A Broad-Spectrum Humanlung Fibroblast-Derived Mitogen Is A
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Proc. Natl. Acad. Sci. USA Vol. 88, pp. 415-419, January 1991 Biochemistry A broad-spectrum human lung fibroblast-derived mitogen is a variant of hepatocyte growth factor (heparin-binding growth factor/plasminogen/epithelial cells/endothelial ceils/melanocytes) JEFFREY S. RUBIN*, ANDREW M.-L. CHAN*, DONALD P. BOTTARO*, WILSON H. BURGESSt, WILLIAM G. TAYLOR*, ALEX C. CECH*, DAVID W. HIRSCHFIELD*, JANE WONG*, TORU MIKI*, PAUL W. FINCH*t, AND STUART A. AARONSON*§ *Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD 20892; and tLaboratory of Molecular Biology, Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, MD 20855 Communicated by William H. Daughaday, October 10, 1990 (receivedfor review July 12, 1990) ABSTRACT A heparin-binding mitogen was isolated from Mitogenic Assays. DNA synthesis in the B5/589, BALB/ conditioned medium of human embryonic king fibroblasts. It MK, CCL208, and NIH 3T3 lines (10) and in primary exhibited broad target-cell specificity whose pattern was dis- melanocytes (11) was measured as described elsewhere. For tinct from that of any known growth factor. It rapidly stimu- proliferation assays (12), HUVECs were plated at 4 X 104 lated tyrosine phosphorylation of a 145-kDa protein in respon- cells per 6-cm tissue culture dish in basal medium (in the sive cells, suggesting that its signaling pathways involved presence or absence of heparin) supplemented with recom- activation of a tyrosine kinase. Purification identified a major binant aFGF or basic FGF (bFGF) (10 ng/ml) or HSAC- polypeptide with an apparent molecular mass of 87 kDa under purified, fibroblast-derived growth factor (-'100 ng/ml). Me- reducing conditions. Partial amino acid sequence analysis and dium was changed every 3 days. After 10 days, the cells were cDNA cloning revealed that it was a variant of hepatocyte trypsinized and counted. growth factor, a mitogen thought to be specific for hepatic cells Microsequencing. Ten micrograms of C4-purified growth and structurally related to plasminogen. Recombinant expres- factor was electrophoresed under reducing conditions in an sion of the cDNA in COS-1 cells established that it encoded the SDS/12.5% polyacrylamide minigel (Hoefer). After transfer purified growth factor. Its site of synthesis and spectrum of to nitrocellulose (13), the protein at 87 kDa (p87) was incu- targets imply that this growth factor may play an important bated with 0.2 1Lg of lysyl endopeptidase (1:20 enzyme/ role as a paracrine mediator of the proliferation ofmelanocytes substrate ratio; Boehringer Mannheim) in 25 mM Tris/1 mM and endothelial cells, as well as cells of epithelial origin. EDTA/5% acetonitrile, pH 8.5, at 37TC for 18 hr. The reaction mixture was loaded onto an RP300 cartridge (2.1 X Growth factors play important roles in normal development 30 mm) and resolved using a linear gradient of acetonitrile in and wound healing (1-3). Their abnormal expression has been 0.1% trifluoroacetic acid (microbore LC, Applied Biosys- implicated in neoplasia as well as a variety of other prolifer- tems model 130). Purified peptide was subjected to several ative disorders (4-6). Accumulating evidence indicates that rounds of Edman degradation using a gas-phase protein mesenchymal interactions presumably mediated by diffusible sequenator (Applied Biosystems model 477), and phenylthio- substances have a major impact on epithelial cell proliferation hydantoin amino acid derivatives were identified with an (7-9). Systematic efforts to isolate and characterize epithe- automated on-line HPLC column (model 120A). lial-acting mitogens produced by stromal cells have led to the Molecular Cloning. Eight pools of 27-mer oligonucleotide of factor a new mem- probes were synthesized on the basis of the amino acid discovery keratinocyte growth (KGF), sequence Leu-Ala-Arg-Pro-Ala-Val-Leu-Asp-Asn deter- ber of the fibroblast growth factor (FGF) family specific for mined by microsequencing of p87. In addition, three 45-mer epithelial cells (10). In this report, we describe the purifica- oligonucleotide probes were synthesized to match different tion, molecular cloning, and recombinant expression of a regions of the reported hepatocyte growth factor (HGF) fibroblast-derived mitogen possessing a spectrum of targets sequence (14): nucleotides -74 to -30, 1099 to 1143, and which includes endothelial cells and melanocytes in addition 2196 to 2240. The oligonucleotide pools and individual probes to epithelial cells. This factor shows striking homology to (50 pmol of each) were 5'-end-labeled with 83 pmol of other proteins involved in growth and tissue remodeling.