Fn Fragments Research Articles

Biochip Surfaces Containing Recombinant Cell-Binding Domains of Fibronectin

Surface immobilization and characterization of the functional activity of fibronectin (Fn) type-III domains are reported. The domains FnIII9-10 or FnIII10 containing the RGD loop and PHSRN synergy site were recombinantly produced and covalently bound to chemically activated PEG methacrylate (MA) hydrogel coatings by microcontact printing. Such fabricated biochip surfaces were 6 mm in diameter and consisted of 190 µm wide protein stripes separated by 200 µm spacing. They were analyzed by imaging null ellipsometry, atomic force microscopy and fluorescence microscopy. Also, the coatings were tested in human foreskin fibroblast and HeLa cultures for at least 96 h, thus evaluating their suitability for controlled cell adhesion and proliferation. However, while HeLa cultures were equally well responsive to the FnIII9-10, FnIII10 and Fn surfaces, the fibroblasts displayed lower cell and lower focal adhesion areas, as well as lower proliferation rates on the Fn fragment surfaces as compared to Fn. Nevertheless, full functional activity of the fibroblasts was confirmed by immunostaining of Fn produced by the cells adherent on the biochip surfaces. The observed interaction differences that were either cell type or surface composition-dependent demonstrate the potential use of specifically engineered Fn and other ECM protein-derived domains in biochip architectures.

Inhibition of the interaction between fibronectin and dermatopontin by Clostridium perfringens fibronectin-binding proteins.

Fibronectin (Fn) is an approximately 450 kDa glycoprotein that consists of 12 type I, 2 type II, and 15-17 type III modules. Fibrillation of Fn is important for tissue reconstitution and wound healing. We previously reported that Clostridium perfringens produces several Fn-binding proteins (Fbps), two of which, FbpA and FbpB, bind to III1 -C (a fragment of Fn derived from the carboxyl-terminal two-thirds of the first-type III module). Dermatopontin (DPT), a 22 kDa noncollagenous extracellular matrix protein, accelerates normal collagen fibrillation and induces Fn fibrillation. DPT interacts with Fn-type III12-14 (III12-14 ), leading to a change in Fn conformation and promoting Fn fibrillation. Here, we investigated the effects of FbpA and FbpB on the binding of Fn and the III12-14 fragment to DPT and on the DPT-induced Fn fibrillation. Both recombinant FbpA (rFbpA) and recombinant FbpB (rFbpB) significantly inhibited Fn binding to DPT and recombinant III12-14 (rIII12-14 ) binding, and inhibited DPT-induced Fn fibrillation. Furthermore, it was found that both rFbpA and rFbpB significantly bound to coated DPT in an enzyme-linked avidin-biotin complex system, whereas rIII12-14 did not bind to either coated rFbpA or rFbpB. In conclusion, both FbpA and FbpB inhibited DPT-induced Fn fibrillation via their interaction with DPT. Both FbpA and FbpB released from lysed C. perfringens cells in wounds and/or infected tissue may prevent Fn fibrillation and delay the wound healing process, subsequently exacerbating infection.

Differential Engagement Of The Integrin Binding Domain Of Fibronectin Impacts Fibroblast Behavior

The switch between α5β1 and αvβ3 integrin engagement due to unfolding of the integrin binding domain (IBD) of fibronectin (Fn) has been theorized for a long time but was observed in vitro and murine models only recently. Fn is largely present in the extra cellular matrix (ECM) during wound healing and fibroproliferative diseases like idiopathic pulmonary fibrosis or cancer. Thus, further understanding the downstream effects of this differential integrin binding could elucidate several diseases’ mechanisms. Specifically, integrin α5β1 requires the regular, closed conformation of Fn's IBD, while αvβ3 can also bind the unfolded, open conformation. Moreover, cells can generate forces sufficient to cause such unfolding. We hypothesized that this change in Fn conformation affects lung fibroblasts by guiding integrin enrichment, pushing them towards a secretory and disease-relevant phenotype. We tested this premise by engineering Fn fragments containing the IBD site that mimicked either the open or closed conformation. Additionally, these fragments were designed to be covalently bound via thiol chemistry to a variety of substrates in order to preserve their structural integrity. Glass dishes coated with either Fn fragment were used as substrates for Single Cell Force Spectroscopy. By using fibroblasts expressing only α5β1, only αvβ3, and naïve murine lung fibroblasts we observed the need of proper α5β1 engagement to generate high adhesion forces even at short time points (<120s). Plating naïve fibroblasts on similarly functionalized dishes for up to 30 minutes enabled Mass Spec analysis of the adhesome and of the phosphorylation pathways emerging from the differential integrin engagement. Particularly, collagen XIIa and tubulin beta3 appeared to be enriched upon increased αvβ3 binding. Even on soft substrates (4kPa representing healthy lung tissue), lung fibroblasts plated on the open IBD Fn fragment engaged αvβ3 and display increased nuclear translocation of transcription factors associated with a secretory and contractile phenotype, after a 24h incubation. Fn (with Extra Domain A), Tropomyosin, Vimentin, Serpine1, and α integrins were among the genes upregulated on the open Fn fragment, compared to the closed fragment. These results were confirmed by qPCR and then bulk RNAseq assays. In conclusion, these data support that differential integrin engagement due to Fn IBD unfolding appears to affect cell phenotype in both the short and long run. Current efforts are focused on evaluating the efficacy of a novel αvβ3 inhibiting antibody on the progression of murine lung fibrosis. This work contributes to defining the link between changes in the ECM and cell behavior in the context of fibroproliferative diseases.

