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.     

Transforming growth factor beta increases cell surface binding and assembly of exogenous (plasma) fibronectin by normal human fibroblasts.

Transforming growth factor beta (TGF-beta) enhances the cell surface binding of 125I-fibronectin by cultured human fibroblasts. The effect of TGF-beta on cell surface binding was maximal after 2 h of exposure to TFG-beta and did not require epidermal growth factor or protein synthesis. The enhancement was dose dependent and was found with the 125I-labeled 70-kilodalton amino-terminal fragment of fibronectin as well as with 125I-fibronectin. Treatment of cultures with TGF-beta for 6 h resulted in a threefold increase in the estimated number of fibronectin binding sites. The increase in number of binding sites was accompanied by an increased accumulation of labeled fibronectin in detergent-insoluble extracellular matrix. The effect of TGF-beta was biphasic; after 6 h of exposure, less labeled fibronectin bound to treated cultures than to control cultures. Exposure of cells to TGF-beta for greater than 6 h caused a two- to threefold increase in the accumulation of cellular fibronectin in culture medium as detected by a quantitative enzyme-linked immunosorbent assay. The second phase of the biphasic effect and the increase in soluble cellular fibronectin were blocked by cycloheximide. Immunofluorescence staining of fibroblast cultures with antifibronectin revealed that TGF-beta caused a striking increase in fibronectin fibrils. The 70-kilodalton amino-terminal fragment of fibronectin, which blocks incorporation of fibronectin into extracellular matrix, blocked anchorage-independent growth of NRK-49F cells in the presence of epidermal growth factor. Our results show that an increase in the binding and rate of assembly of exogenous fibronectin is an early event preceding the increase in expression of extracellular matrix proteins. Such an early increase in cell surface binding of exogenous fibronectin may be a mechanism whereby TGF-beta can modify extracellular matrix characteristics rapidly after tissue injury or during embryonic morphogenesis.     

Transforming growth factor beta and basic fibroblast growth factor synergistically promote early bovine embryo development during the fourth cell cycle.

Developmentally competent bovine blastocysts were produced by adding transforming growth factor beta (TGF beta) and basic fibroblast growth factor (bFGF) to serum-free cultures of in vitro produced, 2-cell bovine embryos. The effects of TGF beta were evaluated because this growth factor signals synthesis and secretion of the extracellular matrix component fibronectin and its receptor. Previous investigations have demonstrated that fibronectin promotes early bovine embryo development in vitro. The effects of TGF beta can be potentiated by bFGF; bFGF itself is an effector of protein synthesis and a potent mitogen. A positive interaction between the 2 growth factors resulted in 38.8% of fertilized oocytes maturing beyond the 16-cell stage; of these, 24.6% formed blastocysts. Transfer of early blastocysts produced using serum-free medium supplemented with growth factors resulted in pregnancy in 3 of 9 recipients. These results support the hypothesis that TGF beta and bFGF act synergistically to promote development of bovine embryos beyond the "8-cell block" observed in vitro.     

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.     

Transforming growth factor beta type 1 binds to collagen IV of basement membrane matrix: implications for development

The interaction of transforming growth factor beta (TGF beta) with extracellular matrix macromolecules was examined by using radiolabeled TGF beta and various matrix macromolecules immobilized on nitrocellulose. TGF beta bound to collagen IV with greater affinity than to other extracellular matrix macromolecules tested. Neither laminin nor fibronectin, both of which bind type IV collagen, interfered with the binding of TGF beta to type IV collagen. TGF beta 2 competed effectively with TGF beta 1 for binding to type IV collagen. The biological effect of TGF beta was tested by an assay based on inhibition of proliferation of an osteoblast cell line, MC3T3-E1. The results demonstrated that the effect of TGF beta 1 was sustained when cells were grown on type IV collagen compared to cells grown on laminin, collagen type I, and plastic. These results demonstrate that extracellular matrix components may function as an affinity matrix for binding and immobilizing soluble growth and differentiation factors. In view of the demonstrated role of basement membranes in development the present results imply an important function for transforming growth factor beta bound to collagen IV in local regulation of cell proliferation and differentiation.     

