Spatial organization of extracellular matrix and fibroblast activity: Effects of serum, transforming growth factor β, and fibronectin

The goal of our research is to understand reciprocal relationships between cell function and tissue organization. We studied the regulation of fibroblast activity in an in vitro culture model that recapitulates in continuous fashion the cycle of events occurring during connective tissue repair. We present evidence that concomitant with spatial reorganization of the extracellular matrix, there was a dramatic decline in extracellular matrix synthesis and cell proliferation. Therefore, spatial reorganization was a crucial turning point for fibroblast activity. Factors that regulated the timing of spatial reorganization included serum, transforming growth factor β, and fibronectin. By accelerating spatial reorganization of the cultures, transforming growth factor β led to a relative decrease in cell proliferation and extracellular matrix synthesis. By retarding spatial reorganization of the cultures, fibronectin led to a relative increase in cell proliferation and extracellular matrix synthesis. The results indicate that spatial information in the three-dimensional cell—matrix interaction permits higher order, tissue-level regulation of fibroblast function.     

Extracellular matrix organization modulates fibroblast growth and growth factor responsiveness

To learn more about the relationship between extracellular matrix organization, cell shape, and cell growth control, we studied DNA synthesis by fibroblasts in collagen gels that were either attached to culture dishes or floating in culture medium during gel contraction. After 4 days of contraction, the collagen density (initially 1.5 mg/ml) reached 22 mg/ml in attached gels and 55 mg/ml in floating gels. After contraction, attached collagen gels were well organized; collagen fibrils were aligned in the plane of cell spreading; and fibroblasts had an elongated, bipolar morphology. Floating collagen gels, however, were unorganized; collagen fibrils were arranged randomly; and fibroblasts had a stellate morphology. DNA synthesis by fibroblasts in contracted collagen gels was suppressed if the gels were floating in medium but not if the gels were attached, and inhibition was independent of the extent of gel contraction. Therefore, growth of fibroblasts in contracted collagen gels could be regulated by differences in extracellular matrix organization and cell shape independently of extracellular matrix density. We also compared the responses of fibroblasts in contracted collagen gels and monolayer culture to peptide growth factors including fibroblast growth factor, platelet-derived growth factor, transforming growth factor-beta, and interleukin 1. Cells in floating collagen gels were generally unresponsive to any of the growth factors. Cells in attached collagen gels and monolayer culture were affected similarly by fibroblast growth factor but not by the others. Our results indicate that extracellular matrix organization influenced not only cell growth, but also fibroblast responsiveness to peptide growth factors.     

Prodomains of transforming growth factor beta (TGFbeta) superfamily members specify different functions: extracellular matrix interactions and growth factor bioavailability

The specific functions of the prodomains of TGFβ superfamily members are largely unknown. Interactions are known between prodomains of TGFβ-1-3 and latent TGFβ-binding proteins and between prodomains of BMP-2, -4, -7, and -10 and GDF-5 and fibrillins, raising the possibility that latent TGFβ-binding proteins and fibrillins may mediate interactions with all other prodomains of this superfamily. This possibility is tested in this study. Results show that the prodomain of BMP-5 interacts with the N-terminal regions of fibrillin-1 and -2 in a site similar to the binding sites for other bone morphogenetic proteins. However, in contrast, the prodomain of GDF-8 (myostatin) interacts with the glycosaminoglycan side chains of perlecan. The binding site for the GDF-8 prodomain is likely the heparan sulfate chain present on perlecan domain V. These results support and extend the emerging concept that TGFβ superfamily prodomains target their growth factor dimers to extracellular matrix macromolecules. In addition, biochemical studies of prodomain·growth factor complexes were performed to identify inactive complexes. For some members of the superfamily, the prodomain is noncovalently associated with its growth factor dimer in an inactive complex; for others, the prodomain·growth factor complex is active, even though the prodomain is noncovalently associated with its growth factor dimer. Results show that the BMP-10 prodomain, in contrast to BMP-4, -5, and -7 prodomains, can inhibit the bioactivity of the BMP-10 growth factor and suggest that the BMP-10 complex is like TGFβ and GDF-8 complexes, which can be activated by cleavage of the associated prodomain.     

