Role of fibronectin in collagenous matrix-induced mesenchymal cell proliferation and differentiation in vivo

The importance of fibronectin in in vivo collagenous matrix-mesenchyme cell interaction was investigated using purified antibodies to rat plasma fibronectin. Subcutaneous implantation of demineralized bone matrix normally resulted in de novo local endochondral ossification. Local injections of the purified antibodies apparently inhibited collagenous matrix-mesenchyme cell interaction by inhibiting the action of endogenous fibronectin. Anti-fibronectin treatment resulted in reduced cell proliferation as assessed by [³H]thymidine incorporation (59%, reduction) and ornithine decarboxylase activity (66%, reduction); and chondrogenesis as measured by proteoglycan synthesis (43%, reduction). Neutralization of fibronectin's biological activity by antibodies also resulted in a qualitative change in the proteoglycan type synthesized. The physiological role of fibronectin in tissue morphogenesis appears to allow for initial extracellular matrix-cell attachment.     

Modulation of fibronectin synthesis and fibronectin binding during transformation and differentiation of mouse AKR fibroblasts

In previous studies it was shown that transformation of AKR fibroblasts with 3-methylcholanthrene was associated with a loss of surface fibronectin and that induction of differentiation of the transformed cells with N,N-dimethylformamide (DMF) was associated with reacquisition of surface fibronectin (Chakrabarty et al., J. Cell. Physiol. 133:415, 1987). It is shown in the present study that changes in surface fibronectin reflect altered fibronectin synthesis and altered fibronectin binding. Both the nontransformed cells (AKR-2B) and their transformed counterparts (AKR-MCA) bound 125I-fibronectin in a receptor-like fashion, but the AKR-MCA cells had only 20% of the receptors found on the AKR-2B cells. Whole cell extracts prepared from the AKR-2B cells and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions were examined for 125I-fibronectin binding. Under these conditions, the majority of binding occurred to moieties with molecular weights of 180 kD, 150 kD, and 97 kD. Binding to similar moieties on the AKR-MCA cells was virtually absent but occurred rapidly after treatment with DMF. The appearance of these moieties paralleled the acquisition of 125I-fibronectin binding activity by whole cells. Antibodies to the fibronectin receptor isolated from human placenta reacted with the DMF-sensitive moieties in immunoblot assays. Both the appearance of the fibronectin binding moieties and the acquisition of 125I-fibronectin binding activity by whole cells occurred within 6 hr of DMF treatment and increased over the subsequent 4 day period. The time course of these events paralleled closely the time course for induction of fibronectin biosynthesis by DMF. These changes in fibronectin binding and fibronectin production were associated with alterations in cell-substrate adhesion. The AKR-2B cells rapidly attached and spread on bovine serum albumin-coated dishes and on fibronectin-coated dishes, whereas the AKR-MCA cells were less adhesive on both substrates. Capacity to attach and spread was regained concomitantly with the induction of fibronectin binding and fibronectin production. Adhesion on both substrates was partially inhibited by antibodies to the fibronectin receptor and by RGDS. These studies suggest that fibronectin production and fibronectin binding are coregulated in AKR fibroblasts and that they function together to bring about changes in cell-substrate adhesion.     

Modulation of cell adhesion, proliferation and differentiation on materials designed for body implants

