Growth factor modulation of the extracellular matrix

Two cell culture models were utilized to characterize the actions of peptide growth factors on the composition of the extracellular matrix of embryonic mesenchymal tissue. To model the three-dimensional architecture of mesenchymal tissue, chick embryonic mesenchymal cells were maintained in organ culture as adherent cell populations in small three-dimensional tissue spheroids and as sparse populations of cells embedded in a mesh of hydrated native collagen fibrils. Cell proliferation was stimulated by a variety of growth factors. All of the growth factors that elicited a mitogenic response in both of these culture systems also stimulated the deposition of an abundant fibronectin-containing extracellular matrix that colocalized with the regions of active cell proliferation. The suggestion that the matrigenic actions of growth factors for intact mesenchymal tissue are an integral part of mitogenic signaling is supported by the observation that surfaces derivatized with ProNectin, an artificial mimic of the RGD attachment domain of fibronectin, stimulated the proliferation of embryonic mesenchyme in the absence of exogenous growth factors. All of the growth factors that activated proliferation and fibronectin matrix accumulation stimulated the transformation of the mesenchymal cells into myofibroblasts that displayed the marker alpha-smooth muscle actin.     

Thrombin stimulates the production and release of a major surface-associated glycoprotein (fibronectin) in cultures of human fibroblasts

Exposure of cultured human skin fibroblasts to thrombin in serum-free medium had several effects on fibronectin, a major cell surface-associated glycoprotein. Pericellular fibronectin fibrils, visualized by immunofluorescence, were lost after exposure for 4–20 h to thrombin (1–8 U/ml). Loss of fibronectin fibrils did not occur if thrombin was inhibited by phenylmethyl-sulfonyl fluoride (PMSF), N-α-tosyl-1-lysyl chloromethane (TosLysCH₂Cl), alpha-1-antithrombin, alpha-2-macroglobulin, or hirudin. Cell surface fibronectin, labeled by lactoperoxidase-catalysed iodination, and newly synthesized fibronectin, metabolically labeled with [³H]mannose, were lost after exposure for 20 h to thrombin. Within 60 min, increased concentrations of fibronectin were detected by radioimmunoassay in media of thrombin-treated cultures. Thrombin increased several-fold the total amount of fibronectin accumulating in cultures over a 20 h period by increasing the amount of fibronectin secreted or shed into the medium. Fetal calf serum, which contained inhibitors of thrombin and hence only low levels of thrombin activity (<0.05 U/ml), also stimulated fibronectin production but did not cause loss of pericellular fibronectin fibrils. Thrombin or serum, under the same experimental conditions, stimulated proliferation of human fibroblasts [46]. The effects of thrombin on fibronectin may be important in wound healing and tissue repair.     

A comparison of the responses of cultured myoblasts and chondrocytes to fibroblast and epidermal growth factors.

The effects of fibroblast and epidermal growth factors on proliferation and differentiation of cultured myoblasts and chondrocytes have been compared. FGF stimulated myoblast proliferation, as determined by monitoring levels of DNA synthesis during seven days growth in vitro and by the morphology of the cultures after myotube formation. EGF has relatively little effect on myoblast proliferation. With chondrocytes, both FGF and EGF are mitogenic and FGF's, but not EGF's effect is potentiated by dexamethasone. One implication of these results is that in the course of differentiation cell types which develop from the same embryonic origin as fibroblasts are controlled by different sets of mitogenic factors. Myoblasts become primarily dependent on mitogenic agents such as FGF while chondrocytes can respond to both FGF and EGF.     

Protease-activated receptor-1 can mediate responses to SFLLRN in thrombin-desensitized cells: evidence for a novel mechanism for preventing or terminating signaling by PAR1's tethered ligand