Mammary epithelial-mesenchymal interaction regulates fibronectin alternative splicing via phosphatidylinositol 3-kinase

The way alternative splicing is regulated within tissues is not understood. A relevant model of this process is provided by fibronectin, an important extracellular matrix protein that plays a key role in cell adhesion and migration and contains three alternatively spliced regions known as EDI, EDII, and IIICS. We used a cell culture system to simulate mammary epithelial-stromal communication, a process that is crucial for patterning and function of the mammary gland, and studied the effects of extracellular signals on the regulation of fibronectin pre-mRNA alternative splicing. We found that soluble factors from a mammary mesenchymal cell-conditioned medium, as well as the growth factors HGF/SF (hepatocyte growth factor/scatter factor), KGF (keratinocyte growth factor), and aFGF (acidic fibroblast growth factor), stimulate EDI and IIICS but not EDII inclusion into fibronectin mRNA in the mammary epithelial cell line SCp2, favoring fibronectin isoforms associated with proliferation, migration, and tissue remodeling. We explored the signaling pathways involved in this regulation and found that the mammary mesenchymal cell-conditioned medium and HGF/SF act through a phosphatidylinositol 3-kinase-dependent cascade to alter fibronectin alternative splicing. This splicing regulation is independent from promoter structure and de novo protein synthesis but does require two exonic elements within EDI. These results shed light on how extracellular stimuli are converted into changes in splicing patterns.     

Differential effects of growth factors and cytokines on the synthesis of SPARC, DNA, fibronectin and alkaline phosphatase activity in human periodontal ligament cells

Growth factors and cytokines play an important role in tissue development and repair. However, it remains unknown how they act on proliferation and differentiation of periodontal ligament cells. In this study, we investigated the effects of several growth factors and cytokines on the synthesis of DNA, alkaline phosphatase (ALPase), fibronectin, and secreted protein acidic and rich in cysteine (SPARC) in human periodontal ligament (HPL) cells. Transforming growth factor-beta (TGF-beta) increased the synthesis of DNA, fibronectin and SPARC, whereas it decreased ALPase activity. Basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and tumor necrosis factor-alpha (TNF-alpha) decreased SPARC and ALPase levels, whereas these peptides increased DNA synthesis and did not affect fibronectin synthesis. Epidermal growth factor (EGF) up-regulated the synthesis of DNA and fibronectin and inhibited SPARC and ALPase levels. Interleukin-1beta (IL-1beta) decreased the synthesis of DNA, ALPase, fibronectin and SPARC. These findings demonstrate that TGF-beta, bFGF, EGF, PDGF, TNF-alpha and IL-1beta have characteristically different patterns of action on DNA, SPARC, fibronectin and ALPase synthesis by HPL cells. The differences in regulation of function of periodontal ligament cells by these peptides may be involved in the regeneration and repair of periodontal tissue.     

ALK1 heterozygosity increases extracellular matrix protein expression,proliferation and migration in fibroblasts

Fibrosis is a pathological situation in which excessive amounts of extracellular matrix (ECM) are deposited in the tissue. Myofibroblasts play a crucial role in the development and progress of fibrosis as they actively synthesize ECM components such as collagen I, fibronectin and connective tissue growth factor (CTGF) and cause organ fibrosis. Transforming growth factor beta 1 (TGF-β1) plays a major role in tissue fibrosis. Activin receptor-like kinase 1 (ALK1) is a type I receptor of TGF-β1 with an important role in angiogenesis whose function in cellular biology and TGF-β signaling is well known in endothelial cells, but its role in fibroblast biology and its contribution to fibrosis is poorly studied. We have recently demonstrated that ALK1 regulates ECM protein expression in a mouse model of obstructive nephropathy. Our aim was to evaluate the role of ALK1 in several processes involved in fibrosis such as ECM protein expression, proliferation and migration in ALK1^+/+ and ALK1^+/− mouse embryonic fibroblasts (MEFs) after TGF-β1 stimulations and inhibitors. ALK1 heterozygous MEFs show increased expression of ECM proteins (collagen I, fibronectin and CTGF/CCN2), cell proliferation and migration due to an alteration of TGF-β/Smad signaling. ALK1 heterozygous disruption shows an increase of Smad2 and Smad3 phosphorylation that explains the increases in CTGF/CCN2, fibronectin and collagen I, proliferation and cell motility observed in these cells. Therefore, we suggest that ALK1 plays an important role in the regulation of ECM protein expression, proliferation and migration.     

