Cell-binding Domain Of Fibronectin Research Articles

Nanoparticles traversing the extracellular matrix induce biophysical perturbation of fibronectin depicted by surface chemistry.

While the filtering and accumulation effects of the extracellular matrix (ECM) on nanoparticles (NPs) have been experimentally observed, the detailed interactions between NPs and specific biomolecules within the ECM remain poorly understood and pose challenges for in vivo molecular-level investigations. Herein, we adopt molecular dynamics simulations to elucidate the impacts of methyl-, hydroxy-, amine-, and carboxyl-modified gold NPs on the cell-binding domains of fibronectin (Fn), an indispensable component of the ECM for cell attachment and signaling. Simulation results show that NPs can specifically bind to distinct Fn domains, and the strength of these interactions depends on the physicochemical properties of NPs. NP-NH3+ exhibits the highest affinity to domains rich in acidic residues, leading to strong electrostatic interactions that induce severe deformation, potentially disrupting the normal functioning of Fn. NP-CH3 and NP-COO- selectively occupy the RGD/PHSRN motifs, which may hinder their recognition by integrins on the cell surface. Additionally, NPs can disrupt the dimerization of Fn through competing for residues at the dimer interface or by diminishing the shape complementarity between dimerized proteins. The mechanical stretching of Fn, crucial for ECM fibrillogenesis, is suppressed by NPs due to their local rigidifying effect. These results provide valuable molecular-level insights into the impacts of various NPs on the ECM, holding significant implications for advancing nanomedicine and nanosafety evaluation.

Engineering of Bio-Adhesive Ligand Containing Recombinant RGD and PHSRN Fibronectin Cell-Binding Domains in Fusion with a Colored Multi Affinity Tag: Simple Approach for Fragment Study from Expression to Adsorption.

Engineering of biomimetic motives have emerged as promising approaches to improving cells’ binding properties of biomaterials for tissue engineering and regenerative medicine. In this study, a bio-adhesive ligand including cell-binding domains of human fibronectin (FN) was engineered using recombinant protein technology, a major extracellular matrix (ECM) protein that interacts with a variety of integrins cell-surface’s receptors and other ECM proteins through specific binding domains. 9th and 10th fibronectin type III repeat containing Arginine-Glycine-Aspartic acid (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) synergic site (FNIII9-10) were expressed in fusion with a Colored Multi Affinity Tag (CMAT) to develop a simplified production and characterization process. A recombinant fragment was produced in the bacterial system using E. coli with high yield purified protein by double affinity chromatography. Bio-adhesive surfaces were developed by passive coating of produced fragment onto non adhesive surfaces model. The recombinant fusion protein (CMAT-FNIII9/10) demonstrated an accurate monitoring capability during expression purification and adsorption assay. Finally, biological activity of recombinant FNIII9/10 was validated by cellular adhesion assay. Binding to α5β1 integrins were successfully validated using a produced fragment as a ligand. These results are robust supports to the rational development of bioactivation strategies for biomedical and biotechnological applications.

A biomimetic hydrogel functionalized with adipose ECM components as a microenvironment for the 3D culture of human and murine adipocytes.

The lack of relevant in vitro models for adipose tissue makes necessary the development of a more physiological environment providing spatial and chemical cues for the effective maturation of adipocytes. We developed a biofunctionalized hydrogel with components of adipose extracellular matrix: collagen I, collagen VI, and the cell binding domain of fibronectin and we compared it to usual 2D cultures on plastic plates. This scaffold allowed 3D culture of mature adipocytes from the preadipocytes cell lines 3T3-L1 and 3T3-F442A, as well as primary Human White Preadipocytes (HWP), acquiring in vivo-like organization, with spheroid shaped adipocytes forming multicellular aggregates. The size of these aggregates increased with time up to 120 μm in diameter after 4 weeks of maturation, with good viability. Significantly higher lipogenic activity (up to 20-fold at day 28 for HWP cultures) and differentiation rates were also observed compared to 2D. Gene expression analyses highlighted earlier differentiation and complete maturation of 3D HWP compared to 2D, reinforced by the expression of Perilipin protein after 21 days of nutrition. This increase in adipocytes phenotypic and genotypic markers made this scaffold-driven culture as a robust adipose 3D model. Retinoic acid inhibition of lipogenesis in HWP or isoprenalin and caffeine induction of lipolysis performed on mouse 3T3-F442A cells, showed higher doses of molecules than typically used in 2D, underlying the physiologic relevance of this 3D culture system. Biotechnol. Bioeng. 2017;114: 1813-1824. © 2017 Wiley Periodicals, Inc.

