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L T Furcht

Publications and source records attributed to L T Furcht.

At least 37 records · Page 2Linked to original sources
PubMed1996

Synthetic fibronectin peptides and ischemic brain injury after transient middle cerebral artery occlusion in rats.

Leukocytes play an important role in the development of ischemia-reperfusion injury. This study was conducted to ascertain whether synthetic peptides corresponding to the cell- and heparin-binding sequences of fibronectin that disturb leukocyte adhesion molecules were effective in neuronal protection after transient focal cerebral ischemia in rats. The authors evaluated the efficacy of peptides on infarction size, leukocyte infiltration in the ischemic tissue, and neurological outcome in rats subjected to 1 hour of cerebral ischemia and 48 hours of reperfusion. Twenty-one animals were divided into three groups: transient ischemia without treatment (Group I), transient ischemia with administration of vehicle (Group II), and transient ischemia with administration of fibronectin peptides (Group III). The mean myeloperoxidase activity (U/g wet wt) in the ischemic area was as follows: Group I, 0.19% +/- 0.05; Group II, 0.21% +/- 0.03; and Group III, 0.08% +/- 0.02. The mean size of the infarction as a percentage of the total hemispheric volume was as follows: Group I, 38.35% +/- 1.34%; Group II, 39.21% +/- 2.42%; and Group III, 25.81% +/- 4.87%. Group III showed a significant decrease in myeloperoxidase activity in the lesion and the infarction size was smaller when compared to Groups I and II (p < 0.05). The neurological grade in Group III was significantly better than in Groups I and II at 48 hours after reperfusion (p < 0.01). This study is the first to explore the therapeutic potential of synthetic fibronectin peptides in brain protection after transient focal ischemia, and the results also serve as a basis for studies of important cellular and molecular events that contribute to tissue damage.

Animals↗
PubMed1995

A peptide model of basement membrane collagen alpha 1 (IV) 531-543 binds the alpha 3 beta 1 integrin.

Tumor cell adhesion to the triple-helical domain of basement membrane (type IV) collagen occurs at several different regions. Cellular recognition of the sequence spanning alpha 1(IV)531-543 has been proposed to be independent of triple-helical conformation (Miles, A. J., Skubitz, A. P. N., Furcht, L. T., and Fields, G. B. (1994) J. Biol. Chem. 269, 30939-30945). In the present study, integrin interactions with a peptide analog of the alpha 1(IV)-531-543 sequence have been analyzed. Tumor cell adhesion (melanoma, ovarian carcinoma) to the alpha 1(IV)531-543 chemically synthesized peptide was inhibited by a monoclonal antibody against the alpha 3 integrin subunit, and to a lesser extent by monoclonal antibodies against the beta 1 and alpha 2 integrin subunits. An anti-alpha 5 monoclonal antibody and normal mouse IgG were ineffective as inhibitors of tumor cell adhesion to the peptide. Two cell surface proteins of 120 and 150 kDa bound to an alpha 1(IV)531-543 peptide affinity column and were eluted with 20 mM EDTA. When the eluted proteins were incubated with monoclonal antibodies against either the alpha 3 or beta 1 integrin subunit, proteins corresponding in molecular weight to alpha 3 and beta 1 integrin subunits were precipitated. No proteins were immunoprecipated with monoclonal antibodies against the alpha 2 or alpha 5 integrin subunits. Thus, the alpha 3 beta 1 integrin from two tumor cell types has been shown to bind directly to the alpha 1 (IV)531-543 peptide. The alpha 1(IV)531-543 peptide is the first collagen-like sequence that has been shown to bind the alpha 3 beta 1 integrin.

Amino Acid Sequence↗
PubMed1995

Identification of a homologous heparin binding peptide sequence present in fibronectin and the 70 kDa family of heat-shock proteins.