Plasma and Cellular Forms of Fibronectin as Prognostic Markers in Sepsis.

Background There is a pressing need for specific prognostic markers that could be used to monitor the severity of sepsis. The aims of our study were to investigate changes in the expression of different molecular forms of fibronectin in sepsis and to assess their relationship to the clinical severity and mortality of patients. Material and Methods. Forms of fibronectin: plasma (pFN), cellular (EDA-FN), FN-fibrin complexes, and fibronectin fragments were analyzed in 71 sepsis patients (survivors and nonsurvivors) and in the control by ELISA and immunoblotting. Results The baseline pFN concentration of patients with sepsis was significantly lower than in the control (133.0 mg/L vs. 231.2 mg/L) (P < 0.001), and in nonsurvivors, it was lower than in survivors (106.0 mg/L vs. 152.8 mg/L) (P = 0.004). The baseline EDA-FN was significantly elevated in both sepsis groups (survivors: 6.7 mg/L; nonsurvivors: 9.4 mg/L) compared to the control (1.4 mg/L) (P < 0.001). It should be noted that among patients with more severe sepsis, the EDA-FN level was higher in nonsurvivors than in survivors. Furthermore, molecular FN-fibrin complexes as well as FN fragments occurred much more frequently in nonsurvivors than in survivors. Conclusion The study showed that in sepsis, changes in plasmatic and cellular form of fibronectin were associated with the severity of sepsis and may be useful predictors of outcome.

Role of Ninth Type-III Domain of Fibronectin in the Mediation of Cell-Binding Domain Adsorption on Surfaces with Different Chemistries.

The orientation and conformation of adhesive proteins after adsorption play a central role in cell-binding bioactivity. Fibronectin (Fn) holds two peptide sequences that favor cell adhesion: the Arg-Gly-Asp (RGD) loop on the tenth type-III domain (Fn-III10) and the Pro-His-Ser-Arg-Asn (PHSRN) synergy site on the ninth type-III domain (Fn-III9). Herein, adsorption of Fn fragments (Fn-III10 and Fn-III9-10) on self-assembled monolayers (SAMs) carrying various functional groups (-COOH, -NH2, -CH3, and -OH) was investigated by the Monte Carlo method and molecular dynamics simulation in order to understand its mediation effect on cell adhesion. The results demonstrated that Fn-III9 could enhance the stiffness of the Fn molecule and further fix the adsorption orientation. The RGD site of the Fn fragment appeared to be deactivated on hydrophobic surfaces (CH3-SAM) because of the binding of adjacent nonpolar residues on surfaces, whereas charged surfaces (COOH-SAM and NH2-SAM) and hydrophilic surfaces (OH-SAM) were conducive to the formation of cell-binding-favorable orientation for Fn fragments. The cell adhesion capability of Fn fragments was highly improved on positively charged surfaces (NH2-SAM) and hydrophilic surfaces because of the advantageous steric structure and orientation of both RGD and PHSRN sites. This work provides an insight into the interplay at the atomic scale between protein adsorption and surface chemistry for designing biologically responsive substrate surfaces.

