Inhibition of bFGF/EGF-dependent endothelial cell proliferation by the hyaluronan-binding protease from human plasma

Recently we identified a plasma serine protease with a high affinity to glycosaminoglycans like heparin or hyaluronic acid, termed hyaluronan-binding protease (HABP). Since glycosaminoglycans are found on cell surfaces and in the extracellular matrix a physiological role of this plasma protease in a pericellular environment was postulated. Here we studied the influence of HABP on the regulation of endothelial cell growth. We found that HABP efficiently prevented the basic fibroblast growth factor/epidermal growth factor (bFGF/EGF)-dependent proliferation of human umbilical vein endothelial cells. Proteolytic cleavage of adhesion molecules was found to be involved, but was not solely responsible for the anti-proliferative activity. Pre-treatment of growth factor-supplemented cell culture medium with HABP indicated that no direct contact between the active protease and cells was required for growth inhibition. In vitro studies revealed a growth factor-directed activity of HABP, resulting in complexation and partial hydrolysis and, thus, inactivation of basic fibroblast growth factor, a potent mitogen for endothelial cells. Heparin and heparan sulfate fully protected bFGF from complexation and cleavage by HABP, although these glycosaminoglycans are known to enhance the proteolytic activity of HABP. This finding suggested that free circulating bFGF rather than bFGF bound to heparan sulfate proteoglycans would be a physiologic substrate. In conclusion, down-regulation of bFGF-dependent endothelial cell growth represents an important mechanism through which HABP could control cell growth in physiologic or pathologic processes like angiogenesis, wound healing or tumor development.     

Induction of Fibroblast Growth Factor Receptor-1 mRNA and Protein by Platelet-Derived Growth Factor BB

Expression levels of growth factor receptors are subject to complex regulation, which is of consequence for their signaling capacity in physiological and pathological processes. We examined the regulation of expression levels of fibroblast growth factor receptor 1 (FGFR-1) in human fibroblasts treated with a panel of growth regulatory factors. Only platelet-derived growth factor BB (PDGF-BB) treatment had a significant effect and induced FGFR-1 mRNA levels fourfold, with a peak around 8 h of stimulation. The increase in mRNA levels was followed by an increased synthesis of FGFR-1 protein, which responded to basic FGF (bFGF) stimulation with induction of kinase activity and biological signaling. Thus, murine brain endothelial cells displayed an augmented induction of plasminogen activator activity in response to bFGF, following treatment with PDGF-BB. These data suggest that PDGF-BB could support FGFR-1-mediated biological responses in processes such as angiogenesis.     

Heparin can activate a receptor tyrosine kinase.

Heparin, a densely sulfated glycosaminoglycan produced by mast cells, is best known for its inhibitory effects on the blood coagulation system. Heparin or heparan sulfate proteoglycans are also essential cofactors for the interaction of fibroblast growth factors (FGFs) with their receptor tyrosine kinases (FGFRs). Here we show that heparin is a growth factor-independent activating ligand for FGFR-4. Heparin stimulates FGFR-4 autophosphorylation on transfected myoblasts, fibroblasts and lymphoid cells, and is most potent on cells lacking surface heparan proteoglycan. Two functional analogs of heparin, fucoidan and dextran sulfate, are also activators of FGFR-4, while neither heparin nor its analogs can stimulate FGFR-1 in the absence of FGF. A mutation in the FGFR-4 ectodomain which impairs receptor activation by FGFs does not interfere with activation by heparin, demonstrating that receptor domains required for heparin or FGF activation are not identical. Heparin activation of FGFR-4 or of a chimeric receptor bearing FGFR-4 ectodomain and FGFR-1 cytodomain triggers downstream tyrosine phosphorylation of several signaling proteins, and induces proliferation of cells bearing the chimeric receptor. Consistent with these findings, a soluble FGFR-4 ectodomain has strong FGF-independent affinity for immobilized heparin resin, while soluble FGFR-1 requires FGF for stable heparin interaction. Heparin activation of FGFR-4 is the first example of a mammalian polysaccharide serving as a signaling ligand.     

