR, fluorescence visualization and quantitative measurements showed that biosignal molecules, RGD and EGF, were successfully immobilized on chitosan scaffolds. ATDC5 murine chondrogenic cells were seeded on both type of scaffolds, chitosan-RGD and chitosan-EGF, and cultured for 28 days in stationary conditions. According to the results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) test, considerable increase in cell proliferation was only detected on chitosan-EGF scaffolds. Biochemical analysis of the chondrocyte seeded scaffolds showed that glycosaminoglycan (GAG) and deoxyribonucleic acid (DNA) content of the scaffolds increases with time. In conclusion, EGF-modified chitosan scaffolds (containing 1.83 microg EGF/3 mg dry scaffold) have been proposed to promote chondrogenesis and to have potential for reticular cartilage regeneration.

Topics: Analysis of Variance; Chitosan; Epidermal Growth Factor; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Oligopeptides; Spectroscopy, Fourier Transform Infrared; Tetrazolium Salts; Thiazoles; Tissue Engineering; Tissue Scaffolds

We have defined the molecular basis of cell adhesion to fibrillin-1, the major structural component of extracellular microfibrils that are associated with elastic fibres. Using human dermal fibroblasts, and recombinant domain swap fragments containing the Arg-Gly-Asp motif, we have demonstrated a requirement for upstream domains for integrin-alpha(5)beta(1)-mediated cell adhesion and migration. An adjacent heparin-binding site, which supports focal adhesion formation, was mapped to the fibrillin-1 TB5 motif. Site-directed mutagenesis revealed two arginine residues that are crucial for heparin binding, and confirmed their role in focal adhesion formation. These integrin and syndecan adhesion motifs juxtaposed on fibrillin-1 are evolutionarily conserved and reminiscent of similar functional elements on fibronectin, highlighting their crucial functional importance.

Topics: Base Sequence; Binding Sites; Cell Adhesion; DNA Primers; Epidermal Growth Factor; Fibrillin-1; Fibrillins; Heparin; Integrin alpha5beta1; Microfilament Proteins; Mutagenesis, Site-Directed; Oligopeptides; Recombinant Proteins

The properties of the insulin-like growth factor-binding proteins (IGFBP-1 to 6) are not limited to modulation of IGF actions. IGFBP-1, which shares an Arg-Gly-Asp (RGD) motif in its C-terminal domain, modulates cell motility by binding to integrin alpha5beta1. The cross-talks between integrins and growth factor receptor signalling pathways are extensively documented, particularly in the case of the epidermal growth factor receptor (EGFR). However, whether IGFBP-1 can modulate growth factor signalling through its interaction with integrin alpha5beta1 has not yet been studied. As EGF is involved in the decidualisation of endometrial stromal cells (ESCs) and as decidualised ESCs are a source of IGFBP-1, we investigated if IGFBP-1 can modulate EGF effects on ESCs. RGD- and IGF-independent inhibition of EGF mitogenic activity and EGFR signalling by IGFBP-1 were demonstrated in ESC primary cultures, A431, cells and in mouse fibroblasts lacking IGF receptors.

Topics: Animals; Cells, Cultured; Endometrium; Epidermal Growth Factor; Female; Fibroblasts; Insulin-Like Growth Factor Binding Protein 1; Integrin alpha5beta1; Mice; Mitosis; Oligopeptides; Receptor, IGF Type 1; Receptors, Fibroblast Growth Factor; Stromal Cells

