Sulfated Derivatives of Escherichia coli K5 Polysaccharides as Modulators of Fibroblast Growth Factor Signaling
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m304208200
ISSN1083-351X
AutoresMarjut Borgenström, Markku Jalkanen, Markku Salmivirta,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoHeparan sulfate (HS) proteoglycans are intimately involved in the regulation of fibroblast growth factor (FGF) signaling. HS and the related glycosaminoglycan heparin interact with FGFs and FGF receptors (FGFRs), and it is believed that both interactions are required for productive FGF signaling. Attempts to inhibit FGF activity have been made with modified heparin preparations, various heparin-like polysaccharide analogues and other polyanionic molecules, which may all act by interfering with the physiological HS-FGF-FGFR interactions on the cell surface. Here, we have studied the potential of sulfated derivatives of a bacterial polysaccharide (capsular polysaccharide from Escherichia coli K5 (K5PS)) in the modulation of FGF-heparin/HS interactions and FGF signaling. We demonstrate that O-sulfated and N,O-sulfated species of K5PS, with high degrees of sulfation, displaced FGF-1, FGF-2, and FGF-8b from heparin. However, only O-sulfated K5PS efficiently inhibited the FGF-induced proliferation of S115 mammary carcinoma cells and 3T3 fibroblasts, whereas N,O-sulfated K5PS had little or no inhibitory effect. Studies with CHO677 cells lacking endogenous HS, as well as with chlorate-treated S115 cells expressing undersulfated HS, indicated that whereas exogenously administered heparin and N,O-sulfated K5PS restored the cellular response toward FGF stimulation, O-sulfated K5PS was largely devoid of such stimulatory activity. Our data suggest that highly O-sulfated species of K5PS may be efficient inhibitors of FGF signaling. Heparan sulfate (HS) proteoglycans are intimately involved in the regulation of fibroblast growth factor (FGF) signaling. HS and the related glycosaminoglycan heparin interact with FGFs and FGF receptors (FGFRs), and it is believed that both interactions are required for productive FGF signaling. Attempts to inhibit FGF activity have been made with modified heparin preparations, various heparin-like polysaccharide analogues and other polyanionic molecules, which may all act by interfering with the physiological HS-FGF-FGFR interactions on the cell surface. Here, we have studied the potential of sulfated derivatives of a bacterial polysaccharide (capsular polysaccharide from Escherichia coli K5 (K5PS)) in the modulation of FGF-heparin/HS interactions and FGF signaling. We demonstrate that O-sulfated and N,O-sulfated species of K5PS, with high degrees of sulfation, displaced FGF-1, FGF-2, and FGF-8b from heparin. However, only O-sulfated K5PS efficiently inhibited the FGF-induced proliferation of S115 mammary carcinoma cells and 3T3 fibroblasts, whereas N,O-sulfated K5PS had little or no inhibitory effect. Studies with CHO677 cells lacking endogenous HS, as well as with chlorate-treated S115 cells expressing undersulfated HS, indicated that whereas exogenously administered heparin and N,O-sulfated K5PS restored the cellular response toward FGF stimulation, O-sulfated K5PS was largely devoid of such stimulatory activity. Our data suggest that highly O-sulfated species of K5PS may be efficient inhibitors of FGF signaling. Fibroblast growth factors (FGFs) 1The abbreviations used are: FGFfibroblast growth factorFGFRFGF receptorCHOChinese hamster ovaryDCCdextran-coated charcoalERKextracellular signal-regulated protein kinaseFCSfetal calf serumGlcNglucosamineGlcAglucuronic acidHSheparan sulfateHSPGheparan sulfate proteoglycanIdoAiduronic acidDMEMDulbecco's modified Eagle's mediumLSDMEMlow sulfate DMEMPBSphosphate-buffered saline. are involved in the proliferation, migration, and differentiation of many cell types. FGFs have been implicated in physiological processes such as embryonic development (1.Szebenyi G. Fallon J.F. Int. Rev. Cytol. 