The fibronectin-binding integrins alpha5beta1 and alphavbeta3 differentially modulate RhoA-GTP loading,organization of cell matrix adhesions,and fibronectin fibrillogenesis

We have studied the formation of different types of cell matrix adhesions in cells that bind to fibronectin via either alpha5beta1 or alphavbeta3. In both cases, cell adhesion to fibronectin leads to a rapid decrease in RhoA activity. However, alpha5beta1 but not alphavbeta3 supports high levels of RhoA activity at later stages of cell spreading, which are associated with a translocation of focal contacts to peripheral cell protrusions, recruitment of tensin into fibrillar adhesions, and fibronectin fibrillogenesis. Expression of an activated mutant of RhoA stimulates alphavbeta3-mediated fibrillogenesis. Despite the fact that alpha5beta1-mediated adhesion to the central cell-binding domain of fibronectin supports activation of RhoA, other regions of fibronectin are required for the development of alpha5beta1-mediated but not alphavbeta3-mediated focal contacts. Using chimeras of beta1 and beta3 subunits, we find that the extracellular domain of beta1 controls RhoA activity. By expressing both beta1 and beta3 at high levels, we show that beta1-mediated control of the levels of beta3 is important for the distribution of focal contacts. Our findings demonstrate that the pattern of fibronectin receptors expressed on a cell dictates the ability of fibronectin to stimulate RhoA-mediated organization of cell matrix adhesions.     

Collagen fibrillogenesis in vitro: comparison of types I, II, and III

The self-assembly of pepsin-extracted types I, II, and III collagen was studied to determine how differences in the triple-helical structure between collagen types influence in vitro collagen fibrillogenesis. Collagen types I, II, and III were extracted and purified from bovine sources, and were studied in solution by laser light scattering, pH titration, and determination of turbidity-time curves. The molecular weights were between 280,000 and 289,000, while the translational diffusion coefficients and particle scattering factors at 175.5 degrees were consistent with those expected for single collagen molecules. Titration of collagen types I, II, and III between pH 7.0 and 2.0 using HCl indicated that type I collagen had the most titratable carboxylic groups with type II and III having significantly fewer titratable groups. The self-assembly of these collagens was studied in vitro in phosphate-buffered saline. The time course and extent of fibril formation were studied turbidimetrically, and were found to be dependent on collagen type. Apparent rate constants were determined for both the lag and growth phases of fibril formation. The rates of both phases were greater for type III than for type I collagen, with the rates for type II collagen being intermediate. The extent of fibril formation was based on the turbidity per unit concentration (specific turbidity) extrapolated to zero concentration (intrinsic turbidity), which was found to be greater for type I than for type III collagen. Type II collagen had the smallest intrinsic turbidity. The specific and intrinsic turbidity values were consistent with the relative fibril diameters seen in dermis and cartilage by transmission electron microscopy. These observations indicate that helix-helix interactions are important in the regulation of the rate and extent of collagen fibrillogenesis and may be involved in the determination of fibril structure.     

Integrin dynamics and matrix assembly: tensin-dependent translocation of alpha(5)beta(1) integrins promotes early fibronectin fibrillogenesis

Fibronectin matrix assembly is a multistep, integrin-dependent process. To investigate the role of integrin dynamics in fibronectin fibrillogenesis, we developed an antibody-chasing technique for simultaneous tracking of two integrin populations by different antibodies. We established that whereas the vitronectin receptor alpha(v)beta(3) remains within focal contacts, the fibronectin receptor alpha(5)beta(1) translocates from focal contacts into and along extracellular matrix (ECM) contacts. This escalator-like translocation occurs relative to the focal contacts at 6.5 +/- 0.7 microm/h and is independent of cell migration. It is induced by ligation of alpha(5)beta(1) integrins and depends on interactions with a functional actin cytoskeleton and vitronectin receptor ligation. During cell spreading, translocation of ligand-occupied alpha(5)beta(1) integrins away from focal contacts and along bundles of actin filaments generates ECM contacts. Tensin is a primary cytoskeletal component of these ECM contacts, and a novel dominant-negative inhibitor of tensin blocked ECM contact formation, integrin translocation, and fibronectin fibrillogenesis without affecting focal contacts. We propose that translocating alpha(5)beta(1) integrins induce initial fibronectin fibrillogenesis by transmitting cytoskeleton-generated tension to extracellular fibronectin molecules. Blocking this integrin translocation by a variety of treatments prevents the formation of ECM contacts and fibronectin fibrillogenesis. These studies identify a localized, directional, integrin translocation mechanism for matrix assembly.     