Integrin α3β1 Binding to Fibronectin Is Dependent on the Ninth Type III Repeat

Fibronectin (Fn) is a promiscuous ligand for numerous cell adhesion receptors or integrins. The vast majority of Fn-integrin interactions are mediated through the Fn Arg-Gly-Asp (RGD) motif located within the tenth type III repeat. In the case of integrins αIIbβ3 and α5β1, the integrin binds RGD and the synergy site (PHSRN) located within the adjacent ninth type III repeat. Prior work has shown that these synergy-dependent integrins are exquisitely sensitive to perturbations in the Fn integrin binding domain conformation. Our own prior studies of epithelial cell responses to recombinant fragments of the Fn integrin binding domain led us to hypothesize that integrin α3β1 binding may also be modulated by the synergy site. To explore this hypothesis, we created a variety of recombinant variants of the Fn integrin binding domain: (i) a previously reported (Leu → Pro) stabilizing mutant (FnIII9'10), (ii) an Arg to Ala synergy site mutation (FnIII9(R)→(A)10), (iii) a two-Gly (FnIII9(2G)10) insertion, and (iv) a four-Gly (FNIII9(4G)10) insertion in the interdomain linker region and used surface plasmon resonance to determine binding kinetics of integrin α3β1 to the Fn fragments. Integrin α3β1 had the highest affinity for FnIII9'10 and FnIII9(2G)10. Mutation within the synergy site decreased integrin α3β1 binding 17-fold, and the four-Gly insertion decreased binding 39-fold compared with FnIII9'10. Cell attachment studies demonstrate that α3β1-mediated epithelial cell binding is greater on FnIII9'10 compared with the other fragments. These studies suggest that the presence and spacing of the RGD and synergy sites modulate integrin α3β1 binding to Fn.

The TLR4 agonist fibronectin extra domain A is cryptic, exposed by elastase-2; use in a fibrin matrix cancer vaccine.

Fibronectin (FN) is an extracellular matrix (ECM) protein including numerous fibronectin type III (FNIII) repeats with different functions. The alternatively spliced FN variant containing the extra domain A (FNIII EDA), located between FNIII 11 and FNIII 12, is expressed in sites of injury, chronic inflammation, and solid tumors. Although its function is not well understood, FNIII EDA is known to agonize Toll-like receptor 4 (TLR4). Here, by producing various FN fragments containing FNIII EDA, we found that FNIII EDA's immunological activity depends upon its local intramolecular context within the FN chain. N-terminal extension of the isolated FNIII EDA with its neighboring FNIII repeats (FNIII 9-10-11) enhanced its activity in agonizing TLR4, while C-terminal extension with the native FNIII 12-13-14 heparin-binding domain abrogated it. In addition, we reveal that an elastase 2 cleavage site is present between FNIII EDA and FNIII 12. Activity of the C-terminally extended FNIII EDA could be restored after cleavage of the FNIII 12-13-14 domain by elastase 2. FN being naturally bound to the ECM, we immobilized FNIII EDA-containing FN fragments within a fibrin matrix model along with antigenic peptides. Such matrices were shown to stimulate cytotoxic CD8+ T cell responses in two murine cancer models.

Shear-Dependent Fibrillogenesis of Fibronectin: Impact of Platelet Integrins and Actin Cytoskeleton