The thrombin receptor PAR1 is activated when thrombin cleaves the receptor's amino-terminal exodomain to reveal the new N-terminal sequence SFLLRN which then acts as a tethered peptide ligand. Free SFLLRN activates PAR1 independent of receptor cleavage and has been used to probe PAR1 function in various cells and tissues. PAR1-expressing cells desensitized to thrombin retain responsiveness to SFLLRN. Toward determining the mechanism of such responses, we utilized fibroblasts derived from a PAR1-deficient mouse. These cells were unresponsive to thrombin and SFLLRN and became sensitive to both ligands after transfection with human PAR1 cDNA. Moreover, PAR1-transfected cells responded to SFLLRN after thrombin-desensitization, indicating that signaling of thrombin-desensitized cells to SFLLRN was mediated by PAR1 itself. SFLLRN caused signaling in thrombin-desensitized cells when no uncleaved PAR1 was detectable on the cell surface; however, cleaved PAR1 was present. To determine whether the cleaved receptors could still signal, fibroblasts were transfected with a PAR1 mutant containing a trypsin site/SFLLRN sequence carboxyl terminal to the native thrombin site. These cells retained responsiveness to trypsin after thrombin-desensitization. Conversely, fibroblasts expressing a PAR1 mutant with the trypsin site/SFLLRN sequence amino terminal to the native thrombin site retained responsiveness to thrombin after trypsin-desensitization. This suggests that a population of thrombin-cleaved PAR1 can respond both to exogenous SFLLRN and to a second tethered ligand. In this population, the tethered ligand unmasked by thrombin cleavage must not be functional, suggesting the possibility of a novel mechanism of receptor shutoff involving sequestration or modification of the tethered ligand to prevent or terminate its function.     

The regulation of DNA synthesis by fibronectin and its proteolytic products in the skin fibroblasts of healthy donors and patients with systemic scleroderma and rheumatoid arthritis

To study the effect of fibronectin isolated from plasma and culture media and the effect of its tryptic hydrolyzates on DNA synthesis, cultured skin fibroblasts of healthy donors and these of patients with systemic scleroderma (SSD) and rheumatoid arthritis (RA) were employed. It was shown that both fibronectin and total products of its proteolysis markedly stimulated DNA synthesis only in skin fibroblasts of patients with SSD. Fibronectin fragments inhibited DNA synthesis in all fibroblast strains studied. The effect of fibronectin and all its Gel fragments on the DNA synthesis in skin fibroblasts of patients with SSD was dose-dependent. The activity of total fibronectin tryptate, Gel-fragment-free tryptate, and Gel fragments themselves depended on the duration of fibronectin proteolysis, i. e. on the size of the fragments obtained. Culture media collected after treatment of fibroblast monolayer with trypsin and subsequent removal of fibronectin Gel fragments had mitogenic effect on skin fibroblasts, especially on those of patients with SSD and RA. It is supposed that fibronectin Gel fragments are inhibitors of growth factors produced by fibroblasts. The results suggest that fibronectin and its fragments have an important regulatory role in fibroblast proliferation.     

TGF-β1 stimulates cultured human fibroblasts to proliferate and produce tissue-like fibroplasia: A fibronectin matrix-dependent event

During wound repair, fibroblasts accumulate in the injured area until any defect is filled with stratified layers of cells and matrix. Such fibroplasia also occurs in many fibrotic disorders. Transforming growth factor-β (TGF-β), a promotor of granulation tissue in vivo and extracellular matrix production in vitro, is expressed during the active fibroplasia of wound healing and fibroproliferative diseases. Under usual tissue culture conditions, normal fibroblasts grow to confluence and then cease proliferation. In this study, culture conditions with TGF-β1 have been delineated that promote human fibroblasts to grow in stratified layers mimicking in vivo fibroplasia. When medium supplemented with serum, ascorbate, proline, and TGF-β was added thrice weekly to normal human dermal fibroblasts, the cells proliferated and stratified up to 16 cell layers thick within the culture dish, producing a tissue-like fibroplasia. TGF-β stimulated both DNA synthesis as measured by 1H-thymidine uptake and cell proliferation as measured by a Hoechst dye DNA assay in these postconfluent cultures. The stratification was dependent on fibronectin assembly, as demonstrated by anti-fibronectin antibodies which inhibited both basal and TGF-β-stimulated cell proliferation and stratification. Suppression of collagen matrix assembly in cell layers with β-amino-proprionitrile (BAPN) did not inhibit basal or TGF-β stimulated in vitro fibroplasia. BAPN did not interfere with fibronectin matrix assembly as judged by immunofluorescence microscopy. Thus, in concert with serum factors, TGF-β stimulates postconfluent, fibronectin matrix-dependent, fibroblast growth creating a fibroplasia-like tissue in vitro. J Cell Physiol 170:69–80, 1997 © 1997 Wiley-Liss, Inc.     