¶ [y-32P]ATP (3000 Ci/mmol, Amersham; 1 Ci = 37 GBq) and METHODS AND 10 units of T4 polynucleotide kinase. Recombinant phages MATERIALS from the M426 cDNA library (15) were replica-plated onto Cells. The source and maintenance of the M426, BALB/ nitrocellulose filters and hybridized for 18 hr at 42°C in 6X MK, B5/589, CCL208, and NIH 3T3 cell lines were de- standard saline citrate (SSC; lx is 0.15 M NaCI/0.015 M scribed (10). Primary cultures of human melanocytes (11) sodium citrate, pH 7) containing 0.2% Ficoll, 0.2% polyvi- were prepared by published techniques. Human umbilical nylpyrrolidone, 0.2% bovine serum albumin, 0.05% sodium vein endothelial cells (HUVECs), from T. Maciag (Jerome H. pyrophosphate, and sonicated salmon sperm DNA (250 ,g/ Holland Laboratory for the Biomedical Sciences, Rockville, MD), were established in the presence ofrecombinant acidic 1 Abbreviations: HGF, hepatocyte growth factor; KGF, keratinocyte FGF (aFGF, ng/ml) and grown as described (12). growth factor; FGF, fibroblast growth factor; aFGF, acidic FGF; Purification and Physical Characterization. Conditioned- bFGF, basic FGF; HSAC, heparin-Sepharose affinity chromatogra- medium collection, ultrafiltration, heparin-Sepharose affinity phy; HUVEC, human umbilical vein endothelial cell; PCR, poly- chromatography (HSAC), reverse-phase C4 HPLC, and merase chain reaction. SDS/PAGE were performed as described for KGF (10). tPresent address: Department of Neurosurgery, Rhode Island Hos- pital, 593 Eddy Street, Providence, RI 02903. §To whom reprint requests should be addressed at: Building 37, The publication costs of this article were defrayed in part by page charge Room 1E24, National Institutes of Health, Bethesda, MD 20892. payment. This article must therefore be hereby marked "advertisement" 1The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M55379). 415 Downloaded by guest on September 26, 2021 416 Biochemistry: Rubin et al. Proc. Natl. Acad. Sci. USA 88 (1991) ml). Filters hybridized with the degenerate pools were washed in 6x SSC/0.1% SDS twice at room temperature and once at 540C, while those hybridized with the individual probes were washed in 2x SSC/0.1% SDS twice at room kDa --I. temperature and once at 550C. 94 - Recombinant Expression. A fragment of cDNA clone Ala . - p87 (nucleotides -27 to 2199) spanning the entire coding se- 67 - quence was generated by use of the polymerase chain reac- D55-6Cj tion (PCR; ref. 16) and subcloned into the BamHI site of 4.- vector pCDV (17) in either the sense or the antisense orien- 43 tation. Ten micrograms of each plasmid DNA was trans- fected by the calcium phosphate method (18) into -2 x 105 I COS-1 cells (19) that had been maintained in Dulbecco's .p-p34 modified Eagle's medium (DMEM) supplemented with 10% I p32 fetal bovine serum. Forty-eight hours after transfection, the 30 medium was changed to 0.1% fetal bovine serum in DMEM, and conditioned medium was harvested 16 hr later. The medium was filtered and concentrated 25-fold in a Centri- con-10 microconcentrator (Amicon), and aliquots were di- 21 5 luted 100-, 300-, and 1000-fold for assay ofmitogenic activity. Biosynthetic Studies. COS-transfected cells (14 hr after FIG. 1. SDS/PAGE of pooled fractions containing mitogenic to medium) and M426 cells grown in 10-cm activity from reverse-phase C4 HPLC. Approximately 0.4 Jg of switch low-serum purified protein was redissolved in sample buffer either lacking (-) dishes were incubated for 30 min in methionine-free DMEM or containing (+) 2.5% (vol/vol) 2-mercaptoethanol as reducing supplemented with heparin (50 ttg/ml; bovine lung, Sigma), agent, boiled for 3 min, and electrophoresed in an SDS/10%/ poly- which was then replaced with fresh medium containing acrylamide gel, which was subsequently silver-stained. Arrows [35S]methionine (1 mCi/5 ml per dish). After 4 hr, the medium indicate bands observed in individual column fractions from different was collected and concentrated >10-fold in Centricon-10 preparations whose intensity correlated with the level of mitogenic microconcentrators. The cells were washed on ice once with activity in these fractions. 10 ml of phosphate-buffered saline, lysed with 0.4 ml of 10 mM Tris, pH 7.4/150 mM NaCI/1 mM EDTA/10 mM chain disulfide bonds. Additional bands at 55-60, 32, and 34 KCI/1% Nonidet P-40/0.1% SDS/0.05% Tween 20, and kDa were observed with varying intensity in different prep- scraped off the dishes, and lysates were centrifuged (14,000 arations (Fig. 1). Proteolytic digestion (Staphylococcus au- x g, 30 min). Immunoprecipitations were performed with 10 reus V8 protease) and peptide mapping of individual bands ,lI of nonimmune or immune serum adsorbed to Gamma supported the conclusion that p34 was a fragment ofp87 (data Bind-G agarose (Genex) and samples were analyzed by not shown). These findings suggested that the broad, -75- SDS/10% PAGE under reducing conditions. kDa band present under nonreducing conditions consisted of Tyrosine Kinase Activity. Stimulation and detection of a mixture ofpolypeptides, including a single-chain form (p87) tyrosine phosphorylation were as described for KGF (20). and a processed, disulfide-linked heterodimer containing HGF Antiserum.