CD97, a membrane protein expressed at high levels on inflammatory cells and some carcinomas, is a member of the adhesion G protein-coupled receptor family, whose members have bipartite structures consisting of an extracellular peptide containing adhesion motifs noncovalently coupled to a class B 7-transmembrane domain. CD97alpha, the extracellular domain of CD97, contains 3 to 5 fibrillin class 1 epidermal growth factor (EGF)-like repeats, an Arg-Gly-Asp (RGD) tripeptide, and a mucin stalk. We show here that CD97alpha promotes angiogenesis in vivo as demonstrated with purified protein in a directed in vivo angiogenesis assay (DIVAA) and by enhanced vascularization of developing tumors expressing CD97. These data suggest that CD97 can contribute to angiogenesis associated with inflammation and tumor progression. Strong integrin alpha5beta1 interactions with CD97 have been identified, but alpha v beta3 also contributes to cell attachment. Furthermore, soluble CD97 acts as a potent chemoattractant for migration and invasion of human umbilical vein endothelial cells (HUVECs), and this function is integrin dependent. CD97 EGF-like repeat 4 is known to bind chondroitin sulfate. It was found that coengagement of alpha5beta1 and chondroitotin sulfate proteoglycan by CD97 synergistically initiates endothelial cell invasion. Integrin alpha5beta1 is the first high-affinity cellular counterreceptor that has been identified for a member within this family of adhesion receptors.

Topics: Animals; Antigens, CD; Cell Adhesion; Chondroitin Sulfates; Endothelium, Vascular; Epidermal Growth Factor; HT29 Cells; Humans; Integrin alpha5beta1; Integrin alphaVbeta3; Membrane Glycoproteins; Mice; Neoplasms; Neovascularization, Physiologic; NIH 3T3 Cells; Oligopeptides; Phenotype; Protein Structure, Tertiary; Rats; Receptors, G-Protein-Coupled; Umbilical Veins

Migrating cells can sustain a relatively constant direction of lamellipodial protrusion and locomotion over timescales ranging from minutes to hours. However, individual waves of lamellipodial extension occur over much shorter characteristic times. Little understanding exists regarding how cells might integrate biophysical processes across these disparate timescales to control the directional persistence of locomotion. We address this issue by examining the effects of epidermal growth factor (EGF) stimulation on long-timescale directional persistence and short-timescale lamellipodial dynamics of EGF receptor-transfected Chinese hamster ovary cells migrating on fibronectin-coated substrata. Addition of EGF increased persistence, with the magnitude of increase correlating with fibronectin coating concentration. Kymographic analysis of EGF-stimulated lamellipodial dynamics revealed that the temporal stability of lamellipodial protrusions similarly increased with fibronectin concentration. A soluble RGD peptide competitor reduced both the persistence of long-timescale cell paths and the stability of short-timescale membrane protrusions, indicating that cell-substratum adhesion concomitantly influences lamellipodial dynamics and directional persistence. These results reveal the importance of adhesion strength in regulating the directional motility of cells and suggest that the short-timescale kinetics of adhesion complex formation may play a key role in modulating directional persistence over much longer timescales.

Topics: Animals; Cell Adhesion; Cell Movement; CHO Cells; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Fibronectins; Oligopeptides; Pseudopodia; Recombinant Proteins

Endostatin, a fragment of collagen XVIII, is a potent antagonist of angiogenesis and inhibitor of tumor growth in mouse models. At present, the mechanism of action of endostatin is unknown. We show here that recombinantly produced human endostatin interacts with alpha(5)- and alpha(v)-integrins on the surface of human endothelial cells. We further demonstrate that the endostatin-integrin interaction is of functional significance in vitro, as we found that immobilized endostatin supports endothelial cell survival and migration in an integrin-dependent manner. Soluble endostatin in turn inhibits integrin-dependent endothelial cell functions, such as cell migration. Taken together, these results implicate integrins as potential targets for endostatin function and support the importance of integrins in endothelial cell biology and angiogenesis.

Topics: Angiogenesis Inhibitors; Animals; Antigens, CD; Cell Adhesion; Cell Movement; Cells, Cultured; Collagen; Collagen Type XVIII; Edetic Acid; Endostatins; Endothelium, Vascular; Epidermal Growth Factor; Fibroblast Growth Factor 2; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Integrin alpha5; Integrin alphaV; Kinetics; Mice; Oligopeptides; Peptide Fragments; Phosphorylation; Protein-Tyrosine Kinases; Recombinant Proteins; Umbilical Veins