The opposing effects of basic fibroblast growth factor and transforming growth factor beta on the regulation of plasminogen activator activity in capillary endothelial cells

Basic fibroblast growth factor (bFGF), a potent inducer of angiogenesis in vivo, stimulates the production of both urokinase- and tissue-type plasminogen activators (PAs) in cultured bovine capillary endothelial cells. The observed increase in proteolytic activity induced by bFGF was effectively diminished by picogram amounts of transforming growth factor beta (TGF beta), but could not be abolished by increasing the amount of TGF beta. However, the inhibition by TGF beta was greatly enhanced if the cells were pretreated with TGF beta before addition of bFGF. After prolonged incubation of cultures treated simultaneously with bFGF and TGF beta, the inhibitory effect of TGF beta diminished and the stimulatory effect of the added bFGF dominated as assayed by PA levels. TGF beta did not alter the receptor binding of labeled bFGF, nor did a 6-h pretreatment with TGF beta reduce the amount of bFGF bound. The major difference between the effects of bFGF and TGF beta was that while bFGF effectively enhanced PA activity expressed by the cells, TGF beta decreased the amounts of both cell-associated and secreted PA activity by decreasing enzyme production. Both bFGF and TGF beta increased the secretion of the endothelial-type plasminogen activator inhibitor.     

Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix

We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol. Chem. 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization. Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium. Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular collagen and fibronectin fibril deposition. Matrix fibronectin and collagen deposition remained decreased in cultures containing anti-60k Fab' until cells became bilayered or more dense, when fibronectin and collagen began to appear in lower cell layers. Anti-60k Fab' added to confluent cultures 24 h before fixation and staining had no effect on matrix fibronectin or collagen, so anti- 60k Fab' did not simply block immunostaining. Confluent cultures grown in anti-60k Fab' and labeled for 24 h with [3H]proline incorporated identical amounts of [3H]proline and [3H]hydroxyproline, but [3H]hydroxyproline deposition in the cell layer was significantly decreased by anti-60k Fab' (P less than 0.01). Extracellular matrix collagen does not appear to form a scaffold for fibronectin deposition, as neither gelatin nor a gelatin-binding fragment of plasma fibronectin inhibited deposition of matrix fibronectin. Our results suggest that interstitial collagens and fibronectin interact to form a fibrillar component of the extracellular matrix, and that fibronectin is required for normal collagen organization and deposition by fibroblasts in vitro. Domain-specific antibodies to fibronectin are powerful tools to study the biological role of fibronectin in extracellular matrix organization and other processes.     

Synergistic interactions between transforming growth factor beta and fibroblast growth factor regulate Schwann cell mitosis

Cultured Schwann cells divide in response to a limited repertoire of mitogens. In addition to cyclic AMP analogs and reagents that raise intracellular cyclic AMP, the only purified mitogens for Schwann cells are transforming growth factor beta (TGFβ), acidic (a) and basic (b) fibroblast growth factor (FGF), and the BB and AB dimers of platelet-derived growth factor (PDGF). Although individually each one of these growth factors is only weakly mitogenic, it is shown here that when TGFβ and bFGF are added to Schwann cell cultures together, they interact to produce a mitogenic response that is much greater than that produced by either growth factor alone. Both the absolute concentration of each protein and the molar ratio of TGFβ to bFGF determines the magnitude of the Schwann cell response.     