Phenotypic modulation of endothelial cells by transforming growth factor-beta depends upon the composition and organization of the extracellular matrix

Transforming growth factor beta (TGF-beta) is angiogenic in vivo. In vitro, endothelial cell proliferation is inhibited by TGF-beta. We have correlated this inhibitory effect with an increase in cellular fibronectin synthesis and deposition in a two-dimensional culture system using specific matrix coatings. The inhibitory effect was mimicked by addition of soluble fibronectin to cultures. In contrast, TGF-beta was found to elicit the formation of tube-like structures (mimicking angiogenesis) when microvascular endothelial cells were grown in three-dimensional collagen gels. In this culture system TGF-beta elicited rapid extensive formation of complex, branching, tube-like structures, while cell proliferation was not inhibited. These data confirm and support the hypothesis that TGF-beta is angiogenic and may exert some of its effects through modulation of matrix synthesis and are consistent with the hypothesis that the organization of the extracellular environment influences cellular responses to this "panregulin."     

Transforming growth factor beta (TGF beta) causes a persistent increase in steady-state amounts of type I and type III collagen and fibronectin mRNAs in normal human dermal fibroblasts.

It has been previously shown that transforming growth factor beta (TGF beta) is capable of stimulating fibroblast collagen and fibronectin biosynthesis. The purpose of this study was to examine the mechanisms involved in TGF beta stimulation of fibroblast biosynthetic activity. Our results indicate that TGF beta causes a marked enhancement of the production of types I and III collagens and fibronectin by cultured normal human dermal fibroblasts. The rate of collagen production by fibroblasts exposed to TGF beta was 2-3-fold greater than that of control cells. These effects were associated with a 2-3-fold increase in the steady-state amounts of types I and III collagen mRNAs and a 5-8-fold increase in the amounts of fibronectin mRNAs as determined by dot-blot hybridization with specific cloned cDNA probes. In addition, the increased production of collagen and fibronectin and the increased amounts of their corresponding mRNAs remained elevated for at least 72 h after removal of TGF beta. These findings suggest that TGF beta may play a major role in the normal regulation of extracellular matrix production in vivo and may contribute to the development of pathological states of fibrosis.     

Culture and properties of cells derived from Kaposi sarcoma

We describe the establishment of four continuous cell cultures isolated from pleural or peritoneal fluid of patients with Kaposi sarcoma (KS) and show evidence that these cells are derived from vascular endothelium. Although provision of an extracellular matrix (fibronectin, laminin, or matrigel) was essential, the cell cultures were not dependent on exogenously added growth factors (platelet-derived growth factor, epidermal growth factor with or without heparin) for continuous culture. Specific staining for endothelial cell (EC) markers (factor VIII, Ulex europaeus type 1 lectin) and the secretion of endothelin, a vascular EC product, were demonstrated. The KS cells secreted large amounts of cytokines (granulocyte-macrophage-CSF, TNF-alpha, IL-1 beta, and especially IL-6). Conditioned media from the KS cells caused normal capillary EC to proliferate. The KS cells synthesized fibroblast growth activity in amounts sufficient to induce the proliferation of normal EC and fibroblasts. These data support the existence of a paracrine pathway of EC proliferation in KS and suggest that KS cells could sustain their own growth via an autocrine mechanism.     

Cellular ageing of Alzheimer's disease and Down syndrome cells in culture.

In Alzheimer's disease, the typical clinical symptoms and the pathological findings are restricted to the nervous system. Nevertheless, like in some other neurologic-metabolic disorders, several alterations are found in peripheral tissues. The aim of this study was to examine whether cellular properties which can be studied in vitro on skin fibroblast cultures obtained from Alzheimer's disease patients differ from those of age-matched controls. Down syndrome patients were also included, since the same neuropathological findings are present in nearly 100% of Down syndrome patients. Since Alzheimer's disease is an age-related disorder, we examined the growth characteristics of skin fibroblast cultures. The in vitro senescence of cultured fibroblasts is widely accepted as a model for in vivo ageing. Normal growth properties were found. We can conclude that there is no premature ageing in Alzheimer's disease nor in Down syndrome and that the abnormalities found in peripheral tissues are related to the disease itself. The beta amyloid precursor protein (beta APP) has been shown to have adhesive interactions. We therefore investigated several parameters of adhesion in the skin fibroblast cultures: adhesion to a fibronectin coat, adhesion to extracellular matrix of Alzheimer's disease cultures and semi-quantification of adhesion-related molecules (beta 1-integrin, cell surface proteoglycans, extracellular matrix proteoglycans, extracellular matrix fibronectin). No significant difference was found in the parameters examined.     

