S100A13 Is Involved in the Regulation of Fibroblast Growth Factor-1 and p40 Synaptotagmin-1 Release in Vitro
1998; Elsevier BV; Volume: 273; Issue: 35 Linguagem: Inglês
10.1074/jbc.273.35.22224
ISSN1083-351X
AutoresCarla Mouta Carreira, Theresa LaVallee, Francesca Tarantini, Anthony Jackson, Julia Tait Lathrop, Brian Hampton, Wilson H. Burgess, Thomas Maciag,
Tópico(s)Fibroblast Growth Factor Research
ResumoWe have previously characterized the release of the signal peptide sequence-less fibroblast growth factor (FGF) prototype, FGF-1, in vitro as a stress-induced pathway in which FGF-1 is released as a latent homodimer with the p40 extravesicular domain of p65 synaptotagmin (Syn)-1. To determine the biologic relevance of the FGF-1 release pathway in vivo, we sought to resolve and characterize from ovine brain a purified fraction that contained both FGF-1 and p40 Syn-1 and report that the brain-derived FGF-1:p40 Syn-1 aggregate is associated with the calcium-binding protein, S100A13. Since S100A13 binds the anti-inflammatory compound amlexanox and FGF-1 is involved in inflammation, we examined the effects of amlexanox on the release of FGF-1 and p40 Syn-1 in response to stress in vitro. We report that while amlexanox was able to repress the heat shock-induced release of FGF-1 and p40 Syn-1 in a concentration-dependent manner, it had no effect on the constitutive release of p40 Syn-1 from p40 Syn-1 NIH 3T3 cell transfectants. These data suggest the following: (i) FGF-1 is associated with Syn-1 and S100A13 in vivo; (ii) S100A13 may be involved in the regulation of FGF-1 and p40 Syn-1 release in response to temperature stress in vitro; and (iii) the FGF-1 release pathway may be accessible to pharmacologic regulation. We have previously characterized the release of the signal peptide sequence-less fibroblast growth factor (FGF) prototype, FGF-1, in vitro as a stress-induced pathway in which FGF-1 is released as a latent homodimer with the p40 extravesicular domain of p65 synaptotagmin (Syn)-1. To determine the biologic relevance of the FGF-1 release pathway in vivo, we sought to resolve and characterize from ovine brain a purified fraction that contained both FGF-1 and p40 Syn-1 and report that the brain-derived FGF-1:p40 Syn-1 aggregate is associated with the calcium-binding protein, S100A13. Since S100A13 binds the anti-inflammatory compound amlexanox and FGF-1 is involved in inflammation, we examined the effects of amlexanox on the release of FGF-1 and p40 Syn-1 in response to stress in vitro. We report that while amlexanox was able to repress the heat shock-induced release of FGF-1 and p40 Syn-1 in a concentration-dependent manner, it had no effect on the constitutive release of p40 Syn-1 from p40 Syn-1 NIH 3T3 cell transfectants. These data suggest the following: (i) FGF-1 is associated with Syn-1 and S100A13 in vivo; (ii) S100A13 may be involved in the regulation of FGF-1 and p40 Syn-1 release in response to temperature stress in vitro; and (iii) the FGF-1 release pathway may be accessible to pharmacologic regulation. The FGF 1The abbreviations used are: FGFfibroblast growth factorGalgalactosidaseRP-HPLCreversed phase-high pressure liquid chromatographySynsynaptotagminPAGEpolyacrylamide gel electrophoresisERendoplasmic reticulumILinterleukin.prototype, FGF-1, functions as an extracellular mitogen for a diverse population of target cells, yet it lacks a classical signal peptide sequence for secretion (1Burgess W.H. Winkles J.A. Pusztai L. Lewis C.E. Yap E. Cell Proliferation in Cancer: Regulatory Mechanisms of Neoplastic Cell Growth. Oxford University Press, Oxford1996: 154-217Google Scholar). FGF-1 is released in response to temperature stress in vitro (2Jackson A. Friedman S. Zhan X. Engleka K.A. Forough R. Maciag