Role of chemical crosslinking in material-driven assembly of fibronectin (nano)networks: 2D surfaces and 3D scaffolds

Poly(ethyl acrylate) (PEA) induces the formation of biomimetic fibronectin (FN) (nano)networks upon simple adsorption from solutions, a process referred to as material-driven FN fibrillogenesis. The ability of PEA to organize FN has been demonstrated in 2D and 2.5D environments, but not as yet in 3D scaffolds, which incorporate three-dimensionality and chemical crosslinkers that may influence its fibrillogenic potential. In this paper we show for the first time that while three-dimensionality does not interfere with PEA-induced FN fibrillogenesis, crosslinking does, and we determined the maximum amount of crosslinker that can be added to PEA to maintain FN fibrillogenesis. For this, we synthesised 2D substrates with different amounts of crosslinker (1–10% of ethylene glycol dimethacrylate) and studied the role of crosslinking in FN organization using AFM. The glass transition temperature was seen to increase with crosslinking density and, accordingly, polymer segmental mobility was reduced. The organization of FN after adsorption (formation of FN fibrils) and the availability of the FN cell-binding domain were found to be dependent on crosslinking density. Surface mobility was identified as a key parameter for FN supramolecular organization. PEA networks with up to 2% crosslinker organize the FN in a similar way to non-crosslinked PEA. Scaffolds prepared with 2% crosslinker also had FN (nano)networks assembled on their walls, showing PEA’s ability to induce FN fibrillogenesis in 3D environments as long as the amounts of crosslinker is low enough.

Fibronectin adsorption on surface modified polyetherurethanes for blood-contacting applications