The interaction of cells and tissues with artificial materials designed for applications in biotechnologies and in medicine is governed by the physical and chemical properties of the material surface. There is optimal cell adhesion to moderately hydrophilic and positively charged substrates, due to the adsorption of cell adhesion-mediating molecules (e.g. vitronectin, fibronectin) in an advantageous geometrical conformation, which makes specific sites on these molecules (e.g. specific amino acid sequences) accessible to cell adhesion receptors (e.g. integrins). Highly hydrophilic surfaces prevent the adsorption of proteins, or these molecules are bound very weakly. On highly hydrophobic materials, however, proteins are adsorbed in rigid and denatured forms, hampering cell adhesion. The wettability of the material surface, particularly in synthetic polymers, can be effectively regulated by physical treatments, e.g. by irradiation with ions, plasma or UV light. The irradiation-activated material surface can be functionalized by various biomolecules and nanoparticles, and this further enhances its attractiveness for cells and its effectiveness in regulating cell functions. Another important factor for cell-material interaction is surface roughness and surface topography. Nanostructured substrates (i.e. substrates with irregularities smaller than 100nm), are generally considered to be beneficial for cell adhesion and growth, while microstructured substrates behave more controversially (e.g. they can hamper cell spreading and proliferation but they enhance cell differentiation, particularly in osteogenic cells). A factor which has been relatively less investigated, but which is essential for cell-material interaction, is material deformability. Highly soft and deformable substrates cannot resist the tractional forces generated by cells during cell adhesion, and cells are not able to attach, spread and survive on such materials. Local variation in the physical and chemical properties of the material surface can be advantageously used for constructing patterned surfaces. Micropatterned surfaces enable regionally selective cell adhesion and directed growth, which can be utilized in tissue engineering, in constructing microarrays and in biosensorics. Nanopatterned surfaces are an effective tool for manipulating the type, number, spacing and distribution of ligands for cell adhesion receptors on the material surface. As a consequence, these surfaces are able to control the size, shape, distribution and maturity of focal adhesion plaques on cells, and thus cell adhesion, proliferation, differentiation and other cell functions.     

Coordinating cell proliferation and differentiation

Cell proliferation and differentiation are highly coordinated processes during development. Recent studies have revealed that this coordination may result from dual functions residing in the central regulators of proliferation, allowing them to also regulate differentiation. Studies have also shown that some terminally differentiated cells can be made to divide beyond their normal capacity.     

Modulation of turkey myogenic satellite cell differentiation through the shedding of glypican-1

Glypican-1 is a cell membrane heparan sulfate proteoglycan. It is composed of a core protein with covalently attached glycosaminoglycan, and N-linked glycosylated (N-glycosylated) chains, and is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) linkage. Glypican-1 plays a key role in the growth and development of muscle by regulating fibroblast growth factor 2 (FGF2). The GPI anchor of glypican-1 can be cleaved, resulting in glypican-1 being secreted or shed into the extracellular matrix environment. The objective of the current study was to investigate the role of glypican-1 shedding and the glycosaminoglycan and N-glycosylated chains in regulating the differentiation of turkey myogenic satellite cells. A glypican-1 construct without the GPI anchor was cloned into the mammalian expression vector pCMS-EGFP, and glypican-1 without the GPI anchor and glycosaminoglycan and N-glycosylated chains were also cloned. These constructs were co-transfected into turkey myogenic satellite cells with a small interference RNA targeting the GPI anchor of endogenous glypican-1. The soluble glypican-1 mutants were not detected in the satellite cells but in the cell medium, suggesting the secretion of the soluble glypican-1 mutants. Soluble glypican-1 increased satellite cell differentiation and enhanced myotube formation in the presence of exogenous FGF2. The increase in differentiation was supported by the elevated expression of myogenin. In conclusion, the shedding of glypican-1 from the satellite cell surface acts as a positive regulator of satellite cell differentiation and sequesters FGF2, permitting further differentiation.     

Functional changes in the conformation of thrombospondin-1 during complexation with fibronectin or heparin

Thrombospondin-1 (TSP-1) interacts specifically with heparin and fibronectin in vitro and colocalizes with fibronectin and heparan sulfate in the extracellular matrix (ECM). Its conformation is strongly dependent on Ca2+ concentration. We have previously shown that both heparin and fibronectin have two binding sites on the TSP-1 subunit which may require conformational change for their occupancy (R. Dardik and J. Lahav, 1987, Eur. J. Biochem. 168, 347; ibid 1989, 185, 581). To investigate the effect of TSP-1 binding to fibronectin and heparin on its functional conformation, TSP-1 was subjected to proteolysis in the presence and absence of ligands and of Ca2+. We found that while trypsin cleavage of free TSP-1 resulted in the inactivation of ligand binding, TSP-1 bound to either fibronectin or heparin remained stably associated with these ligands. Cleavage by thrombin or tissue plasminogen activator (tPA) showed that Ca2+-depleted TSP-1, when bound to fibronectin or to heparin, yielded proteolytic cleavage patterns typical of the Ca2+-containing form. Cleavage by chymotrypsin was not affected by binding to fibronectin or heparin; hence loss of proteolytic susceptibility was not due to steric hindrance by the ligands. Taken together, these results indicate that: (A) binding of TSP-1 to fibronectin or heparin is a two-step mechanism where binding to one site leads to conformational changes that enable binding to the second site; (B) TSP-1 in complex with fibronectin or heparin adopts the Ca2+-containing conformation in the absence of Ca2+; and (C) such complexes are highly resistant to cleavage by tPA and, if cleaved by other enzymes, the TSP-1 fragments remain bound to other ECM components. These characteristics have profound significance for platelet adhesion and cell migration into wounds where Ca2+ concentrations are reduced.     