Cell adhesion molecules are involved in a number of biological functions, such as cell survival, cell differentiation, tissue repair, and development. A novel molecule, POEM (preosteoblast epidermal growth factor-like repeat protein with meprin, A5 protein, and receptor protein-tyrosine phosphatase mu domain), was isolated by reverse transcription-polymerase chain reaction using a set of degenerate primers designed after other known epidermal growth factor (EGF)-like motifs. From its structure, POEM was suggested to be a novel adhesion molecule with five EGF-like domains, an Arg-Gly-Asp (RGD) cell binding motif, and a meprin, A5 protein, and receptor protein-tyrosine phosphatase mu (MAM) domain. By in situ hybridization using embryonic day 16.5 (E16.5) mouse embryos, strong expression of POEM mRNA was observed in developing kidney renal tubules, parathyroid and thyroid glands, developing bone, tooth germ, and endocrine organs of the brain. The inner ear, skeletal muscle, smooth muscle (except for the vascular system), and skin were also positive for POEM expression. Bacterial recombinant POEM protein containing the RGD sequence and MAM domain showed strong cell adhesion, spreading, and survival-promoting activities. By mutating the RGD sequence to RGE, the cell spreading and survival activities were significantly decreased, but the MAM domain was shown to contribute only to cell adhesion and not to cell spreading and survival-promoting activities. The distribution of POEM in several tissues was close to that of alpha(8)beta(1) integrin. Therefore, we conducted cell adhesion assays using KA8 cells, a K562 leukemia clone stably expressing alpha(8) integrin. Parental K562 cells, which expressed alpha(5)beta(1) integrin, bound to fibronectin but not to POEM. On the other hand, KA8 cells showed strong binding and spreading on both fibronectin and POEM. These results suggest that POEM is a novel ligand for alpha(8)beta(1) integrin and that POEM may be involved in the development and function of various tissues, such as kidney, bone, muscles, and endocrine organs.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Adhesion; Cell Adhesion Molecules; Cell Line; Cloning, Molecular; Embryo, Mammalian; Epidermal Growth Factor; Integrins; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Oligopeptides; Osteoblasts; Repetitive Sequences, Amino Acid; RNA, Messenger; Stem Cells; Tissue Distribution

Integrin adhesion receptors play a crucial role in regulating interactions between cells and extracellular matrix (ECM). Integrin activation initiates multiple intracellular signaling pathways and results in regulation of cell functions such as motility, proliferation and differentiation. Two key observations regarding the biophysical nature of integrin-mediated cell-matrix interactions motivated the present study: (1) cell motility can be regulated by modulating the magnitude of cell-substratum adhesion, by varying cell integrin expression level, integrin-ECM binding affinity or substratum ECM surface density; and (2) integrin clustering enables assembly of multiple cytoplasmic regulatory and structural proteins at sites of aggregated integrin cytoplasmic domains, activating certain intracellular signalling pathways. Here, using a minimal integrin adhesion ligand, YGRGD, we test the hypothesis that ligand clustering can affect cell migration in a manner related to its modulation of cell-substratum adhesion. We employ a synthetic polymer-linking method, which allows us to independently and systematically vary both the average surface density and the local (approx. 50 nm scale) spatial distribution of the YGRGD peptide, against a background otherwise inert with respect to cell adhesion. In this system, the ligand was presented in three alternative spatial distributions: singly, in clusters with an average of five ligands per cluster, or in clusters with an average of nine ligands per cluster; for each of these spatial distributions, a range of average ligand densities (1,000-200,000 ligands/micrometer(2)) were examined. Cluster spacing was adjusted in order to present equivalent average ligand densities independently of cluster size. The murine NR6 fibroblast cell line was used as a model because its migration behavior on ECM in the presence and absence of growth factors has been well-characterized and it expresses integrins known to interact with the YGRGD peptide. Using time-lapse videomicroscopy and analysis of individual cell movement paths, we find that NR6 cells can migrate on substrata where adhesion is mediated solely by the YGRGD peptide. As previously observed for migration of NR6 cells on fibronectin, migration speed on YGRGD is a function of the average surface ligand density. Strikingly, clustering of ligand significantly reduced the average ligand density required to support cell migration. In fact, non-clustered integrin ligands support cell

