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. A specific neutralizing antiserum was p55-60 and p32 or p34 chains. prepared by injecting -10 ug ofC4-purified growth factor into Unique Target Cell Specificity and Evidence of Mitogenic the inguinal lymph nodes of a rabbit and giving intranodal as Signaling Involving Tyrosine Phosphorylation. A summary of well as intramuscular booster injections of highly purified C4 the responsive cells for this and other growth factors is fractions containing microgram quantities of the factor. provided in Table 1. Significant stimulation was evident on mammary (B5/589) and bronchial (CCL208) epithelial cells RESULTS as well as keratinocytes, while there was no detectable activity on fibroblasts. Of note, the factor elicited as strong Purification and Physical Characterization of a Fibroblast- a response as the FGFs on endothelial cells and was even Derived Mitogen. A growth factor with activity on mouse more potent than the FGFs on human melanocytes. Thus, the keratinocytes (BALB/MK) was isolated from conditioned a cell whose pattern medium of M426 human embryonic lung fibroblasts by a factor exhibited broad target specificity, combination of ultrafiltration, HSAC, and reverse-phase C4 was distinct from known growth factors. In contrast to the HPLC. The growth factor activity was eluted from heparin- FGFs, whose activity on endothelial cells was enhanced or Sepharose with 0.8-0.9 M NaCI and from the C4 resin with unaltered by heparin, proliferation in response to the new -48% acetonitrile in 0.1% trifluoroacetic acid. Recovery of growth factor was markedly inhibited by heparin. A similar activity after C4 HPLC required prompt dilution and neu- inhibitory effect of heparin was observed with other target tralization or immediate drying of fractions in vacuo. The cells (data not shown). We estimated that the half-maximal purification routinely yielded 20-40 ,ug ofprotein from 8 liters mitogenic effect of the C4-purified factor, when expressed in ofmedium. Assuming no contribution from other mitogens or terms ofmolarity (for a molecular mass of87 kDa), was -250 inhibitors in the medium, we estimated an enrichment of pM, well within the range of growth factors. -1500-fold and yield of 5-10% activity. Because many growth factor receptors possess tyrosine Analysis of the C4-purified pool by SDS/PAGE under kinase activity (21, 22), we investigated whether the factor nonreducing conditions revealed two broad bands at -75 and triggered tyrosine phosphorylation in target cells. When -34 kDa (Fig. 1). SDS/PAGE of individual HSAC and B5/589 cells were exposed to the growth factor, anti- C4-HPLC fractions established that the intensity of the phosphotyrosine immunoprecipitation/immunoblotting de- 75-kDa but not the 34-kDa band correlated with mitogenic tected the rapid appearance of a 145-kDa band (ppl45, Fig. activity (data not shown). Under reducing conditions, the 2). Preincubation of the phosphotyrosine antiserum with an contaminant 34-kDa protein shifted to a higher apparent excess of phenyl phosphate, an analog of phosphotyrosine, molecular mass (Fig. 1). The pattern observed with the larger eliminated the ppl45 band (Fig. 2). These findings are con- protein was more complex. A substantial portion ofthe larger sistent with the possibility that pp145 represents an auto- protein migrated more slowly, consistent with loss of intra- phosphorylated receptor with tyrosine kinase activity. In any Downloaded by guest on September 26, 2021 Biochemistry: Rubin et al. Proc. Natl. Acad. Sci. USA 88 (1991) 417 Table 1. Target-cell specificity of growth factors Fold stimulation of [3H]thymidine incorporation* or cell numbert Growth Epithelial Fibroblast Endothelial Melanocyte factor B5/589 CCL208 BALB/MK (NIH 3T3) (HUVECs) (primary) C4 prep 10 10 40 <1 3 180 KGF 3 10 800 <1 ND <1 aFGFt 3 10 800 60 3 100 bFGF 3 5 200 60 3 100 EGF 15 20 200 15 ND <1 TGFa ND ND 300 15 ND <1 EGF, epidermal growth factor; TGFa, transforming growth factor a; ND, not determined. *Maximal thymidine incorporation stimulated by the newly identified growth factor and other well-characterized factors is expressed as fold