Regulation of extracellular matrix proteins by transforming growth factor beta1 in cultured pulmonary endothelial cells

Transforming growth factor beta-1 (TGF-beta1), which is present in lung tissue, has been suggested to play a role in modulating vascular cell function in vivo. The action of TGF-beta1 in vivo, especially at the local site of application to connective tissue, is anabolic and leads to pulmonary fibrosis and angiogenesis, strongly indicating that TGF-beta may have practical applications in repair of tissue injury caused by burns, trauma, or surgery. In the present study, we have used cultured bovine pulmonary artery endothelial (BPAE) cells as a model system. Expression of various proteins, including SPARC (secreted protein acidic and rich in cysteines), type IV procollagen and fibronectin (FN) was examined by radiolabeling the cells with [3H]proline, immunoprecipitation with specific antibodies, and Northern blot analyses by using specific cDNA probes. Cultured cells were labeled with [3H]proline for 24 h in either the absence or in the presence of TGF-beta1 (0-20 ng/ml). Incorporation of radioactivity was observed in a concentration-dependent manner, maximal at 5 ng/ml. Northern blot hybridization demonstrated that TGF-beta1 (5 ng/ml) treatment of BPAE cells caused an increase in steady-state levels     

Basic fibroblast growth factor-induced activation of latent transforming growth factor beta in endothelial cells: regulation of plasminogen activator activity

Plasmodium falciparum malaria parasites invade human erythrocytes by means of a parasite receptor for erythrocytes, the 175-kD erythrocyte binding antigen (EBA-175). Similar to invasion efficiency, binding requires N-acetylneuraminic acid (Neu5Ac) on human erythrocytes, specifically the glycophorins. EBA-175 bound to erythrocytes with receptor-like specificity and was saturable. The specificity of EBA-175 binding was studied to determine if its binding is influenced either by simple electrostatic interaction with the negatively charged Neu5Ac (on the erythrocyte surface); or if Neu5Ac indirectly affected the conformation of an unknown ligand, or if Neu5Ac itself in specific linkage and carbohydrate composition was the primary ligand for EBA-175 as demonstrated for hemagglutinins of influenza viruses. Most Neu5Ac on human erythrocytes is linked to galactose by alpha 2-3 and alpha 2-6 linkages on glycophorin A. Soluble Neu5Ac by itself in solution did not competitively inhibit the binding of EBA-175 to erythrocytes, suggesting that linkage to an underlying sugar is required for binding in contrast to charge alone. Binding was competitively inhibited only by Neu5Ac(alpha 2-3)Gal-containing oligosaccharides. Similar oligosaccharides containing Neu5Ac(alpha 2-6)Gal-linkages had only slight inhibitory effects. Binding inhibition assays with modified sialic acids and other saccharides confirmed that oligosaccharide composition and linkage were primary factors for efficient binding. EBA-175 bound tightly enough to glycophorin A that the complex could be precipitated with an anti-glycophorin A monoclonal antibody. Selective cleavage of O-linked tetrasaccharides clustered at the NH2 terminus of glycophorin A markedly reduced binding in inhibition studies. We conclude that the Neu5Ac(a2,3)-Gal- determinant on O-linked tetrasaccharides of glycophorin A appear to be the preferential erythrocyte ligand for EBA-175.     

Synergistic interactions between transforming growth factor beta and fibroblast growth factor regulate Schwann cell mitosis.

Cultured Schwann cells divide in response to a limited repertoire of mitogens. In addition to cyclic AMP analogs and reagents that raise intracellular cyclic AMP, the only purified mitogens for Schwann cells are transforming growth factor beta (TGF beta), acidic (a) and basic (b) fibroblast growth factor (FGF), and the BB and AB dimers of platelet-derived growth factor (PDGF). Although individually each one of these growth factors is only weakly mitogenic, it is shown here that when TGF beta and bFGF are added to Schwann cell cultures together, they interact to produce a mitogenic response that is much greater than that produced by either growth factor alone. Both the absolute concentration of each protein and the molar ratio of TGF beta to bFGF determines the magnitude of the Schwann cell response.     

Dynamics-modulated biological activity of transforming growth factor beta3

Transforming growth factor beta3 (TGF-beta3) is an important mediator of growth, maintenance, and repair processes in human cells. Internal dynamic properties have been derived from (15)N NMR relaxation data and mapped onto the spatial structure of TGF-beta3. The pattern of internal dynamics in the structure identifies potential "hot spots" of binding free energy and reveals the importance of conformational entropy in the interaction of TGF-beta3 with the receptors. The observed internal dynamics set TGF-beta3 apart from other TGF-beta isoforms, with which it shares the same fold. These findings may explain functional differences among the various TGF-beta isoforms and thus prove essential in the search for related therapeutic agents.     

