School of Chemical Engineering, Georgia Institute of Technology, Atlanta 30332; and Departments of Surgery and Bioengineering, Emory University, Atlanta, Georgia 30322
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ABSTRACT |
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The ability of a soluble heparin-binding peptide sequence derived from fibronectin to modulate the adhesion and chemokinetic migration behavior of arterial smooth muscle cells was assessed using a novel glass microsphere centrifugation assay and automated time-lapse fluorescence videomicroscopy, respectively. Treatment of cells grown on fibronectin-coated substrates with the soluble heparin-binding peptide resulted in the disassembly of focal adhesions, as assessed by immunohistochemical staining. These observations were consistent with an observed dose-dependent two- to fivefold reduction in cell-substrate adhesive strength (P < 0.001) and a biphasic effect on migration speed (P < 0.05). Moreover, heparin-binding peptides induced a twofold reduction (P < 0.01) in two-dimensional cell dispersion in the presence of a non-heparin-binding growth factor, platelet-derived growth factor-AB (PDGF-AB). Heparin-binding peptides were unable to mediate these effects when cells were grown on substrates lacking a heparin-binding domain. These data support the notion that competitive interactions between cell surface heparan sulfates with heparin-binding peptides may modulate chemokinetic cell migration behavior and other adhesion-related processes.
heparan sulfates; focal contact; extracellular matrix
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INTRODUCTION |
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CURRENT EVIDENCE SUGGESTS that cell surface heparan sulfate proteoglycans (HSPG) promote focal adhesion formation, thereby enhancing cell-substrate interactions. The role of cell surface heparan sulfates in the formation of focal adhesions was initially suggested by the inability of two mutant cell lines that lacked or had altered HSPGs to form focal adhesions in response to a fibronectin substrate (10, 21). Before these observations, Woods et al. (37) had demonstrated that substrata coated with the isolated cell-binding domain of fibronectin were not sufficient for complete cell adhesion; cells attached and spread but did not form focal contacts unless heparin-binding fibronectin sequences were added. Others (4, 35) have also observed that endothelial cells, vascular smooth muscle cells, and human fibroblasts are able to spread on cell-binding RGD containing fibronectin fragments but are unable to form actin stress fibers or focal contacts if the glycosaminoglycan (GAG)-binding domain is absent. Pretreatment of cells with heparitinase I and III also blocks focal contact formation (35, 39).
To date, most studies that have investigated the potential of soluble peptides to effectively compete for cell surface adhesion receptors have focused on peptides or protein fragments that contain integrin-binding RGD sequences (14, 17). For example, Wu et al. (40) demonstrated that an RGD-containing peptide was capable of enhancing chemokinetic cell motility on substrates containing high surface concentrations of fibronectin. These results were consistent with an observed reduction in the strength of short-term adhesive interactions in the presence of a competing integrin-binding peptide. Because the interaction of cell surface HSPGs with matrix-bound heparin-binding domains promotes focal adhesion formation, we speculated that the presence of soluble heparin-binding peptides might also influence cell locomotion through direct competitive interactions. Specifically, we have investigated the ability of heparin-binding fragments to alter cell adhesion strength and chemokinetic cell motility by either limiting the formation of focal adhesions and/or by inducing their disassembly. We have observed that a heparin-binding peptide derived from fibronectin is capable of inducing a dose-dependent decrease in cell adhesive strength and a biphasic effect on chemokinetic cell migration speed. This phenomenon appears to be related, in part, to the disassembly of focal adhesions.
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MATERIALS AND METHODS |
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Cell culture techniques. Immortalized rat pulmonary arterial smooth muscle cells were a gift of Dr. A. Rothman (University of California San Diego Medical Center, San Diego. CA; see Ref. 29). Cells were grown in growth media composed of medium 199 (M199; Mediatech) supplemented with 10% FBS (Hyclone), 2 mM L-glutamine (GIBCO-BRL Life Technologies), and 1% antibiotic-antimycotic mixture (GIBCO-BRL Life Technologies). Cell quiescence was established by incubation for 24 h in M199 with 0.5% FBS (quiescence medium). All migration and adhesion assays were performed in serum-free medium. Serum-free medium consisted of MCDB-104 (GIBCO-BRL Life Technologies) supplemented with 1 mM CaCl2, 10 mg/l insulin, 6.7 µg/l sodium selenite, 5.5 mg/l transferrin, 2 mM L-glutamine, and 1% antibiotic-antimycotic mixture.