Modulation of extracellular-matrix synthesized by cultured stromal cells from normal human breast tissue by epidermal growth factor

A routine, reproducible procedure was developed for the preparation and characterization of stromal cells from normal human breast tissue obtained by reduction mammaplasty. Isolates (n = 15) all exhibited enhanced rates of proliferation, even in the presence of 20% fetal calf serum, when exposed to epidermal growth factor or transforming growth factor a (both 10(-8) M). Cellular responsiveness to these growth factors was consistent with expression of specific surface receptors for epidermal growth factor (approximately 10(4)/cell). In cultures, stromal cells elaborated an extensive, cross-linked, insoluble extracellular matrix which remained firmly associated with the plastic surface of tissue culture ware upon lysis of cells. The insoluble matrix material was analyzed using enzymatic digestion procedures following incorporation of radiolabelled precursors into macromolecular material prior to lysis and preparation. The relative proportion of glycoconjugate (glycopeptides and proteoglycans) and collagenous material present in matrix material was approximately 45% and approximately 55%, respectively, and this was modulated by inclusion of epidermal growth factor into culture medium to approximately 60% and approximately 40%, respectively. Under similar culture conditions stromal cells synthesized twice as much hyaluronate as was produced by control cultures. By use of specific antibody preparations we identified at least four species of glycopeptide present in stromal matrices (namely, fibronectin, laminin, tenascin, and thrombospondin) as well as three types of collagen (types I, III, and IV). The rapid and reproducible procedure for the preparation of radiolabelled insoluble matrix material from normal human breast tissue allows for the study of cellular interaction involving extracellular matrix turnover and degradation.     

Modulation of cultured chicken growth plate chondrocytes by transforming growth factor-beta 1 and basic fibroblast growth factor.

Expression of several cellular and matrix proteins which increase significantly during the maturation of growth plate cartilage has been shown to be affected by various endocrine and autocrine factors. In the studies reported here, transforming growth factor-beta (TGF-beta 1) and basic fibroblast growth factor (bFGF) were administered to primary cultures of avian growth plate chondrocytes at pre- or post-confluent stages to study the interplay that occurs between these factors in modulating chondrocytic phenotype. Added continuously to pre-confluent chondrocytes, TGF-beta 1 stimulated the cells to produce abundant extracellular matrix and multilayered cell growth; cell morphology was altered to a more spherical configuration. These effects were generally mimicked by bFGF, but cell shape was not affected. Administered together with TGF-beta 1, bFGF caused additive stimulation of protein synthesis, and alkaline phosphatase (AP) activity was markedly, but transiently enhanced. During this pre-confluent stage, TGF-beta 1 also increased fibronectin secretion into the culture medium. Added to post-confluent cells, TGF-beta 1 alone caused a dosage-dependent suppression of AP activity, but bFGF alone did not. Under these conditions, TGF-beta 1 and bFGF had little effect on general protein synthesis, but TGF-beta 1 alone caused large, dosage-dependent increases in synthesis of fibronectin, and to some extent type II and X collagens. Given together with bFGF, TGF-beta 1 synergistically increased secretion of fibronectin. These findings reveal that regulation of phenotypic expression in maturing growth plate chondrocytes involves complex interactions between growth factors that are determined by timing, level, continuity, and length of exposure.     

Alpha4beta1 integrin and erythropoietin mediate temporally distinct steps in erythropoiesis: integrins in red cell development

Erythropoietin (Epo) is essential for the terminal proliferation and differentiation of erythroid progenitor cells. Fibronectin is an important part of the erythroid niche, but its precise role in erythropoiesis is unknown. By culturing fetal liver erythroid progenitors, we show that fibronectin and Epo regulate erythroid proliferation in temporally distinct steps: an early Epo-dependent phase is followed by a fibronectin-dependent phase. In each phase, Epo and fibronectin promote expansion by preventing apoptosis partly through bcl-xL. We show that alpha(4), alpha(5), and beta(1) are the principal integrins expressed on erythroid progenitors; their down-regulation during erythropoiesis parallels the loss of cell adhesion to fibronectin. Culturing erythroid progenitors on recombinant fibronectin fragments revealed that only substrates that engage alpha(4)beta(1)-integrin support normal proliferation. Collectively, these data suggest a two-phase model for growth factor and extracellular matrix regulation of erythropoiesis, with an early Epo-dependent, integrin-independent phase followed by an Epo-independent, alpha(4)beta(1)-integrin-dependent phase.     