Event Abstract Back to Event Fibronectin adsorption on surface modified polyetherurethanes for blood-contacting applications Ludivine Hugoni1, 2, Vanessa Montaño-Machado1, 2, Emmanuel Pauthe2, Diego Mantovani1 and J Paul Santerre3 1 CHU de Québec Research Center, Laval University, Lab. Biomaterials and Bioengineering, CRC-I, Dept Min-Met-Materials Eng, Canada 2 University of Cergy Pontoise, Equipe de Recherche sur les Relations Matrice Extracellulaire-Cellules, France 3 University of Toronto, Institute of Biomaterials and Biomedical Engineering, Canada Introduction: After the implantation of a medical device, adhesive proteins such as fibronectin (FN) are adsorbed to the surface of the material[1]. These are the biomolecules that monocytes (MCs) will interact with, to adopt a pro-inflammatory or pro-wound healing phenotype[2]. Hence, it was desired to study FN coatings on two surface modified polyetherurethanes (PEUs) with potential blood-contacting applications, to mimic the biological environment. A fluorinated surface modifying oligomer (PPO212L), known to reduce platelet adhesion[3] and an anionic/dihydroxy oligomeric (ADO) additive, known to enable cell adhesion[4] were used to modify surface blood cell compatibility of a commercial PEU. FN adsorption and the subsequent MC inflammatory response was investigated on the two modified PEUs. Materials and Methods: The PEU was prepared by dissolving Tecoflex® 65D in 10 % (wt/v) chloroform. PPO212L or ADO was added at 0.5 % (wt/wt). Solutions were casted on PTFE for 24 h followed by 2 h in vacuum (30 mmHg), both at room temperature. Protein adsorption was performed with a FN solution (50 μg.mL-1 in HEPES 10 mM, pH 7.4) at 37 °C for 30 min. Surfaces were characterized by water contact angle (WCA), atomic force microscopy (AFM) and immunofluorescence staining (IFS). Adsorbed FN was quantified by a bicinchoninic acid assay. MCs, isolated from blood of healthy donors (University of Toronto ethics protocol #22203) were seeded on PEUs (106 cells.mL-1). After 72 h of culture, the viability of adhered MCs was evaluated by Alamar blue® assay and cell morphology was analyzed by scanning electron microscopy (SEM). Results were given as mean ± standard deviation (SD) and were statistically analyzed by one-way analysis of variance (ANOVA), with significant differences reported for p<0.05. Results and Discussion: On all PEUs, FN adsorption significantly decreased the WCA whereas surface roughness increased approximately 2 fold (Table 1). No statistically significant difference was observed for the amount of adsorbed FN (Table 1) corroborating the presence of a protein monolayer to surfaces[5]. The difference in WCA prior vs after FN adsorption was greatest for the base PEU and PEU-ADO. This result suggested a different FN conformation on PEU-PPO212L than on the two other surfaces. The availability of FN cell binding domains for all coated PEUs was observed after IFS (data not shown). Preliminary tests with MCs showed that all FN-coated PEUs significantly increased MC viability, without affecting cell morphology except for PEU-ADO (Figure 1). These results suggested that FN activated MCs more extensively on PEU-ADO than on the base PEU and PEU-PPO212L, which showed the least activated morphology of all surfaces. Conclusions: A comparative study of two surface modified PEUs, PEU-PPO212L and PEU-ADO, intended for blood-contacting applications was performed. Investigations suggested that FN-coated PEU-PPO212L has less influence on MC activation than FN-coated PEU-ADO. Further investigations are ongoing to determine the effect of FN coating on cell production of cytokines such as pro-inflammatory tumor necrosis factor-α (TNF-α) and anti-inflammatory interleukin 10 (IL-10). This work was supported by a MSc Scholarship from NSERC CREATE Program in Regenerative Medicine (www.ncprm.ulaval.ca), NSERC, CIHR, Fonds Samuel de Champlain, FQRS, and FQRNT; The authors would like to thank Pascale Chevallier and Lucie Levesque for their technical assistance

A comparative study of polyethylene glycol hydrogels derivatized with the RGD peptide and the cell-binding domain of fibronectin

The goal of our study was to compare the biological responses of cells cultured on polyethylene glycol (PEG) hydrogels functionalized with varying concentrations of the widely used adhesion peptide, RGD, and the cell-binding domain of fibronectin, III(9-10). We used Michael addition chemistry to covalently link cysteines in GRGDSPC and glutathione S-transferase (GST) tagged III(9-10) (GST-III(9-10)), to the acrylate groups in PEG diacrylate (PEGDA). Conjugation of GST-III(9-10) to PEGDA occurred through cysteine residues in GST. Ellman's reagent and immunoblotting studies demonstrated an efficiency of 90% or more for PEG conjugation of 1 μM GST-III(9-10) or GRDGSPC in 10% (wt/vol) PEGDA at 37°C for 1 h. Circular dichroism and limited proteolysis demonstrated that conjugating PEGDA to GST-III(9-10) did not significantly perturb its native secondary structure. Sodium dodecyl sulfate polyacrylamide gel electrophoresis characterization of the wash solution of PEG hydrogels after photopolymerization demonstrated that >95% of the 1 μM GST-III(9-10) was incorporated into the PEG hydrogels after cross-linking. PEG hydrogels derivatized with 1 μM GST-III(9-10) had significantly higher cell adhesion and spreading than PEG hydrogels with 1 μM GRGDSPC. A comparable adhesion response between GRGDSPC and GST-III(9-10) was obtained when the former was at millimolar and the latter at micromolar concentration. The amount and type of conjugate in the PEG hydrogel derivative was statistically more significant than hydrogel rigidity in stimulating the biological responses observed. This report presents new evidence of the robustness of III(9-10) in mediating cell adhesion and spreading on PEG hydrogels.