Peptides. All peptides were synthesized in the Emory University Winship Cancer Center Microchemistry Center. The heparin-binding peptides used in this study are derived from the known primary amino acid sequence of fibronectin designated as FN-C/H-V (WQPPRARI) and scrambled FN-C/H-V (RPQIPWAR; see Refs. 24 and 39). In selective cases, an additional tyrosine or cysteine residue was added to the COOH-terminus of the peptides to facilitate iodination or fluorescein labeling, respectively. The sequence for the scrambled FN-C/H-V peptide is identical to that reported by Huebsch et al. (18, 19). Human plasma fibronectin (pFN; Sigma), the 110-kDa fibronectin fragment (FN-110; Upstate Biotechnology), which has an RGD cell-binding domain but lacks a heparin-binding domain, collagen type I (Vitrogen), and recombinant human platelet-derived growth factor-AB (PDGF-AB; GIBCO-BRL) were obtained from commercial sources.
Protein adsorption assay. Fibronectin, collagen type I, and heparin-binding peptides were radiolabeled with 125I using Iodobeads (Pierce) following the manufacturer's protocol. Both the percentage of iodine incorporation and specific activity were determined. We have previously reported detailed methods for determining the fibronectin adsorption profile on non-tissue culture-treated polystyrene discs (7, 8). The surface density (d) of other proteins or peptide sequences adsorbed on glass coverslips and glass microspheres (d 30-50 µm; Polysciences) was ascertained in a similar fashion. Briefly, glass or polystyrene surfaces were incubated with protein or peptide for 24 h at 4°C followed by three washes with PBS, blocking with 1% BSA for 45 min at room temperature, and three more washes with PBS. Adhesion and migration assays, as well as correlative immunohistochemical staining studies, were conducted on a variety of fibronectin-coated substrate types (e.g., glass vs. nontissue culture polystyrene) and configurations (e.g., spherical vs. planar surfaces). The determination of an absolute fibronectin surface density on each surface type ensured that consistent correlations between assays could be ascertained.
Immunostaining. Focal adhesion formation and disassembly in the presence of soluble heparin-binding peptides were examined in cells cultured on fibronectin-coated glass coverslips. Coverslips (Fisher Scientific) were incubated with pFN for 24 h at 4°C, followed by three washes with PBS, blocking with 1% BSA for 45 min at room temperature, and three more washes with PBS. Cells at 80% confluency were detached with trypsin/EDTA and resuspended in serum-free migration medium. Approximately 10,000 cells/cm2 were seeded on each glass coverslip and were allowed to adhere and spread overnight before any further treatment. Plated cells were then incubated for an additional 24 h with a test peptide. To visualize focal adhesions, cells were stained for vinculin with hVIN-1 monoclonal anti-vinculin antibody (Sigma). Briefly, the cells were fixed with a 5-min incubation in 3.7% formaldehyde (Sigma) at room temperature followed by permeabilization with 1% Triton X-100 for 5 min at room temperature and three washes for 15 min each with PBS. Slides were incubated with the anti-vinculin monoclonal antibody (1:250 dilution) in a humidified chamber at 37°C for 45 min. After three washes with PBS for 15 min each, slides were incubated with donkey anti-mouse antibody conjugated with rhodamine (PharMingen) in a humidified chamber at 37°C for 45 min. After three washes with PBS for 15 min each, coverslips were mounted on precleaned microscope slides and viewed with a Nikon TMD epifluorescence microscope under a ×100 objective.