stimulation over background. Typical background levels were as follows: B5/589, 3000 cpm; CCL208, 1000 cpm; BALB/MK, 200 cpm; NIH 3T3, 3000 cpm; melanocytes, 500 cpm. Assays were routinely performed with C4-purified factor (C4 prep; purity .70%o); when HSAC-purified material was employed, activity was shown to be specific by use of a neutralizing antiserum. These data are representative of several experiments. tResults for endothelial cells were from proliferation assays. tMaximal stimulation by aFGF required the presence of heparin. case, the appearance of pp145 argues that the growth factor screened in the M426 cDNA library. One was large enough rapidly activates tyrosine phosphorylation in its target cells. [-6 kilobases (kb)] to be almost full-length according to the Molecular Identification and Cloning Reveals Homology to reported size of the HGF mRNA (14, 23) and our own results HGF. To establish the identity of this broad-spectrum mito- (Fig. 3 A and B). Several -3-kb clones were found by gen, we attempted to obtain amino acid sequence informa- restriction mapping to match the 5' half of the largest insert tion. While p87 appeared to have a blocked amino terminus, (Fig. 3A). Northern blot analysis of M426 cellular RNA digestion with lysyl endopeptidase yielded soluble peptides revealed, in addition to a 6-kb transcript, a 3-kb transcript not that were resolved by reverse-phase HPLC for sequence reported for HGF (Fig. 3B). Thus, the 3-kb clones could have analysis. From one, positive identifications were made arisen from the 3-kb transcript or from the 6-kb mRNA by through the first nine cycles; the amino-terminal assignment internal priming. was inferred from the cleavage specificity of lysyl endopep- We chose a representative 3-kb cDNA clone (Ala) for tidase: (Lys)-Leu-Ala-Arg-Pro-Ala-Val-Leu-Asp-Asn. dideoxy chain-termination sequencing (30) of overlapping During efforts to isolate cDNA clones by using degenerate fragments that had been subcloned into M13 vectors. Con- oligonucleotide probes based on this amino acid sequence, firmatory data were obtained with clones A9 and A22. It two reports appeared concerning the predicted protein se- contained an open reading frame of2172 nucleotides predict- quence of HGF (14, 23), a mitogen purified from human ing a protein of82,606 Da. There were four potential N-linked plasma (24) or rat platelets (25). This growth factor, also glycosylation sites (Asn-289, -397, -561, and -648) and a referred to as hepatopoietin A (26), acts on hepatocytes and putative signal-peptide sequence at the amino terminus (Fig. is thought to be important for liver regeneration (14, 23-26). 3C). With these potential posttranslational modifications, thb The first nine amino acids of our protein sequence were predicted protein corresponded in size to p87. Comparison identical to a region of the predicted HGF sequence. By use with the HGF sequence of Miyazawa et al. (14) revealed of our oligonucleotide probes as well as those corresponding complete identity including four kringle structures and a to three different regions of the HGF cDNA sequence, we protease-like domain, except for the absence of a 5-amino identified 35 positive clones out of an estimated 500,000 acid stretch caused by a 15-base-pair deletion (Fig. 3C). The sequence also differed from the HGF sequence of Nakamura 1 2 3 4 5 et al. (23) in this region and showed 14 other single amino acid kDa substitutions scattered throughout the molecule (Fig. 3C). PCR analysis revealed that the 6-kb cDNA clone as well as 200 - about halfofthe 3-kb cDNA clones exhibited the 15-base-pair - pp145 deletion, while the other half did not. When we performed PCR analysis on RNA prepared from M426 fibroblasts and 92.5 - from the fibroblasts of 18 other individuals, we detected both