Interdomain association in fibronectin: insight into cryptic sites and fibrillogenesis

The process by which fibronectin (FN), a soluble multidomain protein found in tissue fluids, forms insoluble fibrillar networks in the extracellular matrix is poorly understood. Cryptic sites found in FN type III domains have been hypothesized to function as nucleation points, thereby initiating fibrillogenesis. Exposure of these sites could occur upon tension-mediated mechanical rearrangement of type III domains. Here, we present the solution structures of the second type III domain of human FN ((2)FNIII), and that of an interaction complex between the first two type III domains ((1-2)FNIII). The two domains are connected through a long linker, flexible in solution. A weak but specific interdomain interaction maintains (1-2)FNIII in a closed conformation that associates weakly with the FN N-terminal 30 kDa fragment (FN30 kDa). Disruption of the interdomain interaction by amino-acid substitutions dramatically enhances association with FN30 kDa. Truncation analysis of (1-2)FNIII reveals that the interdomain linker is necessary for robust (1-2)FNIII-FN30 kDa interaction. We speculate on the importance of this interaction for FN function and present a possible mechanism by which tension could initiate fibrillogenesis.     

Collagen crosslinks: occurrence in basement membrane collagens

Basement membrane preparations from anterior lens capsule, Descemet's membrane, and renal glomerulus were reduced with NaB³H₄ in order to label carbonyl-derived crosslinks. Quantitative incorporation of tritium into the basement membranes was found, similar to the levels observed in fibrous collagens. A considerable proportion of the label was chromatographically identical with the reduced aldehydes and crosslinks of collagen, suggesting that is is the collagenous portions of basement membrane which contain these compounds. This was substantiated by showing that the isolated collagenous proteins contained substantial amounts of reduced aldehydes and crosslinks.     

Motility of fibronectin receptor-deficient cells on fibronectin and vitronectin: collaborative interactions among integrins.

Cells are capable of adhering to and migrating on protein components of the extracellular matrix. These cell-matrix interactions are thought to be mediated largely through a family of cell surface receptors termed integrins. However, the manner in which individual integrins are involved in cell adhesion and motility has not been fully determined. To explore this issue, we previously selected a series of CHO variants that are deficient in expression of the integrin alpha 5 beta 1, the "classical" fibronectin receptor. Two sets of subclones of these variants were defined which respectively express approximately 20% or 2% of fibronectin receptor on the cell surface when compared to wild-type cells (Schreiner, C. L., J. S. Bauer, Y. N. Danilov, S. Hussein, M. M. Sczekan, and R. L. Juliano. 1989. J. Cell Biol. 109:3157-3167). In the current study, the variant clones were tested for haptotactic motility on substrata coated with fibronectin or vitronectin. Data from assays using fibronectin show that cellular motility of the 20% variants was substantially decreased (30-75% of wild type), while the motility of the 2% variants was nearly abolished (2-20% of wild type). Surprisingly, a similar pattern was seen for haptotactic motility of both 2% and 20% variants when vitronectin was used (approximately 20-30% of wild type). The reduced haptotactic motility of the fibronectin receptor-deficient variant clones on vitronectin was shown not to be due to reduced vitronectin receptor (alpha v beta 3) expression nor to a failure of these variants to adhere to vitronectin substrata. Transfection of the deficient variants with a cDNA for the human alpha 5 subunit resulted in normal levels of fibronectin receptor expression (as a human alpha 5/hamster beta 1 chimera) and restored the motility of the CHO variants on fibronectin and vitronectin. This indicates that expression of the alpha 5 subunit is required for normal haptotactic motility on vitronectin substrata and suggests that the fibronectin receptor (alpha 5 beta 1) plays a cooperative role with vitronectin receptors in cell motility.     