Adhesion assay. Adhesive strength of attached and spread cells was quantified using a modified form of the centrifugation adhesion assay. Specifically, rather than pulling cells off of a substrate, adhesion strength was determined by pulling the test substrate, in this instance, protein-coated glass microspheres, off of a cell monolayer. Details of assay development and optimization are described elsewhere (6). Briefly, cells grown to 80% confluency were detached using trypsin/EDTA and seeded in the presence of growth media on the inner six wells of sterile tissue culture-treated detachable eight-well strips (Corning Costar). Cells were allowed to grow to confluence, at which time the monolayer was washed three times with PBS, followed by the addition to each well of 200 µl of fibronectin-coated glass microspheres (mean d 38.5 µm; specific gravity 2.48, Polysciences) in serum-free medium and test peptide, where indicated. After a 6-h incubation period at 37°C, 5% CO2 humidified atmosphere, individual wells were detached and placed inverted in a 12-well plate prefilled with 0.1% BSA in PBS. Typically, beads covered >95% of the well surface area, which represents ~33,000 beads/well. The plate was centrifuged for 1 min at speeds ranging from 500 to 3,500 rpm, and each well then was viewed under a microscope using a ×4 objective. The percentage of beads remaining attached to the cell layer was characterized by computing the fraction of the well surface that remained covered by beads. This was facilitated by visualizing the wells with high ambient light and performing routine image analysis on the captured image (IPLab Spectrum; Signal Analytics).
The centrifugal force at which 50% of the beads are detached from the cell surface was computed by plotting the percentage of attached beads as a function of centrifugal force. The Levenberg-Marquardt method for nonlinear least squares fitting was used to fit the data to
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(1) |
Single-cell migration assay. All migration assays were performed using a computer-assisted, fluorescence videomicroscopy system, as described previously (7, 8). Briefly, cells at ~80% confluency were labeled with 0.625 µg/ml 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) in quiescence medium for 24 h. Cells were then seeded in serum-free medium at a density of ~1,000 cells/cm2 in non-tissue culture-treated polystyrene multiwell plates coated at a defined surface density with a specific matrix protein. Cells were observed for 24 h with images obtained at 30-min intervals. The images were analyzed to determine the location of the centroids of each cell at each observation time point.
Average squared displacement as a function of time interval n
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(2) |
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(3) |
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(4) |
Statistical analysis. The data were analyzed using the Student-Newman-Keuls test. The SE for the percentage of motile cells was calculated by analyzing independent Bernoulli trials. Statistical significance of these data was determined by utilizing the z-test with the Yates correction for continuity. Unless otherwise indicated, statistical significance was determined between a treatment group and untreated cells that were not exposed to test peptide.
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RESULTS |
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Adsorption of heparin-binding peptides to fibronectin-coated substrates. To ensure that soluble heparin-binding peptide sequences were not adsorbed to any significant degree on test well surfaces, 125I-labeled peptides were used in an adsorption study. Glass coverslips, coated with human pFN and blocked with BSA, were incubated overnight with 100 µM peptide solutions. Minimal adsorption of FN-C/H-V was observed on either fibronectin- or BSA-adsorbed glass coverslips (<0.5 nmol/cm2 or <0.4% of total added peptide).
Disassembly of focal adhesions is induced by heparin-binding
peptides.
Cells were allowed to attach and spread on pFN-coated glass coverslips
and subsequently incubated overnight with 1 or 100 µM of FN-C/ H-V
or scrambled peptides. Large, well-organized clusters of vinculin,
representing the formation of focal adhesion contacts, were visible on
cells seeded on pFN substrate in the absence of soluble peptide
treatment (Fig. 1). Focal adhesions were
uniformly distributed over the entire cell area.
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Smooth muscle cell adhesion is inhibited in a dose-dependent manner
by heparin-binding peptides.
The adhesive strength between cells and adsorbed fibronectin-coated
glass microspheres, in the presence or absence of test peptide and the
respective scrambled sequence, was defined using a modified centrifugal
bead assay. This assay facilitated the generation of an adhesion
profile as a function of soluble peptide concentration, and logistic
regression allowed expression of data in terms of F50 (Fig.
2 and Table
1).
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Soluble heparin-binding peptides modulate chemokinetic smooth
muscle cell migration.
To ensure comparable fibronectin surface densities, the adsorption
isotherms of fibronectin on glass and non-tissue culture-treated polystyrene surfaces were determined and noted to be similar on both
surfaces (Fig. 3). Conformational
differences between fibronectin adsorbed on glass and polystyrene may
exist; however, the effect on heparan sulfate binding is likely small.
Furthermore, all assays were performed using surfaces coated at a
concentration of pFN that exceeded monolayer coverage (0.5 µg/cm2).
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Soluble heparin-binding peptides alter cell adhesion and migration
by competitive interactions with heparin-binding domains located within
insoluble extracellular matrix proteins.