69 - sequences in every instance, arguing strongly that the differ- ent forms were due to alternative RNA splicing (31). Recombinant cDNA Expression Confirms the Identity and 46 - Properties ofthe Mitogen. To directly establish that the cloned sequence encoded the broad-spectrum mitogen, we intro- 30 - duced the Ala coding sequence into the pCDV expression Time(min) 0 1 3 10 10 vector and analyzed cell lysates and conditioned medium coms following transient expression in COS-1 cells. Medium from cells transfected in the "sense," but not the "antisense," FIG. 2. Tyrosine phosphorylation in response to the newly iso- orientation caused potent stimulation of [3H]thymidine in- lated growth factor. After exposure ofB5/589 cells to HSAC-purified corporation by B5/589 human mammary epithelial cells (Fig. growth factor (100 ng/ml) for various intervals at 37°C, 1.5 mg of 4A). It also was active on BALB/MK cells and melanocytes protein from each cell lysate was immunoprecipitated with phos- but was inactive on fibroblasts not photyrosine antiserum, electrophoresed in an SDS/8% polyacryl- NIH 3T3 (data shown). amide gel, and immunoblotted with phosphotyrosine antiserum (20). Moreover, the recombinant activity was neutralized by an- Antiserum preincubated with 2 mM phenyl phosphate as competitor tiserum that efficiently blocked mitogenic activity of M426- was used as a control (lane 5). The major band seen in response to derived HGF (Fig. 4A). Metabolic labeling of M426 and the growth factor, ppl45, is indicated by an arrow. transfected COS cells revealed that p87 was present in cell Downloaded by guest on September 26, 2021 418 Biochemistry: Rubin et al. Proc. Natl. Acad. Sci. USA 88 (1991) A B kb 0 1 2 3 4 5 6 kb - 7.5 A 9 6.0 a ______I A la I_ A 22 _4.6 5' 3' X Rv X K 3.0o K3 - 2.4 ESIGfISIGIN-TKRN-TERM | 1| K2I2JI _3~ K4 ILI PROTEASE-LIKE

A A A 0 31 129 202207 284301 379387 465 489 723 -1 .4 C 10 20 30 40 50 60 70 80 90 _RKRRN-dmiRKNIT HEFKKSAKTTLI KIDPALKtIKTltKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWFP ' 100 HK N FNSMSSGVKKEFGHEFDLYENKDY IRNCI IGKGRSYKGTVS ITKSGIKCQPWSSMIPHEH.%YRGKDLQENYCPRGEEGGPWCFTSNPEVRYEVCD IP ! 200

CSEVECMTCNGESYRGLMDHTESGKICQRNDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTIJ)PHTRNEYCAIKTCADNTMNDTDVP LETTEC 300 V M IQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKcKDLRENCtRNPDGSESPNcFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQ 400 A K N TRSGLTCSMWDINMEDLHRHI FWEPDASKLNENYCRNPDDDAiGPWCYTGNPLI PDYCP ISRCEGDTTPTIVN{LDHVI SCAKTKQLfRVVNG IPTRTNI 500 N V GWMSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDYEANGIHDVHGRGDEICQVLNVSQLVYGPEGSDLVUIUAAVLDDFVSTIDLPNYGCT 600 I N IPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAGAEKIGSGPCEGDYGGPLVCEGHKMMVL-GVIVPGRGCAIPNRPGI 700 FVRVAYYAMWIHKIILTYKVPQS FIG. 3. (A) cDNA clones encoding the mitogen. The largest cDNA insert, A9, and representative inserts of 3 kb, Ala and A22, are shown above a diagram of the complete coding sequence and the adjacent 5' and 3' untranslated regions. The coding sequence is boxed; untranslated regions are represented by a line. X, Xho I; Rv, EcoRV; K, Kpn I. An expanded view of the coding sequence shows the major domains and the amino acid residues defining their borders: SIG, signal peptide; N-TERM, amino terminus; K1-K4, kringles [-80-amino acid motifs containing three disulfide bridges (27)]; L, linker ending at internal cleavage site (arrow); PROTEASE-LIKE, serine protease domain. Open arrowhead indicates the site ofthe 5-amino acid deletion relative to the published HGF sequences. (B) Identification ofthe growth factor mRNAs by RNA blot analysis. A Northern blot ofM426 total cellular RNA was hybridized with a 32P-labeled 2.2-kb fragment ofAla containing the entire coding sequence (nucleotides -27 to 2199) according to standard procedures. (C) Predicted amino acid sequence (standard single-letter code). The peptide sequence obtained from the purified growth factor is underlined. The putative signal peptide is shaded. The 5-amino acid segment absent frnm nur sequence as compared with both reported HGF sequences (14, 23) is shown in a box below the deletion site. The additional differences in the HGF sequence reported by Nakamura et al. (23) are noted below our sequence. Highlighted among these differences are a boxed C-. R substitution and an overlined RGD segment. The internal cleavage site, conserved relative to plasminogen (28, 29), is identified by an open arrowhead; the substitutions for catalytically active residues are marked with asterisks.