Ultrastructural immunolocalization of basic fibroblast growth factor in fibroblasts and extracellular matrix

Basic fibroblast growth factor (bFGF) is thought to play an important role in normal tissue repair and wound healing. It is a potent mitogenic and chemotactic factor for fibroblasts, regulating proliferation and extracellular matrix (ECM) production by these cells. In this study, we present morphologic evidence of the ultrastructural location of bFGF in fibroblasts and ECM using several antibodies, tissues, and species. Distinct labeling is seen in the nuclei of fibroblasts and some labeling in the cytosol. Immunolabeling of the cytosol excludes organelles involved in the usual secretory pathway, such as rough endoplasmic reticulum, Golgi apparatus, and secretory vacuoles. The same labeling is observed with either polyclonal or monoclonal antibodies. We suggest that bFGF functions as a nuclear protein in fibroblasts and is not secreted by a normal secretory pathway. Fibroblasts may export bFGF via unique cellular pathways that are clearly distinct from classic signal peptide mediated secretion. This may provide a source for ECM-resident bFGF. The same antibodies show different labeling intensity in the ECM. This protein, through integration into the ECM, may act as a local regulator and promote regeneration of these tissues after wounding. Direct evidence is the dramatic reduction of bFGF labeling in axotomized rat ECM collagen fibers versus control animals. Accepted: 20 December 1999     

The transforming growth factor-beta-inducible matrix protein (beta)ig-h3 interacts with fibronectin

Proper growth and development require the orderly synthesis and deposition of individual components of the extracellular matrix (ECM) into well ordered networks. Once formed, the ECM maintains tissue structure and houses resident cells. One ECM component, (beta)ig-h3, is a highly conserved transforming growth factor-beta-inducible protein that has been hypothesized to function as a bifunctional linker between individual matrix components and resident cells. To gain insights into its physiological function, full-length (beta)ig-h3 protein was produced using a baculovirus expression system and purified under native conditions. Human fibroblasts attached and spread on (beta)ig-h3-coated plates and developed actin stress fibers. Purified (beta)ig-h3 binds fibronectin (FN) and type I collagen (Col I) but does not bind gelatin. Using defined fragments of FN, we localized the (beta)ig-h3 recognition region to the gelatin/collagen binding domain present in the N-terminal region of the FN molecule. Our results identify FN and Col I as two ligands of (beta)ig-h3 in the ECM.     

Heparanase activity expressed by platelets, neutrophils, and lymphoma cells releases active fibroblast growth factor from extracellular matrix.

Incubation of platelets, neutrophils, and lymphoma cells with Descemet's membranes of bovine corneas and with the extracellular matrix (ECM) produced by cultured corneal endothelial cells resulted in release of basic fibroblast growth factor (bFGF), which stimulated the proliferation of 3T3 fibroblasts and vascular endothelial cells. Similar requirements were observed for release of endogenous bFGF stored in Descemet's membrane and of exogenous bFGF sequestered by the subendothelial ECM. Release of ECM-resident bFGF by platelets, neutrophils, and lymphoma cells was inhibited by carrageenan lambda, but not by protease inhibitors, in correlation with the inhibition of heparanase activity expressed by these cells. Degradation of the ECM-heparan sulfate side chains by this endo-beta-D-glucuronidase is thought to play an important role in cell invasion, particularly in the extravasation of blood-borne tumor cells and activated cells of the immune system. We propose that both heparanase and ECM-resident bFGF may modulate the cell response to contact with its local environment. Heparanase-mediated release of active bFGF from storage in basement membranes provides a novel mechanism for a localized induction of neovascularization in various normal and pathological processes, such as wound healing, inflammation, and tumor development.     