Modulation of cell surface fibronectin assembly sites by lysophosphatidic acid

Lysophosphatidic acid is a product of activated platelets and has diverse actions on cells. We have characterized the effect of lysophosphatidic acid on cell-mediated binding and assembly of fibronectin, an extracellular matrix protein. Serum made from whole blood, but neither platelet-poor plasma nor serum made from platelet- poor plasma, caused enhanced binding of fibronectin to cultured fibroblastic cells. The ability of whole blood serum to enhance binding of fibronectin was abolished by phospholipase B. These results indicate that lysophosphatidic acid derived from platelets is the principal component in whole blood serum that is active in the fibronectin binding assay. 1-oleoyl lysophosphatidic acid, 20-200 nM, was as active as 0.1-0.2% whole blood serum. The stimulatory effect of lysophosphatidic acid on the binding of fibronectin or the amino- terminal 70-kD fragment of fibronectin was rapid, sustained, and lost upon removal of lysophosphatidic acid. The stimulatory effect on binding could not be duplicated by bradykinin, platelet-activating factor, bombesin, or a peptide agonist of the thrombin receptor. Enhanced binding of the 70-kD fragment was due to increases in both the number and affinity of binding sites. Enhanced binding and assembly of fibronectin correlated with changes in cell shape and actin-containing cytoskeleton. The binding sites for fibronectin on lysophosphatidic acid-stimulated cells, as assessed by fluorescence, video, and scanning electron microscopy, were on areas of cell membrane containing numerous filopodia that extended between cells or between cells and substratum. These observations suggest that lysophosphatidic acid functions as a powerful and specific modulator of cell shape and early matrix assembly during wound healing.     

Regulation of hemopoietic cell differentiation and proliferation

Differentiation and proliferation of almost all hemopoietic cell lines can now be studied in vitro. Cloning techniques and suspension cultures allow the study of proliferation of the multipotential hemopoietic progenitor cell and the committed progenitors for granulocytes, macrophages, eosinophils, megakryocytes, and erythrocytes. The proliferation of each of the committed progenitor cells is controlled by specific glycoproteins and two of these have recently been purified: granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin. The rate of proliferation of the GM-progenitor cells and their pattern of differentiation depends on the concentration of the hormone. At low concentrations of GM-CSF (10^?11 M) fewer progenitor cells are stimulated and macrophage colonies rather than granulocyte colonies develop. The change in the direction of granulocyte-macrophage differentiation appears to be related to (a) the concentration of GM- CSF and (b) the different sensitivity of a subpopulation of monocyte colony-forming cells which are responsive to GM-CSF even at low concentrations of the regulator. Analysis of the rate of RNA synthesis by bone marrow cells has shown that GM-CSF stimulates the mature nondividing end cells of differentiation (ie, polymorphs) as well as the progenitor cells. Although GM-CSF and erythropoietin have been radiolabeled, binding studies have been hampered by the loss of biologic activity during the labeling procedure and the heterogeneity of the target cells to which the regulators bind. Surface proteins and receptors for erythrocytes have been well characterized but the relationships between these proteins and the cell surface proteins of nucleated blood cells is not well understood. It appears that some proteins are lost from the cell surface during the development of granulocytes, which are retained on the surface of the B lymphocyte. Other proteins such as chemotactic receptors and complement receptors only appear on the mature cells. External radiolabeling of the granulocyte surface using iodogen yielded a simple profile of ¹²⁵I-labeled proteins when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis.     