Directing epithelial to mesenchymal transition through engineered microenvironments displaying orthogonal adhesive and mechanical cues

Cell interactions with their extracellular matrix (ECM) microenvironments play a major role in directing cellular processes that can drive wound healing and tissue regeneration but, if uncontrolled, lead to pathological progression. One such process, epithelial to mesenchymal transition (EMT), if finely controlled could have significant potential in regenerative medicine approaches. Despite recent findings that highlight the influence of biochemical and mechanical properties of the ECM on EMT, it is still unclear how these two orthogonal cues act synergistically to control epithelial cell phenotype. Here, we cultured lung epithelial cells on combinations of different mutants of fibronectin's cell binding domain that preferentially engage specific integrins and substrates of varying stiffness. Our results suggest that while stiff substrates induce spontaneous EMT, this response can be overcome by with fragments of fibronectin that support α3 and α5 integrin engagement. Furthermore, we found that substrate-induced EMT correlates with transforming growth factor beta activation by resident epithelial cells and is dependent on Rho/ROCK signaling. Suppressing cell-contractility was sufficient to maintain an epithelial phenotype. Our results suggest that integrin-specific engagement of fibronectin adhesive domains and the mechanics of the ECM act synergistically to direct EMT.

Influence of crystallite size of nanophased hydroxyapatite on fibronectin and osteonectin adsorption and on MC3T3-E1 osteoblast adhesion and morphology

The characteristic topographical features (crystallite dimensions, surface morphology and roughness) of bioceramics may influence the adsorption of proteins relevant to bone regeneration. This work aims at analyzing the influence of two distinct nanophased hydroxyapatite (HA) ceramics, HA725 and HA1000 on fibronectin (FN) and osteonectin (ON) adsorption and MC3T3-E1 osteoblast adhesion and morphology. Both substrates were obtained using the same hydroxyapatite nanocrystals aggregates and applying the sintering temperatures of 725°C and 1000°C, respectively. The two proteins used in this work, FN as an adhesive glycoprotein and ON as a counter-adhesive protein, are known to be involved in the early stages of osteogenesis (cell adhesion, mobility and proliferation). The properties of the nanoHA substrates had an important role in the adsorption behavior of the two studied proteins and clearly affected the MC3T3-E1 morphology, distribution and metabolic activity. HA1000 surfaces presenting slightly larger grain size, higher root-mean-square roughness (Rq), lower surface area and porosity, allowed for higher amounts of both proteins adsorbed. These substrates also revealed increased number of exposed FN cell-binding domains as well as higher affinity for osteonectin. Regarding the osteoblast adhesion results, improved viability and cell number were found for HA1000 surfaces as compared to HA725 ones, independently of the presence or type of adsorbed protein. Therefore the osteoblast adhesion and metabolic activity seemed to be more sensitive to surfaces morphology and roughness than to the type of adsorbed proteins.

Fibronectin Adsorption, Cell Adhesion, and Proliferation on Nanostructured Tantalum Surfaces