This study was undertaken to characterize the potential heparin affinity of an amino-acid sequence within the 70 kDa heat-shock family of proteins (HSPs) that shares homology with a heparin-binding sequence present in the carboxy-terminus of fibronectin (FN), defined by the synthetic peptide, FN-C/H-II (KNNQKSEPLIGRKKT). To first define the heparin binding sequence within FN-C/H-II, solid phase binding assays were performed using overlapping, short (7 amino acids) synthetic peptides corresponding to the amino-acid sequence within FN-C/H-II. Only the sequence LIGRKKT bound [3H] heparin, and the LIGRKKT peptide blocked heparin binding to intact fibronectin by 47% (+/- 0.4, p < 0.001). A computer-generated homology search revealed that two members of the 70 kDa HSP family, HSP70 and HSC70, contain the sequences LIGRK and LIGRR, respectively. Examination of heparin binding using affinity chromatography indicated that while native HSC70 binds heparin, native HSP70 does not. Treatment of the heparin-unbound fraction with heat or urea led to enhanced HSP70 binding to heparin affinity columns. Soluble LIGRKKT peptide or anti-FN-C/H-II IgG also significantly inhibited heparin binding to HSC70 that had been purified by heparin affinity chromatography. Finally, Western blot analysis of HSC70 purified by heparin affinity chromatography demonstrated that polyclonal anti-FN-C/H-II IgG could recognize HSC70. These data demonstrate that LIGRK or LIGRR represent a a common heparin binding motif in fibronectin, HSP70, and HSC70, and are consistent with a proposed role for heparin or similar polyanionic structures in the function of the 70 kDa heat-shock proteins.

Amino Acid Sequence↗
PubMed1995

Spreading and focal contact formation of human melanoma cells in response to the stimulation of both melanoma-associated proteoglycan (NG2) and alpha 4 beta 1 integrin.

In this study, we evaluated the potential role for a specific melanoma-associated chondroitin sulfate proteoglycan core protein, termed NG2, to collaborate with alpha 4 beta 1 integrin in focal contact formation in human melanoma cells. Although melanoma cells adhered to substrata coated with either the alpha 4 beta 1 integrin binding fibronectin synthetic peptide CS1-OVA or anti-NG2 mAbs, no spreading or focal contact formation was observed on either substratum. However, melanoma cells spread and formed focal contacts on "chimeric substrata" coated with CS1-OVA and the anti-NG2 mAb, 9.2.27, indicating that engaging both adhesion receptors changes the adhesion phenotype of melanoma cells by reorganizing the cytoskeleton. The collaboration between the two receptors is specific to fibronectin, since cells adherent on substrata coated with low concentrations of either laminin and 9.2.27 or type IV collagen and 9.2.27 failed to spread, while cells adherent on low concentrations of fibronectin and 9.2.27 exhibited a fully spread morphology. Two selective tyrosine kinase inhibitors, genistein and herbimycin A, totally inhibited cell spreading on the substrata coated with CS1-OVA and 9.2.27, indicating that tyrosine kinase(s) is important for cell spreading and focal contact formation. When cells were cultured on substrata coated with CS1-OVA and 9.2.27, two proteins (M(r) 130,000 and 120,000) were tyrosine phosphorylated in a genistein- and herbimycin A-sensitive fashion. These proteins were not immunologically related to pp125FAK or alpha 4 beta 1 integrin. Importantly, when melanoma cells were cultured on substrata coated with CS1 and then stimulated with 9.2.27-conjugated microsphere beads, formation of focal contacts and stress fibers was also observed, indicating that NG2 can collaborate with alpha 4 beta 1 integrin when each receptor is engaged on distinct and separate substrata. These results demonstrate that NG2 acts as a coreceptor for spreading and focal contact formation in association with alpha 4 beta 1 integrin in melanoma cells and suggest a model in which the NG2 core protein communicates to alpha 4 beta 1 integrin by an inside-out signaling mechanism.

Antibodies, Monoclonal↗
PubMed1995

Functional involvement of sciatic nerve-derived versican- and decorin-like molecules and other chondroitin sulphate proteoglycans in ECM-mediated cell adhesion and neurite outgrowth.