Heparin-dependent regulation of fibronectin matrix conformation

Extracellular matrix (ECM) conformation is regulated by a variety of stimuli in vivo, including mechanical forces and allosteric binding partners, and these conformational changes contribute to the regulation of cell behavior. Heparin and heparan sulfate, for example, have been shown to regulate the sequestration and presentation of numerous growth factors, including vascular endothelial growth factor, on the heparin 2 binding domain in fibronectin (Fn). However, mechanical force also alters Fn conformation, indicating that the growth factor binding region may be co-regulated by both heparin and mechanical force. Herein, we describe a simple antibody-based method for evaluating the conformation of the heparin 2 binding domain in Fn, and use it to determine the relative contributions of heparin and mechanical strain to the regulation of Fn conformation. We achieved specificity in quantifying conformational changes in this region of Fn by measuring the ratio of two fluorescent monoclonal antibodies, one that is insensitive to Fn conformational changes and a second whose binding is reduced or enhanced by non-equilibrium conformational changes. Importantly, this technique is shown to work on Fn adsorbed on surfaces, single Fn fibers, and Fn matrix fibers in cell culture. Using our dual antibody approach, we show that heparin and mechanical strain co-regulate Fn conformation in matrix fibrils, which is the first demonstration of heparin-dependent regulation of Fn in its physiologically-relevant fibrillar state. Furthermore, the dual antibody approach utilizes commercially available antibodies and simple immunohistochemistry, thus making it accessible to a wide range of scientists interested in Fn mechanobiology.     

Statistical conformation of human plasma fibronectin

Fibronectin is a multifunctional glycoprotein (molecular mass, M = 530 kg/mol) of the extra cellular matrix (ECM) having a major role in cell adhesion. In physiological conditions, the conformation of this protein still remains debated and controversial. Here, we present a set of results obtained by scattering experiments. In "native" conditions, the radius of gyration (R(g) = 15.3 +/- 0.3 nm) was determined by static light scattering as well as small-angle neutron scattering. The hydrodynamic radius (R(H) = 11.5 +/- 0.1 nm) was deduced from quasi-elastic light scattering measurements. These results imply a low internal concentration compared to that of usual globular proteins. This is also confirmed by the ratio R(H)/R(g) = 0. 75 +/- 0.02 consistent with a Gaussian chain, whereas R(H)/R(g) = 1. 3 for spherical shaped molecules. However, adding a denaturing agent (urea 8 M) increases R(g) by a factor 2. This means that fibronectin "native" chain is not either completely unfolded. The average shape of fibronectin conformation was also probed by small-angle neutron scattering performed for reverse scattering vector q(-)(1) smaller than R(g) (0.2 < q(-)(1) < 15 nm). The measured form factor is in complete agreement with the form factor of a random string of 56 beads of 5 nm diameter. It rules out the possibility of unfolded chain as well as globular structures. These results have structural and biological implications as far as ECM organization is concerned.     

Caspases in cell survival, proliferation and differentiation

Caspases, a family of evolutionarily, conserved cysteinyl proteases, mediate both apoptosis and inflammation through aspartate-specific cleavage of a wide number of cellular substrates. Most substrates of apoptotic caspases have been conotated with cellular dismantling, while inflammatory caspases mediate the proteolytic activation of inflammatory cytokines. Through detailed functional analysis of conditional caspase-deficient mice or derived cells, caspase biology has been extended to cellular responses such as cell differentiation, proliferation and NF-kappaB activation. Here, we discuss recent data indicating that non-apoptotic functions of caspases involve proteolysis exerted by their catalytic domains as well as non-proteolytic functions exerted by their prodomains. Homotypic oligomerization motifs in the latter mediate the recruitment of adaptors and effectors that modulate NF-kappaB activation. The non-apoptotic functions of caspases suggest that they may become activated independently of--or without--inducing an apoptotic cascade. Moreover, the existence of non-catalytic caspase-like molecules such as human caspase-12, c-FLIP and CARD-only proteins further supports the non-proteolytic functions of caspases in the regulation of cell survival, proliferation, differentiation and inflammation.     