Introduction: Soluble plasma fibronectin (Fn) with its inactive compact structure requires unfolding to assemble into active fibrils. Fibril formation of Fn is cell-mediated (Mao and Schwarzbauer, 2005) and depending on interactions of Fn with integrin receptors, through binding to αIIbβ3, α5β1, or αvβ3. Less is known about the contribution of biomechanical forces on the fibrillogenesis of Fn. Tension forces generated by cells via cytoskeleton could modulate Fn fibrillogenesis. The aim of this study was to investigate conformational changes of Fn, as induced by (1) platelet integrins, (2) cytoskeletal forces and/or (3) shear rates simulating venous or arterial flow conditions.Methods: Human plasma Fn (100 μg/ml) was added to plates pre-coated with 100 μg/ml of Fn or collagen in the presence or absence of washed platelets (2.5 x 107/ml). Subsequently, the solutions were exposed to shear using a cone-plate rheometer (Haake Rheostress 1). For microscopic analysis (LSM 510, Carl Zeiss), Alexa flour 488-conjugated Fn was used. In parallel experiments, a N-terminal 70kDa fragment of Fn (70 μg/ml) was incubated with soluble Fn at room temperature for 20 min before exposure to shear. To examine the role of distinct platelet integrins on fibril formation of Fn, washed platelets were incubated with monoclonal antibodies LM609, P1D6, 10E5, or abciximab (10 μg/ml, each) for 30 min at room temperature to block αvβ3, α5β1, αIIbβ3, or both αIIbβ3 and αvβ3, prior to the addition of Fn (100 μg/ml) and subsequent exposure to shear. In parallel experiments, washed platelets were pre-incubated with actin-modifying reagents, jasplakinolide (1 μM) or cytochalasin D (10 μM). In all experiments, flow conditions were simulated by shear rates, stepwise increasing from 50 s-1 to 5000 s-1 within 5 min and subsequently decreasing from 5000 s-1 to 50 s-1 within 5 min. Viscosities (mPa’s) of shear-exposed solutions were recorded over 10 min. To study the structure of Fn fibrils, solutions were examined by laser scanning microscopy after exposure to shear. To quantify the amount of fibril formation, deoxycholate solubility assays and densitometric analysis of Western blots were performed. Control experiments were conducted under static conditions.Results: Microscopic analyses showed that exposing Fn solutions to shear resulted in fibril formation. Fn fibril diameter varied from 0.5 to 5 μm. Observed fibrils were linked with each other and varied in length (from 50 to 300 μm). Addition of washed platelets to Fn solution resulted in a higher intertwined matrix of fibrils. Treatment of Fn with the N-terminal 70 kDa fragment of Fn, which is known to inhibit Fn matrix assembly, blocked fibril formation of Fn. Western blotting and densitometric analyses revealed that, in the absence of washed platelets, fibril formation (calculated as the ratio of insoluble to soluble Fn) on plates coated with collagen were 2-fold higher than on Fn-immobilized plates (n = 4, p < 0.05). Addition of washed platelets to Fn solution (100 μg/ml) resulted in increases of 20- or 7-fold in fibril formation of Fn, generated by shear on Fn- and collagen-immobilized plates, respectively (p < 0.05, n = 3). In contrast, 10E5 or abxicimab blocking αIIbβ3, or both αIIbβ3 and αvβ3 caused a reduction by 82% or 74% in fibril formation of Fn (p < 0.05, n = 3 each), in comparison to samples without antibodies. Blocking α5β1 or αvβ3by P1D6 or LM609 only caused a reduction by 17% or 56% (p > 0.05, n = 3 each). Incubation of platelets with jasplakinolide, which stabilizes actin, caused an increase in fibril formation by 41%, as compared to samples with untreated platelets (p > 0.05, n = 3). In contrast, disruption of actin by cytochalasin D resulted in a decrease by 86% (p < 0.05, n = 3). Under static conditions, no fibril formation was detected.Conclusions: Our results indicate that fibrillogenesis of Fn is modulated by shear conditions in a surface-dependent manner. Furthermore, formation of fibrils is induced by platelet integrins and actin cytoskeleton. Hereby, αIIbβ3 plays a predominant role, while α5β1 has a minor part among the three examined platelet integrins in terms of Fn fibril formation. DisclosuresNo relevant conflicts of interest to declare.

Determination of the Clostridium perfringens-binding site on fibronectin

The extracellular matrix protein fibronectin (Fn) is known to bind to the surface of Clostridium perfringens cells. Fn is a disulfide-linked homodimer protein, with each Fn polypeptide consisting of three types of repeating modules: 12 type I, 2 type II, and 15-17 type III modules. To determine the epitope on Fn recognized by C. perfringens cells, anti-Fn monoclonal antibodies (mAbs) and various Fn fragments (III2-10, rIII2-4, rIII5-7, rIII8, rIII9, rIII10) were employed. Although two C. perfringens-derived Fn-binding proteins, FbpA and FbpB, have been reported, they appear not to be the bacterium's surface Fn receptor. Moreover, both FbpA and FbpB were found to bind to C. perfringens cells. To avoid confusion, a mutant C. perfringens lacking both the fbpA and fbpB genes (MW5) was prepared using an in-frame deletion system. MW5 cells bound Fn on their surface, suggesting the presence of a putative Fn receptor(s) on C. perfringens cells. Of several anti-Fn mAbs, both HB39 and MO inhibited the binding of Fn to MW5 cells. HB39 reacted strongly with III2-10 and rIII9, and weakly with rIII2-4, rIII10 and rIII5-7 in Western blotting analysis. Binding of HB39 to Fn was inhibited in the presence of either rIII9 or rIII10, but not in the presence of rIII2-4, rIII5-7, or rIII8. Binding of Fn to MW5 cells was strongly inhibited by both III2-10 and rIII9, marginally inhibited by rIII2-4, but not affected by rIII5-7, rIII8, or rIII10. Significant binding of MW5 cells to immobilized rIII9 and rIII10 as well as immobilized III2-10 was observed. The region of Fn recognized by C. perfringens was thus mapped to the region encompassed by III9 and III10.