lysates and was the predominant species secreted by the cells - DISCUSSION (Fig. 4B). Thus, the recombinant factor was largely synthe- sized and secreted as a single polypeptide chain. All of the We have identified a fibroblast-derived mitogen with activity above findings established that the molecular clone Ala on melanocytes, epithelial cells, and endothelial cells. Its encoded the purified, broad-spectrum mitogen. near identity to the published sequences of HGF was sur-

FIG. 4. (A) Mitogenic activity of the recombinant A B factor on B5/589 cells. Conditioned medium from M426 COS-S COS-AS COS-1 cells transfected with the Ala coding sequence in the sense (o) or antisense (A) orientation was 0 CL CM CL CM CL CM collected, filtered, and assayed on B5/589 cells for - kDa N N N N N N stimulation of [3H]thymidine incorporation. Medium 0. from cells transfected with the coding sequence in the 200 - w of s sense orientation was also tested in the presence ofan z%-i antiserum (final dilution, 1:50) that specifically neu- uz 925- 4 p87 tralized the activity of the purified growth factor (s). z Each point is the mean of duplicate measurements Q 69- that varied <3500 cpm. (B) Biosynthesis of the growth factor in M426 and transfected COS cells. [35S]Methionine-labeled proteins from cell lysates 46- (CL) or conditioned medium (CM) were immunopre- cipitated with nonimmune serum (N) or neutralizing antiserum prepared against the purified growth factor (I), electrophoresed under reducing conditions in an 30- SDS/l0o polyacrylamide gel, and detected by auto- radiography. COS-S and COS-AS, cells transfected CONDIONED MEDIUM with cDNA in the sense or antisense orientation, (DILUTION) respectively. Downloaded by guest on September 26, 2021 Biochemistry: Rubin et al. Proc. Natl. Acad. Sci. USA 88 (1991) 419 prising in view of the latter's initially reported properties. immortalized mammary epithelial cell line B5/589; T. Maciag for HGF was isolated from plasma (24, 26) or platelets (25) and providing HUVECs; H. Osada, S. Rudikoff, and D. Ron for helpful was considered to be a hormone-like activity released in discussions; S. R. Tronick and K. Usmani for assistance in running response to liver injury, with a narrowly defined target, the sequence-analysis computer programs; and N. Lichtenberg and F. hepatocyte (24-26). We have observed expression of the Hyman for preparation of the manuscript. A.C.C. and J.W. are Howard Hughes Institute/National Institutes of Health Research factor in stromal fibroblasts derived from adult skin, lung, Scholars. gastrointestinal tract, and prostate and in embryonic lung fibroblasts (unpublished observations). Others have recently 1. James, R. & Bradshaw, R. A. (1984) Annu. Rev. Biochem. 53, 259-292. reported detection of an HGF transcript (32) or immunolog- 2. Deuel, T. F. (1987) Annu. Rev. Cell Biol. 3, 443-492. ically crossreactive material (33) in several organs. More- 3. Barbul, A., Pines, E., Caldwell, M. & Hunt, T. K., eds. (1988) Prog. over, expression of the transcript in liver was demonstrated Clin. Biol. Res. 266. E. D. & H. L. as to 4. Goustin, A. S., Leof, B., Shipley, G. Moses, (1986) in the nonparenchymal component opposed hepato- Cancer Res. 46, 1015-1029. cytes (34). Thus, this factor appears to be widely expressed 5. Sporn, M. B. & Roberts, A. B. (1986) J. Clin. Invest. 78, 329-332. and likely to act in a paracrine fashion on a wide variety of 6. Ross, R. (1986) New Engl. J. Med. 314, 488-500. cell types. 7. Cunha, G. R., Chung, L. W. K., Shannon, J. M., Taguchi, 0. & Fujii, In contrast to published reports about HGF, we detected H. (1983) Recent Prog. Horm. Res. 39, 559-598. 8. Sawyer, R. H. & Fallow, 1. F., eds. (1983) Epithelial-Mesenchymal multiple transcripts that varied both in coding and in non- Interactions During Development (Praeger, New York). coding regions and could account for some of the heteroge- 9. Schor, S. L., Schor, A. M., Howell, A. & Crowther, D. (1987) Exp. Cell neity in the predicted HGF protein sequences. One cDNA Biol. 55, 11-17. had a coding sequence identical to that reported by Miyazawa 10. Rubin, J. S., Osada, H., Finch, P. W., Taylor, W. G., Rudikoff, S. & et while another differed from those of both Aaronson, S. A. (1989) Proc. Natl. Acad. Sci. USA 86, 802-806. al. (14), 11. Halaban, R., Ghosh, S. & Baird, A. (1987) In Vitro Cell. Dev. Biol. 23, Miyazawa et al. (14) and Nakamura et al. (23) due to the 47-52. absence of five amino acids: Phe-Leu-Pro-Ser-Ser. Of note, 12. Maciag, T., Hoover, G. A., Stemerman, M. B. & Weinstein, R. (1981) J. the