The interaction between dental pulp derived mesenchymal stem cells (DP-MSCs) and three different tantalum nanotopographies with and without a fibronectin coating is examined: sputter-coated tantalum surfaces with low surface roughness <0.2 nm, hut-nanostructured surfaces with a height of 2.9 +/- 0.6 nm and a width of 35 +/- 8 nm, and dome structures with a height of 13 +/- 2 nm and a width of 52 +/- 14 nm. Using ellipsometry, the adsorption and the availability of fibronectin cell-binding domains on the tantalum surfaces were examined, as well as cellular attachment, proliferation, and vinculin focal adhesion spot assembly on the respective surfaces. The results showed the highest fibronectin mass uptake on the hut structures, with a slightly higher availability of cell-binding domains and the most pronounced formation of vinculin focal adhesion spots as compared to the other surfaces. The proliferation of DP-MSCs was found to be significantly higher on dome and hut surfaces coated with fibronectin compared to the uncoated flat tantalum surfaces. Consequently, the results presented in this study indicate that fibronectin-coated nanotopographies with a vertical dimension of less than 5 nm influence cell adhesion. This rather interesting behavior is argued to originate from the more available fibronectin cell-binding domains observed on the hut structures.

An integrin-activating peptide, PHSRN, ameliorates inhibitory effects of conventional peritoneal dialysis fluids on peritoneal wound healing

Bioincompatible peritoneal dialysis fluids (PDFs) cause pathological changes in the peritoneal membrane, related to membrane dysfunction and progressive peritoneal fibrosis. We investigated the effects of Pro-His-Ser-Arg-Asn (PHSRN) peptide, one of the fibronectin cell-binding domains that activates integrins and reinforces wound healing, on peritoneal remodelling in a rat peritoneal injury model undergoing peritoneal dialysis. The peritoneal mesothelial monolayer was removed by a stripping procedure in rats receiving conventional high glucose-containing PDF supplemented with or without PHSRN or control His-Ser-Pro-Asn-Hrg (HSPNR) peptides. Effects of PHSRN on cell motility and signalling molecules were examined in cultured rat peritoneal mesothelial cells (RPMCs) and normal rat kidney fibroblasts (NRKs). The cytokeratin- and HBME-1-positive mesothelial cell monolayer was selectively removed by the procedure. By day 6, HBME-1-positive cells had regenerated to 53.3 +/- 6.5% of the peritoneal surface in the control group. Regeneration of the mesothelial layer was delayed in the PDF group (35.2 +/- 10.2%, P < 0.05), but PHSRN reversed the effects of PDF (51.7 +/- 9.6%, P < 0.05). PDF treatment increased thickening of granulomatous submesothelial tissue and numbers of ED1-, CD31- and alpha-smooth muscle actin-positive cells, but PHSRN ameliorated these effects. HSPNR had no effects on mesothelial regeneration or peritoneal wound healing. PHSRN, but not HSPNR, recovered glucose-induced inhibition of cell motility and phosphorylation of focal adhesion kinase and its downstream p130(Cas) in RPMCs and NRKs. These results suggest that PHSRN has beneficial effects on peritoneal regeneration by reducing the inhibitory effects of conventional PDF on integrin-mediated wound healing.

Fibronectin-mediated endothelialisation of chitosan porous matrices

Chitosan (Ch) porous matrices were investigated regarding their ability to be colonized by human microvascular endothelial cells (HPMEC-ST1.6R cell line) and macrovascular endothelial cells namely HUVECs. Specifically we assessed if previous incubation of Ch in a fibronectin (FN) solution was effective in promoting endothelial cell (EC) adhesion to Ch matrices with different degrees of acetylation (DAs). Upon FN physiadsorption, marked differences were found between the two DAs investigated, namely DA 4% and 15%. While cell adhesion was impaired on Ch with DA 15%, ECs were able to not only adhere to Ch with DA 4%, but also to spread and colonize the scaffolds, with retention of the EC phenotype and angiogenic potential. To explain the observed differences between the two DAs, protein adsorption studies using 125I-FN and immunofluorescent labelling of FN cell-binding domains were carried out. In agreement with the higher cell numbers found, scaffolds with DA 4% revealed a higher number of exposed FN cell-binding domains as well as greater ability to adsorb FN and to retain and exchange adsorbed FN in the presence of competitive proteins. These findings suggest that the DA is a key parameter modulating EC adhesion to FN-coated Ch by influencing the adsorbed protein layer.