Combinatorial Design of an Anticalin Directed against the Extra-Domain B for the Specific Targeting of Oncofetal Fibronectin

The oncofetal isoform of the extracellular matrix protein fibronectin (Fn), which carries the extra-domain B (ED-B) and is exclusively expressed in neovasculature, has gained interest for tumor diagnosis and therapy using engineered antibody fragments. We have employed the human lipocalin 2 (Lcn2) as a small and robust non-immunoglobulin scaffold to select ED-B-specific Anticalins from a new advanced random library using bacterial phage display and ELISA screening against appropriately engineered Fn fragments. As a result, we have isolated and biochemically characterized four different Anticalins that all show low nanomolar affinities for ED-B, right in the range between the monomeric and dimeric forms of the single-chain variable antibody fragment L19 that has been widely applied in this area before. All Anticalins can be readily expressed in Escherichia coli as soluble and strictly monomeric proteins, and they show specific staining of ED-B-positive tumor cells in immunofluorescence microscopy while BIAcore affinity analyses indicate recognition of distinct ED-B epitopes. The crystal structure for one Anticalin, N7A, in complex with the Fn7B8 fragment, was solved at 2.6Å resolution and reveals binding to the gfcc′ sheet and cc′ loop on ED-B. This is the second example of a protein-specific Lcn2-based Anticalin, which illustrates the remarkable plasticity of the calyx-like ligand pocket of lipocalins with their four structurally hypervariable loops supported by a highly conserved β-barrel. The ED-B-specific Anticalins resulting from this study should provide useful reagents in research and biomedical drug development, both for in vivo imaging and for directed cancer therapy.

Ail Protein Binds Ninth Type III Fibronectin Repeat (9FNIII) within Central 120-kDa Region of Fibronectin to Facilitate Cell Binding by Yersinia pestis

The Yersinia pestis adhesin molecule Ail interacts with the extracellular matrix protein fibronectin (Fn) on host cells to facilitate efficient delivery of cytotoxic Yop proteins, a process essential for plague virulence. A number of bacterial pathogens are known to bind to the N-terminal region of Fn, comprising type I Fn (FNI) repeats. Using proteolytically generated Fn fragments and purified recombinant Fn fragments, we demonstrated that Ail binds the centrally located 120-kDa fragment containing type III Fn (FNIII) repeats. A panel of monoclonal antibodies (mAbs) that recognize specific epitopes within the 120-kDa fragment demonstrated that mAb binding to (9)FNIII blocks Ail-mediated bacterial binding to Fn. Epitopes of three mAbs that blocked Ail binding to Fn were mapped to a similar face of (9)FNIII. Antibodies directed against (9)FNIII also inhibited Ail-dependent cell binding activity, thus demonstrating the biological relevance of this Ail binding region on Fn. Bacteria expressing Ail on their surface could also bind a minimal fragment of Fn containing repeats (9-10)FNIII, and this binding was blocked by a mAb specific for (9)FNIII. These data demonstrate that Ail binds to (9)FNIII of Fn and presents Fn to host cells to facilitate cell binding and delivery of Yops (cytotoxins of Y. pestis), a novel interaction, distinct from other bacterial Fn-binding proteins.

Abstract 426: The molecular microenvironment critically affects the formation of isoDGR-dependent integrin binding sites in fibronectin

Abstract Fibronectin (FN) is an adhesive protein present in most body fluids and in the extracellular matrix of many tissues. This protein mediates a variety of cellular interactions with extracellular matrix and play important roles in haemostasis, thrombosis, inflammation, wound repair and angiogenesis. FN is constituted of three types of repeating homologous modules termed FN-I, FN-II and FN-III. Each module contains binding sites for molecules of the extracellular matrix and for cell adhesion receptors, such as integrins. Moreover, FN contains four NGR sites located in the: 5th type I, 2nd type II, 7th type I repeat, 9th type III repeats. We have previously shown that the NGR site of the 5th type I repeat (FN-I5) rapidly undergoes deamidation leading to formation of isoDGR. A deamidated FN fragment corresponding to this domain binds αvβ3, inhibits endothelial cell adhesion and proliferation and inhibits tumor growth in animal models. In the present study we have investigated the molecular mechanisms that regulate isoDGR formation in FN in vivo. We provide evidence to suggest that FN purified from plasma contains very low amounts of isoDGR (&amp;lt;3%) despite its long half life (48h). In contrast, using anti-isoDGR antibodies, we observed that isoDGR is formed in significant amounts in small FN fragments injected in mice and left to circulate for 4 or 6 h (&amp;gt;17% in 4 h). In vitro cell adhesion assays, antibody binding assays and integrin binding studies showed that FN deamidation can rapidly occur when FN is bound to microtiter plates, a condition known to cause conformational changes in this protein, but not with FN in solution. Rapid deamidation was observed to occur also with short fragments (FN-I5), either adsorbed on microtiter plates or in solution. These results suggest that the kinetics of NGR-to-isoDGR transition in plasma FN is slower than its clearance rate. Furthermore, the finding that conformational changes and/or protein fragmentation increase the kinetics of NGR-to-isoDGR transition suggest that changes in the molecular microenvironment of NGR sites, potentially occurring after FN deposition in tissues, could be critical for the formation of isoDGR integrin binding sites. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 426. doi:10.1158/1538-7445.AM2011-426