Topics: 3T3 Cells; Actins; Animals; Antineoplastic Agents; Binding Sites; Cell Adhesion; Cell Count; Cell Membrane; Cell Movement; Culture Media; Epidermal Growth Factor; Extracellular Matrix; Integrins; Ligands; Mice; Molecular Conformation; Oligopeptides; Polymers; Protein Structure, Tertiary; Time Factors

A novel protein was engineered by inserting the GRGDS motif of fibronectin within the 14-residue loop of the EGF-like module from human complement protease C1r. The resulting chimeric EGF-RGD module (52 residues, three disulfide bridges) was assembled by automated solid-phase synthesis using the t-Boc strategy. Using reduced/oxidized glutathione, the EGF-RGD module was folded as efficiently as the natural C1r-EGF module, resulting in formation of the appropriate disulfide bridge pattern as shown by mass spectrometry and N-terminal sequence analyses of thermolytic fragments. Circular dichroism and NMR measurements provided further indication that introduction of the GRGDS motif had no significant effect on the folding. Using Chinese Hamster Ovary (CHO) cells bearing the integrin receptors specific for fibronectin and vitronectin, EGF-RGD was shown to induce cell adhesion via the introduced GRGDS motif. Cell binding was inhibited specifically and efficiently by the synthetic peptide GRGDSP and by fibronectin, and to a much lesser extent by vitronectin, whereas the monoclonal antibody PB1 directed to the alpha5 subunit of alpha5beta1 integrin had no effect. The ability of EGF-RGD to trigger significant cell spreading and intracellular signaling was also demonstrated using immunofluorescence and confocal microscopy.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Cell Adhesion; CHO Cells; Circular Dichroism; Complement C1r; Cricetinae; Epidermal Growth Factor; Fibronectins; Fluorescent Antibody Technique; Mass Spectrometry; Models, Molecular; Molecular Sequence Data; Oligopeptides; Recombinant Fusion Proteins; Sequence Analysis

Insulin-like growth factor binding protein-1 (IGFBP-1) can bind to the alpha5beta1 integrin and stimulate cellular migration in Chinese hamster ovary (CHO) cells. IGFBP-1 is a major product of the endometrium of pregnancy (decidua), and may interact with invading cytotrophoblasts expressing alpha5beta1 integrin to modulate their invasion. The present study investigated IGFBP-1 interaction with cytotrophoblast alpha5beta1 integrin, and its effects on trophoblast attachment to fibronectin and invasion into decidualized endometrial stromal cell multilayers. IGFBP-1 incubated with cytotrophoblast extracts was co-precipitated by an antibody to the alpha5 integrin subunit. Up to 55% of radiolabeled IGFBP-1 bound to cytotrophoblasts was displaced by excess non-radioactive IGFBP-1, but not by IGFBP-3. Cytotrophoblast attachment to fibronectin was inhibited by an RGD-containing octapeptide, by antibodies to the alpha5 subunit or the alpha5beta1 heterodimer, and by IGFBP-1. Cytotrophoblasts showed limited invasion into endometrial stromal multilayers decidualized in vitro secreting abundant IGFBP-1, but invaded multilayers when IGFBP-1 production was inhibited by insulin. Invasion into insulin-treated multilayers was prevented by addition of exogenous IGFBP-1 but not by IGFBP-3. These findings suggest IGFBP-1 may modulate trophoblast invasiveness.