Influence of thrombin on proliferation and prostaglandin production in cultured bovine endometrial cells

The control of cell proliferation by thrombin was studied in vitro in cultured epithelial and stromal cells of the endometrium. The effect of thrombin was studied after chronic treatment (72 hr) in medium containing 10% fetal bovine serum (FBS) combined or not with sex steroids. Thrombin inhibited slightly the proliferation (based on DNA measurements) only in epithelial cells (P < 0.05). 17β-estradiol (E) and progesterone (P4) had no mitogenic effects. The presence of functional thrombin receptors was estimated by stimulation of second messenger generation in response to increasing doses of thrombin (0-1,500 ng/ml). In confluent cultures of epithelial cells, the addition of thrombin for 10 min stimulated cAMP production by 50% with a maximal response at 500 ng/ml (P < 0.05). Similarly, in stromal cells, thrombin stimulated cAMP production in a dose-dependent manner (P < 0.01). Generation of inositol-phosphates was also stimulated by 50% in epithelial cells (P < 0.03), with a maximal response at 500 ng/ml, and by 45% in stromal cells (P < 0.01), with a maximal response at 50 ng/ml. The effect of thrombin on cell proliferation was investigated by ³H-thymidine incorporation in serum-free medium for 24 hr. Thrombin inhibited incorporation in epithelial cells (P < 0.0001) in a dose-dependent manner. Conversely, thrombin stimulated significantly incorporation of stromal cells (P < 0.05) at 50 ng/ml. The effect of sex steroids was also evaluated and it was found that E had no effect on cell proliferation, while P4 inhibited the incorporation in both epithelial (P < 0.001) and stromal cells (P < 0.001). The effect of a combined treatment with thrombin and E inhibited both epithelial (P < 0.001) and stromal cell (P < 0.001) growth, but a combination of thrombin and P4 had no additional effect on growth compared to P4 alone. Further investigation of the role of thrombin has been carried out by measuring prostaglandin (PG) responses. Addition of thrombin for 24 hr inhibited PGF_2α production by epithelial cells (P < 0.0001) but had no effect on PGE₂ production by stromal cells. Therefore, functional receptors for thrombin appear to be present in epithelial and stromal cells of the bovine endometrium. The minimal effect of thrombin alone or in combination with sex steroids on endometrial cell proliferation in vitro combined with the evidence of functional thrombin receptor in these cells, suggest that: (1) the effect of sex steroids in cultured endometrial cells is not modulated by the presence of thrombin, and (2) other factors are necessary for the full expression of mitogenic responses to sex steroids in vitro. © 1996 Wiley-Liss, Inc.     

Macrocyclic hexapeptide analogues of the thrombin receptor (PAR-1) activation motif SFLLRN

The thrombin receptor (PAR-1) is activated by alpha-thrombin to stimulate various cell types, including platelets, through the tethered-ligand sequence SFLLRN. Macrocyclic peptide analogues of SFLLRN were synthesized and evaluated in vitro. In general, the compounds were much less potent in inducing platelet aggregation relative to SFLLRN-NH2 and did not act as antagonists of alpha-thrombin. Derivative 3c was the most potent macrocycle in activating PAR-1, with an EC50 of 24 microM.     

Effect of polydeoxyribonucleotides on human fibroblasts in primary culture

The effects of a mixture of oligo‐ and polydeoxyribonucleotides (PDRN) on the growth and protein secretion of cultured human skin fibroblasts were investigated. Both intact and DNAase‐digested PDRN stimulated cell proliferation to a similar extent. When cultured fibroblasts were incubated with radioactive amino acids in the presence of intact or digested PDRN the incorporation of the tracer into secreted proteins increased significantly. This stimulation appears to be specific for certain protein components, including fibronectin. These results are interpreted assuming that PDRN and the nucleotides and nucleosides resulting from its degradation, can act as signal transducers or, alternatively, can be internalized and utilized to provide purine and pyrimidine rings for the salvage pathways. Copyright © 1999 John Wiley & Sons, Ltd.     