We have previously described two proteoglycans from human sciatic nerve which are immunochemically related to the chondroitin sulphate proteoglycans versican and decorin. The chondroitin sulphate of the versican-like molecule and the core protein of the decorin-like molecule have been found previously to be up-regulated after lesioning the adult mouse sciatic nerve. To investigate if the versican- and decorin-like molecules are involved in cell-extracellular matrix interactions, we studied the effect of both molecules on cell adhesion. The versican- and decorin-like molecules, substrate-coated in a mixture with fibronectin, but not with laminin or collagen types I or IV, inhibited the adhesion of several cell lines, neonatal dorsal root ganglion neurons and Schwann cells. The inhibitory activity was concentration-dependent and mediated by the chondroitin sulphate. Furthermore, when different proteoglycans were incubated with fibronectin, only the versican- and decorin-like molecules and the chondroitin sulphate proteoglycan aggrecan, but not the heparan sulphate proteoglycan perlecan, were able to inhibit fibronectin-mediated cell adhesion. The versican- and decorin-like molecules, substrate-coated alone or in a mixture with fibronectin or laminin, were at most slightly inhibitory to neurite outgrowth from PC12 phaeochromocytoma cells and neonatal dorsal root ganglion neurons. In a solid-phase ligand-binding assay the versican- and decorin-like molecules interacted with fibronectin, but not with laminin or collagen types I and IV. Binding of the versican-like molecule to fibronectin and inhibition of cell adhesion by this molecule was mediated via the heparin and cell-binding domains of fibronectin. These observations suggest that binding of the two proteoglycans to fibronectin is involved in the modulation of adhesion of cells to fibronectin.

Animals↗
PubMed1995

Epidermal growth factor stimulates integrin-mediated cell migration of cultured human corneal epithelial cells on fibronectin and arginine-glycine-aspartic acid peptide.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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:

Amino Acid Sequence↗
PubMed1995

Involvement of integrins with adhesion-promoting, heparin-binding peptides of type IV collagen in cultured human corneal epithelial cells.

PURPOSE: The aim of this work was to identify the integrin subunits present on the cell surface of human corneal epithelial cells. The authors determined to show whether type IV collagen, heparin-binding peptides of type IV collagen (Hep-I, Hep-II, and Hep-III), fibronectin, and GRGDSP promote cell adhesion of human corneal epithelial cells. Type IV collagen and heparin-binding peptides of type IV collagen may be important in corneal epithelial cell adhesion in normal and pathologic conditions and reepithelialization. The authors assess the role of cell surface integrins in mediating cell adhesion to these proteins and peptides. METHODS: Fluorescence-activated cell sorter (FACS) analysis was used to determine the integrin subunits expressed at the cell surface of the cultured human corneal epithelial cells. Cell adhesion was assessed with type IV collagen, heparin-binding peptides of type IV collagen, fibronectin, and GRGDSP: Antibodies to the integrin subunits were used to determine the potential role of integrins in cell adhesion to the above proteins and peptides. RESULTS: FACS analysis identified the beta 1, beta 4, alpha 2, alpha 3, alpha 5, alpha 6, and alpha v integrin subunits on human corneal epithelial cells grown as primary cultures. The anti-beta 1 antibody inhibited cell adhesion to heparin-binding peptides of type IV collagen, type IV collagen, fibronectin, and GRGDSP: Antibodies to the alpha 2 integrin subunit inhibited cell adhesion to the heparin-binding peptides of type IV collagen and slightly inhibited cell adhesion to intact type IV. Antibodies to the alpha 3 integrin subunit exhibited a somewhat lesser effect compared to the anti-alpha 2 integrin antibody. CONCLUSIONS: These data show that the alpha 2 beta 1 integrin of human corneal epithelial cells recognize heparin-binding peptide sequences derived from human type IV collagen. It seems likely that these sequences play an important role in integrin-mediated corneal epithelial cell adhesion. In addition, the alpha 3 beta 1 integrin may mediate similar events.

Amino Acid Sequence↗
PubMed1994

Promotion of cell adhesion by single-stranded and triple-helical peptide models of basement membrane collagen alpha 1(IV)531-543. Evidence for conformationally dependent and conformationally independent type IV collagen cell adhesion sites.