Synergistic activity of fibronectin and fibroblast growth factor receptors on neuronal adhesion and neurite extension through extracellular signal-regulated kinase pathway

Fibroblast growth factor (FGF) is an important modulator of cell growth and differentiation of various cells including neuron. Cells need to adhere specifically to cellular and extracellular components of their environment to carry out diverse physiological functions. Here, we examined whether fibronectin (FN) and FGF can cooperate for neuronal adhesion and neurite outgrowth. Using recombinant FN peptide (FNIII9-10), we found that FNIII9-10-mediated adhesion promotes the effect of FGF1 on neurite outgrowth of PC12 cells, while FGF1 enhances the FNIII9-10-mediated neuronal adhesion of PC12 cells. This collaboration of FNIII9-10 and FGF1 was the result of the sustained activation of extracellular signal-regulated kinase (ERK)-type MAP kinase. Finally, the synergistic activity of FGF1 and FN was inhibited by PD98059, an MEK inhibitor. Taken together, these findings indicate that FN-mediated signaling can collaborate with FGFRs signaling for neurite outgrowth through selective activation of ERK-type MAP kinase in PC12 cells, and suggest that FN and FGF act in concert to regulate cell differentiation in the nervous system.     

Biophysical integration of effects of epidermal growth factor and fibronectin on fibroblast migration

Cell migration is regulated simultaneously by growth factors and extracellular matrix molecules. Although information is continually increasing regarding the relevant signaling pathways, there exists little understanding concerning how these pathways integrate to produce the biophysical processes that govern locomotion. Herein, we report the effects of epidermal growth factor (EGF) and fibronectin (Fn) on multiple facets of fibroblast motility: locomotion speed, membrane extension and retraction activity, and adhesion. A surprising finding is that EGF can either decrease or increase locomotion speed depending on the surface Fn concentration, despite EGF diminishing global cell adhesion at all Fn concentrations. At the same time, the effect of EGF on membrane activity varies from negative to positive to no-effect as Fn concentration and adhesion range from low to high. Taking these effects together, we find that EGF and Fn regulate fibroblast migration speed through integration of the processes of membrane extension, attachment, and detachment, with each of these processes being rate-limiting for locomotion in sequential regimes of increasing adhesivity. Thus, distinct biophysical processes are shown to integrate for overall cell migration responses to growth factor and extracellular matrix stimuli.     

Influence of fibroblast growth factor, insulin-like growth factor I, and transforming growth factor beta on satellite cell type I collagen expression and localization during differentiation

Expression, and temporal and spatial distribution of type I collagen were investigated in chicken satellite cell cultures during differentiation. There was no difference in the relative amounts of type I collagen after treatment with basic fibroblast growth factor (FGF), insulin-like growth factor-I (IGF-I), or transforming growth factor beta 1 (TGF-beta 1). However, myotube morphology was influenced by the presence of the growth factors. The temporal and spatial distribution of type I collagen was also modified. Control cultures maintained a predominant distribution of type I collagen surrounding the cellular area until approximately 48 h after the initiation of fusion whereas cultures with FGF or IGF-I maintained a cellular localization of type I collagen throughout the fusion process. TGF-beta 1 resulted in the early formation of an extracellular network of type I collagen preceding control cultures by approximately 24 h. These results suggest that type I collagen expression but not localization is independent of satellite cell proliferation and differentiation.     

Protamine sulfate inhibits mitogenic activities of the extracellular matrix and fibroblast growth factor, but potentiates that of epidermal growth factor

Protamine sulfate, an inhibitor of angiogenesis in vivo, markedly inhibits the ability of angiogenic factors such as acidic or basic fibroblast growth factor (aFGF, bFGF) to stimulate the proliferation in vitro of either BHK-21 cells or vascular endothelial cells. The inhibition is reversible, and cells remain viable even after prolonged exposure to protamine sulfate. Protamine sulfate inhibits the mitogenic effects of both growth factors by preventing them from binding to their common cell surface receptors. It also inhibits the mitogenic activity of the extracellular matrix produced by bovine corneal endothelial cells. This substrate has been shown in previous studies to replace the requirement for FGF of many cell types. In contrast, protamine sulfate potentiates the mitogenic activity of epidermal growth factor (EGF). This indicates that protamine sulfate also acts at cellular sites which are not associated with FGF receptors.     