Induction of vascular smooth muscle cell growth by selective activation of the thrombin receptor. Effect of heparin.

The synthetic peptide, SFLLRNPNDKYEPF, has been recently described as a peptide mimicking the new amino-terminus created by cleavage of the thrombin receptor, therefore acting as an agonist of the thrombin receptor. This peptide was a potent mitogen for rabbit arterial smooth muscle cells (SMC) and exhibited the same activity as that of native alpha-thrombin. Both compounds stimulated the proliferation of growth-arrested SMCs with half-maximum mitogenic responses at 1 nM. NAPAP, a synthetic inhibitor of the enzymatic activity of thrombin, specifically inhibited thrombin-induced SMC growth (IC50 = 0.35 +/- 0.04 microM) but was without effect on the mitogenic effect of the agonist peptide. These results therefore demonstrate that the mitogenic effect of alpha-thrombin for SMCs is intimately linked to its esterolytic activity. Heparin, which inhibited fetal calf serum-induced SMC growth, was without effect on thrombin-induced SMC growth but strongly reduced the mitogenic effect of the agonist peptide (IC50 = 32 +/- 5 micrograms/ml). This effect was not related to the anti-coagulant activity of heparin but was highly dependent on molecular mass and on the global charge of the molecule and was also observed for other sulphated polysaccharides such as pentosan polysulphate.     

Regulation of cell growth and the expression of extracellular matrix proteins in colorectal adenocarcinoma: a fibroblast-tumor cell coculture model to study tumor-host interactions in vitro

The production of abundant connective tissue within malignant tumors, the so-called desmoplastic stromal reaction, is a hallmark of colorectal adenocarcinomas. This stroma is produced to a large extent by myofibroblasts and contains various amounts of collagens (type I, III, and V), chondroitin sulfate proteoglycan, hyaluronic acid, fibronectin, and tenascin-C. In this study we have established a monolayer coculture model between two different colorectal adenocarcinoma cell lines (HRT-18, and CX-2) and colonic fibroblasts (CCD-18) to investigate the mechanisms regulating (i) the production of extracellular matrix (ECM) components, (ii) the induction of myofibroblastic differentiation, and (iii) cellular proliferation. We found that TGFbeta1 and FGF-2 stimulated ECM synthesis of fibroblasts. Myofibroblastic differentiation was stimulated by TGFbeta1 but suppressed by FGF-2. There was a mutual stimulation of proliferation between fibroblasts and carcinoma cells. The analogies with ECM components expressed in cocultures and colorectal adenocarcinoma samples suggest that the coculture model used in this study is useful to study tumor cell-fibroblast interactions.     

Human alpha-thrombin stimulates proliferation of interferon-gamma differentiated,growth-arrested U937 cells,overcoming differentiation-related changes in expression of p21CIP1/WAF1 and cyclin D1

In addition to its central role in blood coagulation and hemostasis, human alpha-thrombin is a growth factor for a variety of cell types. We recently demonstrated that interferon-gamma (IFNgamma)-differentiated U937 cells show increased expression of the proteolytically activated receptor for thrombin (PAR-1) relative to undifferentiated U937. In the present study we show that cell proliferation is inhibited in IFNgamma-differentiated cells relative to undifferentiated U937. Addition of thrombin to the differentiated cells, however, overcomes the inhibition and restores the cells to a highly proliferative state. Ribonuclease protection assays indicate that the IFNgamma-induced growth arrest is associated with an increased expression of the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) and downregulation of cyclin D(1). Treatment of cells with thrombin downregulates p21(CIP1/WAF1) expression in these cells and upregulates cyclin D(1) mRNA expression, thus overcoming the differentiation-related effects in a coordinated manner. Treating differentiated cells with the PAR-1 activation peptide, SFLLRN, stimulates proliferation and has effects similar to those of thrombin on expression of p21(CIP1/WAF1). Thus, it appears that these thrombin stimulated proliferative effects are mediated through activation of PAR-1. These results may help explain how thrombin can overcome growth arrest in normal tissue to initiate tissue repair and why thrombin and thrombin-like enzymes may contribute to unrestricted proliferation observed in certain malignancies.     