We postulated that heparin-binding peptides alter smooth muscle cell
adhesion and migration behavior by competitively inhibiting interactions of cell surface heparan sulfates with heparin-binding domains located within insoluble matrix proteins. This hypothesis was
tested by defining the effect of the FN-C/H-V sequence on cell adhesion
and motility on substrates coated with FN-110, which lacks the
heparin-binding domain. Characteristically, focal adhesions are not
observed when cells are plated on FN-110. In fact, measured adhesion
strength between protein-coated beads and a cell monolayer was
significantly lower when beads were coated with FN-110 compared with
those beads coated with either native fibronectin or collagen type I
(Fig. 5). Incubation of cells with the
FN-C/H-V sequence had no additional effect on adhesion between beads
coated with FN-110 and the underlying cell monolayer. Likewise, no
change in cell motility on FN-110-coated surfaces was observed in the presence or absence of the FN-C/H-V sequence (Fig.
6).
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Effect of soluble heparin-binding peptides is enhanced in the
context of PDGF-AB-stimulated cell migration.
PDGF-AB does not bind to cell surface heparan sulfates, nor does it
alter syndecan, glypican, or cell surface heparan sulfate expression.
However, PDGF-AB does increase chemokinetic cell motility through a
mechanism probably related to dynamic cytoskeletal assembly processes.
We speculated that at increased cell speeds the impact of competing
soluble heparin-binding sequences might be enhanced. The effect of
FN-C/H-V sequences on PDGF-AB-stimulated cell locomotion is summarized
in Fig. 7. Cell speed, persistence
time, the dispersion coefficient, and the percentage of
motile cells were all increased by PDGF-AB. Notably, treatment with
FN-C/H-V had no effect on the proportion of migrating cells. However, a
significant reduction in both cell speed and persistence time led to a
twofold reduction in the dispersion coefficient (P < 0.01), which is indicative of a marked reduction in cell flux across a
region of space.
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DISCUSSION |
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Recent evidence suggests that cell surface HSPG, in particular, syndecans 1 and 4, are important determinants of cell-matrix adhesive processes. For example, Liebersbach and Sanderson (22) have demonstrated that the expression of syndecan 1 in a human myeloma cell line limits its ability to form tumors in vivo. In addition, Carey et al. (5) have shown that overexpressing syndecan 1 induces cell spreading and the development of microfilament bundles that terminate in focal adhesions. This behavior has been correlated further with an inability of syndecan 1-expressing cells to invade collagen matrixes (34). These reports have suggested that the migration of at least some cell types may require the loss of syndecan 1 HSPG receptors (22, 28).
Although syndecan 1 may have a role in modulating cell-matrix adhesive
processes, syndecan 4 is the only HSPG incorporated in focal adhesions,
where it colocalizes with 1- and
3-integrin subunits and secreted fibronectin fibrils
(1, 30). Indeed, during the formation of focal adhesions,
syndecan 4 becomes clustered into higher-order, self-associating
structures that appear to be linked to the cytoskeleton. With this in
mind, a current view holds that focal adhesions form in two stages
(4). The first stage is dependent on integrin activation
and clustering, includes aggregation of vinculin, talin, paxillin,
tensin, filamin, and
-actinin, and requires the activation of
protein kinase C (PKC) and focal adhesion kinase. The second stage is
mediated by the interaction of syndecan 4 with heparin-binding domains
in the extracellular matrix (ECM) and, likewise, involves PKC signaling (1). In support of a possible role of syndecan 4 in cell
locomotion, Woods et al. (38) have recently reported that
overexpression of syndecan 4 in CHO-K1 cells limits cell migration.
These studies have provided significant insight into a potentially
important function of cell surface HSPG. Nonetheless, it bears emphasis that a primary change in substratum adhesiveness could initiate significant changes in motile cell behavior, even in the absence of a
requisite change in the surface concentration or binding affinity of
heparan sulfate. Specifically, the functional consequences of syndecan
1 or 4 expression must be interpreted within the context of the type,
distribution, and spatial density of their respective ligands.
Moreover, this process will also be influenced by the ability of these
cell surface receptors to effectively interact with available
matrix-bound ligands.