IGFBP-3 and IGFBP-5 associate with the cell binding domain (CBD) of fibronectin

We have used Surface Plasmon Resonance (SPR) – based biosensor technology to investigate the interaction of the six high affinity insulin-like growth factor binding proteins (IGFBP 1–6) with the cell binding domain (CBD) of fibronectin. Using a biotinylated derivative of the ninth and tenth TypeIII domains of FN (9–10FNIII), we show that IGFBP-3 and -5 bind to FN-CBD. We show that this binding is inhibited by IGF-I and that, for IGFBP-5, binding occurs through the C-terminal heparin binding domain of the protein. Using site-directed mutagenesis of 9–10FNIII, we show both the “synergy” and RGD sites within these FN domains are required for maximum binding of both IGFBPs. We discuss the possible biological consequences of our results.

Orientation and surface coverage of adsorbed fibronectin cell binding domains and bound integrin α5β1 receptors

Integrin α5β1 binds the human 9th–10th type III fibronectin domain pair (FIII9–10) to mediate cell attachment and spreading. FIII9–10 mutants with increased conformational stability (FIII9′10) or highly restricted interdomain mobility (FIII9′10-CC) support cell spreading to greater and lesser extents, respectively. We have used neutron reflectivity to show that the surface adsorbed layers of the wild-type and mutant proteins are remarkably different. At bulk concentrations of protein equivalent to those used in cell spreading assays, the surface coverage of FIII9–10 was 14% compared to 31% for FIII9′10 and 100% for FIII9′10-CC. For FIII9′10-CC, three distinct transitions in the packing and orientation of the domain pair were observed. No similar transitions were observed for FIII9–10 and only a transition to bilayer was observed for FIII9′10. We discuss these observations by analogy to the surface pressure area isotherm of surfactants, with reference to the electrostatic surface potentials and conformational stabilities of the domain pairs. Data for the binding of purified integrin α5β1 receptors to adsorbed FIII9′10-CC were fitted with an integrin layer thickness of 130 Å. This indicates a movement of the integrin α5β1 headpiece away from its position in the compact ‘bent’ conformation. Thus, neutron reflectivity should prove to be a useful technique for the determination of the averaged integrin conformation upon binding to various ligands.

Cell Response to RGD Density in Cross-Linked Artificial Extracellular Matrix Protein Films

This study examines the adhesion, spreading, and migration of human umbilical vein endothelial cells on cross-linked films of artificial extracellular matrix (aECM) proteins. The aECM proteins described here were designed for application in small-diameter grafts and are composed of elastin-like structural repeats and fibronectin cell-binding domains. aECM-RGD contains the RGD sequence derived from fibronectin; the negative control protein aECM-RDG contains a scrambled cell-binding domain. The covalent attachment of poly(ethylene glycol) (PEG) to aECM substrates reduced nonspecific cell adhesion to aECM-RDG-PEG but did not preclude sequence-specific adhesion of endothelial cells to aECM-RGD-PEG. Variation in ligand density was accomplished by the mixing of aECM-RGD-PEG and aECM-RDG-PEG prior to cross-linking. Increasing the density of RGD domains in cross-linked films resulted in more robust cell adhesion and spreading but did not affect cell migration speed. Control of cell-binding domain density in aECM proteins can thus be used to modulate cell adhesion and spreading and will serve as an important design tool as these materials are further developed for use in surgery, tissue engineering, and regenerative medicine.

Up-regulation of HSP70 by the fibronectin-derived peptide PHSRN in human corneal epithelial cells

HSP70 is a member of the heat shock protein family and is induced by various types of cellular stress. We examined whether HSP70 might play a role in the healing of corneal epithelial wounds. Given that the PHSRN peptide, which corresponds to the second cell-binding domain of fibronectin, promotes corneal epithelial migration, we investigated the effect of this peptide on HSP70 expression in cultured human corneal epithelial cells. Reverse transcription–polymerase chain reaction and immunoblot analyses revealed that PHSRN increased the amounts of HSP70 mRNA and protein in these cells in a concentration- and time-dependent manner, whereas a control peptide (NRSHP) had no such effects. Furthermore, the PHSRN-induced up-regulation of HSP70 was blocked by SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), but it was not affected by PD98059 or SP600125, inhibitors of signaling by the MAPKs ERK and JNK, respectively. Our results suggest that induction of HSP70 expression may contribute to the promotion of corneal epithelial migration by PHSRN and hence to corneal epithelial wound healing.