Characterizing conformation changes in proteins through the torsional elastic response

The relationship between functional conformation changes and thermal dynamics of proteins is investigated with the help of the torsional network model (TNM), an elastic network model in torsion angle space that we recently introduced. We propose and test a null-model of “random” conformation changes that assumes that the contributions of normal modes to conformation changes are proportional to their contributions to thermal fluctuations. Deviations from this null model are generally small. When they are large and significant, they consist in conformation changes that are represented by very few low frequency normal modes and overcome small energy barriers. We interpret these features as the result of natural selection favoring the intrinsic protein dynamics consistent with functional conformation changes. These “selected” conformation changes are more frequently associated to ligand binding, and in particular phosphorylation, than to pairs of conformations with the same ligands. This deep relationship between the thermal dynamics of a protein, represented by its normal modes, and its functional dynamics can reconcile in a unique framework the two models of conformation changes, conformational selection and induced fit. The program TNM that computes torsional normal modes and analyzes conformation changes is available upon request. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.     

Peroxisome through cell differentiation and neoplasia

Summary— Peroxisomes are essential in cellular metabolism as their dysgenesis or defects in single enzymes or impairment of multiple peroxisomal enzymatic functions have been found in several inherited metabolic diseases with serious clinical sequelae. The assembly and formation of these cytoplasmic organelles constitute a major and intringuing research topic. In the present study the biogenesis of peroxisomes and the developmental patterns of their enzymes have been reviewed during embryonic and/or post-embryonic ontogenesis of lower (amphibians) and higher (avians, mammals) vertebrates. In developing vertebrates, epithelial cell differentiation is accompanied by increases in frequency and size of peroxisomes. The tissue-specific expression of peroxisomal enzymes contributes substantially to the biochemical maturation of epithelial cells. The relationship between biogenesis of peroxisomes, expression of peroxisomal enzymes and structural and functional cellular phenotype has also been investigated in differentiating epithelial cells along the crypt-villus axis of the adult rat intestine. Cytochemical studies at the ultrastructural level have provided evidence that peroxisomes are already present in proliferating cells of the intestinal crypt region before they begin to differentiate. Migration and differentiation of intestinal epithelial cells from crypt to villus compartments are marked by significant increases in number and size of catalase-positive structures. Increasing activity gradients from crypt to surface areas are found for the peroxisomal oxidases examined (enzymes of the peroxisomal β-oxidation system, d -amino acid oxidase and polyamine oxidase). Thus, peroxisomes are more and more involved in oxidative metabolic pathways as intestinal epithelial cells differentiate. Finally, we have analyzed the peroxisomal behaviour in human neoplastic epithelial cells. The presence of peroxisomes has been cytochemically revealed in human breast and colon carcinomas. Peroxisomal enzyme specific activities are significantly lower in human breast and colon carcinomas than in the adjacent healthy mucosa. Furthermore, a relationship is found between the specific activities of some peroxisomal enzymes and the histological tumour grades.     

Stromelysin generates a fibronectin fragment that inhibits Schwann cell proliferation

Our previous report (Muir, D., S. Varon, and M. Manthorpe. 1990. J. Cell Biol. 109:2663-2672) described the isolation and partial characterization of a 55-kD antiproliferative protein found in Schwann cell (SC) and schwannoma cell line-conditioned media and we concluded that SC proliferation is under negative autocrine control. In the present study the 55-kD protein was found to possess metalloprotease activity and stromelysin immunoreactivity. The SC-derived metalloprotease shares many properties with stromelysin isolated from other sources including the ability to cleave fibronectin (FN). Furthermore, limited proteolysis of FN by the SC-derived protease generated a FN fragment which itself expresses a potent antiproliferative activity for SCs. The active FN fragment corresponds to the 29-kD amino-terminal region of the FN molecule which was also identified as an active component in SC CM. Additional evidence that a proteolytic fragment of FN can possess antiproliferative activity for SCs was provided by the finding that plasmin can generate an amino-terminal FN fragment which mimicked the activity of the SC metalloprotease-generated antiproliferative FN fragment. Both the 55-kD SC metalloprotease and the 29-kD FN fragment could completely and reversibly inhibit proliferation of SCs treated with various mitogens and both were largely ineffective at inhibiting proliferation by immortalized or transformed SC lines. Normal and transformed SC types do secrete the proform of stromelysin, however, transformed cultures do not produce activated stromelysin and thus cannot generate the antiproliferative fragment of FN. These results suggest that, once activated, a SC-derived protease similar to stromelysin cleaves FN and generates an antiproliferative activity which can maintain normal SC quiescence in vitro.