Thrombin, epidermal growth factor, and phorbol myristate acetate stimulate tubulin polymerization in quiescent cells: a potential link to mitogenesis.

Previous studies suggest that alterations in the microtubule (MT)-tubulin equilibrium during G0/G1 affect mitogenesis. To determine the effect of growth factors on the MT-tubulin equilibrium, we developed a radioactive monoclonal antibody binding assay (Ball et al.: J. Cell. Biol. 103:1033-1041, 1986). With this assay, 3H-Ab 1-1.1 binding to cytoskeletons in confluent populations of cultured cells is proportional to the number of tubulin subunits polymerized into MTs. We now show that purified alpha-thrombin increases 3H-Ab 1-1.1 binding to cytoskeletons of serum-arrested mouse embryo (ME) fibroblasts from 1.5- to 3-fold. This stimulation is dose-dependent and correlates with concentrations of thrombin required for initiation of DNA synthesis. Other mitogenic factors, epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA), also stimulate MT polymerization. Addition of colchicine (0.3 microM) eight hours after growth factor addition, blocks stimulation of 3H-thymidine incorporation by thrombin, EGF, or PMA, suggesting that tubulin polymerization or subsequent events triggered by MT polymerization are required for cells to enter a proliferative cycle. Consistent with models for autoregulation of tubulin synthesis, thrombin, EGF, and PMA all increase tubulin synthesis 9 to 15 hr after growth factor addition, raising the possibility that the decrease in free tubulin and subsequent stimulation of tubulin synthesis is linked to progression of cells into a proliferative cycle. Colchicine addition to these cells also stimulates DNA synthesis, but colchicine-stimulated cells enter S phase 6 to 8 hr later than those stimulated by growth factors. This delayed stimulation may be related to the time required for degradation of tubulin-colchicine complexes below a critical level. These data suggest that regulation of cell proliferation may be linked to increased MT polymerization and the resulting decrease in free tubulin pools.     

Effects of EGF and thrombin on inositol-containing phospholipids of cultured fibroblasts: stimulation of phosphatidylinositol synthesis by thrombin but not EGF

The effects of growth factors on inositol-containing phospholipids were investigated to test the hypothesis that alterations in their metabolism are involved in mitogenic stimulation. Thrombin and EGF stimulated comparable increases in the synthesis (30-50%) and degradation (20-40%) of phosphatidylinositol 4-monophosphate (DPI) and phosphatidylinositol 4,5-bisphosphate (TPI) in a cell line which is mitogenically responsive to both growth factors. The increases in synthesis were time and dose dependent in a manner which was consistent with their involvement in mitogenesis; the increases were observed only under conditions where a mitogenic response occurred. While it has been suggested that an increased synthesis of phosphatidylinositol (PI) is coupled to the stimulation of DPI and TPI synthesis, we found that thrombin stimulated an early synthesis PI but EGF did not. To further evaluate the involvement of PI in thrombin-stimulated cell division we determined the time and dose dependence of the stimulated PI synthesis and found that it also occurred in a manner which was consistent with its involvement in thrombin-stimulated cell division. Furthermore, the stimulated PI synthesis was not observed with nonmitogenic proteases or in cell lines which were not responsive to thrombin. These results demonstrate that the metabolism of DPI and TPI appears closely related to the mitogenic response generated by EGF and thrombin. However, an early stimulation of PI synthesis is not coupled to this metabolism and is not necessary for mitogenic stimulation by EGF. Thus, a stimulation of PI synthesis is not a valid measure of alterations in inositol-containing phospholipids and what has been termed the "PI response."     

Growth-promoting effects of iron- and cobalt- protoporphyrins on cultured embryonic cells

The effects of hemin (Fe-protoporphyrin) and Co-protoporphyrin on cellular growth have been investigated principally in cultured fibroblasts, but also in myoblasts and hepatocytes from chick embryos. In the presence of horse serum in the culture medium, which by itself did not stimulate cell growth appreciably, Co-protoporphyrin stimulated cell attachment while hemin stimulated cell proliferation of fibroblasts. When Co-protoporphyrin and hemin were added together, the most potent stimulation of cell growth, consisting of increases in cell attachment and rapid cell proliferation, was observed. These findings indicate that the two metalloporphyrins have differential and complementary effects on cellular growth in culture, with synthetic Co-protoporphyrin principally affecting cellular attachment and Fe-protoporphyrin stimulating cellular proliferation.     