1999; 185: 45-106Crossref PubMed Google Scholar) and tissue regeneration but also in a number of pathological conditions including cancer, inflammatory diseases, fibrosis, etc. (2.Basilico C. Moscatelli D. Adv. Cancer Res. 1992; 59: 115-165Crossref PubMed Scopus (1057) Google Scholar). FGF signals are thought to stimulate tumor cell proliferation in an autocrine or paracrine fashion and various types of malignant tumors have been shown to overexpress FGFs and FGF receptors (FGFRs) (2.Basilico C. Moscatelli D. Adv. Cancer Res. 1992; 59: 115-165Crossref PubMed Scopus (1057) Google Scholar, 3.McKeehan W.L. Wang F. Kan M. Prog. Nucleic Acids Res. Mol. Biol. 1998; 59: 135-176Crossref PubMed Scopus (363) Google Scholar). FGFs are also potent angiogenic factors that promote tumor neovascularization by stimulating the proliferation and migration of endothelial cells (4.Folkman J. Klagsbrun M. Science. 1987; 235: 442-447Crossref PubMed Scopus (4049) Google Scholar). Conceivably, development of agents that antagonize FGF signaling might provide means to influence multiple aspects of tumor progression. fibroblast growth factor FGF receptor Chinese hamster ovary dextran-coated charcoal extracellular signal-regulated protein kinase fetal calf serum glucosamine glucuronic acid heparan sulfate heparan sulfate proteoglycan iduronic acid Dulbecco's modified Eagle's medium low sulfate DMEM phosphate-buffered saline. FGFs exert their biological activities by binding to FGFRs that leads to FGFR dimerization, phosphorylation of the cytoplasmic receptor domains, and activation of intracellular signal transduction pathways (5.Klint P. Claesson-Welsh L. Front. Biosci. 1999; 4: 165-177Crossref PubMed Google Scholar). FGF activity is critically dependent on the sulfated glycosaminoglycan heparan sulfate (HS) (6.Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1707Crossref PubMed Scopus (1291) Google Scholar, 7.Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Abstract Full Text PDF PubMed Scopus (2089) Google Scholar), a component of HS proteoglycans (HSPGs) found on cell surfaces and in the extracellular matrix (8.Bernfield M. Götte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2330) Google Scholar, 9.Iozzo R.V. Annu. Rev. Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1346) Google Scholar). HS has been demonstrated to interact with FGFs as well as with the extracellular domains of FGFRs (10.Powers C.J. McLeskey S.W. Wellstein A. Endocr. Relat. Cancer. 2000; 7: 165-197Crossref PubMed Scopus (1129) Google Scholar). HS likely plays a critical role in the formation of "ternary complexes" consisting of FGF, FGFR, and HS, in which HS concomitantly interacts with both the growth factor and receptor components of the complex (11.Pellegrini L. Burke D.F. von Delft F. Mulloy B. Blundell T.L. Nature. 2000; 407: 1029-1034Crossref PubMed Scopus (630) Google Scholar, 12.Schlessinger J. Plotnikov A.N. Ibrahimi O.A. Eliseenkova A.V. Yeh B.K. Yayon A. Linhardt R.J. Mohammadi M. Mol. Cell. 2000; 6: 743-750Abstract Full Text Full Text PDF PubMed Scopus (973) Google Scholar). Cells lacking endogenous HS thus respond poorly to FGFs, whereas the response can be readily restored by addition of heparin (6.Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1707Crossref PubMed Scopus (1291) Google Scholar, 7.Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Abstract Full Text PDF PubMed Scopus (2089) Google Scholar, 13.Moscatelli D. J. Biol. Chem. 1992; 267: 25803-25809Abstract Full Text PDF PubMed Google Scholar, 14.Fannon M. Forsten K.E. Nugent M.A. Biochemistry. 