重组人碱性成纤维细胞生长因子(bFGF)融合蛋白的高效表达及鉴定

碱性成纤维细胞生长因子（ｂＦＧＦ）参与了许多细胞生长和分化的调控过程。本文采用重组ＤＮＡ技术在大肠杆菌中高效表达了人ｂＦＧＦ。首先将编码人ｂＦＧＦ基因克隆到ｐＸＴ表达载体中与其上游的一短Ｓ导肽共一阅读框架,ｂＦＧＦ基因的表达受强的Ｔ７启动子调控。采用ＢＬ２１（ＤＥ３）大肠杆菌作为宿主菌,用ＩＰＴＧ诱导ＢＬ２１（ＤＥ３）细菌合成的Ｔ７ＲＮＡ聚合酶,后者可催化高水平的ｂＦＧＦ基因表达,其ｂＦＧＦ产量可占总菌体蛋白的４２.５％。采用肝素Ｓｅｐｈａｒｏｓｅ一步亲和层析法直接从诱导后的细菌裂解产物中得到纯化的重组人ｂＦＧＦ蛋白。经Ｗｅｓｔｅｒｎ印迹分析证明该蛋白可被人ｂＦＧＦ特异性单克隆抗体所识别。进一步研究证明该蛋白具有刺激ＮＲ６Ｒ－３Ｔ３成纤维细胞增殖的生物学活性,并且这一活性可被人ｂＦＧＦ特异性中和抗体所中和。     

Paracrine control of myoblast proliferation and differentiation by fibroblasts

Double-muscling (DM) is a hereditary (apparently single-gene) skeletal muscle hyperplasia which occurs in beef cattle. In order to investigate the cellular basis of this phenotype, cell cultures from developing muscle tissue of normal and DM fetal calves were studied. In cultures composed of both myogenic cells and nonmyogenic, fibroblast-like cells, DM myoblasts exhibited a prolonged proliferative phase. This resulted in delayed, but increased production of fused myotubes in the DM cultures. "Conditioned" media experiments indicated that the fibroblast-like cells in the cultures produced soluble myoblast growth factor activity. Both normal and DM fibroblast-like cells produced the growth factor activity, but the mutant fibroblast-like cells produced a greater level of such activity. The conditioned media failed to increase proliferation of bovine muscle fibroblasts and did not stimulate quiescent Swiss 3T3 cells to divide, indicating that the myoblast trophic activity is distinct from bFGF or PDGF. Also, the myotrophic activity present in the conditioned media acted in an additive fashion with saturating doses of bFGF and of IGF-1, suggesting that the activity is not due to either of these known myogenic growth factors. Both normal and DM fibroblast-like cells produced myoblast trophic activity when the cells were proliferating, but did not produce myotrophic activity when the fibroblasts were mitotically quiescent. These findings indicate that the proliferative state of the connective tissue cells in muscle may have a controlling influence on myoblast proliferation and differentiation during development.     

Constitutive expression of hyaluronan binding protein 1 (HABP1/p32/gC1qR) in normal fibroblast cells perturbs its growth characteristics and induces apoptosis

Hyaluronan binding protein 1 (HABP1) is a ubiquitously expressed multifunctional phospho-protein that interacts with a wide range of ligands and is implicated in cell signalling. Recently, we have reported that HABP1 is an endogenous substrate for MAP kinase and upon mitogenic stimulation it is translocated to the nucleus in a MAP kinase-dependent manner (Biochem. Biophys. Res. Commun. 291(4) (2002) 829-837). This prompted us to investigate the role of HABP1 in cell growth or otherwise in low MAP kinase background. We demonstrate that HABP1, when overexpressed in normal rat skin fibroblasts, remained in the cytosol, primarily concentrated around the nuclear periphery. However, HABP1 overexpressing cells showed extensive vacuolation and reduced growth rate, which was corrected by frequent medium replenishment. Further investigation revealed that HABP1 overexpressing cells undergo apoptosis, as detected by TUNEL assay, induction of Bax expression, and FACS analysis, and they failed to enter into the S-phase. Periodic medium supplementation prevented these cells from undergoing apoptotic death. We also demonstrate that upon induction of apoptosis in HeLa cells by cisplatin, HABP1 level is upregulated, indicating a correlation between HABP1 and cell death in a normal cellular environment.     