Guiding Epithelial Cell Phenotypes with Engineered Integrin-Specific Recombinant Fibronectin Fragments

The extracellular matrix (ECM) provides important cues for directing cell phenotype. Cells interact with underlying ECM through cell-surface receptors known as integrins, which bind to specific sequences on their ligands. During tissue development, repair, and regeneration of epithelial tissues, cells must interact with an interstitial fibronectin (Fn)-rich matrix, which has been shown to direct a more migratory/repair phenotype, presumably through interaction with Fn's cell binding domain comprised of both synergy Pro-His-Ser-Arg-Asn (PHSRN) and Arg-Gly-Asp (RGD) sequences. We hypothesized that the Fn synergy site is critical to the regulation of epithelial cell phenotype by directing integrin specificity. Epithelial cells were cultured on Fn fragments displaying stabilized synergy and RGD (FnIII9'10), or RGD alone (FnIII10) and cell phenotype analyzed by cytoskeleton changes, epithelial cell-cell contacts, changes in gene expression of epithelial and mesenchymal markers, and wound healing assay. Data indicate that epithelial cells engage RGD only with αv integrins and display a significant shift toward a mesenchymal phenotype due, in part, to enhanced transforming growth factor-β activation and/or signaling compared with cells on the synergy containing FnIII9'10. These studies demonstrate the importance of synergy in regulating epithelial cell phenotype relevant to tissue engineering as well as the utility of engineered integrin-specific ECM fragments in guiding cell phenotype.

The Streptococcal Binding Site in the Gelatin-binding Domain of Fibronectin Is Consistent with a Non-linear Arrangement of Modules

Fibronectin-binding proteins (FnBPs) of Staphylococcus aureus and Streptococcus pyogenes mediate invasion of human endothelial and epithelial cells in a process likely to aid the persistence and/or dissemination of infection. In addition to binding sites for the N-terminal domain (NTD) of fibronectin (Fn), a number of streptococcal FnBPs also contain an upstream region (UR) that is closely associated with an NTD-binding region; UR binds to the adjacent gelatin-binding domain (GBD) of Fn. Previously, UR was shown to be required for efficient streptococcal invasion of epithelial cells. Here we show, using a Streptococcus zooepidemicus FnBP, that the UR-binding site in GBD resides largely in the 8F19F1 module pair. We also show that UR inhibits binding of a peptide from the α1 chain of type I collagen to 8F19F1 and that UR binding to 8F1 is likely to occur through anti-parallel β-zipper formation. Thus, we propose that streptococcal proteins that contain adjacent NTD- and GBD-binding sites form a highly unusual extended tandem β-zipper that spans the two domains and mediates high affinity binding to Fn through a large intermolecular interface. The proximity of the UR- and NTD-binding sequences in streptococcal FnBPs is consistent with a non-linear arrangement of modules in the tertiary structure of the GBD of Fn.

Influence of primary adhesive interactions with fibronectin on clonal growth and osteogenic potential of rat mesenchymal stromal cells

Interactions with extracellular matrix including fibronectin (Fn) play an important role in regulation of cell growth and differentiation. Influence of Fn and its individual domains on adhesion and osteogenic potencies of rat mesenchymal stromal cells (MSCs) was estimated. Investigation of bone marrow or fetal liver MSCs adhesion dynamics showed that after 7 days of cultivation on Fn the number of adhered clonogenic cells derived from both sources was comparable to their number observed on plastic but their content in suspension was commonly decreased. Population of fetal liver MSCs differed from bone marrow-derived population by greater fraction of cells that adhered for the first 7 days. Bone marrow MSC cultures on Fn were characterized by reduced activity of alkaline phosphatase as compared with cultivation on plastic; furthermore, they deposed significantly smaller amount of calcium salts under cultivation in osteogenic medium. Cultivation of MSCs on Fn fragments demonstrated the primary role of its cell-binding domain in the inhibition of osteogenesis.