Several regions within the triple-helical domain of type IV collagen function as cellular recognition sites. We have demonstrated previously that melanoma cell activities promoted by the alpha 1(IV)1263-1277 sequence are enhanced by triple helicity (Fields, C. G., Mickelson, D. J., Drake, S.L., McCarthy, J.B., and Fields, G.B. (1993) J. Biol. Chem. 268, 14153-14160), whereas Eble et al. reached similar conclusions for alpha 1 beta 1 integrin-mediated fibrosarcoma cell adhesion to [alpha 1(IV)]2 alpha 2(IV)434-472 (Eble, J. A., Golbik, R., Mann, K., and Kühn, K. (1993) EMBO J. 12, 4795-4802). In the present study, we have examined the cell adhesion activities of a third region in type IV collagen. A single-stranded peptide (SSP) incorporating the alpha 1(IV)531-543 sequence promoted the adhesion of melanoma, ovarian carcinoma, and Jurkat cells in a dose-dependent manner, with 40% cell adhesion observed at [SSP] = 1.8, 11.5, and 42.2 microM, respectively. Nearly identical results were obtained for cell adhesion to an all-D-enantiomer of the SSP, suggesting that the cell surface receptor(s) for this site do not discriminate based on chirality. The alpha 1(IV)531-543 sequence maintained its cell adhesion promoting activity when incorporated into a homotrimeric triple-helical polypeptide, although relative levels of adhesion were either slightly enhanced or slightly diminished compared with the SSP. Triple-helical conformation was thus not critical for cellular recognition of the alpha 1(IV)531-543 sequence. Single-site substitution experiments of the SSP showed no overall correlation between the biological effects of substitutions and SSP conformation. The SSP, D-SSP, triple-helical polypeptide, and SSP substitution results suggest that cell recognition of the alpha 1(IV)531-543 sequence is generally independent of substrate conformation. The present and prior studies indicate that "conformationally dependent" and "conformationally independent" cellular recognition sites exist within the triple-helical domain of type IV collagen.

Amino Acid Sequence↗
PubMed1994

Synthetic fibronectin peptides interrupt inflammatory cell infiltration in transforming growth factor beta 1 knockout mice.

Pronounced mononuclear leukocyte (MNL) infiltration occurs in multiple organs of mice homozygous for a transforming growth factor beta 1 (TGF-beta 1) loss-of-function gene mutation [TGF-beta 1 (-/-)], followed by cachexia and eventually death. Consistent with the increased leukocyte adhesion and tissue infiltration, MNLs isolated from spleen, thymus, and peripheral blood of symptomatic TGF-beta 1 (-/-) mice, as compared to littermate controls, exhibited increased adhesion to extracellular matrix proteins and to endothelial cells in vitro. Incubation of TGF-beta 1 (-/-) MNLs with selected synthetic peptides corresponding to cell- and heparin-binding sequences of fibronectin (FN) significantly attenuated adhesion of these cells not only to FN but also to endothelial cells in vitro. Based on these observations, mice were treated with the FN peptides in an attempt to rescue them from tissue inflammation and cardiopulmonary failure. Daily injections of a combination of four synthetic FN peptides that interact with beta 1-integrins and/or cell surface proteoglycans blocked the massive infiltration of MNLs into the heart and lungs of TGF-beta 1 (-/-) mice. Peptide treatment initiated on day 8, coincident with the first evidence of increased leukocyte-endothelial cell interactions, not only blocked tissue infiltration but also moderated the lethal wasting syndrome.

Amino Acid Sequence↗
PubMed1994

Synthetic fibronectin peptides suppress arthritis in rats by interrupting leukocyte adhesion and recruitment.