2000; 39: 1434-1445Crossref PubMed Scopus (140) Google Scholar, 15.Forsten K.E. Fannon M. Nugent M.A. J. Theor. Biol. 2000; 205: 215-230Crossref PubMed Scopus (35) Google Scholar). By contrast, in cells which express endogenous HSPGs, exogenously administered heparin may inhibit rather than stimulate FGF signaling, presumably by perturbing the interactions of cellular HSPGs with FGFs/FGFRs (13.Moscatelli D. J. Biol. Chem. 1992; 267: 25803-25809Abstract Full Text PDF PubMed Google Scholar, 14.Fannon M. Forsten K.E. Nugent M.A. Biochemistry. 2000; 39: 1434-1445Crossref PubMed Scopus (140) Google Scholar, 15.Forsten K.E. Fannon M. Nugent M.A. J. Theor. Biol. 2000; 205: 215-230Crossref PubMed Scopus (35) Google Scholar). In this paper, we have investigated the potential of semisynthetic, heparin-like polysaccharides as inhibitors of FGF signaling. The capsular polysaccharide of Escherichia coli K5 bacteria (hereafter denoted as K5 polysaccharide or K5PS) has the same structure as the primary polymerization product of heparin/HS biosynthesis, consisting of alternating d-glucuronic acid and d-N-acetylglucosamine units (GlcAα1,4GlcNAc)n (16.Vann W.F. Schmidt M.A. Jann B. Jann K. Eur. J. Biochem. 1981; 116: 359-364Crossref PubMed Scopus (213) Google Scholar). K5PS can be subjected to various degrees of chemical N-deacetylation/N-sulfation as well as O-sulfation (17.Casu B. Grazioli G. Razi N. Guerrini M. Naggi A. Torri G. Oreste P. Tursi F. Zoppetti G. Lindahl U. Carbohydr. Res. 1994; 263: 271-284Crossref PubMed Scopus (99) Google Scholar, 18.Leali D. Belleri M. Urbinati C. Coltrini D. Oreste P. Zoppetti G. Ribatti D. Rusnati M. Presta M. J. Biol. Chem. 2001; 276: 37900-37908Abstract Full Text Full Text PDF PubMed Google Scholar), to yield different heparin/HS-like semisynthetic polysaccharides (Fig. 1). Highly sulfated species of K5PS have been shown to bind to FGF-2 and inhibit FGF-2 induced endothelial cell proliferation, probably by interfering with the formation of FGF-2·FGFR·HS complexes (18.Leali D. Belleri M. Urbinati C. Coltrini D. Oreste P. Zoppetti G. Ribatti D. Rusnati M. Presta M. J. Biol. Chem. 2001; 276: 37900-37908Abstract Full Text Full Text PDF PubMed Google Scholar). Interestingly, the N,O-sulfated polysaccharide species have also displayed anti-angiogenic activity in chick chorioallontoic membrane assays (18.Leali D. Belleri M. Urbinati C. Coltrini D. Oreste P. Zoppetti G. Ribatti D. Rusnati M. Presta M. J. Biol. Chem. 2001; 276: 37900-37908Abstract Full Text Full Text PDF PubMed Google Scholar) (see "Discussion"). The results presented in this paper provide novel data with regard to the properties of sulfated K5PSs as FGF inhibitors. We have assessed the previously described series (18.Leali D. Belleri M. Urbinati C. Coltrini D. Oreste P. Zoppetti G. Ribatti D. Rusnati M. Presta M. J. Biol. Chem. 2001; 276: 37900-37908Abstract Full Text Full Text PDF PubMed Google Scholar) of chemically sulfated derivatives of K5PS for their capability to interact with FGFs and modulate FGF-induced cellular responses. Our data suggest that whereas various species of sulfated K5PS bind FGF-1, FGF-2 and FGF-8b, only a highly O-sulfated preparation was an efficient inhibitor of the biological activity of FGFs at low (≤1 μg/ml) concentrations. Experiments in cells lacking endogenous active HS suggest that the differential inhibitory capacity of the preparations derive from differences in their agonist/antagonist properties with regard to FGF signaling. Thus, compounds such as O-sulfated K5PS deserve to be further explored as inhibitors of FGF-dependent tumor cell proliferation. Materials—Cell culture media, chemicals for the preparation of low sulfate DMEM (LSDMEM) (6.Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1707Crossref PubMed Scopus (1291) Google Scholar), testosterone, and the monoclonal antibodies against ERKs were from Sigma. Sodium chlorate was purchased from Merck, and [methyl-3H]thymidine was from PerkinElmer Life Sciences. FGF-1 and FGF-2 were from Peprotech. The expression vector for FGF-8b was kindly provided by Dr. Graig MacArthur (Washington University). FGF-8b was produced and purified as described elsewhere (19.MacArthur C.A. Lawshé A. Xu J. Santos-Ocampo S. Heikonheimo M. Chellaiah A.T. Ornitz D.M. Development (Camb.). 1995; 121: 3603-3613PubMed Google Scholar, 20.Loo B.-M. Salmivirta M. J. Biol. Chem. 2002; 277: 32616-32623Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The K5PS preparations used in this study were kindly provided by Drs. Giorgio Zoppetti and Pasqua Oreste. The synthesis and characterization of the K5PSs have been described elsewhere (18.Leali D. Belleri M. Urbinati C. Coltrini D. Oreste P. Zoppetti G. Ribatti D. Rusnati M. Presta M. J. Biol. Chem. 2001; 276: 37900-37908Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly, a series of sulfated K5PSs has been generated by chemical N-deacetylation/N-sulfation and/or O-sulfation of the unmodified K5PS structure (GlcA-GlcNAc)n (18.Leali D. Belleri M. Urbinati C. Coltrini D. Oreste P. Zoppetti G. Ribatti D. Rusnati M. Presta M. J. Biol. Chem. 2001; 276: 37900-37908Abstract Full Text Full Text PDF PubMed Google Scholar). The modified compounds included fully N-sulfated K5PS without (K5-NS) or with (K5-NSOS) O-sulfation as well as O-sulfated K5PS (K5-OS). The O-sulfation of K5-NSOS was somewhat lower than that of K5-OS, such that their overall degrees of sulfation were similar (3.84 versus 3.77 sulfate groups/disaccharide unit, respectively). However, in both N-sulfated and N-acetylated preparations the degree of GlcN 6-O-sulfation was nearly 100%, such that the differences in O-sulfation were found in the 2-O- and 3-O-sulfation of the GlcA units and in the 3-O-sulfation of the GlcN residues. The mean molecular masses of the preparations were in the range of 10–15 kDa. Cell Cultures—S115 mouse mammary carcinoma cells (21.Leppä S. Härkönen P. Jalkanen M. Cell Regul. 1991; 2: 1-11Crossref PubMed Scopus (64) Google Scholar) were maintained in DMEM supplemented with 5% heat-inactivated fetal calf serum (FCS), 1 mm sodium pyruvate, 1 mm l-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 10 nm testosterone. For experiments, the cells were grown in medium containing 4% dextran-coated charcoal-treated FCS (DCC-FCS) (22.Noguchi S. Uchida N. Sato B. Koyama H. Matsumoto K. J. Steroid Biochem. 1989; 32: 479-483Crossref PubMed Scopus (9) Google Scholar) instead of FCS, followed by a 24-h serum starvation in a 1:1 mixture of serum-free Ham's F-12 and DMEM (23.Tanaka A. Matsumoto K. Nishizawa Y. Lu J. Yamanishi H. Maeyama M. Nonomura N. Uchida N. Sato B. J. Steroid Biochem. Mol. Biol. 1990; 37: 23-29Crossref PubMed Scopus (11) Google Scholar) and treatment with testosterone/FGF and K5PSs in various combinations, as specified in the figure legends. To assess the cellular DNA synthesis, [methyl-3H]thymidine (2 μCi/ml) was added to the culture medium for the last2hofthe incubation. Subsequently, the cells were washed and solubilized into 1 m NaOH followed by quantification of the incorporated radioactivity in a beta counter (Wallac, Turku, Finland). Alternatively, cell growth was assessed by staining the cells with crystal violet (24.Gillies R.J. Didier N. Denton M. Anal. Biochem. 