PEGylated human plasma fibronectin is proteolytically stable,supports cell adhesion,cell migration,focal adhesion assembly,and fibronectin fibrillogenesis

Delayed wound healing in many chronic wounds has been linked to the degradation of fibronectin (FN) by abnormally high protease levels. We sought to develop a proteolytically stable and functionally active form of FN. For this purpose, we conjugated 3.35 kDa polyethylene glycol diacrylate (PEGDA) to human plasma fibronectin (HPFN). Conjugation of PEGDA to HPFN or HPFN PEGylation was characterized by an increase of approximately 16 kDa in the average molecular weight of PEGylated HPFN compared to native HPFN in SDS‐PAGE gels. PEGylated HPFN was more resistant to α chymotrypsin or neutrophil elastase digestion than native HPFN: after 30 min incubation with α chymotrypsin, 56 and 90% of native and PEGylated HPFN respectively remained intact. PEGylated HPFN and native HPFN supported NIH 3T3 mouse fibroblast adhesion and spreading, migration and focal adhesion formation in a similar manner. Fluorescence microscopy showed that both native and PEGylated HPFN in the culture media were assembled into extracellular matrix (ECM) fibrils. Interestingly, when coated on surfaces, native but not PEGylated HPFN was assembled into the ECM of fibroblasts. The proteolytically stable PEGylated HPFN developed herein could be used to replenish FN levels in the chronic wound bed and promote tissue repair. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 493–504, 2013     

Collagen fibrillogenesis in tendon development: current models and regulation of fibril assembly

Tendons are collagen-based fibrous tissues that connect and transmit forces from muscle to bone. These tissues, which are high in collagen type I content, have been studied extensively to understand collagen fibrillogenesis. Although the mechanisms have not been fully elucidated, our understanding has continued to progress. Here, we review two prevailing models of collagen fibrillogenesis and discuss the regulation of the process by candidate cellular and extracellular matrix molecules. Although numerous molecules have been implicated in the regulation of collagen fibrillogenesis, we focus on those that have been suggested to be particularly relevant to collagen type I fibril formation during tendon development, including members of the collagen and small leucine-rich proteoglycan families, as well as other molecules, including scleraxis, cartilage oligomeric matrix protein, and cytoskeletal proteins.     

Polymerization of type I and III collagens is dependent on fibronectin and enhanced by integrins alpha 11beta 1 and alpha 2beta 1

Polymerization of the ECM proteins fibronectin and laminin has been shown to take place in close vicinity to the cell surface and be facilitated by beta(1) integrins (Lohikangas, L., Gullberg, D., and Johansson, S. (2001) Exp. Cell Res. 265, 135-144 and Wennerberg, K., Lohikangas, L., Gullberg, D., Pfaff, M., Johansson, S., and Fassler, R. (1996) J. Cell Biol. 132, 227-238). We have studied the role of collagen receptors, integrins alpha(11)beta(1) and alpha(2)beta(1), and fibronectin in collagen polymerization using fibronectin-deficient mouse embryonic fibroblast cell lines. In contrast to the earlier belief that collagen polymerization occurs via self-assembly of collagen molecules we show that a preformed fibronectin matrix is essential for collagen network formation and that collagen-binding integrins strongly enhance this process. Thus, collagen deposition is regulated by the cells, both indirectly through integrin alpha(5)beta(1)-dependent polymerization of fibronectin and directly through collagen-binding integrins.     

Immunofluorescent localization of collagens,fibronectin, and laminin during terminal differentiation of odontoblasts

The localization of collagens types I, II, III, IV, laminin, and fibronectin was analyzed in mouse embryonic molars by indirect immunofluorescence. Using affinity-purified antibodies, all these antigens except collagen type II were detected in tooth germs and particularly at the epithelio-mesenchymal junction. Collagens type I, type IV, and laminin were localized at the junction before, during, and after odontoblasts terminal differentiation. The staining patterns corresponding to type III collagen and fibronectin were modified during the polarization of odontoblasts. Collagen type III present at the epithelio-mesenchymal junction could no longer be detected in this region when odontoblasts were polarized. Fibronectin, surrounding preodontoblasts, was confined to the epithelio-mesenchymal junction when odontoblasts were fully polarized. Previous studies had shown that the presence of a basement membrane and associated material was a prerequisite for the polarization of odontoblasts. Therefore, the redistribution of collagen type III and fibronectin was discussed in terms of fibronectin-collagen interactions and transmembranous control of the cytoskeleton activity in the differentiating odontoblasts.     

Mac-2 binding protein is a cell-adhesive protein of the extracellular matrix which self-assembles into ring-like structures and binds beta1 integrins, collagens and fibronectin.