In an experimental model of arthritis, increased leukocyte adhesion is associated with the evolution of acute and chronic synovial inflammation. Whereas peripheral blood mononuclear cells (PBMC) from control animals bind minimally to fibronectin matrices, PBMC from animals receiving arthropathic doses of bacterial cell walls demonstrate increased integrin mRNA expression and enhanced adhesion. To determine whether this augmented adhesion was causal in the development of synovial pathology, peptides synthesized from several fibronectin domains which inhibited leukocyte adhesion in vitro were administered to arthritic animals either as free peptides or coupled to a carrier molecule. Not only were peptides containing either the RGD or CS-1 cell-binding domains inhibitory to chronic synovial pathology (articular index = 10.5 +/- 0.3 for untreated animals compared to 1.25 +/- 0.25 for RGD and 2.5 +/- 0.7 for CS-1), but three peptides synthesized from the carboxy-terminal 33-kD heparin-binding domain of fibronectin were also found to significantly inhibit leukocyte recruitment and the evolution of arthritis. Based on these data, which are the first to explore the therapeutic potential of heparin-binding fibronectin peptides in chronic inflammation, it appears that antagonism of cellular adhesion and recruitment by fibronectin peptides may provide an important mechanism for modulating the multi-step adhesion process and attenuating aberrant inflammatory responses.

Amino Acid Sequence↗
PubMed1994

Tenascin-R (J1 160/180 inhibits fibronectin-mediated cell adhesion--functional relatedness to tenascin-C.

Cell adhesion and neurite outgrowth on fibronectin is a multistep process modulated by different extra- and intracellular signals. Fibronectin-mediated cell attachment and spreading can be affected in a negative way by tenascin-C, an extracellular matrix glycoprotein expressed in a temporally and spacially restricted manner during early morphogenesis. Tenascin-R (J1-160/180), consisting of two major isoforms of 160 kDa (tenascin-R 160) and 180 kDa (tenascin-R 180) in mammals, is an extracellular matrix glycoprotein of the central nervous system that shares high structural homologies with tenascin-C. Here we show that in relation to fibronectin-mediated adhesion, the two extracellular matrix molecules are also functionally closely related. When offered as mixed substrata with other extracellular matrix molecules, the two tenascin-R isoforms and tenascin-C derived from mouse brain selectively inhibit fibronectin-dependent cell adhesion and neurite outgrowth, and affect cell morphology of different mesenchymal and neural cells. This effect is partially due to interactions at the substrate level that result in a steric hindrance and/or conformational change of the cell binding sites of the fibronectin molecule. In addition, tenascin-R 180 and tenascin-C interact with cells by an RGD- and beta 1 integrin-independent mechanism, leading to cell rounding and detachment from such substrata. The expression of tenascin-R and tenascin-C in the nervous system at times and locations where fibronectin-mediated cellular processes take place may be related to the role of inhibitory signals in the extracellular matrix in the regulation of cell migration and differentiation in general.

Animals↗
PubMed1993

Structural features of fibronectin synthetic peptide FN-C/H II, responsible for cell adhesion, neurite extension, and heparan sulfate binding.

FN-C/H II (KNNQKSEPLIGRKKT), a heparin-binding peptide derived from the COOH-terminal heparin-binding domain of fibronectin, mediates cell adhesion for a variety of cell types and promotes neurite outgrowth. By systematic amino acid substitution of synthetic peptide analogues of FN-C/H II, the basic structural features necessary for activity have been identified in the COOH-terminal residues LIGRKK. This biologically "active" sequence has been located in several other heparin/heparan sulfate-binding proteins and may represent a potential binding motif for sulfated polyanions. NMR structural studies indicate that the COOH-terminal segment of FN-C/H II displays significant multiple-turn or helix-like character suggesting that the RKK sequence may lie on the same surface of the protein, as opposed to alternating in an extended chain motif.

Amino Acid Sequence↗
PubMed1993

A synthetic peptide from the COOH-terminal heparin-binding domain of fibronectin promotes focal adhesion formation.

Cell adhesion to extracellular matrix molecules such as fibronectin involves complex transmembrane signaling processes. Attachment and spreading of primary fibroblasts can be promoted by interactions of cell surface integrins with RGD-containing fragments of fibronectin, but the further process of focal adhesion and stress fiber formation requires additional interactions. Heparin-binding fragments of fibronectin can provide this signal. The COOH-terminal heparin-binding domain of fibronectin contains five separate heparin-binding amino acid sequences. We show here that all five sequences, as synthetic peptides coupled to ovalbumin, can support cell attachment. Only three of these sequences can promote focal adhesion formation when presented as multicopy complexes, and only one of these (WQPPRARI) retains this activity as free peptide. The major activity of this peptide resides in the sequence PRARI. The biological response to this peptide and to the COOH-terminal fragment may be mediated through cell surface heparan sulfate proteoglycans because treatment of cells with heparinase II and III, or competition with heparin, reduces the response. Treatment with chondroitinase ABC or competition with chondroitin sulfate does not.