T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10691-10695Crossref PubMed Scopus (226) Google Scholar) as a latent FGF-1 Cys-30 homodimer (3Jackson A. Tarantini F. Gamble S. Friedman S. Maciag T. J. Biol. Chem. 1995; 270: 33-36Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 4Tarantini F. Gamble S. Jackson A. Maciag T. J. Biol. Chem. 1995; 270: 29039-29042Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) through a secretion pathway that is independent of the conventional route mediated by the endoplasmic reticulum (ER)-Golgi apparatus (3Jackson A. Tarantini F. Gamble S. Friedman S. Maciag T. J. Biol. Chem. 1995; 270: 33-36Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) but is sensitive to inhibition by actinomycin D and cycloheximide (2Jackson A. Friedman S. Zhan X. Engleka K.A. Forough R. Maciag T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10691-10695Crossref PubMed Scopus (226) Google Scholar). fibroblast growth factor galactosidase reversed phase-high pressure liquid chromatography synaptotagmin polyacrylamide gel electrophoresis endoplasmic reticulum interleukin. We have partially characterized the latent FGF-1 species releasedin vitro in response to temperature stress. The extracellular FGF-1 homodimer is a component of a high molecular weight aggregate that contains the extravesicular p40 domain of synaptotagmin (Syn)-1 (5Tarantini F. LaVallee T. Jackson A. Gamble S. Garfinkel S. Mouta Carreira C. Burgess W.H. Maciag T. J. Biol. Chem. 1998; 273: 22209-22216Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The full-length Syn-1 translation product (p65) has been implicated in the regulation of exocytotic (6Perin M.S. Brose N. Jahn R. Sudhof T.C. J. Biol. Chem. 1991; 266: 623-629Abstract Full Text PDF PubMed Google Scholar) and endocytotic (7Zhang J.Z. Davletov B.A. Sudhof T.C. Anderson R.G.W. Cell. 1994; 78: 751-760Abstract Full Text PDF PubMed Scopus (435) Google Scholar) traffic and, like FGF-1 (4Tarantini F. Gamble S. Jackson A. Maciag T. J. Biol. Chem. 1995; 270: 29039-29042Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), Syn-1 is a phosphatidylserine- (8Perin M.S. Fried V.A. Mignery G.A. Jahn R. Sudhof T.C. Nature. 1990; 345: 260-263Crossref PubMed Scopus (650) Google Scholar) and heparin-binding protein (9Nishiki T. Kamata Y. Nemoto Y. Omori A. Ito T. Takahashi M. Kozaki S. J. Biol. Chem. 1994; 269: 10498-10503Abstract Full Text PDF PubMed Google Scholar). Whereas the stress-induced FGF-1 and p40 Syn-1-containing extracellular aggregate is not biologically active and binds poorly to immobilized heparin (5Tarantini F. LaVallee T. Jackson A. Gamble S. Garfinkel S. Mouta Carreira C. Burgess W.H. Maciag T. J. Biol. Chem. 1998; 273: 22209-22216Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), the biological and heparin-binding properties of FGF-1 can be recovered if the aggregate is treated with a reducing agent such as reduced glutathione (3Jackson A. Tarantini F. Gamble S. Friedman S. Maciag T. J. Biol. Chem. 1995; 270: 33-36Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) or with ammonium sulfate (2Jackson A. Friedman S. Zhan X. Engleka K.A. Forough R. Maciag T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10691-10695Crossref PubMed Scopus (226) Google Scholar). It is possible that the latent character of this FGF-1 aggregate may represent a physiologic safeguard to ensure that extracellular FGF-1 can only signal if the extracellular environment is appropriate for the cell to respond to its stimuli; otherwise, it would be rapidly cleared. Indeed, cell-surface-associated heparin-sulfate proteoglycans are thought to not only present FGF-1 to its high affinity receptor tyrosine kinase (10Friesel R.E. Maciag T. FASEB J. 1995; 9: 919-925Crossref PubMed Scopus (406) Google Scholar) but also to protect FGF-1 from degradation by proteolytic enzymes present in the extracellular milieu during cell migration and proliferation (11Gospodarowicz D. Cheng J. J. Cell. Physiol. 