A Fibronectin Fragment Elicits Vasodilatation and Alters Myogenic Responsiveness of Skeletal Muscle Arterioles

Interactions between integrin‐extracellular matrix proteins are important modulators of vascular tone that are altered during vascular remodeling, wound healing, and inflammation. Degraded collagen, which contains integrin‐binding sequences Arg‐Gly‐Asp (RGD), has been shown to modulate vascular tone by altering calcium entry. In this study, we investigated the effects of a proteolytic fragment of fibronectin (FN120) on vascular tone in isolated, cannulated, and pressurized rat skeletal muscle arterioles. Abluminal incubation with FN120 (1, 10, 100 μM) for 15 minutes elicited a dose‐dependent, transient dilatation at 90 cmH2O intraluminal pressure, with 10 μM FN120 producing significant dilation (n=9, p=0.03). FN120 (10 μM) also significantly increased the steady‐state myogenic index (−0.3; control: −0.13; n=9, p=0.02), as measured over the pressure range of 60–180 cmH2O. After wash‐off and re‐application of FN120, the time to peak transient dilation at 60 cmH2O was markedly shortened (from 8 to 1.5 minutes for 10 μM FN120), compared to the first application. To determine if these effects depended on FN‐integrin interactions, arterioles were pre‐incubated with a β1 integrin function blocking antibody (26 μg/mL) prior to addition of 10 μM FN120. The β1 integrin antibody blunted the steady‐state dilation to FN120 (25% decrease; p=0.039) and attenuated the increased myogenic index (−0.3 to −0.1; n=3, p=0.03). Our data suggest that the interaction between this FN fragment and β1 integrin may be important components of vascular remodeling. Supported by NIH 072989 to MJD.

Fibronectin-binding proteins ofClostridium perfringensrecognize the III1-C fragment of fibronectin

The Clostridium perfringens strain 13 genome contains two genes (fbpA, fbpB) that encode putative Fbp. Both rFbpA and rFbpB were purified and their reactivity with human serum Fn was analyzed. To determine the region of the Fn molecule recognized by rFbp, a plate binding assay using N-terminal 70-kDa peptide, III1-C peptide, and 110-kDa peptide containing III2-10 of Fn was performed. Both rFbp bound to the III1-C peptide of Fn but not to the other peptides. However, the III1-C fragment of Fn is known to be cryptic in serum Fn. Then, rFbp-BP from Fn were purified by rFbp-affinity chromatography. The yield of purified proteins was approximately 1% of the applied Fn on a protein basis. Western blotting analysis of the rFbp-BP, using four different anti-Fn monoclonal antibodies, revealed that the rFbp-BP carried partial Fn antigenicity. Bindings of rFbp to rFbp-BP were inhibited by the presence of the III1-C peptide, suggesting that rFbp-BP express the III1-C fragment. The binding of Fn to III1-C was inhibited by the presence of either rFbpA or rFbpB. This result that suggests C. perfringens Fbps may inhibit the formation of Fn-matrix in vivo.

Fibronectin-binding proteins of Clostridium perfringens recognize the III1-C fragment of fibronectin

The Clostridium perfringens strain 13 genome contains two genes (fbpA, fbpB) that encode putative Fbp. Both rFbpA and rFbpB were purified and their reactivity with human serum Fn was analyzed. To determine the region of the Fn molecule recognized by rFbp, a plate binding assay using N-terminal 70-kDa peptide, III1-C peptide, and 110-kDa peptide containing III2–10 of Fn was performed. Both rFbp bound to the III1-C peptide of Fn but not to the other peptides. However, the III1-C fragment of Fn is known to be cryptic in serum Fn. Then, rFbp-BP from Fn were purified by rFbp-affinity chromatography. The yield of purified proteins was approximately 1% of the applied Fn on a protein basis. Western blotting analysis of the rFbp-BP, using four different anti-Fn monoclonal antibodies, revealed that the rFbp-BP carried partial Fn antigenicity. Bindings of rFbp to rFbp-BP were inhibited by the presence of the III1-C peptide, suggesting that rFbp-BP express the III1-C fragment. The binding of Fn to III1-C was inhibited by the presence of either rFbpA or rFbpB. This result that suggests C. perfringens Fbps may inhibit the formation of Fn-matrix in vivo.

Involution Is an Inflammatory Tumor Promoting Event That Increases Metastasis in Pregnancy Associated Breast Cancer.