Angiogenic response of endothelial cells to heparin-binding domain of fibronectin

Interaction of endothelial cells with cell-binding domain of fibronectin through integrin receptors is important in the process of angiogenesis. The present study was designed to examine the role of heparin-binding domain of fibronectin in angiogenesis using human umbilical vein endothelial cells. Attachment of endothelial cells in vitro to heparin-binding domain of fibronectin was inhibited by heparin. Chick chorioallantoic membrane assay revealed the proangiogenic nature of heparin-binding domain. Analysis by reverse transcription-polymerase chain reaction showed an increase in the expression of vascular endothelial growth factor and its receptor mRNA. Enzyme-linked immunosorbent assay showed a significant increase in the level of vascular endothelial growth factor secreted by cells maintained on heparin-binding domain. Treatment with calphostin C, an inhibitor of protein kinase C, decreased the expression of vascular endothelial growth factor receptor 2. Chick chorioallantoic membrane assay showed that the vascular endothelial growth factor secreted by cells maintained on heparin-binding domain was biologically more active, which appeared to be due to a decrease in its poly-adenosine diphosphate ribosylation. Binding assays showed that heparin-binding domain preferably binds unmodified vascular endothelial growth factor as compared to intact fibronectin. It is concluded that the heparin-binding domain of fibronectin by itself can promote angiogenesis in endothelial cells possibly by interaction with cell surface heparan sulphate proteoglycans involving protein kinase C dependent signaling and making available more active form of vascular endothelial growth factor to the cells.

Stimulation of Corneal Epithelial Migration by a Synthetic Peptide (PHSRN) Corresponding to the Second Cell-Binding Site of Fibronectin

Fibronectin plays an important role in the migration of corneal epithelial cells in vivo. The Arg-Gly-Asp (RGD) sequence in the principal cell binding domain of fibronectin mediates the interaction of fibronectin with integrins, whereas the Pro-His-Ser-Arg-Asn (PHSRN) sequence of fibronectin is thought to modulate this interaction. The authors examined the effects of a PHSRN peptide on corneal epithelial migration in vitro and in vivo. Epithelial migration in vitro was examined with the rabbit cornea in organ culture. The motility and phenotype of simian virus 40-transformed human corneal epithelial (HCE) cells were evaluated by time-lapse and immunofluorescence microscopy, respectively. Tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin was examined by immunoprecipitation and immunoblot analysis. The healing of rabbit corneal epithelial wounds induced by 1-heptanol was evaluated by fluorescein staining. The PHSRN peptide stimulated corneal epithelial migration in organ culture in a concentration-dependent manner, and it increased HCE cell motility in vitro. The peptide induced the accumulation of F-actin and the formation of focal adhesions at the leading edge of HCE cells. It also upregulated the tyrosine phosphorylation of FAK and paxillin in HCE cells, but it did not affect HCE cell proliferation or attachment to a fibronectin matrix. Administration of the PHSRN peptide in eye drops promoted corneal epithelial wound closure in vivo in a dose-dependent manner. None of these effects of the PHSRN peptide were induced by a control NRSHP peptide. The PHSRN peptide mimics many of the effects of fibronectin on corneal epithelial cells and may prove suitable as a substitute for fibronectin in the treatment of persistent corneal epithelial defects.