1986; 128: 475-484Crossref PubMed Scopus (683) Google Scholar,12Damon D.H. Lobb R.R. D'Amore P.A. Wagner J.A. J. Cell. Physiol. 1989; 138: 221-226Crossref PubMed Scopus (142) Google Scholar). Because FGF-1 is released in response to heat shock in vitroas a reducing agent and denaturant-sensitive aggregate with p40 Syn-1 (5Tarantini F. LaVallee T. Jackson A. Gamble S. Garfinkel S. Mouta Carreira C. Burgess W.H. Maciag T. J. Biol. Chem. 1998; 273: 22209-22216Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), and Syn-1 is required for FGF-1 secretion in response to heat shock (13LaVallee T. Tarantini F. Gamble S. Mouta Carreira C. Jackson A. Maciag T. J. Biol. Chem. 1998; 273: 22217-22223Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), we sought to determine whether FGF-1 and Syn-1 also exist as an aggregate in vivo in order to provide a physiologic correlate to the in vitro data. Since neural tissue has served as the traditional source of native FGF-1 (14Maciag T. Cerundolo J. Ilsley S. Kelley P.R. Forand R. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 5674-5678Crossref PubMed Scopus (577) Google Scholar) and early evidence suggested that FGF-1 is present in neural tissue as an acid-sensitive high molecular weight aggregate (15Maciag T. Hoover G.A. Weinstein R. J. Biol. Chem. 1982; 257: 5333-5336Abstract Full Text PDF PubMed Google Scholar), we sought to determine whether FGF-1 and p40 Syn-1 could be resolved as a heparin-binding aggregate from neutral extracts of neural tissue. We report that brain-derived FGF-1 exists as a multiprotein aggregate with p40 Syn-1 and S100A13, a member of the S100 gene family of calcium-binding proteins (16Wicki R. Schafer B.W. Erne P. Heizmann C.W. Biochem. Biophys. Res. Commun. 1996; 227: 594-599Crossref PubMed Scopus (63) Google Scholar, 17Schafer B.W. Heizmann C.W. Trends Biochem. Sci. 1996; 21: 134-140Abstract Full Text PDF PubMed Scopus (1035) Google Scholar) and have been able to utilize a pharmacologic strategy to illustrate that S100A13 may be a functional component of the FGF-1 release pathway. Amlexanox (also known as AA673, Amoxanox, and Solfa) and its three derivatives were a very generous gift of Dr. G. Goto from Takeda Chemical Industries, Osaka, Japan. All other chemicals were reagent-grade and obtained from Sigma except where otherwise indicated. Chromatography solvents were HPLC-grade and obtained from Burdick and Jackson (Muskegon, MI). The rabbit polyclonal antibodies against recombinant human FGF-1 and rat p40 Syn-1 were prepared as described previously (5Tarantini F. LaVallee T. Jackson A. Gamble S. Garfinkel S. Mouta Carreira C. Burgess W.H. Maciag T. J. Biol. Chem. 1998; 273: 22209-22216Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 13LaVallee T. Tarantini F. Gamble S. Mouta Carreira C. Jackson A. Maciag T. J. Biol. Chem. 1998; 273: 22217-22223Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). All antibodies were used for immunoblot analysis at a concentration of 4 μg/ml in blocking buffer as described previously (2Jackson A. Friedman S. Zhan X. Engleka K.A. Forough R. Maciag T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10691-10695Crossref PubMed Scopus (226) Google Scholar) except that an enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech) was used for protein detection. Ten (1.5 kg) unstripped ovine brains (Pel-Freeze® Biologicals, Rogers, AR) were homogenized in 1.3 volumes of 50 mm Tris-HCl, pH 7.4, for 2 min in a Waring blender. The homogenate was centrifuged at 10,000 × gfor 1 h, and the supernatant was filtered through sterile gauze. The filtrate was subjected to stepwise salt fractionation with 50 and 95% (NH4)2SO4 saturation, and the precipitates were collected by centrifugation as described (14Maciag T. Cerundolo J. Ilsley S. Kelley P.R. Forand R. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 5674-5678Crossref PubMed Scopus (577) Google Scholar). The 95% (NH4)2SO4 saturation precipitate was resuspended in 100 ml of 50 mm Tris-HCl, pH 7.4, and dialyzed for 18 h against 50 volumes of the resuspension buffer using a Spectra/Por (Mr 12–14,000) dialysis membrane (Spectrum Medical Industries Inc., Houston, TX). All purification procedures were performed at 4 °C. A 2.5 × 22-cm plastic column containing 25 ml of hydrated heparin-Sepharose CL-6B was equilibrated with 10 volumes of 50 mm Tris-HCl, pH 7.4, and the brain extract was adsorbed twice over the immobilized heparin. The column was washed with at least 10 bed volumes of the resuspension buffer until the absorbance of the eluate at λ = 280 nm was less than 0.01. Three batch fractions were eluted with 100 ml of 50 mm Tris-HCl, pH 7.4, containing 0.4 m NaCl, 0.7 m NaCl, and 1.5 mNaCl, and samples from each NaCl eluate (25 ml) were adsorbed to a C4 column (VydacTM, Hesperia, CA) conditioned in 0.1% trifluoroacetic acid (Pierce). RP-HPLC was performed as described (18Burgess W.H. Mehlman T. Friesel R. Johnson W. Maciag T. J. Biol. Chem. 1985; 260: 11389-11392Abstract Full Text PDF PubMed Google Scholar) using a linear gradient of acetonitrile (40 to 100%) in 0.1% (v/v) trifluoroacetic acid at a flow rate of 1 ml/min, and the effluent was monitored at λ = 214 nm. Samples were collected as absorbance peaks independent of volume in 1.0 m Tris-HCl, pH 7.4, in an attempt to maintain aggregate integrity and analyzed by FGF-1 and Syn-1 immunoblot analysis as described (2Jackson A. Friedman S. Zhan X. Engleka K.A. Forough R. Maciag T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10691-10695Crossref PubMed Scopus (226) Google Scholar, 5Tarantini F. LaVallee T. Jackson A. Gamble S. Garfinkel S. Mouta Carreira C. Burgess W.H. Maciag T. J. Biol. Chem. 1998; 273: 22209-22216Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) except that the ECL system was used for protein detection. Although many peaks exhibited the presence of both FGF-1 and Syn-1 by immunoblot analysis, only the 1.5m NaCl heparin-Sepharose elution fraction contained a unique absorbance peak that contained both FGF-1 and Syn-1 by immunoblot analysis at a dilution of 1:100. This peak was re-chromatographed on a microbore 300-Å C4 Aquapore RP300 column (Perkin-Elmer), and bound proteins were eluted as absorbance peaks at λ = 214 with a linear gradient (40–100%) of 70% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid and a flow rate of 0.2 ml/min. Approximately 10 μg of protein from the indicated peaks in Fig. 1, B and C, were subjected to proteolytic digestion using lysyl endopeptidase C (Boehringer Mannheim) as described (73Egerton M. Burgess W.H. Chen D. Druker B.J. Bretscher A.S. Samelson L.E. J. Immunol. 1992; 149: 1847-1852PubMed Google Scholar). Peptides were isolated by RP-HPLC using an Applied Biosystems model 130 separation system. Isolated peptides were subjected to automated Edman degradation using either an Applied Biosystems model 473A or 477A protein sequenator. Proteins were identified by comparison of the amino acid sequences obtained for several of these peptides against an NCBI (National Center for Biotechnology Information) protein sequence data base using the BLAST (Basic Local Alignment Search Tool) program. NIH 3T3 cell FGF-1 [2] transfectants and FGF-1:β-gal and p65 Syn-1 co-transfectants (13LaVallee T. Tarantini F. Gamble S. Mouta Carreira