Latent transforming growth factor-beta 1 associates to fibroblast extracellular matrix via latent TGF-beta binding protein

The role of latent transforming growth factor-beta (TGF-beta) binding protein (LTBP) in the association of TGF-beta 1 to the extracellular matrix of cultured fibroblasts and HT-1080 fibrosarcoma cells was studied by immunochemical methods. The matrices were isolated from the cells, and the levels of LTBP and TGF-beta 1 were estimated by immunoblotting and immunoprecipitation. LTBP, TGF-beta 1, and its propeptide (latency-associated peptide, LAP) were found to associate to the extracellular matrix. Immunoblotting analysis indicated that treatment of the cells with plasmin resulted in a concomitant time and dose dependent release of both LTBP and TGF-beta 1 from the extracellular matrix to the supernatant. Comparison of molecular weights suggested that plasmin treatment resulted in the cleavage of LTBP from the high molecular weight fibroblast form to a form resembling the low molecular weight LTBP found in platelets. Pulse- chase and immunoprecipitation analysis indicated that both the free form of LTBP and LTBP complexed to latent TGF-beta were efficiently incorporated in the extracellular matrix, from where both complexes were slowly released to the culture medium. Addition of plasmin to the chase solution resulted, however, in a rapid release of LTBP from the matrix. Fibroblast derived LTBP was found to associate to the matrix of HT-1080 cells in a plasmin sensitive manner as shown by immunoprecipitation analysis. These results suggest that the latent form of TGF-beta 1 associates with the extracellular matrix via LTBP, and that the release of latent TGF-beta 1 from the matrix is a consequence of proteolytic cleavage(s) of LTBP.     

Transforming growth factor beta 1 modulates extracellular matrix organization and cell-cell junctional complex formation during in vitro angiogenesis.

Transforming growth factor-beta 1 (TGF-beta 1) is angiogenic in vivo. In two-dimensional (2-D) culture systems microvascular endothelial cell proliferation is inhibited up to 80% by TGF-beta 1; however, in three-dimensional (3-D) collagen gels TGF-beta 1 is found to have no effect on proliferation while eliciting the formation of calcium and magnesium dependent tube-like structures mimicking angiogenesis. DNA analyses performed on 3-D cell cultures reveal no significant difference in the amount of DNA or cell number in control versus TGF-beta 1 treated cultures. In 2-D cultures TGF-beta 1 is known to increase cellular fibronectin accumulation; however, in 3-D cultures no difference is seen between control and TGF-beta 1 treated cells as established by ELISA testing for type IV collagen, fibronectin, and laminin. In 3-D cultures there is increased synthesis and secretion of type V collagen in both control and TGF-beta 1 treated cultures over 2-D cultures. Even though an equal amount of type V collagen is seen in both 3-D conditions, there is a reorganization of the protein with concentration along an organizing basal lamina in TGF-beta 1 treated cultures. EM morphological analyses on 3-D cultures illustrate quiescent, control cells lacking cell contacts. In contrast, TGF-beta 1 treated cells show increased pseudopod formation, cell-cell contact, and organized basal lamina-like material closely apposed to the "abluminal" plasma membranes. TGF-beta 1 treated cells also appear to form junctional complexes between adjoining cells. Immunofluorescence using specific antibodies to the tight junction protein ZO-1 results in staining at apparent cell-cell junctions in the 3-D cultures. Northern blots of freshly isolated microvascular endothelium, 2-D and 3-D cultures, using cDNA and cRNA probes specific for the ZO-1 tight junction protein, reveal the presence of the 7.8 kb mRNA. Western blots of rat epididymal fat pad endothelial cells (RFC) monolayer lysates probed with anti-ZO-1 label a 220 kd band which co-migrates with the bonafide ZO-1 protein. These data confirm and support the hypothesis that TGF-beta 1 is angiogenic in vitro, eliciting microvascular endothelial cells to form tube-like structures with apparent tight junctions and abluminal basal lamina deposition in three-dimensional cultures.