Inhibition of tumor growth with a vaccine based on xenogeneic homologous fibroblast growth factor receptor-1 in mice

Angiogenesis is important for the growth of solid tumors. The breaking of the immune tolerance against the molecule associated with angiogenesis should be a useful approach for cancer therapy. However, the immunity to self-molecules is difficult to elicit by a vaccine based on autologous or syngeneic molecules due to immune tolerance. Basic fibroblast growth factor (bFGF) is a specific and potent angiogenic factor implicated in tumor growth. The biological activity of bFGF is mediated through interaction with its high-affinity receptor, fibroblast growth factor receptor-1 (FGFR-1). In this study, we selected Xenopus FGFR-1 as a model antigen by the breaking of immune tolerance to explore the feasibility of cancer therapy in murine tumor models. We show here that vaccination with Xenopus FGFR-1 (pxFR1) is effective at antitumor immunity in three murine models. FGFR-1-specific autoantibodies in sera of pxFR1-immunized mice could be found in Western blotting analysis. The purified immunoglobulins were effective at the inhibition of endothelial cell proliferation in vitro and at the antitumor activity in vivo. The antitumor activity and production of FGFR-1-specific autoantibodies could be abrogated by depletion of CD4+ T lymphocytes. Histological examination revealed that the autoantibody was deposited on the endothelial cells within tumor tissues from pxFR1-immunized mice, and intratumoral angiogenesis was significantly suppressed. Furthermore, the inhibition of angiogenesis could also be found in alginate-encapsulate tumor cell assay. These observations may provide a new vaccine strategy for cancer therapy through the induction of autoimmunity against FGFR-1 associated with angiogenesis in a cross-reaction.     

Enhanced MYCN oncogene expression in human neuroblastoma cells is associated with altered FGF receptor expression and cellular growth response to basic FGF.

We have examined the effect of human basic fibroblast growth factor (bFGF) on the proliferation of human neuroblastoma cells with normal and enhanced MYCN oncogene expression. bFGF stimulated the proliferation of the neuroblastoma cells with enhanced, but not normal, MYCN expression. Both cell species express FGFR-1, but not FGFR-2, receptors and both harbor FGF receptor species of Mr 145.000, but they differ in their pattern of lower and higher-molecular weight FGF receptor species. Our results demonstrate that enhanced MYCN expression confers to neuroblastoma cells the ability to respond to bFGF, possibly by inducing functional FGF receptors. This mechanism may contribute to the advanced malignant phenotype of human neuroblastomas with enhanced MYCN expression.     

Activation of a transfected FGFR-1 receptor in Madin-Darby epithelial cells results in a reversible loss of epithelial properties