Human Mac-2 binding protein (M2BP) was prepared in recombinant form from the culture medium of 293 kidney cells and consisted of a 92 kDa subunit. The protein was obtained in a native state as indicated by CD spectroscopy, demonstrating alpha-helical and beta-type structure, and by protease resistance and immunological analysis. It was highly modified by N- and O-glycosylation but not by glycosaminoglycans. Ultracentrifugation showed non-covalent association into oligomers with molar masses of 1000-1500 kDa. Electron microscopy showed ring-like shapes with diameters of 30-40 nm. M2BP bound in solid-phase assays to collagens IV, V and VI, fibronectin and nidogen, but not to fibrillar collagens I and III or other basement membrane proteins. The protein also mediated adhesion of cell lines at comparable strength with laminin. Adhesion to M2BP was inhibited by antibodies to integrin beta1 subunits but not to alpha2 and alpha6 subunits, RGD peptide or lactose. This distinguishes cell adhesion of M2BP from that of laminin and excludes involvement of lactose-binding galectin-3. Immunological assays demonstrated variable secretion by cultured human cells of M2BP, which was detected in the extracellular matrix of several mouse tissues.     

Immunohistochemical localization of collagens and fibronectin in human breast neoplasms

Forty four specimens from neoplastic, hyperplastic and normal human breast tissues were studied for localization of collagens and fibronectin. Affinity purified antihuman type I, III and IV collagens and antifibronectins were utilized by the indirect immunoperoxidase technique on fixed and paraffin-embedded sections. 86% of the cell cytoplasm of infiltrating ductal and 83% of the lobular cancers were positively stained for collagen type I and III. Collagen type IV, however, was detected in 100% of infiltrating ductal and 83% of lobular carcinomas. Focal cytoplasmic staining is a predominant feature for all antigens in the intraduct carcinoma while a diffuse pattern is encountered in the infiltrating types. Intact basement membranes in various lesions always stained for type IV collagen and showed variable staining for type III collagen and fibronectin. Epithelia of normal, benign, hyperplastic breast and most medullary carcinoma were negative for the three collagen types. Our results are in favour of the view that infiltrating breast carcinoma cells produce inappropriately the majority of collagens and inconsistently other proteins such as fibronectin.     

Nanoscale features of fibronectin fibrillogenesis depend on protein-substrate interaction and cytoskeleton structure

Cell-reorganized fibronectin layers on polymer films providing a gradation of the binding strength between protein and substrate were analyzed by combined fluorescence and scanning force microscopy. The nanoscale fibronectin patterns exhibited paired parallel fibrils with characteristic spacings of 156, 233, 304, and 373 nm. These spacings depend on the interaction of fibronectin with the substrate: at enhanced fibronectin-substrate anchorage the cells form larger stress fibers, which are assembled by alpha-actinin cross-linked pairs of actin filaments subunits at the focal adhesions. A ubiquitous repeating unit of approximately 71 nm was found within these characteristic distances. We conclude that the dimensions of the actin stress fibers reflect the binding strength of fibronectin to the polymer substrate and act--in turn--as a template for the reorganization of fibronectin into surface-bound nanofibrils with characteristic spacings. This explanation was confirmed by data showing the alpha-actinin/fibronectin colocalization.     

Molecular interactions between fibronectin and integrins during mouse blastocyst outgrowth

To investigate the mechanism of trophoblast adhesion to fibronectin, we cultured blastocysts in serum-free medium on proteolytic fibronectin fragments containing its major functional domains, and localized fibronectin-binding integrins in outgrowing trophoblast cells by immunofluorescent staining. Outgrowth comparable to that obtained with intact fibronectin was observed using a 120 kD chymotryptic fragment containing the central cell-binding domain (FN-120) and the Arg-Gly-Asp (RGD) recognition sequence. A 40 kD COOH-terminal chymotryptic fragment of fibronectin containing both a heparin-binding region and an alternate (non-RGD) cell-binding site was inactive in supporting trophoblast adhesion. Three synthetic peptides derived from the heparin-binding domain, including the CS1 alternate cell-binding site, were also unable to promote trophoblast cell adhesion. A 75 kD recombinant protein, ProNectin F, containing 13 copies of the cell recognition epitope of fibronectin, Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser, vigorously supported blastocyst outgrowth. Blastocyst outgrowth was not significantly different when surfaces were precoated with cellular fibronectin, which contains an alternatively spliced type III repeat and is the form actually encountered in vivo. Several putative fibronectin receptors were localized in trophoblast outgrowths by immunofluorescent labeling. Antibodies reactive with integrin subunits α3, α5, αllb, αv, β1 and β3, but not α4, all bound to trophoblast cells. Antibodies raised against either the β1 or β3 integrin subunits significantly inhibited fibronectin-mediated outgrowth. These findings demonstrate the key role of the central cell-binding domain of fibronectin in trophoblast adhesion, and suggest four RGD-binding integrins, α3β1, α5β1, αllbβ3, and αvβ3, that could mediate trophoblast adhesion in vitro and may play an important role during implantation. © 1995 Wiley-Liss, Inc.     