Amino Acid Sequence↗
PubMed1993

Human keratinocytes adhere to and spread on synthetic peptide FN-C/H-V derived from fibronectin.

In a previous study, we reported that two synthetic peptides derived from the 33-kD carboxyl terminal cell/heparin-binding fragment of fibronectin A chain promoted keratinocyte adhesion but not spreading. Because keratinocytes are capable of spreading on the 33/66-kD fragments, we focused on identifying additional chemically synthesized peptides from the cell/heparin-binding fragments of fibronectin that might promote cell spreading. When plastic substrata were coated with peptide FN-C/H-V (WQPPRARI), which is derived from the carboxyl-terminal heparin-binding domain of all plasma fibronectin isoforms, keratinocytes adhered and displayed a spread morphology. In solution, soluble peptide FN-C/H-V inhibited cell spreading on intact fibronectin and on the 33/66-kD fragments. Furthermore, polyclonal antibodies raised against peptide FN-C/H-V also inhibited keratinocyte spreading on fibronectin and the 33/66-kD fragments. These data support the hypothesis that keratinocyte cell adhesion and cell spreading on fibronectin are mediated by multiple distinct domains and different regulatory processes.

Amino Acid Sequence↗
PubMed1993

Type IV collagen, laminin, and fibronectin promote the adhesion and migration of rabbit lens epithelial cells in vitro.

PURPOSE: To assess the ability of basement membrane and extracellular matrix proteins to promote rabbit lens epithelial cell adhesion and migration, which may play a role in the development of secondary cataract. METHODS: Rabbit lens epithelial cells were isolated and grown in tissue culture for use in standardized assays to study adhesion and migration of rabbit lens epithelial cells in response to type IV collagen, laminin, fibronectin, and ovalbumin. RESULTS: Under these conditions, the adhesion of rabbit lens epithelial cells to surfaces coated with varying concentrations of type IV collagen, laminin, and fibronectin was shown to be dependent on concentration. Rabbit lens epithelial cells did not adhere to ovalbumin-coated surfaces at any concentration tested. Type IV collagen promotes maximal in vitro adhesion of rabbit lens epithelial cells at lower coating concentrations in comparison to laminin and fibronectin. In cell migration experiments, fibronectin promoted maximal migration at lower concentrations in comparison with laminin and type IV collagen. This was shown both in haptotaxis experiments (the migration of cells to surfaces coated with protein) and in chemotaxis experiments (the migration of cells to attractants in solution). Lens epithelial cells did not migrate in response to ovalbumin under the conditions of this study. CONCLUSION: The results of these studies indicate that adhesion and migration of lens epithelial cells occurs in response to the lens capsule proteins type IV collagen and laminin and in response to fibronectin, a protein found in the lens during embryologic development. Because fibronectin plays a role in the embryologic development of the lens but is not normally present in the adult lens, the possible introduction of fibronectin into the eye after surgery may play a critical role in the posterior migration of lens epithelial cells and the development of posterior capsular opacification or secondary cataract.

Animals↗
PubMed1993

Characterization of FN-C/H-V, a novel synthetic peptide from fibronectin that promotes rabbit corneal epithelial cell adhesion, spreading, and motility.