1986; 159: 109-113Crossref PubMed Scopus (607) Google Scholar) after various periods of incubation. In some experiments, the added saccharide was withdrawn after a 24-h incubation by change of the culture medium, after which the cells were allowed to grow for 48 h before crystal violet staining. The treatment of S115 cells with sodium chlorate has been described elsewhere (25.Borgenström M. Tienhaara A. Spillmann D. Salmivirta M. Jalkanen M. Exp. Cell Res. 2001; 264: 307-314Crossref PubMed Scopus (8) Google Scholar). Briefly, cells were cultured in LSDMEM containing 5% DCC-FCS and 30 mm sodium chlorate, followed by trypsinization, replating, and starvation in LSDMEM supplemented with 0.1% DCC-FCS and 30 mm sodium chlorate. Testosterone and K5PSs were added to the cells that were subsequently assessed for [methyl-3H]thymidine incorporation as described above. HS-deficient Chinese hamster ovary (CHO) cells (line 677; Ref. 26.Esko J. Curr. Opin. Cell Biol. 1991; 3: 805-816Crossref PubMed Scopus (183) Google Scholar), and the same cells stably transfected with cDNA encoding FGFR-1 or FGFR-4 (20.Loo B.-M. Salmivirta M. J. Biol. Chem. 2002; 277: 32616-32623Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), were cultured in α-minimum Eagle's medium containing 5% FCS, whereas mouse NIH-3T3 fibroblasts were cultured in DMEM supplemented with 5% FCS. For the [methyl-3H]thymidine incorporation experiments, both types of cells were serum-starved and FGF-stimulated according to the protocol described above. Analysis of ERK Phosphorylation—To analyze the phosphorylation of ERK 1/2, S115 cells were cultured as described above and lysed in Laemmli buffer at the time points of 2, 10, 20, and 60 min followed by fractionation of the lysates on 10% SDS-PAGE. After electrophoresis, the samples were transferred onto nitrocellulose by semidry blotting. Sample loading was studied by protein staining with Ponceau S. The membranes were blocked in PBS (0.15 m NaCl in 20 mm phosphate buffer, pH 7.4) containing 5% nonfat dry milk and 0.4% Tween 20 at room temperature for 1 h followed by incubation with mouse monoclonal antibodies against phosphorylated ERK 1/2 (0.1–0.2 μg/ml) overnight at 4 °C. After several washes with 0.4% Tween 20 in PBS, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies against mouse immunoglobulins (dilution 1:3000) for 1 h at room temperature. The membranes were washed several times followed by visualization of the bound antibodies using an enhanced chemiluminescence detection kit (Amersham Biosciences). Subsequently, the membranes were soaked in 0.1 m glycine, pH 2.5, and re-probed with monoclonal antibodies against ERK 2. FGF Binding Assay—To study the ability of sulfated K5PSs to displace [3H]heparin from FGF, [3H]heparin (20,000 cpm), sulfated K5 polysaccharides (0.05–5 μg/ml), and FGFs (2.5 μg/ml) were incubated in PBS containing 0.1 mg/ml bovine serum albumin at room temperature for 2 h. The reaction mixtures were rapidly passed through PBS-washed nitrocellulose filters (Sartorius, 25-mm diameter, pore size: 0.45 μm) using a vacuum suction apparatus followed by two washes of the filters with PBS. Proteins and protein-bound saccharides bind to the filter, whereas unbound saccharides pass through (27.Maccarana M. Lindahl U. Glycobiology. 1993; 3: 271-277Crossref PubMed Scopus (110) Google Scholar). The filter-bound [3H]heparin was released with 2 m NaCl and quantified by scintillation counting. Inhibition of Testosterone-induced Proliferation of S115 Cells by Sulfated K5PSs—Stimulation of S115 cells with testosterone leads to a morphological change of the cells from epithelial to fibroblast-like phenotype, increased proliferation, ability to grow in soft agar, and disorganization of the actin filament network (28.Darbre P.D. King R.J.B. Cancer Treat. Res. 1988; 40: 307-341Crossref PubMed Scopus (42) Google Scholar). The process has been shown to involve testosterone-induced expression of FGF-8b (29.Tanaka A. Miyamoto K. Minamino N. Takeda M. Sato B. Matsuo H. Matsumoto K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8928-8932Crossref PubMed Scopus (388) Google Scholar, 30.Ruohola J.K. Valve E.M. Vainikka S. Alitalo K. Härkönen P.K. Endocrinology. 