Epithelial-mesenchymal interactions and lung branching morphogenesis. Role of polyamines and transforming growth factor beta1

Lung branching morphogenesis is a result of epithelial-mesenchymal interactions, which are in turn dependent on extracellular matrix composition and cytokine regulation. Polyamines have recently been demonstrated as able to modify chick embryo skin differentiation. In this work we have examined the effects of putrescine and spermidine during chick embryo lung morphogenesis in organotypic cultures by morphological, histochemical and biochemical examination. To verify the role of polyamines, we used specific inhibitors, such as bis-cyclohexylammonium sulphate and alfa-difluoromethylornithine, and transforming growth factor beta1, an ornithine decarboxylase and polyamine stimulator. Our data show that lung morphogenesis is significantly altered following the induced mesenchymal glycosaminoglycan changes. The increase of mesenchymal glycosaminoglycans is correlated with a stimulation of lung development in the presence of polyamines, and with its inhibition when transforming growth factor beta1 is added to the culture medium. The morphometric data show a uniform increase of both the mesenchyme and epithelial branching with spermidine and putrescine stimulus, whereas the mesenchymal substance alone is significantly increased in apical-median lung sections with transforming growth factor beta1 and transforming growth factor beta1 + spermidine lung cultures. Transforming growth factor beta1 and transforming growth factor beta1 + spermidine confirm the blocking of epithelial branching formations and fibroblast activation, and show that polyamines are unable to prevent the blocking of epithelial cells due to the inhibitory effect of transforming growth factor beta1.     

Induction of extracellular matrix gene expression in normal human keratinocytes by transforming growth factor beta is altered by cellular differentiation

Changes in epithelial substrate have been related to the cellular capacity for proliferation and to changes in cellular behavior. The effect of TGF beta 1 on the expression of the basement membrane genes, fibronectin, laminin B1, and collagen alpha 1 (IV), was examined. Northern analysis revealed that treatment of normal human epidermal keratinocytes with 100 pM TGF beta 1 increased the expression of each extracellular matrix (ECM) gene within 4 h of treatment. Maximal induction was reached within 24 h after treatment. The induction of ECM mRNA expression was dose dependent and was observed at doses as low as 1-3 pM TGF beta 1. Incremental doses of TGF beta 1 also increased cellular levels of fibronectin protein in undifferentiated keratinocytes and resulted in increased secretion of fibronectin. Squamous-differentiated cultures of keratinocytes expressed lower levels of the extracellular matrix RNAs than did undifferentiated cells. Treatment of these differentiated cells with TGF beta 1 induced the expression of fibronectin mRNA to levels seen in TGF beta-treated, undifferentiated keratinocytes but only marginally increased the expression of collagen alpha 1 (IV) and laminin B1 mRNA. The increased fibronectin mRNA expression in the differentiated keratinocytes was also reflected by increased accumulation of cellular and secreted fibronectin protein. The inclusion of cycloheximide in the protocol indicated that TGF beta induction of collagen alpha 1 (IV) mRNA was signaled by proteins already present in the cells but that TGF beta required the synthesis of a protein(s) to fully induce expression of fibronectin and laminin B1 mRNA. The differential regulation of these genes in differentiated cells may be important to TGF beta action in regulating reepithelialization.     

Peptide growth factor interactions in embryonic and fetal growth.

Peptide growth factors are expressed by multiple tissues in the animal and human embryo and fetus. They undergo specific interactions which control the rate of cellular proliferation, tissue differentiation and the induction of specific morphogenic events such as mesoderm formation in the embryo. Biologic control may not only be exerted at the level of growth factor synthesis and receptor expression but by the sequestration and storage of growth factors by extracellular matrix molecules. In the case of insulin-like growth factors (IGFs), storage maybe mediated by attachment to specific IGF-binding proteins which may additionally modulate biological potency. Basic fibroblast growth factor (basic FGF) and transforming growth factor-beta (TGF beta) directly bind to glycosaminoglycan molecules. Release of growth factors from these stores may be by local proteolytic action. A sequential expression of basic FGF, IGF-II and TGF beta occurs in the ovine fetal epiphyseal growth plate as chondrocytes progress from a proliferative to a postmitotic, hypertrophic state. Cellular phenotype may be largely explained by the relative amounts of these autocrine growth factors within the growth plate.