Induction of a high affinity fibronectin receptor inCandida albicansby caspofungin: requirements for β (1,6) glucans and the developmental regulator Hbr1p

Candida albicans expresses at least two biochemically distinct fibronectin receptors. Hemoglobin induces expression of a low affinity receptor recognizing the fibronectin cell-binding domain, whereas growth in complex media induces a high affinity receptor recognizing the collagen-binding domain. We now show that sub-inhibitory concentrations of caspofungin and nikkomycin Z, but not fluconazole, induce the high affinity fibronectin receptor in a dose-dependent manner. Macromolecular complexes mechanically sheared from caspofungin-treated cells retained high affinity fibronectin binding that was sensitive to protease, disulfide reduction, and beta (1,3) glucanase digestion. The high affinity fibronectin receptor was not inducible in a Kre9 mutant strain of C. albicans deficient in beta (1,6) glucans. Conversely, a mutant strain lacking the fibronectin binding protein Als5p showed no defects in induction of high or low affinity fibronectin receptors. Heterozygous mutants of a regulator of white-opaque phenotypic switching, HBR1, lacked any detectable high affinity fibronectin receptor expression in response to caspofungin, and re-introduction of the gene restored activity. Therefore, sub-inhibitory dosages of caspofungin induce a high affinity fibronectin receptor that is distinct from the known receptor Als5p and is dependent on beta (1,6) glucans and HBR1.

Ligand density and integrin repertoire regulate cellular response to LPA

Engagement of integrin receptors by the extracellular matrix (ECM) protein fibronectin (FN) activates intracellular signaling, cytoskeletal reorganization and cellular tension. The soluble factor lysophosphatidic acid (LPA) acts through Rho GTPase and its effector Rho kinase (ROCK) to enhance alpha5beta1 integrin-mediated cell spreading on the Arg-Gly-Asp (RGD) cell-binding domain of FN. A second cell-binding site for alpha4 integrins resides in the CS1 segment of the alternatively spliced V region of FN. We show here that LPA treatment of alpha4beta1-expressing CHOalpha4 cells on FN induced a significant decrease in spread cell area. LPA also decreased apoptosis induced by serum-deprivation in CHOalpha4 and human A375 melanoma cells in an alpha4beta1-dependent manner. Improvement in cell viability and changes in cell morphology were dependent on ROCK and on the number of substrate binding sites for alpha4beta1. LPA signaling combined with alpha4beta1-mediated adhesion appears to sustain cell viability in situations where FN matrix is limiting. Such cooperation may impact dynamic cellular events such as wound healing, fibrosis, and metastasis.

Integrin binding specificity regulates biomaterial surface chemistry effects on cell differentiation

Biomaterial surface chemistry has profound consequences on cellular and host responses, but the underlying molecular mechanisms remain poorly understood. Using self-assembled monolayers as model biomaterial surfaces presenting well defined chemistries, we demonstrate that surface chemistry modulates osteoblastic differentiation and matrix mineralization independently from alterations in cell proliferation. Surfaces were precoated with equal densities of fibronectin (FN), and surface chemistry modulated FN structure to alter integrin adhesion receptor binding. OH- and NH(2)-terminated surfaces up-regulated osteoblast-specific gene expression, alkaline phosphatase enzymatic activity, and matrix mineralization compared with surfaces presenting COOH and CH(3) groups. These surface chemistry-dependent differences in cell differentiation were controlled by binding of specific integrins to adsorbed FN. Function-perturbing antibodies against the central cell binding domain of FN completely inhibited matrix mineralization. Furthermore, blocking antibodies against beta(1) integrin inhibited matrix mineralization on the OH and NH(2) surfaces, whereas function-perturbing antibodies specific for beta(3) integrin increased mineralization on the COOH substrate. These results establish surface-dependent differences in integrin binding as a mechanism regulating differential cellular responses to biomaterial surfaces. This mechanism could be exploited to engineer materials that control integrin binding specificity to elicit desired cellular activities to enhance the integration of biomaterials and improve the performance of biotechnological culture supports.