Basic fibroblast growth factor (bFGF) is a potent mitogen for a wide variety of cell types derived from mesoderm and neuroectoderm. The activity of bFGF is mediated by several types of closely related receptors belonging to the tyrosine-kinase family of receptors. We have found that Madin-Darby epithelial cells (MDCK) do not seem to produce bFGF or bFGF receptors. High level expression of human bFGF cDNA in these cells did not produce any mitogenic or morphological effects. Expression of the mouse-derived cDNA encoding FGF receptor-1 (FGFR-1) in MDCK cells resulted in the acquisition of a fibroblast-like morphology when the transfected cells were cultured at low density in the presence of 0.6% fetal calf serum and 20 ng/ml bFGF. Acidic fibroblast growth factor (aFGF) also induced these morphological changes but not keratinocyte growth factor. The morphological effect was not accompanied by increased bFGF-induced cell proliferation and did not result in the loss of epithelial cell markers such as cytokeratins. However, the morphological transition was accompanied by changes in the intracellular distribution of actin. In spite of these changes the transfected cells formed monolayers even in the presence of bFGF. Coexpression of bFGF and FGFR-1 in the MDCK cells resulted in similar morphological effects that were not dependent upon exogenous bFGF. These morphological effects were mimicked by exposure of MDCK cells to either orthovanadate or phorbol ester. Parental and FGFR-1 -expressing MDCK cells formed monolayers tht displayed high electrical resistance. Incubation of monolayers of FGFR-1-transfected cells with bFGF resulted in the loss of trans-epithelial resitance. Monolayers of parental MDCK cells did not lose their trans-epithelial resistance in response to bFGF, although exposure to phorbol ester did result in the loss of their trans-epithelial resistance, indicating that the effects on the trans-epithelial resistance are mediated by protein kinase C activation. Interestingly, orthovanadate did not cause a loss of transepithelial resistance, suggesting that the loss of trans-epithelial resistance is separable from the morphological transition. © 1995 Wiley-Liss, Inc.     

Growth factor-initiated proliferation of mouse embryonic fibroblasts induces cytotoxicity by natural killer cells and by a non-cytolysin cytotoxin in natural killer granules

NK cells preferentially kill normal embryonic fibroblasts. Because embryonic cells are growth factor responsive and maintain high proliferative rates, we examined the requirement for growth factor-initiated proliferation for NK susceptibility. Murine embryonic fibroblasts made quiescent in defined medium lacking growth factors were relatively resistant to NK cytolysis. However, reinitiation of proliferation with basic fibroblast growth factor (bFGF) or epidermal growth factor enhanced lysis in a dose-dependent fashion. TGF-beta, which blocked cell division, did not enhance cytotoxicity. Additionally, growth inhibition by prolonged incubation at confluence suppressed lysis. The enhanced NK cytotoxicity of bFGF-stimulated fibroblasts was caused by a post-binding event because no difference in cold target inhibition could be demonstrated with bFGF-treated cells. NK cytotoxicity has largely been attributed to the action of cytotoxins released from cytoplasmic granules. In a 51Cr release assay, bFGF-treated fibroblasts were insensitive to NK granules isolated from the RNK large granular lymphocyte leukemia. However, these same cells exhibited marked sensitivity to lysis in an 18-h adhesion assay normally utilized to detect TNF-alpha. With the use of this assay, a dose-dependent increase in sensitivity of bFGF-treated fibroblasts was observed, whereas quiescent fibroblasts were resistant to the action of isolated NK granules. Granule cytotoxicity was not caused by cytolysin/perforin because inactivation of granule hemolytic activity with CaCl2 did not affect fibroblast killing, and bFGF-treated cells were insensitive to purified cytolysin/perforin. This suggested that another granule associated cytotoxin was responsible for enhanced NK sensitivity of actively proliferating fibroblasts.     

Growth-stimulatory action of plasma membrane-associated growth factor. A synergistic effect with platelet-poor plasma

Growth factor activity was partially purified from mouse liver plasma membranes and its growth-stimulatory action on cultured mouse fibroblasts was studied. The plasma membrane-associated growth factor (PMGF) was unable to support the proliferation of mouse fibroblasts in monolayer when added as the sole source of growth factor. However, it stimulated the growth of fibroblasts in the presence of CM-Sephadex-treated human platelet-poor plasma (h-CMP) which by itself is not growth-stimulatory. The stimulation of DNA synthesis in quiescent fibroblasts was also observed upon the addition of PMGF and h-CMP. Under the same conditions, both platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) showed the same effect as did PMGF. The synergistic action of h-CMP with PMGF on quiescent cells was partially reproduced by insulin at microgram quantities or by insulin-like growth factor I(IGF-I) at nanogram quantities. Thus, the data presented here indicates that the action of PMGF is similar to that of the family of growth factors termed 'competence factor', and distinct from that of plasma growth factors termed 'progression factor'.     