It is likely that syndecan 1 and 4 influence cell motility and
remodeling processes by direct contact-mediated interactions between
core protein-associated heparan sulfate chains with heparin-binding domains found within matrix glycoproteins. For example, at least two
domains within each fibronectin monomer bind to heparan sulfate (31). The HEP I site is located at the
NH2-terminus, is composed of type I repeat units, and is
associated with a relatively weak heparin dissociation constant in the
range 106 to 10
5 M (2, 3). The
other major heparin-binding site, known as HEP II, is located in three
type III repeat units (nos. 12-14) between the cell-binding (RGD)
site and the variably spliced IIICS region (domain V) near
the COOH-terminus. At least four independent sites consisting of
basically charged amino acids may mediate heparin binding within the
HEP II domain; none of which overlap with integrin-binding sites. The
heparin dissociation constant for HEP II is considerably greater than
HEP I, in the range of 10
8 to 10
7 M
(2, 3). Recently, Walker and Gallagher (33)
have identified a likely HEP II binding site in heparan sulfate
characterized by sequences of N-sulfated disaccharides in which a
proportion of the iduronate residues are sulfated at C-2. All of this
suggests that competing interactions may take place between
heparin-binding proteins with common high-affinity sites on the heparan
sulfate chain. Likely, both protein affinity and concentration will
determine the end result of these interactions. It has been postulated
that competing mechanisms of this kind may play a significant role in
the control and integration of cellular responses to a variety of
heparin-binding growth factors and matrix proteins. However, confirmatory evidence in support of this hypothesis has been limited.
Our results indicate that heparin-binding peptides derived from the HEP II domain may limit the formation and/or induce the disassembly of focal adhesions, as assessed by immunohistochemical staining. These observations were consistent with an observed dose-dependent decrease in cell-substrate adhesive strength and a biphasic effect on migration speed. The competitive nature of these interactions is also demonstrated by the inability of heparin-binding peptides to mediate these effects when cells were grown on substrates lacking a heparin-binding domain. Finally, a scrambled peptide sequence that contains a high density of cationic residues had no significant effect. It is notable that the capacity of heparin-binding peptides to inhibit two-dimensional cell dispersion was enhanced significantly in the presence of a non-heparin-binding growth factor, PDGF-AB. Significantly, PDGF-AB does not effect the expression or shedding of cell surface HSPGs or their interactions with ECM heparin-binding domains (9, 32). PDGF-AB likely influences adhesion strength through a variety of mechanisms, including an effect on integrin expression, activation, or clustering, and may have an impact on other factors that are important to the process of cell migration, such as intracellular force generation or lamellipod extension. Indeed, PDGF-AB significantly increased cell dispersion. Nonetheless, blocking heparan sulfate-matrix interactions through exposure to FN-C/H-V inhibited cell migration, despite the presence of this soluble motogenic factor.
Cell locomotion is a dynamic process involving the formation and breakage of attachments to an underlying substrate (20, 25). In this regard, a large body of data has confirmed that the ability of cells to migrate on a given substrate depends on several variables related to integrin-ligand interactions, including ligand levels, integrin levels, and integrin-ligand binding affinities. Conceptually, integrin-ligand interactions appear to affect the way intracellular pathways integrate, so as to effect and regulate cell migration. As an outgrowth of this notion, Palecek et al. (27) and others (12, 13) have suggested that short-term cell-substratum adhesiveness is rate limiting in determining cell migration speed, as the linkage between integrin and the ECM is altered. This postulate is based on experimental studies that have measured the attachment strength of cells after a 20- to 30-min incubation period with substrates coated with varying surface densities of matrix proteins. In correlating the strength of short-term adhesive interactions with cell migration, intermediate cell attachment strength was associated with both maximal cell speed and directional persistence. Importantly, mathematical models that have evolved in the context of these observations predict that cell speed is a function of both cell motile force and short-term binding strength to the substratum (12, 27). These studies have established an important conceptual framework for understanding the role of adhesive interactions in cell locomotion. Nonetheless, there remain a number of important considerations that have received less attention, including the relative contribution of different time scale adhesive events and the proportionate effect of nonintegrin adhesion receptors in determining cell binding strength to the underlying matrix.