Topics: Animals; Antibodies, Monoclonal; Binding, Competitive; Cell Movement; Cells, Cultured; CHO Cells; Coculture Techniques; Cricetinae; Cricetulus; Decidua; Endometrium; Epidermal Growth Factor; Estradiol; Female; Fibronectins; Humans; Insulin; Insulin-Like Growth Factor Binding Protein 1; Insulin-Like Growth Factor Binding Protein 3; Oligopeptides; Progesterone; Receptors, Fibronectin; Stromal Cells; Trophoblasts

Cell-extracellular matrix interactions support the ability of cells to migrate into areas of inflammation and injury. The present study evaluated the ability of different matrix proteins to support bronchial epithelial cell attachment and survival. Collagens were able to support attachment and survival of normal cultured human bronchial epithelial cells but only in the presence of added soluble growth factors such as insulin, epidermal growth factor, platelet-derived growth factor, and bovine pituitary extract. In contrast, fibronectin was able to support attachment and survival of normal human bronchial epithelial cells in growth factor-deficient medium. In addition, fibronectin, in the absence of added growth factors, was able to induce integrin clustering, focal adhesion formation, and phosphorylation of focal adhesion kinase. A 120-kDa chymotryptic fragment of fibronectin containing the Arg-Gly-Asp peptide sequence was able to reproduce the effects of the whole fibronectin molecule. This study supports the concept that fibronectin has specialized roles in injury and repair.

Topics: Animals; Bronchi; Cattle; Cell Adhesion; Cell Adhesion Molecules; Cell Survival; Cells, Cultured; Collagen; Epidermal Growth Factor; Epithelial Cells; Epithelium; Extracellular Matrix Proteins; Fibronectins; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Growth Substances; Humans; Insulin; Integrins; Oligopeptides; Peptide Fragments; Phosphorylation; Pituitary Gland; Platelet-Derived Growth Factor; Protein-Tyrosine Kinases; Tissue Extracts

The BA46 antigen of the human milk fat globule (HMFG) membrane is expressed in human breast carcinomas and has been used successfully as a target for experimental breast cancer radioimmunotherapy. To characterize this antigen further, we obtained the entire cDNA sequence and focused on its possible role in cell adhesion. The derived protein sequence of BA46 encodes a 387-residue precursor composed of a putative signal peptide, an amino-terminal epidermal growth factor (EGF)-like domain containing the cell adhesion tripeptide arginine-glycine-aspartic acid (RGD), and human factor V and factor VIII C1/C2-like domains. The EGF-like domain of BA46 is similar to the calcium-binding EGF-like domains of several coagulation factors, but the BA46 domain lacks a residue required for calcium binding and the coagulation factor domains do not include an RGD sequence. Assuming that all EGF-like domains fold into a similar structure, the RGD-containing sequence in BA46 is inserted between two antiparallel beta strands. This positioning suggests a novel function for the EGF-like domain as a scaffold for RGD presentation.

Topics: Amino Acid Sequence; Animals; Antigens, Surface; Base Sequence; Blood Coagulation Factors; Breast; Breast Neoplasms; Cattle; Cloning, Molecular; Consensus Sequence; Conserved Sequence; DNA Primers; Epidermal Growth Factor; Epithelium; Factor V; Factor VIII; Female; Gene Library; Humans; Milk Proteins; Molecular Sequence Data; Mucin-1; Oligopeptides; Polymerase Chain Reaction; Protein Structure, Secondary; Recombinant Proteins; Sequence Homology, Amino Acid

Various human carcinomas overexpress epidermal growth factor receptor, and the degree of the expression correlates with their malignant phenotype. Because phenotypic transformation of cells involves qualitative and quantitative alteration of integrin function, we compared the effects of exogenous epidermal growth factor on cell-matrix interactions between HSC-1 human cutaneous squamous carcinoma cells overexpressing epidermal growth factor receptor and their revertant cells. Epidermal growth factor impaired RGD-sensitive cell spreading on fibrinogen, fibronectin, or vitronectin in the parent cells in a concentration-dependent manner; 50 ng epidermal growth factor per ml treatment for 1-24 h reduced cell spreading on these substrata by 75-95%. In the presence of epidermal growth factor, the parent HSC-1 cells lost their epithelial phenotype and did not form coherent colonies. This might involve the impaired RGD-sensitive integrin function, because treatment of the cells with the peptide GRGDS mimicked the effects of epidermal growth factor on cell and colony morphology. The revertant cells expressing about one-tenth the amount of epidermal growth factor receptor did not show reduced RGD-sensitive cell spreading or loss of epithelial phenotype in response to epidermal growth factor. Epidermal growth factor did not downregulate the subunits for the RGD-sensitive integrin receptors for fibrinogen, fibronectin, or vitronectin. Tyrosine phosphorylation of integrin beta subunits might be involved in the impairment of integrin function, because EGF tyrosine phosphorylated beta1, subunit in the parent, but not in the revertant cells. Our results suggest that the ligand activation of overexpressed epidermal growth factor receptor results in impairment of RGD-sensitive integrin function and loss of epithelial phenotype. This may be advantageous to epithelial tumor cells progressing along malignant pathways.