Isolation and characteristics of urokinase-type plasminogen activator from a culture of human embryo lung fibroblasts

Plasminogen activator from conditioned medium of human embryonal lung fibroblasts was purified by phosphocellulose P11 chromatography, followed by p-aminobenzamidine-agarose chromatography. Two forms of plasminogen activators were separated by chromatography on the heparin-sepharose. The high molecular weight form (53 kDa) with specific activity 130 000 IU/mg consists of two polypeptide chains (31 kDa and 20 kDa) and exhibits strong affinity for fibrin-celite, lysine-sepharose and heparin-sepharose. The low molecular weight form (32 kDa, 190 000 IU/mg) also binds to these sorbents, but more weakly, and its properties are very similar to those of low molecular weight urokinase. Activity of both forms of plasminogen activators are inhibited by monoclonal antibodies against urokinase. A number of enzymological chromatographic and immunological properties indicates, that the plasminogen activator from lung fibroblasts is of urokinase type.     

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.     

Fibroblast growth factor stimulates protein kinase C in quiescent 3T3 cells without Ca2+ mobilization or inositol phosphate accumulation

To elucidate the transmembrane signalling processes initiated by fibroblast growth factor (FGF), we have studied the effect of recombinant basic FGF (bFGF) on various early events associated with mitogenesis in Swiss 3T3 fibroblasts. bFGF, at mitogenic concentrations, neither induced Ca2+ mobilization from intracellular stores nor increased the accumulation of inositol phosphates. In contrast, bFGF stimulated the phosphorylation of the Mr 80,000 (80K) cellular protein which is a major substrate of protein kinase C. This effect was potentiated by the diacylglycerol kinase inhibitor R59022. Two-dimensional polyacrylamide gel electrophoresis and phosphopeptide mapping showed that the 80K phosphoproteins generated in response to bFGF, bombesin, and phorbol 12,13-dibutyrate were indistinguishable. Down-regulation of protein kinase C prevented bFGF stimulation of 80K phosphorylation. Other protein kinase C-dependent early events such as transmodulation of the epidermal growth factor receptor, cytoplasmic alkalinization, inhibition of vasopressin induced increase in cytosolic [Ca2+], and enhancement of cAMP accumulation in response to forskolin were also induced by bFGF. Similar results were obtained when bFGF was added to quiescent cultures of tertiary mouse embryo fibroblasts. We conclude that bFGF stimulates protein kinase C through a signal transduction pathway distinct from inositol phospholipid turnover and Ca2+ mobilization.     

Noninvasive dynamic fluorescence imaging of human melanomas reveals that targeted inhibition of bFGF or FGFR-1 in melanoma cells blocks tumor growth by apoptosis