PURPOSE: Fibronectin promotes corneal epithelial cell adhesion and motility in vitro and plays an important role in corneal re-epithelialization during corneal wound healing. Multiple domains contribute to the adhesion- and motility-promoting activity of fibronectin. The aim of this study was to identify amino acid sequences that contribute to the rabbit corneal epithelial (RCE) cell adhesion- and motility-promoting activity of the 33 and 66 kD carboxy-terminal heparin-binding fragments of fibronectin. METHODS: Synthetic peptides derived from the 33/66 kD fragments of fibronectin were tested for their ability to directly promote RCE cell adhesion, spreading, and motility. To assess the contribution of these peptides to the activity of fibronectin and the 33/66 kD fragments of fibronectin, synthetic peptides, and antibodies against these peptides were tested for their ability to block RCE cell adhesion, spreading, and motility. RESULTS: In this study, we identified a novel peptide sequence derived from the 33/66 kD fragments of fibronectin, FN-C/H-V (WQPPRARI), that directly promotes the adhesion, spreading, and migration of RCE cells in a concentration-dependent manner. A second peptide from the 33/66 kD fragments of fibronectin, FN-C/H-IV (SPPRRARVT), promoted RCE cell adhesion and spreading, but did not promote RCE cell migration. In contrast, two synthetic peptides from the 33/66 kD fragments of fibronectin that were previously shown to promote RCE cell adhesion (FN-C/H-I and FN-C/H-III) did not promote RCE cell spreading or migration. Soluble FN-C/H-V inhibited RCE cell adhesion to surfaces coated with FN-C/H-V, the 33/66 kD fragments of fibronectin, and to fibronectin. In addition, polyclonal anti-FN-C/H-V IgG inhibited RCE cell adhesion to FN-C/H-V, the 33/66 kD fragments of fibronectin, and to fibronectin. Finally, polyclonal anti-FN-C/H-V IgG also inhibited RCE cell haptotactic migration on the 33/66 kD fragments. CONCLUSIONS: These data suggest that the amino acid sequence defined by peptide FN-C/H-V contributes to the adhesion-, spreading-, and motility-promoting activity of the 33/66 kD carboxy-terminal heparin-binding fragments of fibronectin. Given the important role of fibronectin in corneal wound healing, these findings provide additional insight into the complex molecular basis of corneal epithelial cell interactions with fibronectin and may be important in the context of corneal wound healing.

Amino Acid Sequence↗
PubMed1992

Effects of transforming growth factor-beta 1 on human pulmonary adenocarcinoma cell adhesion, motility, and invasion in vitro.

BACKGROUND: Transforming growth factor-beta 1 (TGF-beta 1), a potent growth modulator produced by a variety of tumor cells, as well as by platelets, has pleiotropic effects on cell-extracellular matrix interactions and may influence tumor cell invasion and metastasis. PURPOSE: Our purpose was to characterize the effects of TGF-beta 1 on the adhesion, motility, and invasiveness of a metastatic human pulmonary carcinoma (A549 cell line) in vitro. METHODS: A549 cells were seeded onto type I collagen gels, and invasion over a 9-day period was measured in the presence or absence of TGF-beta 1 (0.1-10 ng/mL). In addition, cell adhesion to substrata coated with type I collagen (1-100 nM) as well as haptotactic migration through filters coated with type I collagen (100 micrograms/mL) were measured following a 24-hour treatment with TGF-beta 1 (1-10 ng/mL). RESULTS: TGF-beta 1 stimulated the invasion of A549 cells into type I collagen gels in a dose-dependent manner. Both the number of cells entering the gel and the depth of invasion into the gel were increased. In addition, the effects of TGF-beta 1 were blocked in a dose-dependent manner by a purified polyclonal IgG against TGF-beta 1 but not by normal rabbit IgG. A549 cell invasion was accompanied by dramatic changes in A549 cell morphology that included the appearance of numerous long pseudopodia, consistent with a change in the motile behavior of these cells. TGF-beta 1 stimulated by approximately fourfold the haptotactic migration of A549 cells on polycarbonate filters coated with type I collagen. The TGF-beta 1-mediated increase in invasion and motility was accompanied by a fourfold increase in A549 cell adhesion to type I collagen. CONCLUSIONS: The results suggest that TGF-beta 1 can influence cellular recognition of extracellular matrix components and can modulate cellular adhesion and migration on these components, leading to increased invasive potential. IMPLICATIONS: Given the wide-spread tissue distribution of TGF-beta 1 and its secretion by a variety of tumor cells as well as by platelets, TGF-beta 1 may be an important autocrineparacrine regulator of the invasive phenotype in vivo.

Adenocarcinoma↗