C. Jackson A. Maciag T. J. Biol. Chem. 1998; 273: 22217-22223Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) were grown to 80% confluence on fibronectin-coated dishes and subjected to temperature stress (41.5 °C, 90 min) under the conditions previously described (74Shi J.P. Friedman S. Maciag T. J. Biol. Chem. 1997; 272: 1142-1147Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Where indicated, various concentrations of either amlexanox or one of its chemical derivatives were present during the entire heat shock period. A stock solution of amlexanox was freshly prepared in equimolar NaOH, and serial dilutions in phosphate-buffered saline were prepared prior to addition to the cells as recommended by Takeda chemical Industries Ltd. The compounds were added in volumes of 160 μl/20 ml of medium. The same volume of NaOH without drug was also added to the cells and found to have no effect on either protein release, membrane permeability (lactate dehydrogenase activity), or cell viability. In addition, cells maintained at 37 °C in the presence or absence of amlexanox for the same period were used as a control. Conditioned media were collected and filtered through 0.2-μm cellulose acetate. Aliquots (300 μl) were taken for analysis of lactate dehydrogenase enzymatic activity in the conditioned media according to an adaptation of the original method of Bergmeyer (75Bergmeyer H.-U. Bergmeyer H.-U. Methods of Enzymatic Analysis. Academic Press, New York1965: 736-743Crossref Google Scholar) and Sigma procedure DG1340-UV. The remaining filtrate was treated with fresh 0.1% (w/v) dithiothreitol for 2 h at 37 °C and processed by heparin-Sepharose chromatography as described (2Jackson A. Friedman S. Zhan X. Engleka K.A. Forough R. Maciag T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10691-10695Crossref PubMed Scopus (226) Google Scholar). Extracts of ovine brain were prepared at a neutral pH and subjected to (NH4)2SO4precipitation (50 and 95% saturation) as described under "Experimental Procedures." The 95% (NH4)2SO4 saturation precipitate was resuspended as described previously (18Burgess W.H. Mehlman T. Friesel R. Johnson W. Maciag T. J. Biol. Chem. 1985; 260: 11389-11392Abstract Full Text PDF PubMed Google Scholar) and separated into three batch-eluted fractions (0.4, 0.7, and 1.5 m NaCl) by heparin-Sepharose chromatography. Whereas immunoblot analysis of each eluate revealed the presence of both FGF-1 and p40 Syn-1 in all three fractions, only the 1.5 m NaCl post-heparin-Sepharose eluate was able to produce a reversed phase (RP)-HPLC peak (Fig. 1A) that contained both FGF-1 and p40 Syn-1 by immunoblot analysis (data not shown). The presence of p40 Syn-1 in the 1.5 m NaCl post-heparin-Sepharose elution fraction was unexpected since recombinant p40 Syn-1 has been observed to elute at a lower (∼0.6m NaCl) ionic strength [5]. To ensure that the post-RP-HPLC heparin-binding fraction containing both FGF-1 and p40 Syn-1 did not contain additional non-associated proteins, this fraction was again subjected to analysis by RP-HPLC. As shown in Fig.1 B, RP-HPLC analysis revealed a single symmetrical peak with a retention time identical to that previously observed (Fig.1 A). Immunoblot analysis using FGF-1 and Syn-1 antibodies confirmed the presence of both FGF-1 and p40 Syn-1 in this sample (Fig. 2, A andB). Since this fraction (Fig. 1 B) contained both FGF-1 and p40 Syn-1 and the electrophoretic mobility of the FGF-1:Syn-1 aggregate released in response to temperature stress is denaturant-sensitive (5Tarantini F. LaVallee T. Jackson A. Gamble S. Garfinkel S. Mouta Carreira C. Burgess W.H. Maciag T. J. Biol. Chem. 1998; 273: 22209-22216Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), we anticipated that treatment