1995; 136: 2179-2188Crossref PubMed Google Scholar) and to require the presence of sulfated HSPGs (25.Borgenström M. Tienhaara A. Spillmann D. Salmivirta M. Jalkanen M. Exp. Cell Res. 2001; 264: 307-314Crossref PubMed Scopus (8) Google Scholar). Previous data indicate that such proliferation, as measured by incorporation of [3H]thymidine into cellular DNA, can be inhibited by heparin (25.Borgenström M. Tienhaara A. Spillmann D. Salmivirta M. Jalkanen M. Exp. Cell Res. 2001; 264: 307-314Crossref PubMed Scopus (8) Google Scholar, 31.Kasayama S. Sumitani S. Tanaka A. Yamanishi H. Nakamura N. Matsumoto K. Sato B. J. Cell. Physiol. 1991; 148: 260-266Crossref PubMed Scopus (19) Google Scholar). We studied whether sulfated K5PSs were able to influence the testosterone-induced proliferation of S115 cells. The results indicated that K5-OS inhibited the proliferation efficiently (Fig. 2A), whereas K5-NS, K5-NSOS, or unmodified K5PS had little or no inhibitory activity. To assess the inhibitory capacity of K5-OS in a more detail, a range of polysaccharide concentrations was studied in the same cell proliferation assay and their inhibitory capacities were compared with that of heparin (Fig. 2B). The results showed that K5-OS caused a dose-dependent growth inhibition at low concentrations (0.1–1 μg/ml), whereas ∼10-fold higher concentrations of K5-NSOS were required for corresponding inhibitory effects. Notably, heparin had only a limited effect on cell proliferation. These data suggest that the structurally distinct preparations of sulfated K5PSs differed markedly in their inhibitory capacity, albeit their overall degrees of sulfation were similar. The results also demonstrate that sulfated K5PSs inhibit the testosterone-induced proliferation of S115 cells much more efficiently than heparin. We further assessed the effect of K5-OS on cell proliferation by treating S115 cells with K5-OS and testosterone for various periods of time and determining the relative cell numbers by crystal violet staining. Whereas testosterone treatment induced cell proliferation, cells treated with a combination of K5-OS and testosterone displayed less proliferation than control cells without testosterone treatment (Fig. 3A), confirming the idea that K5-OS inhibited testosterone-induced proliferation of S115 cells. Unmodified K5PS did not affect the testosterone response and heparin had only a minor effect, in agreement with the [3H]thymidine incorporation data presented above. To study whether the growth inhibition by K5-OS was reversible, cells were grown in the presence of K5-OS and testosterone for 24 h, after which the medium were changed, and the cells were cultured in the presence of testosterone alone for additional 48 h. Following the withdrawal of K5-OS, increased cell proliferation was observed (Fig. 3B) suggesting that the antiproliferative effect was reversible in nature. Inhibition of FGF-8b-induced Proliferation and ERK Phosphorylation by K5-OS—The transforming effect of testosterone on S115 cells has been demonstrated to involve up-regulation of FGF-8b expression (29.Tanaka A. Miyamoto K. Minamino N. Takeda M. Sato B. Matsuo H. Matsumoto K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8928-8932Crossref PubMed Scopus (388) Google Scholar). We proceeded to test the effects of K5PSs on S115 cells specifically stimulated with FGF-8b, which induced an increased DNA synthesis in serum-starved