Changes in the gene expression of collagens, fibronectin, integrin and proteoglycans during matrix-induced bone morphogenesis

Subcutaneous implantation of demineralized bone matrix in rat results in the local cartilage and bone development. This in vivo model of bone formation was used to examine the expression patterns of cartilage and bone specific extracellular matrix genes. The steady state levels of mRNA in implants for cartilage specific type II collagen, type IX collagen, proteoglycan link protein and cartilage proteoglycan core protein (aggrecan) were increased during chondrogenesis and cartilage hypertrophy. Fibronectin mRNA levels were high during mesenchymal cell migration, attachment and chondrogenesis. Integrin (beta 1 chain) mRNA was expressed throughout the endochondral bone development. Type I collagen mRNA levels in implants increased as early as day 3, reached its peak during osteogenesis. These gene markers will be useful in the study of the mechanism of action of bone morphogenetic proteins present in the demineralized bone matrix.     

Adhesive surface proteins of Erysipelothrix rhusiopathiae bind to polystyrene,fibronectin, and type I and IV collagens

Erysipelothrix rhusiopathiae is a gram-positive bacterium that causes erysipelas in animals and erysipeloid in humans. We found two adhesive surface proteins of E. rhusiopathiae and determined the nucleotide sequences of the genes, which were colocalized and designated rspA and rspB. The two genes were present in all of the serovars of E. rhusiopathiae strains examined. The deduced RspA and RspB proteins contain the C-terminal anchoring motif, LPXTG, which is preceded by repeats of consensus amino acid sequences. The consensus sequences are composed of 78 to 92 amino acids and repeat 16 and 3 times in RspA and RspB, respectively. Adhesive surface proteins of other gram-positive bacteria, including Listeria monocytogenes adhesin-like protein, Streptococcus pyogenes protein F2 and F2-like protein, Streptococcus dysgalactiae FnBB, and Staphylococcus aureus Cna, share the same consensus repeats. Furthermore, the N-terminal regions of RspA and RspB showed characteristics of the collagen-binding domain that was described for Cna. RspA and RspB were expressed in Escherichia coli as histidine-tagged fusion proteins and purified. The recombinant proteins showed a high degree of capacity to bind to polystyrene and inhibited the binding of E. rhusiopathiae onto the abiotic surface in a dose dependent manner. In a solid-phase binding assay, both of the recombinant proteins bound to fibronectin, type I and IV collagens, indicating broad spectrum of their binding ability. It was suggested that both RspA and RspB were exposed on the cell surface of E. rhusiopathiae, as were the bacterial cells agglutinated by the anti-RspA immunoglobulin G (IgG) and anti-RspB IgG. RspA and RspB were present both in surface-antigen extracts and the culture supernatants of E. rhusiopathiae Fujisawa-SmR (serovar 1a) and SE-9 (serovar 2). The recombinant RspA, but not RspB, elicited protection in mice against experimental challenge. These results suggest that RspA and RspB participate in initiation of biofilm formation through their binding abilities to abiotic and biotic surfaces.     

Avian vasculogenesis and the distribution of collagens I, IV, laminin, and fibronectin in the heart primordia

The heart-forming regions of the early embryo are composed of splanchnic mesoderm, endoderm, and the associated ECM. The ECM of the heart-forming regions in stage 7-9 chicken embryos was examined using immunofluorescence. Affinity purified antibodies to chicken collagens type I and IV, chicken fibronectin, and mouse laminin were used as probes. We report that (1) the basement membrane of the endoderm contains immunoreactive laminin and collagen IV; (2) the nascent basement membrane of the heart splanchnic mesoderm contains immunoreactive laminin, but not type IV collagen, and (3) the prominent ECM between the splanchnic mesoderm and the endoderm (the primitive-heart ECM) contains collagen IV, collagen I, fibronectin, but not laminin. In addition, we describe microscopic observations on the spatial relationship of cardiogenic cells to the primitive-heart ECM and the endodermal basement membrane.