of this fraction with a chaotropic agent should resolve the FGF-1 and Syn-1 components as individual peaks with RP-HPLC retention times identical to their retention times defined by both the native (Fig. 1 A) and recombinant (Fig.1 D) proteins. Therefore, the brain-derived, post-RP-HPLC fraction (Fig. 1 B) containing both FGF-1 and p40 Syn-1 was heated (5 min, 95 °C) in the presence of 8.0 m guanidine HCl and analyzed by RP-HPLC. As shown in Fig. 1 C, the fraction containing both FGF-1 and p40 Syn-1 was present, but its absorbance was reduced significantly. In addition, numerous fractions with distinct retention times were readily visible including a major absorption peak and fractions previously defined as FGF-1 and p40 Syn-1 (Fig. 1, A and D). These data suggest that thermal and guanidine HCl denaturation of the brain-derived post-RP-HPLC fraction (Fig. 1 B) containing both FGF-1 and p40 Syn-1 generates additional fractions with different retention times including peaks with retention times identical to FGF-1 and p40 Syn-1. Thus it is likely that this brain-derived, heparin-binding fraction represents an aggregate that contains FGF-1 and p40 Syn-1 as well as other temperature- and chaotropic-sensitive components. Since the major absorption fraction containing both FGF-1 and p40 Syn-1 (Fig.1 B) demonstrated an unknown retention time, we sought to determine its structure. However, automated Edman degradation of this peak failed to yield any information. Therefore, this fraction (Fig.1 B) was subjected to LysC digestion, and the peptides were resolved by RP-HPLC. Automated Edman degradation of these peptides demonstrated that this fraction contained a member of the S100 gene family (17Schafer B.W. Heizmann C.W. Trends Biochem. Sci. 1996; 21: 134-140Abstract Full Text PDF PubMed Scopus (1035) Google Scholar), the ovine homolog of human S100A13 (16Wicki R. Schafer B.W. Erne P. Heizmann C.W. Biochem. Biophys. Res. Commun. 1996; 227: 594-599Crossref PubMed Scopus (63) Google Scholar). Interestingly, the structure of the S100A13 protein predicted from the bovine (GenBankTM accession number AB001567), murine, and human cDNA sequences (16Wicki R. Schafer B.W. Erne P. Heizmann C.W. Biochem. Biophys. Res. Commun. 1996; 227: 594-599Crossref PubMed Scopus (63) Google Scholar) suggests that S100A13 contains 98 amino acid residues, 9 of which have cyclic side chains that absorb in the far-UV area used for detection (19Goldfarb A.R. Saidel L.J. Mosovich E. J. Biol. Chem. 1951; 193: 397-404Abstract Full Text PDF PubMed Google Scholar, 20Wetlaufer D.R. Adv. Protein Chem. 1962; 17: 303-390Crossref Scopus (798) Google Scholar). Thus, the high UV absorbance feature of the FGF-1 and p40 Syn-1 aggregate (Fig. 1 B) may be due in part to the extinction coefficient of S100A13 (Fig. 1, C andD). Since it was possible to resolve a single fraction from RP-HPLC whose retention time was altered by treatment with temperature and the chaotropic agent, guanidine HCl, we suggest that S100A13 is a component of a multiprotein FGF-1- and p40 Syn-1-containing aggregate and that S100A13 is also present in this aggregate as a protein with a blocked amino terminus. It is important to note that the major absorption peak in Fig.1 C, resulting from the denaturation of the fraction (Fig.1 B) containing FGF-1, p40 Syn-1, and S100A13, exhibited a distinct retention time, yet immunoblot analysis of this major absorption peak demonstrated the presence of low levels of FGF-1 and p40 Syn-1 (data not shown). Since members of the S100 gene family are known to self-associate (21Naka M. Qing Z.X. Sasaki T. Kise H. Tawara I. Hamaguchi S. Tanaka T. Biochim. Biophys. Acta. 1994; 1223: 348-353Crossref PubMed Scopus (54) Google Scholar) and to associate with membrane phospholipids (22Donato R. Cell Calcium. 