Investigations into the nature of growth-related proteolysis in human fibroblasts

Previous experiments have shown that cultured human fibroblasts possess a cell-surface proteinase (the growth-related proteinase; GRP) which is essential to cell proliferation. In the present work, proteinase inhibition in defined and complex serum-free media and in pre-conditioned normal medium, still resulted in a corresponding inhibition of cell proliferation. Proteinase inhibition also blocked the action of a range of peptide growth factors and of a phorbol ester. Elevated extracellular calcium concentrations were still mitogenic in the presence of proteinase inhibitors. Proteinase inhibition did not affect the mobilisation of intracellular calcium, nor the metabolism of inositol phosphate derivatives in response to a mitogenic stimulus.     

Factor XIII cross-linking of fibronectin at cellular matrix assembly sites

We describe the effect of activated Factor XIII (Factor XIIIa, plasma transglutaminase) on the incorporation of plasma fibronectin into extracellular matrix by cultured human fibroblasts. In the absence of added Factor XIIIa, fibronectin binds to cultured fibroblast cell layers and is assembled into disulfide-bonded multimers of the extracellular matrix. When Factor XIIIa was included in the binding medium of skin fibroblasts, accumulation of 125I-fibronectin in the deoxycholate-insoluble matrix was increased. Fibronectin accumulating in the cell layer was cross-linked into nonreducible high molecular weight aggregates. The 70-kDa amino-terminal fragment of fibronectin inhibited the binding and cross-linking of 125I-fibronectin to cell layers, whereas fibrinogen had little effect. When 125I-fibronectin was incubated with isolated matrices or with cell layers pretreated with cytochalasin B, it did not bind and could not be cross-linked by Factor XIIIa into the matrix. HT-1080 human fibrosarcoma cells bound exogenous fibronectin following treatment with dexamethasone; Factor XIIIa cross-linked the bound fibronectin and caused its efficient transfer to the deoxycholate-insoluble matrix. These results indicate that exogenous fibronectin is susceptible to Factor XIIIa-catalyzed cross-linking at cellular sites of matrix assembly. Thus, Factor XIIIa-mediated fibronectin cross-linking complements disulfide-bonded multimer formation in the stabilization of assembling fibronectin molecules and thus enhances the formation of extracellular matrix.     

MAP kinase-mediated proliferation of DLD-1 carcinoma by the stimulation of protease-activated receptor 2

Protease-activated receptor-2 (PAR-2) has been demonstrated to be highly expressed in the gastrointestinal tract. In the present study, we investigated the effects of PAR-2 stimulation on the cell signaling and proliferation of DLD-1, a human colon carcinoma cell line, in comparison with the PAR-1 stimulation. PAR-2 stimulation by agonist peptide SLIGKV concentration-dependently induced the increase in [Ca2+]i and the proliferation of DLD-1 whereas the inverse peptide LSIGKV did not. Trypin (10(-9) M), an agonist protease for PAR-2, also enhanced the proliferation of DLD-1. The proliferative response of DLD-1 to PAR-2 stimulation was associated with the transient phosphorylation of MEK and MAP kinase, but not p38 MAP kinase and JNK. Inhibition of MEK by PD98059 (50 microM) completely inhibited the proliferation-stimulating effects as well as the phosphorylation of MAP kinase induced by PAR-2 agonist peptide (100 microM) and trypsin (10(-9) M). The prolonged treatment with PAR-2 agonist peptide for more than one hour was required for the enhanced proliferative response, suggesting the existence of unknown long-lasting cooperative signaling with MAP kinase cascade. PAR-1 stimulation by the agonist peptide SFLLRN (100 microM) or thrombin (10(-8) M) produced Ca2+ signaling, however, the stimulation neither produced the cell proliferative response nor the activation of MEK-MAP kinase cascade. These results indicated that Ca2+ signaling induced by PARs activation was not enough for inducing the cell proliferation in DLD-1 cells and that stimulation of PAR-2 can induce the activation of MEK-MAP kinase cascade, leading to the growth promoting response.