S115 cells (Fig. 4A). The effect of FGF-8b was efficiently inhibited by low concentrations of K5-OS (0.1–1 μg/ml), whereas K5-NSOS caused only a partial inhibition (Fig. 4B). The non-O-sulfated species of K5PS did not inhibit the proliferative effect of FGF-8b. To study the effect of K5-OS on the downstream signaling initiated by FGF stimulation, we analyzed its ability to inhibit FGF-8b induced phosphorylation of ERK, a component of the mitogen-activated protein kinase pathway. Serum-starved S115 cells were treated with FGF-8b and K5-OS, followed by fractionation of cell lysates on SDS-PAGE and Western blotting using antibodies against phosphorylated ERK 1/2. Stimulation with FGF-8b induced rapid ERK phosphorylation, such that the maximal induction was seen 10 min after the start of the FGF treatment (Fig. 4B). Concomitant treatment with K5-OS prevented the ERK phosphorylation completely, whereas heparin or unmodified K5PS did not inhibit ERK phosphorylation. Collectively, the above data indicate that K5-OS antagonizes FGF-8b signaling in S115 cells and further suggest that the inhibitory effect of K5-OS on the proliferation of testosterone-stimulated S115 cells may be mediated through inhibition of FGF-8b activity. FGF-Antagonist Activity of K5-OS Is Influenced by the FGF Species and Cell Type Involved—Previous data suggest that the proliferation of S115 cells may be stimulated also by FGF-1 or FGF-2 (32.Nakamura N. Yamanishi H. Lu J. Uchida N. Nonomura N. Matsumoto K. Sato B. J. Steroid Biochem. 1989; 33: 13-18Crossref PubMed Scopus (27) Google Scholar), and we thus assessed the effect of K5PSs on the biological activity of these FGF species. The results indicated that whereas both FGF-1 and FGF-2 enhanced the DNA synthesis of S115 cells, their effects were almost completely inhibited by K5-OS at a concentration of 1 μg/ml (Fig. 5). By contrast, K5-NSOS appeared to stimulate rather than inhibit the corresponding FGF responses. To assess whether sulfated K5PSs were able to influence the FGF response in other cells than S115, we performed studies with 3T3 mouse fibroblasts that were stimulated with FGF-1, FGF-2, or FGF-8b and treated with K5PSs. Analogously to the results with S115 cells, the proliferative effects of FGF-2 and FGF-8b were inhibited by K5-OS, although the extent of inhibition remained lower than that seen in S115 cells. Notably, the effect of FGF-1 was not influenced by K5-OS in 3T3 cells. Collectively, these results suggest that different cell types may differ in their sensitivity toward the inhibitory effect of K5-OS upon FGF stimulation and further that the inhibitory capacity of the polysaccharide may differ according to the target FGF species. K5-OS and K5-NSOS Inhibit Binding of FGFs to Heparin— A possible mechanism of how sulfated K5PSs might inhibit FGF signaling involves their binding to the growth factors, hence interference with the cellular HS-FGF-FGFR interactions. We studied the binding of K5PSs to FGF-1, FGF-2, or FGF-8b by assessing their capacity to displace [3H]heparin from FGF. FGF was incubated in solution with [3H]heparin and different concentrations of K5PSs, after which the FGF-bound [3H]heparin was quantified by trapping the formed protein-saccharide complexes on nitrocellulose filters (see "Experimental Procedures"). Unlabeled heparin, K5-OS, and K5-NSOS all competed with [3H]heparin for binding to the various FGFs species in a concentration dependent manner (Fig. 6), whereas unmodified K5PS had no inhibitory capacity. Although the sulfated K5PSs differed somewhat in their inhibitory potency with regard to the polysaccharide structure and also the FGF species involved, both the O-sulfated and N,O-sulfated preparations were efficient inhibitors of the [3H]heparin-FGF interactions at the concentration of 1 μg/ml used in the cell culture assays. These data suggested that other reasons than differential FGF binding capacity should be explored to satisfactorily explain the different capacities of K5-OS and K5-NSOS in the inhibition of FGF-induced cell growth. Different Effects of K5-OS and K5-NSOS on FGF Activity in Cells Lacking Endogenous HS—Given the findings that both K5-OS and K5-NSOS bound avidly to FGFs, whereas only the former preparation inhibited FGF-induced cell proliferation, we proceeded to study whether K5-OS and K5-NSOS might differ in their effects on FGF signaling in cells lacking endogenous HSPGs. We have earlier demonstrated that S115 cells treated with sodium chlorate, an inhibitor of cellular sulfation reactions including the sulfation of HSPGs (6.Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1707Crossref PubMed Scopus (1291) Google Scholar), require exogenous heparin/HS for their proliferation upon testosterone stimulation (25.Borgenström M. Tienhaara A. Spillmann D. Salmivirta M. Jalkanen M. Exp. Cell Res. 2001; 264: 307-314Crossref PubMed Scopus (8) Google Scholar). Accordingly, the DNA synthesis of chlorate-treated S115 cells was not enhanced by testosterone treatment, whereas the testosterone response was restored by addition of heparin (Fig. 7A). Addition of K5-NSOS also restored the testosterone response in a concentration dependent manner such that K5-NSOS appeared at least as potent as heparin in this regard. By contrast, K5-OS had only a weak positive effect on the [3H]thymidine incorporation by chlorate-treated S115 cells (Fig. 7A) indicating that its ability to facilitate the testosterone-induced proliferative response was clearly distinct from that of K5-NSOS. We further studied the effects of K5PSs on FGF signaling in CHO677 cells, which are deficient in endogenous HS but responsive to FGF in the presence of exogenous heparin (33.Lundin L. Larsson H. Kreuger J. Kanda S. Lindahl U. Salmivirta M. Claesson-Welsh L. J. Biol. Chem. 2000; 275: 24653-24660Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). CHO677 cells were treated with FGF-8b alone or together with heparin/K5PSs and subjected to the [3H]thymidine incorporation assay. The results were essentially similar to those obtained with chlorate-treated S115 cells in that heparin and K5-NSOS elicited a marked FGF response, while K5-OS had a much weaker effect (Fig. 7B). However, the differences between the stimulatory capacities of K5-NSOS and K5-OS were more pronounced in chlorate-treated S115 cells than in CHO677 cells. To assess whether the differential ability of K5-OS and K5-NSOS to enhance FGF signaling was dependent on the FGF species used to stimulate the cells, similar experiments were performed with CHO677 cells treated with FGF-1 and FGF-2. The results indicated that K5-NSOS was consistently more potent in stimulating the FGF responses regardless of the FGF species (Fig. 7, C and D). Unmodified K5 did not enhance thymidine incorporation in either of the model systems studied (data not shown). The FGF responses of the wild-type CHO677 are presumably mediated by FGFR-1, which is expressed at low levels by the cells (33.Lundin L. Larsson H. Kreuger J. Kanda S. Lindahl U. Salmivirta M. Claesson-Welsh L. J. Biol. Chem. 2000; 275: 24653-24660Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). To investigate how the cellular FGFR expression influenced the capacity of K5PSs to modulate FGF signaling, their effects on the proliferative FGF response were studied in CHO677 cells transfected to express a higher level of FGFR-1 or FGFR-4 (20.Loo B.-M. Salmivirta M. J. Biol. Chem. 2002; 277: 32616-32623Ab
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