The implications of the experimental studies reported herein are twofold. First, it is clear that heparin-binding domains are critical modulators of both cell adhesion and locomotion. Increasing the surface concentration of the FN-110, which contains a cell-binding (RGD) sequence but lacks a heparin-binding domain, had little effect on cell migration parameters despite the increased availability of surface-bound integrin ligands. Furthermore, loss of the heparin-binding domain was associated with a threefold reduction in cell speed (15 vs. 5 µm/h). Thus, if one accepts that short-term cell-substratum adhesiveness is rate limiting in determining cell migration speed, these results suggest that cell surface heparan sulfate-matrix interactions may be important determinants of the rate-limiting process. It is of interest that Lotz et al. (23) observed a significant increase in adhesive "strengthening" in the first 15 min after initial cell binding to a surface, presumably due to the movement of additional adhesive receptors into the adhesive site and the coupling of the actin cytoskeleton to the adhesion complex. Therefore, our data suggest that dissection of the role of specific cell surface HSPG in these critical early events is worthy of further investigation, particularly with quantitative adhesion assays of high sensitivity. Second, the results of our investigations serve to reemphasize that longer time-scale adhesive events, such as the late clustering of receptors to focal contacts or the formation of ECM or matrix contacts, are relevant to cell movement. Specifically, cell-bead adhesive strength, measured after a 6-h incubation period, was fivefold less to surfaces coated with FN-110 compared with those coated with native fibronectin. Likewise, a fivefold reduction in cell adhesion to fibronectin-coated beads was also observed after the addition of FN-C/H-V with an associated loss in focal adhesions. It is noteworthy that the capacity of fibronectin-derived heparin-binding peptides, and in particular FN-C/H-V, to alter the adhesion of endothelial cells, keratinocytes, and corneal epithelial cells to fibronectin has been investigated extensively, as summarized in prior reports, with little or no effect observed (19, 24, 34). In these reports, standard adhesion assay methodology was used in which cells were incubated for 30-90 min with fibronectin-coated surfaces. Thus short-term cell-substrate-binding assays may not necessarily provide an accurate assessment of all adhesive events that influence cell locomotion, either as a result of inadequate assay sensitivity or as a consequence of being unable to capture all rate-limiting steps that characterize the adhesive process.
The integration of chemical and mechanical signals from the matrix directly influences cell adhesive and motility behavior and, as an end result, tissue morphogenesis. Other mechanisms that effect focal adhesion formation and disassembly have been well documented, including thrombospondin-calreticulin and tenascin C-annexin II interactions, among others (16). Our data suggest that competitive binding interactions between heparin-binding peptides and cell surface GAG chains may also influence wound healing and other processes, in part, by modulating cell adhesion and migration behavior. Although the physiological significance of our observations has not been defined fully, new opportunities may exist for drug development that specifically targets heparan sulfate-matrix interactions.
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ACKNOWLEDGEMENTS |
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We thank J. T. Gallagher, A. Woods, and J. R. Couchman for helpful discussions.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-56819 and HL-60963.
Address for reprint requests and other correspondence: E. L. Chaikof, 1639 Pierce Dr., Rm. 5105, Emory University, Atlanta, GA 30322 (E-mail: echaiko{at}emory.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 27 September 2000; accepted in final form 22 December 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Baciu, PC,
and
Goetinck PF.
Protein kinase C regulates the recruitment of syndecan-4 into focal contacts.
Mol Biol Cell
6:
1503-1513,
1995[Abstract].
2.
Benecky, MJ,
Kolvenbach CG,
Amrani DL,
and
Mosesson MW.
Evidence that binding to the carboxyl-terminal heparin-binding domain (Hep II) dominates the interaction between plasma fibronectin and heparin.
Biochemistry
27:
7565-7571,
1988[ISI][Medline].
3.
Bentley, KL,
Klebe RJ,
Hurst RE,
and
Horowitz PM.
Heparin binding is necessary, but not sufficient, for fibronectin aggregation. A fluorescence polarization study.
J Biol Chem
260:
7250-7256,
1985
4.
Burridge, K,
Turner CE,
and
Romer LH.
Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly.
J Cell Biol
119:
893-903,
1992[Abstract].
5.
Carey, DJ,
Stahl RC,
Tucker B,
Bendt KA,
and
Cizmeci-Smith G.
Aggregation-induced association of syndecan-1 with microfilaments mediated by the cytoplasmic domain.
Exp Cell Res
214:
12-21,
1994[ISI][Medline].
6.
Chon, JH.