1986; 7: 123-145Crossref PubMed Scopus (207) Google Scholar), it is possible that the major absorption peak in Fig. 1 B may contain S100A13 aggregates as well as other peptidic and non-protein components such as acidic phospholipids. Thus, denaturation of the multimeric aggregate in Fig. 1 Bcontaining p40 Syn-1, S100A13, and FGF-1, with temperature and treatment with guanidine HCl resulted in only a partial disruption of the aggregate. In addition, we cannot eliminate the possibility that the FGF-1, p40 Syn-1, and S100A13-containing peak resolved in Fig. 1,A and B, is the result of nonspecific protein aggregation under RP-HPLC conditions. However, it is noteworthy that recombinant S100A13 elutes from immobilized heparin between 0.2 and 0.4m NaCl (data not shown), and recombinant p40 Syn-1 elutes from heparin-Sepharose at 0.7 m NaCl (5Tarantini F. LaVallee T. Jackson A. Gamble S. Garfinkel S. Mouta Carreira C. Burgess W.H. Maciag T. J. Biol. Chem. 1998; 273: 22209-22216Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Since the brain-derived, post-heparin-Sepharose fraction containing p40 Syn-1, FGF-1, and S100A13 was isolated as a high affinity (1.5 mNaCl elution peak) heparin-binding fraction prior to resolution by RP-HPLC, it is likely that S100A13 and p40 Syn-1 gained high heparin binding affinity through their ability to associate with FGF-1 prior to analysis by RP-HPLC. Interestingly, automated Edman degradation of the FGF-1 fraction (Fig.1 C) derived from treatment of the FGF-1, S100A13, and p40 Syn-1 aggregate (Fig. 1 B) with temperature and guanidine HCl also failed to yield information. However, automated Edman degradation of LysC fragments derived from the FGF-1 peak (Fig. 1 C) demonstrated that it was present as a protein with a blocked amino terminus that has previously been characterized as FGF-1β (residues 1–154) (23Burgess W.H. Maciag T. Annu. Rev. Biochem. 1989; 58: 575-606Crossref PubMed Google Scholar). Unfortunately, however, it was not possible to collect sufficient material from the p40 Syn-1 peak described in Fig.1 C for structural analysis, but it was possible to confirm the identity of this peak by Syn-1 immunoblot analysis (data not shown). Likewise, automated Edman degradation of the remaining peaks described in Fig. 1 C did not yield any information, and sufficient material was not available for analysis by LysC digestion. These data imply that the ovine brain-derived, heparin-binding and denaturant-sensitive aggregate resolved by RP-HPLC (Fig. 1 B) contains at least FGF-1β, p40 Syn-1, and S100A13. Furthermore, these data also provide an in vivo correlate to the presence of FGF-1 and p40 Syn-1 as a denaturant-sensitive aggregate that is released into the extracellular compartment in response to temperature stress in vitro (5Tarantini F. LaVallee T. Jackson A. Gamble S. Garfinkel S. Mouta Carreira C. Burgess W.H. Maciag T. J. Biol. Chem. 1998; 273: 22209-22216Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Because (i) S100A13 was purified as an aggregate with FGF-1 and p40 Syn-1 from ovine brain, (ii) elevated FGF-1 levels are associated with inflammatory environments in vivo (24Sano H. Forough R. Maier J.A. Case J.P. Jackson A. Engleka K. Maciag T. Wilder R.L. J. Cell Biol. 1990; 110: 1417-1426Crossref PubMed Scopus (161) Google Scholar), (iii) Syn-1 is released as a p40 fragment in response to temperature stress (5Tarantini F. LaVallee T. Jackson A. Gamble S. Garfinkel S. Mouta Carreira C. Burgess W.H. Maciag T. J. Biol. Chem. 1998; 273: 22209-22216Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) and is required for heat shock-induced FGF-1 secretion (13LaVallee T. Tarantini F. Gamble S. Mouta Carreira C. Jackson A. Maciag T. J. Biol. Chem. 1998; 273: 22217-22223Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), (iv) the anti-inflammatory and anti-allergic compound, amlexanox, binds S100A13 (Ref. 25Oyama Y. Shishibori T. Yamashita K. Naya T. Nakagiri S. Maeta H. Kobayashi R. Biochem. Biophys. Res. Commun. 