Abstract Breast cancer diagnosed up to 6 years after a completed child-birth has an increased risk of metastatic recurrence and death, which is independent of current prognostic factors. These pregnancy-associated breast cancers (PABC) account for 45% of all young women's breast cancers and have a 10 year survival rate of 44% compared with 69% for non-PABC. After parturition or lactation, a physiologically normal tissue remodeling program is activated that mimics wound healing and inflammation, including high matrix metalloproteinase -2, -3, and -9 activities, release of bioactive fragments of FN and LN, deposition of fibrillar collagen, and increased cytokine levels. We hypothesize that the normal physiologic, but pro-inflammatory remodeling of breast during involution accounts for increased breast cancer metastasis of PABC.To demonstrate that the human breast is characterized by a pro-inflammatory microenvironment with wound-healing characteristics, formalin fixed breast biopsy tissues were obtained for women who were age-matched nulliparous, pregnant, lactating, whose breast tissue was actively involuting at the time of biopsy or who were &amp;gt; 5 years from a completed pregnancy. Using a novel imaging program, over 1500 images of human breast tissue have been quantitated for CD45 (common leukocyte antigen) and CD68 (macrophage lysosomal marker). CD45 and CD68 staining in the human breast demonstrates 4 times more staining in involuting compared to pregnant tissue. Further, CD45 and CD68 staining returns to baseline level in the fully regressed breast. Further characterization of the macrophages in involution demonstrates an M2 phenotype, similar to immune suppressive tumor associated macrophages. Likewise, increased collagen deposition is present with involution in comparison to the other tissue subgroups, which may help promote immune cell recruitment.To test for the impact of involution on invasion and metastasis of PABC, we have developed the MCF-10DCIS human cell line into a murine model for PABC. In this model, the cancer cells will form in situ ductal carcinoma like lesions that progress over time with loss of their myoepithelial barrier and subsequent invasion present. Orthotopic injection of the MCF-10DCIS cells into SCID mice that whose mammary glands were nulliparous versus actively involuting following timed weaning demonstrated an increased incidence of tumor of 78% involution group versus 50% in the nulliparous group. Tumor size was increased 4 fold and presence of invasive lesions increased 2 fold for the involution group. Moreover, increased lung metastasis that was independent of the primary tumor size was seen in the involuting mice compared with nulliparous controls, isolating the event of involution (independent of the potential potentiating effect of the pregnancy state on the tumor) as the driving feature for the metastatic increase.These data demonstrate for the first time that human mammary gland involution is characterized by a pro-inflammatory microenvironment, implicating physiologic tissue inflammation in the poor prognosis of PABC. Likewise, in a murine model for PABC, involution is associated with increased tumor invasion and metastasis. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 50.

Common wound healing signature in murine and human involuting breast, implications for pregnancy-associated breast cancer.

Abstract Abstract #1049 Full term pregnancy increases breast cancer risk for up to ten years after childbirth and breast cancer diagnosed during this time has been referred to as pregnancy associated breast cancer (PABC). PABC patients have high rates of metastasis, which is independent of known prognostic factors. Following parturition or lactation, during involution, the breast returns to its pre-pregnant state and in the rat model, we found mammary gland involution employs tissue remodeling programs activated during wound healing and inflammation including high matrix metalloproteinase -2, -3, and -9 activities, release of bioactive fragments of FN and LN, deposition of fibrillar collagen, and increased cytokine levels, including TGFβ1, TGFβ3 and CTGF. We hypothesize that this physiologically normal, but pro-inflammatory remodeling of the gland accounts for the high rate of metastases seen in PABC.&amp;#x2028; To further define involution features common to wound healing that may account for the metastasis promoting attributes of PABC, the question of whether macrophages are recruited to the involuting gland was investigated in both rat mammary and human breast tissue. Using the macrophage marker CD68, a lysosomal associated protein, the number of CD68 positive cells in rat mammary glands increased more than 6 fold during involution. Involution-associated macrophages were identified as subtype M2-like by arginase-1 expression and lack of subtype M1-marker iNOS expression. To address the question of whether breast involution in women also is characterized by a wound-healing signature, formalin fixed breast biopsy tissues were obtained for women who were either pregnant, lactating, or whose breast tissue was actively involuting at the time of biopsy. Using a novel computer assisted imaging program, over 200 images of human breast tissue have been quantitated for CD68. CD68 staining in the human breast followed the same trend as in rat, with 5 times more staining in involuting compared to pregnant or lactating tissue. Total leukocyte number, as assessed by CD45 antibody, and oncofetal ECM protein tenascin-C staining were also increased in involuting human breast tissue. With respect to these three stromal markers, involuting human breast tissue was more similar to breast cancer stroma (N=50 images) than stroma from breasts of nulliparous women (N=30 images). These data demonstrate for the first time that human mammary gland involution is characterized by a pro-inflammatory microenvironment, implicating physiologic tissue inflammation in the poor prognosis of PABC.&amp;#x2028; In a newly developed animal model for PABC, MCF10DCIS cells were injected orthotopically into SCID mice whose mammary glands were either quiescent (virgin mice) or actively remodeling following weaning (involuting mice). Tumor incidence was 78% in involuting and 50% in virgin mice. Further, tumor multiplicity, size and DCIS conversion to locally invasive lesions were significantly higher in the involuting mice than control virgin mice. This model is being utilized for mechanistic and chemopreventive studies of PABC. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1049.