Topics: Down-Regulation; Epidermal Growth Factor; Epithelium; ErbB Receptors; Humans; Integrin beta1; Integrins; Ligands; Oligopeptides; Phenotype; Phosphorylation; Tumor Cells, Cultured; Tyrosine

The adhesion of different epidermal growth factor (EGF) receptor (EGFR) expressing cell lines to various extracellular matrix (ECM) proteins is influenced by EGF. To investigate a putative receptor crosstalk between EGFR and integrins we chose two cell lines for a more detailed analysis: the highly metastatic rat mammary carcinoma clone MTLn3 that showed increased adhesion to a panel of ECM proteins in the presence of 10 ng/ml EGF and the nonmetastatic human vulva carcinoma cell line A431 which showed a decreased adhesion under the same conditions. These EGF-mediated stimulatory or inhibitory effects on adhesion were observed within a few minutes. On human A431 cells the inhibitory effect was blocked by an EGFR specific antibody that interferes with ligand binding. In cell adhesion assays performed in the presence of divalent cations MTLn3 and A431 cells exhibited the typical behavior described for integrin-dependent matrix adhesion: Mn2+ enhanced binding to collagen IV and fibronectin whereas Ca2+ inhibited adhesion to collagen IV but not to fibronectin. Adhesion-inhibition assays with anti-human integrin antibodies revealed that A431 cells adhere to collagen via alpha 1 beta 1 and alpha 2 beta 1, and that adhesion to fibronectin is mediated predominantly through alpha 5 beta 1. The interaction of MTLn3 cells with fibronectin was in part RGD dependent, indicating the involvement of either alpha 3 beta 1 or alpha 5 beta 1. Addition of EGF in these assays showed that affecting the integrin extracellular domains by addition of either bivalent cations, RGD peptides, or function-blocking integrin antibodies did not prevent the effects mediated by EGF. We conclude that signals downstream of EGFR can modulate integrin-mediated adhesion to ECM proteins in both an inhibitory and a stimulatory manner.

Topics: Animals; Antibodies, Monoclonal; Calcium; Cations, Divalent; Cell Adhesion; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Extracellular Matrix Proteins; Humans; Integrins; Magnesium; Manganese; Oligopeptides; Rats; Signal Transduction; Time Factors; Tumor Cells, Cultured

The aim of this work was to show epidermal growth factor (EGF)-dependent migration of human corneal epithelial cells to fibronectin and GRGDSP peptide. The authors assessed the role of cell surface integrin heterodimer alpha 5 beta 1 in mediating haptotactic cell migration to fibronectin by the use of specific function-blocking integrin antibodies.. A haptotactic cell migration assay in a Boyden chamber was used to compare the relative migration of the cultured human corneal epithelial cells in the presence of fibronectin and GRGDSP peptide-coated filters. Epithelial cells were incubated in the presence of function-blocking integrin antibodies or anti-EGF-receptor antibodies to determine their role in haptotactic cell migration.. Human corneal epithelial cells grown as primary cultures migrated in the presence of fibronectin or GRGDSP peptide, but only on stimulation with EGF. Antibodies to the EGF receptor blocked the EGF-mediated stimulation of haptotactic cell migration. Anti-beta 1 and anti-alpha 5 antibodies each inhibited haptotactic cell migration to fibronectin and GRGDSP peptide.. Epidermal growth factor provides an important stimulus of haptotactic cell migration of human corneal epithelial cells. Stimulation of cell migration by EGF was maximal in the range of 5 to 10 ng/ml; this response was completely blocked by incubation with an anti-EGF receptor antibody. Function-blocking integrin antibodies, specifically anti-beta 1 and anti-alpha 5, inhibited integrin-mediated cell migration to fibronectin and GRGDSP peptide. These data suggest that EGF represents an essential initial stimulus for haptotactic cell migration of human corneal epithelial cells; furthermore, integrins are important in mediating cell migration to fibronectin and GRGDSP:

Topics: Amino Acid Sequence; Antibodies; Cells, Cultured; Chemotaxis; Cornea; Dose-Response Relationship, Drug; Epidermal Growth Factor; Epithelium; Fibronectins; Humans; Integrins; Molecular Sequence Data; Oligopeptides; Receptors, Fibronectin

Growth factors may play an important role in the repair of the renal proximal tubule epithelium following toxic injury. This study investigated the regeneration of rabbit renal proximal tubule cell (RPTC) monolayers following exposure to the nephrotoxicants tert-butylhydroperoxide (TBHP) and 1,2-dichlorovinyl-L-cysteine (DCVC), and the effect of exogenous growth factors on the regeneration process. Confluent monolayers exposed to TBHP or DCVC for 1.5-2 hr were 23 and 43% confluent, respectively, after 24 hr. Confluency increased to 63 and 80% 4 days after TBHP or DCVC exposure, but decreased to 29 and 24% after 8 days. Monolayer DNA content did not increase after TBHP or DCVC exposure; however, monolayer protein/DNA ratio increased above control values after DCVC exposure. Recovery of confluency was not sensitive to RGD-containing peptides that inhibit the binding of integrins to extracellular matrix. Epidermal growth factor (EGF) treatment resulted in complete recovery of confluency, and protein and DNA contents 4-6 days after injury. Unlike EGF, IGF-1 or insulin treatment produced a small increase in confluency following TBHP exposure. These results suggest that hypertrophy following DCVC exposure and migration/spreading after TBHP and DCVC exposure play a partial and temporary role in the regeneration of RPTC monolayers, that in the absence of exogenous growth factors proliferation and complete regeneration of the monolayer does not occur, that toxicants may alter the production of mitogenic factors, and that EGF is a potent and efficacious growth factor in promoting regeneration.

Topics: Animals; Cells, Cultured; Cysteine; Epidermal Growth Factor; Female; Growth Substances; Insulin; Insulin-Like Growth Factor I; Kidney Tubules, Proximal; Oligopeptides; Peroxides; Rabbits; Reactive Oxygen Species; Regeneration; tert-Butylhydroperoxide

The basement membrane glycoprotein, entactin, has previously been shown to promote cell attachment and chemotaxis. We have constructed a panel of glutathione S-transferase fusion proteins that encompasses the four major structural domains of entactin, G1, G2, E, and G3. These proteins have been synthesized in bacteria and purified by affinity chromatography. The connecting stalk of entactin, E, which contains four cysteine-rich EGF homology repeats and the integrin receptor RGD recognition sequence, has been modified by deletion of the RGD sequence and substituting glutamic acid for aspartic acid. Attachment assays reveal that the RGD sequence is one of the major cell attachment sites in entactin and that this sequence is recognized by the alpha v beta 3 integrin receptor. Analysis of cell attachment on mutant forms of full-length entactin expressed in the baculovirus expression system revealed a second attachment site that was independent of the RGD sequence. This second site was localized to a peptide of 39 amino acid residues in the second globular G2 domain of entactin. This peptide represents a cysteine-rich EGF repeat. Inhibition of cell attachment by anti-integrin receptor antibodies indicates that the second attachment site is recognized by a member of the beta 1 family of integrin receptors, possibly alpha 3 beta 1.