BACKGROUND: Two prominent biological features of the advanced stages of human melanoma are their high degree of vascularity and high-level expression of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGFR-1). Given these characteristics, human melanoma serves as an ideal model to address an important question regarding the efficacy of angiogenesis-based cancer therapy. To induce tumor growth arrest and regression, does it suffice to block expression of bFGF and/or FGFR-1 in only the melanoma cells, or is it essential to inhibit expression of bFGF and/or FGFR-1 in both the melanoma cells and the melanoma cell-interspersing vasculature? MATERIALS AND METHODS: Primary and metastatic human melanomas, grown as subcutaneous tumors in nude mice, were injected twice a week with vector constructs containing the human tyrosinase promoter and antisense- oriented human bFGF or FGFR-1 cDNA. On alternating days, the bFGF and FGFR-1 antisense-targeted tumors received injections of cyanine fluorochrome-conjugated antibodies to a human melanoma and mouse blood vessel marker. Noninvasive, dynamic fluorescence imaging was used to document the cellular events that took place inside the tumors as the result of blocking expression of bFGF or FGFR-1 in the melanoma cells. RESULTS: In vivo, ex vivo, and in vitro fluorescence imaging of the bFGF and FGFR-1 antisense-targeted tumors demonstrated that inhibiting bFGF and FGFR-1 signaling in only the melanoma cells suffices to inhibit tumor growth due to massive induction of melanoma cell apoptosis. CONCLUSIONS: The investigations presented in this study document that inhibiting expression of bFGF or FGFR-1 in only the melanoma cells is as effective in blocking tumor growth as simultaneously inhibiting bFGF or FGFR-1 synthesis in the melanoma cells and the melanoma cell-interspersing vasculature. Furthermore, blocking expression of bFGF or FGFR-1 in the melanoma cells did not lead to activation or increased production of another angiogenic molecule, suggesting the absence of a "salvage pathway" that can circumvent or rescue the blockage of bFGF/FGFR-1 in the melanoma cells.     

Growth of normal human keratinocytes and fibroblasts in serum-free medium is stimulated by acidic and basic fibroblast growth factor

Keratinocytes and fibroblasts isolated from human neonatal foreskin can be plated and grown through multiple rounds of division in vitro under defined serum-free conditions. We utilized these growth conditions to examine the mitogenic potential of acidic and basic fibroblast growth factor (aFGF and bFGF) on these cells. Our results demonstrate that both aFGF and bFGF can stimulate the proliferation of keratinocytes and fibroblasts. aFGF is a more potent mitogen than bFGF for keratinocytes. In contrast, bFGF appears to be more potent than aFGF in stimulating the growth of fibroblast cultures. Heparin sulfate (10 micrograms/ml) dramatically inhibited the ability of bFGF to stimulate the proliferation of keratinocytes. In comparison, heparin slightly inhibited the stimulatory effect of aFGF and had no effect on epidermal growth factor (EGF) stimulation in keratinocyte cultures. In fibroblast cultures the addition of heparin enhanced the mitogenic effect of aFGF, had a minimal stimulatory effect on the mitogenic activity of bFGF, and had no effect on EGF-stimulated growth. Our results demonstrate that the proliferation in vitro of two normal cell types found in the skin can be influenced by aFGF and bFGF and demonstrate cell-type specific differences in the responsiveness of fibroblasts and keratinocytes to these growth factors and heparin.     

Role of senescent fibroblasts on alkali-induced corneal neovascularization

Cellular senescence acts as a potent regulator of tumor suppression and fibrosis limitation; however, its contribution and crosstalk with neovascularization during normal wound healing has not been examined. Here, we explored the role of senescent fibroblasts on neovascularization with a mouse model of alkali-induced corneal wound healing. Senescent cells accumulated in corneal stroma from day 7 to 27 after alkali burn and peaked on day 14, which was consistent with the development of corneal neovascularization (CNV). In vitro and in vivo assays confirmed that the senescent cells were derived primarily from activated corneal fibroblasts. Furthermore, senescent corneal fibroblasts exhibited enhanced synthesis and secretion of extracellular matrix-degrading enzymes (matrix metalloproteinases 2, 3, and 14 and tissue- and urokinase-type plasminogen activators) and angiogenic factors (vascular endothelial growth factor) and decreased expression of anti-angiogenic factors (pigment epithelium-derived factor and thrombospondins), which supported the proliferation, migration, and promotion of tube formation of vascular endothelial cells. Intrastromal injection of premature senescent fibroblasts induced CNV earlier than that of normal fibroblasts, while matrix metalloproteinase inhibitors blocked the early onset of senescent cell-induced CNV. Therefore, senescent fibroblasts promoted the alkali-induced CNV partially via the enhanced secretion of matrix metalloproteases.