1997; 240: 341-347Crossref PubMed Scopus (37) Google Scholar; GenBankTM accession number AB001567), (v) amlexanox is able to interfere with the release of intracellular granules from basophils and mast cells (26Makino H. Saijo T. Ashida H. Kuriki H. Maki Y. Int. Arch. Allergy Appl. Immunol. 1987; 82: 66-71Crossref PubMed Scopus (99) Google Scholar), and (vi) NIH 3T3 cells express the S100A13 transcript (data not shown), we sought to determine whether amlexanox would be able to modify the stress-induced release of FGF-1 and p40 Syn-1 from NIH3T3 cells in vitro. NIH 3T3 cell FGF-1:β-galactosidase (Gal) and p65 Syn-1 co-transfectants were subjected to temperature stress (90 min, 41.5 °C) as described previously (13LaVallee T. Tarantini F. Gamble S. Mouta Carreira C. Jackson A. Maciag T. J. Biol. Chem. 1998; 273: 22217-22223Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) in the absence and presence of amlexanox. The conditioned medium was treated with dithiothreitol, adsorbed to heparin-Sepharose, and the presence of FGF-1:β-gal and Syn-1 assessed by immunoblot analysis as described previously (2Jackson A. Friedman S. Zhan X. Engleka K.A. Forough R. Maciag T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10691-10695Crossref PubMed Scopus (226) Google Scholar). As shown in Fig.3, A and B, amlexanox was able to repress the release of both FGF-1:β-gal and p40 Syn-1 in response to temperature stress. The inhibition of FGF-1:β-gal and p40 Syn-1 release was dependent upon the concentration of amlexanox and was within the concentration range that exhibits pharmacologic effects as an anti-allergic and anti-inflammatory agent (27Saijo T. Kuriki H. Ashida Y. Makino H. Maki Y. Int. Arch. Allergy Appl. Immunol. 1985; 77: 315-321Crossref PubMed Scopus (24) Google Scholar, 28Saijo T. Kuriki H. Ashida Y. Makino H. Maki Y. Int. Arch. Allergy Appl. Immunol. 1985; 78: 43-50Crossref PubMed Google Scholar, 29Saijo T. Makino H. Tamura S. Kuriki H. Ashida Y. Terao S. Maki Y. Int. Arch. Allergy Appl. Immunol. 1986; 79: 231-237Crossref PubMed Scopus (21) Google Scholar). Similar amlexanox concentrations were also able to inhibit the release of FGF-1 from FGF-1 NIH 3T3 cell transfectants in vitro (Fig. 5). Because of the possibility that amlexanox may also possess broad inhibitory activity upon conventional cellular secretion, we evaluated the ability of amlexanox to repress the release of a synthetic form of FGF-1 engineered to enter into the conventional ER-Golgi-mediated secretion pathway (30Forough R. Xi Z. MacPhee M. Friedman S. Engleka K.A. Sayers T. Wiltrout R.H. Maciag T. J. Biol. Chem. 1993; 268: 2960-2968Abstract Full Text PDF PubMed Google Scholar). Exposure of NIH 3T3 cells stably transfected with the FGF-4 signal peptide sequence: FGF-1 chimera (30Forough R. Xi Z. MacPhee M. Friedman S. Engleka K.A. Sayers T. Wiltrout R.H. Maciag T. J. Biol. Chem. 1993; 268: 2960-2968Abstract Full Text PDF PubMed Google Scholar) to amlexanox did not result in inhibition of the constitutive secretion of the FGF-1 chimera (data not shown). Furthermore, the forced secretion of FGF-1 is known to induce a prominent transformed NIH 3T3 cell phenotype in vitro (30Forough R. Xi Z. MacPhee M. Friedman S. Engleka K.A. Sayers T. Wiltrout R.H. Maciag T. J. Biol. Chem. 1993; 268: 2960-2968Abstract Full Text PDF PubMed Google Scholar), and amlexanox was unable to modify this phenotype. Additionally, the p40 extravesicu
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