Topics: Amino Acid Sequence; Base Sequence; Binding Sites; Cell Adhesion; Cysteine; Epidermal Growth Factor; Humans; Integrins; Membrane Glycoproteins; Molecular Sequence Data; Oligopeptides

This study was undertaken to determine the importance of integrin binding and cell shape changes in the control of cell-cycle progression by extracellular matrix (ECM). Primary rat hepatocytes were cultured on ECM-coated dishes in serum-free medium with saturating amounts of growth factors (epidermal growth factor and insulin). Integrin binding and cell spreading were promoted in parallel by plating cells on dishes coated with fibronectin (FN). Integrin binding was separated from cell shape changes by culturing cells on dishes coated with a synthetic arg-gly-asp (RGD)-peptide that acts as an integrin ligand but does not support hepatocyte extension. Expression of early (junB) and late (ras) growth response genes and DNA synthesis were measured to determine whether these substrata induce G0-synchronized hepatocytes to reenter the growth cycle. Cells plated on FN exhibited transient increases in junB and ras gene expression (within 2 and 8 h after plating, respectively) and synchronous entry into S phase. Induction of junB and ras was observed over a similar time course in cells on RGD-coated dishes, however, these round cells did not enter S phase. The possibility that round cells on RGD were blocked in mid to late G1 was confirmed by the finding that when trypsinized and replated onto FN-coated dishes after 30 h of culture, they required a similar time (12-15 h) to reenter S phase as cells that had been spread and allowed to progress through G1 on FN. We have previously shown that hepatocytes remain viable and maintain high levels of liver-specific functions when cultured on these RGD-coated dishes. Thus, these results suggest that ECM acts at two different points in the cell cycle to regulate hepatocyte growth: first, by activating the G0/G1 transition via integrin binding and second, by promoting the G1/S phase transition and switching off the default differentiation program through mechanisms related to cell spreading.

Topics: Amino Acid Sequence; Animals; Cell Adhesion; Cell Cycle; Cell Size; Culture Media, Serum-Free; Epidermal Growth Factor; Extracellular Matrix; Fibronectins; Insulin; Integrins; Liver; Molecular Sequence Data; Oligopeptides; Protein Binding; Rats

We have isolated cDNA clones for mouse tenascin and analyzed expression of tenascin mRNAs during embryonic development of the kidney and gut. The deduced amino acid sequence of the mouse tenascin cDNAs shows a modular structure of repeats similar to chicken and human tenascin. In mouse there are 14.5 cysteine-rich repeats with similarity to the EGF repeat, followed by several repeats with similarity to the type III repeat of fibronectin. A longer variant contains 13 fibronectin type III repeats, whereas a shorter splice variant of mouse tenascin lacks the 5 type III repeats that occur directly after the fifth repeat in the longer variant. Contrary to the chicken and human sequences, mouse tenascin does not contain an RGD sequence in the third type III repeat implicated in cell attachment, or in any other positions. In Northern hybridizations to RNA from primary embryonic fibroblasts, the cDNA clone M 20/1 detects two mRNAs with sizes close to 6 and 8 kb. This, and the other data presented here suggest that the two major mouse tenascin polypeptides arise through an alternative RNA splicing. The two major mRNAs are differentially expressed during development. The 8-kb mRNA is more prominent than the 6-kb mRNA throughout prenatal kidney development, but during postnatal development the ratio of the two mRNAs changes. A different expression pattern is seen in the developing gut where the 6-kb mRNA predominates during embryogenesis with the 8-kb mRNA appearing later. The mRNA data of the developing gut correspond with previous protein data, which showed that the shorter Mr 210,000 polypeptide predominates during earlier developmental stages and the larger Mr 260,000 polypeptide appears later in the embryonic gut (Aufderheide, E., and P. Ekblom. 1988. J. Cell Biol. 107:2341-2349).

Topics: Amino Acid Sequence; Animals; Blotting, Northern; Cell Adhesion Molecules, Neuronal; Embryonic and Fetal Development; Epidermal Growth Factor; Extracellular Matrix Proteins; Fibronectins; Gene Expression Regulation; Glycosylation; Intestinal Mucosa; Intestines; Kidney; Mice; Molecular Sequence Data; Oligopeptides; Protein Sorting Signals; Repetitive Sequences, Nucleic Acid; RNA Splicing; Sequence Homology, Nucleic Acid; Tenascin