Mediation of Vascular Smooth Muscle Cell Adhesion and Migration by Cell Surface Heparan Sulfate Glycosaminoglycans (PhD thesis). Atlanta, GA: Georgia Institute of Technology, 1999.
7.
Chon, JH,
Netzel R,
Rock BM,
and
Chaikof EL.
a4b1 and a5b1 control endothelial cell migration on fibronectin by differentially regulating cell speed and motile cell phenotype.
Ann Biomed Eng
26:
1091-1101,
1998[ISI][Medline].
8.
Chon, JH,
Vizena AD,
Rock BM,
and
Chaikof EL.
Characterization of single-cell migration using a computer-aided fluorescence time-lapse videomicroscopy system.
Anal Biochem
252:
246-254,
1997[ISI][Medline].
9.
Cizmeci-Smith, G,
Langan E,
Youkey J,
Showalter LJ,
and
Carey DJ.
Syndecan-4 is a primary-response gene induced by basic fibroblast growth factor and arterial injury in vascular smooth muscle cells.
Arterioscler Thromb Vasc Biol
17:
172-180,
1997
10.
Couchman, JR,
Austria R,
Woods A,
and
Hughes RC.
Adhesion defective BHK cell mutant has cell cell surface heparan sulphate proteoglycan of altered properties.
J Cell Physiol
136:
226-236,
1988[ISI][Medline].
11.
Cozens-Roberts, C,
Quin JA,
and
Lauffenburger DA.
Receptor-mediated cell attachment and detachment kinetics. II. Experimental model studies with radial-flow detachment assay.
Biophys J
58:
857-872,
1990[Abstract].
12.
DiMilla, PA,
Barbee K,
and
Lauffenburger DA.
Mathematical model for the effects of adhesion and mechanics on cell migration speed.
Biophys J
60:
15-37,
1991[Abstract].
13.
DiMilla, PA,
Stone JA,
Quinn JA,
Albelda SM,
and
Lauffenburger DA.
Maximal migration of human smooth muscle cells on fibronectin and type IV collagen occurs at an intermediate attachment strength.
J Cell Biol
122:
729-737,
1993[Abstract].
14.
Donaldson, DJ,
Mahan JT,
and
Smith GN, Jr.
Newt epidermal cell migration over collagen and fibronectin involves different mechanisms.
J Cell Sci
90:
325-333,
1988[Abstract].
15.
Garcia, AJ,
Ducheyne P,
and
Boettinger D.
Qunatification of cell adhesion using a spinning disc device and application to surface reactive materials.
Biomaterials
18:
1091-1098,
1997[ISI][Medline].
16.
Greenwood, JA,
and
Murphy-Ullrich JE.
Signaling of de-adhesion in cellular regulation and motility.
Microsc Res Tech
43:
420-432,
1998[ISI][Medline].
17.
Hayman, EG,
Pierschbacher MD,
and
Ruoslahti E.
Detachment of cells from culture substrate by soluble fibronectin peptides.
J Cell Biol
100:
1948-1954,
1985[Abstract].
18.
Huebsch, JB,
Fields GB,
Triebes TG,
and
Mooradian DL.
Photoreactive analog of peptide FN-C/H-V from the carboxy-terminal heparin-binding domains of fibronectin supports endothelial cell adhesion and spreading on biomaterial surfaces.
J Biomed Mater Res
31:
555-567,
1996[ISI][Medline].
19.
Huebsch, JC,
McCarthy JB,
Diglio CA,
and
Mooradian DL.
Endothelial cell interactions with synthetic peptides from the carboxyl-terminal heparin-binding domains of fibronectin.
Circ Res
77:
43-53,
1997
20.
Lauffenburger, DA,
and
Linderman JJ.
Receptors. New York: Oxford Univ. Press, 1993.
21.
LeBaron, RG,
Esko JD,
Woods A,
Johansson S,
and
Hook M.
Adhesion of glycosaminoglycan-deficient chinese hamster ovary cell mutants to fibronectin substrata.
J Cell Biol
106:
945-952,
1988[Abstract].
22.
Liebersbach, BF,
and
Sanderson RD.
Expression of syndecan-1 inhibits cell invasion into type I collagen.
J Biol Chem
269:
20013-20019,
1994
23.
Lotz, MM,
Burdsal CA,
Erickson HP,
and
McClay DR.
Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding an strengthening response.
J Cell Biol
109:
1795-1805,
1989[Abstract].
24.
Mooradian, DL,
McCarthy JB,
Skubitz AP,
Cameron JD,
and
Furcht LT.
Characterization of FN-C/H-V, a novel synthetic peptide from fibronectin that promotes rabbit corneal epithelial cell adhesion, spreading, and motility.
Invest Ophthalmol Vis Sci
34:
153-164,
1993[Abstract].
25.
Mow, VC,
Guilak F,
Tran-Soy-Tay R,
and
Hochmuth RM.
Cell Mechanics and Cellular Engineering. New York: Springer-Verlag, 1994.
26.
Othmer, HG,
Dunbar SR,
and
Alt W.
Models of dispersal in biological systems.
J Math Biol
26:
263-298,
1988[ISI][Medline].
27.
Palecek, SP,
Loftus JC,
Ginsberg MH,
Lauffenburger DA,
and
Horwitz AF.
Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness.
Nature
385:
537-540,
1997[ISI][Medline].
28.
Ridley, RC,
Xiao H,
Hata H,
Woodliff J,
Epstein J,
and
Sanderson RD.
Expression of syndecan regulates human myeloma plasma cell adhesion to type I collagen.
Blood
81:
767-774,
1993[Abstract].
29.
Rothman, A,
Kulik TJ,
Taubman MB,
Berk BC,
Smith CWJ,
and
Nadal-Ginard B.
Development and characterization of a cloned rat pulmonary arterial smooth muscle cell line that maintains differentiated properties through multiple subcultures.
Circulation
86:
1977-1986,
1992[Abstract].
30.
Saterback, A,
Kuo SC,
and
Lauffenburger DA.
Heterogeneity and probabilistic binding contributions to receptor-mediated cell detachment kinetics.
Biophys J
65:
243-252,
1993[Abstract].
31.
Sekiguchi, K,
and
Hakomori S.
Functional domain structure of fibronectin.
Proc Natl Acad Sci USA
77:
2661-2665,
1980[Abstract].
32.
Subramanian, SV,
Fitzgerald ML,
and
Bernfield M.
Regulated shedding of syndecan-1 and -4 ectodomains by thrombin and growth factor receptor activation.
J Biol Chem
272:
14713-14720,
1997
33.
Walker, A,
and
Gallagher JT.
Structural domains of heparan sulphate for specific recognition of the C-terminal heparin-binding domain of human plasma fibronectin (HEPII).
Biochem J
317:
871-877,
1996[ISI][Medline].
34.
Wilke, MS,
Vespa J,
Skubitz AP,
Furcht LT,
and
McCarthy JB.
Human keratinocytes adhere to and spread on synthetic peptide FN-C/H-V derived from fibronectin.
J Invest Dermatol
101:
43-48,
1993[Abstract].
35.
Woods, A,
and
Couchman JR.
Protein kinase C involvement in focal adhesion formation.
J Cell
Sci101:
277-290,
1992[ISI].
36.
Woods, A,
and
Couchman JR.
Syndecan 4 heparan sulfate proteoglycan is a selectively enriched and widespread focal adhesion component.
Mol Biol Cell
5:
183-192,
1994[Abstract].
37.
Woods, A,
Couchman JR,
Johnasson S,
and
Hook M.
Adhesion and cytoskeletal organization of fibroblasts in response to fibronectin fragments.
EMBO J
5:
665-670,
1986[Abstract].
38.
Woods, A,
Longley RL,
Tumova S,
and
Couchman JR.
Syndecan-4 binding to the high affinity heparin-binding domain of fibronectin drives focal adhesion formation in fibroblasts.
Arch Biochem Biophys
374:
66-72,
2000[ISI][Medline].
39.
Woods, A,
McCarthy JB,
Furcht LT,
and
Couchman JR.
A synthetic peptide from the COOH-terminal heparin binding domain of fibronectin promotes focal adhesion formation.
Mol Biol Cell
4:
605-613,
1993[Abstract].
40.
Wu, P,
Hoying JB,
Williams SK,
Kozikowski BA,
and
Lauffenburger DA.
Integrin-binding peptide in solution inhibits or enhances endothelial cell migration, predictably from cell adhesion.
Ann Biomed Eng
22:
144-152,
1994[ISI][Medline].