Phe-Leu-Pro sequence is one of the few conserved Cell Biol. 91, 420-426. features in all seven members of the FGF/heparin-binding 13. Friesel, R., Burgess, W. H. & Maciag, T. (1989) Mol. Cell. Biol. 9, to 1857-1865. growth factor family, and its deletion in bFGF is thought 14. Miyazawa, K., Tsubouchi, H., Naka, D., Takahashi, K., Okigaki, M., cause gross conformational changes reflected in altered he- Arakaki, N., Nakayama, H., Hirono, S., Sakiyama, O., Takahashi, K., parin-binding properties (35). Thus, its deletion from HGF Gohda, E., Daikuhara, Y. & Kitamura, N. (1989) Biochem. Biophys. Res. might result in structural changes that could influence bio- Commun. 163, 967-973. logical activity. Among the 14 amino acid substitutions that 15. Miki, T., Matsui, T., Heidaran, M. A. & Aaronson, S. A. (1989) Gene 83, 137-146. further distinguished our HGF sequence from that of Naka- 16. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, mura et al. (23), one involved a potential integrin-binding H. A. & Arnheim, N. (1985) Science 230, 1350-1354. Arg-Gly-Asp (RGD) segment (36) and another added a cys- 17. Okayama, H. & Berg, P. (1983) Mol. Cell. Biol. 3, 280-289. teine residue, which could have an impact on disulfide bridge 18. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A assignments. If not due to sequencing errors, these differ- Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY), 2nd Ed., Vol. 3, pp. 16.1-16.8. ences could alter functional and structural properties. 19. Gluzman, Y. (1981) Cell 23, 175-182. Our studies raise questions concerning the processing of 20. Bottaro, D. P., Rubin, J. S., Ron, D., Finch, P. W., Florio, C. & HGF. Whereas previous reports have described the factor as Aaronson, S. A. (1990) J. Biol. Chem. 265, 12767-12770. a disulfide-linked heterodimer (24-26), we purified a larger 21. Hunter, T. & Cooper, J. A. (1985) Annu. Rev. Biochem. 54, 897-930. 22. Yarden, Y. & Ullrich, A. (1988) Annu. Rev. Biochem. 57, 443-478. single-chain polypeptide, p87, associated with variable and 23. Nakamura, T., Mishizawa, T., Hagiya, M., Seki, T., Shimonishi, M., typically smaller amounts of the heterodimeric form. More- Sugimura, A., Tashiro, K. & Shimizu, S. (1989) Nature (London) 342, over, our biosynthetic experiments demonstrated that the 440-443. recombinant factor was predominantly secreted as p87. 24. Gohda, E., Tsubouchi, H., Nakayama, H., Hirono, S., Sakiyama, O., Takahashi, K., Miyazaki, H., Hashimoto, S. & Daikuhara, Y. (1988) J. Whether mitogenic activity was due to the single-chain form Clin. Invest. 81, 414-419. itself or to processing to yield the disulfide-linked het- 25. Nakamura, T., Nawa, K., Ichihara, A., Kaise, N. & Nishimo, T. (1987) erodimer at the target cell remains to be determined. In this FEBS Lett. 224, 311-316. context, the remarkable similarities in structure ofthe growth 26. Zarnegar, R. & Michalopoulos, G. (1989) Cancer Res. 49, 3314-3320. 27. Patthy, L., Trexler, M., Vali, Z., Banyai, L. & Varadi, A. (1984) FEBS factor and plasminogen merit attention. As reported by Lett. 171, 131-136. Nakamura et al. (23), the sequences ofHGF and plasminogen 28. Sottrup-Jensen, L., Claeys, H., Zajdel, M., Peterson, T. E. & Magnus- are -35% identical, including the presence of kringles and a son, S. (1978) in Progress in Chemical Fibrinolysis and Thrombolysis, serine protease-like domain. In the case of plasminogen, eds., Davidson, J. F., Samama, M. M. & Desnoyers, P. C. (Raven, New York), Vol. 3, pp. 191-209. processing to the heterodimer, plasmin, activates fibrinolytic 29. Robbins, K. C., Summaria, L., Hsich, B. & Shah, R. J. (1967) J. Biol. activity. Because the putative cleavage site of the growth Chem. 242, 2333-2342. factor is identical to that ofplasminogen (Fig. 3C; refs. 34 and 30. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. 35), it seems likely that enzymes such as tissue plasminogen USA 74, 5463-5467. act on to 31. Breitbart, R. E., Andreadis, A. & Nadal-Ginard, B. (1987) Annu. Rev. activator and urokinase could the single-chain form Biochem. 56, 467-495. generate the two-chain structure. If so, the high level of 32. Tashiro, K., Hagiya, M., Nishizawa, T., Seki, T., Shimonishi, M., plasminogen activator expression in neoplasia, wound heal- Shimizu, S. & Nakamura, T. (1990) Proc. Natl. Acad. Sci. USA 87, ing, and tissue remodeling (37, 38) might play a major role in 3200-3204. 33. Zarnegar, R., Muga, S., Rahija, R. & Michalopoulos, G. (1990) Proc. regulating the biological function ofthe growth factor, should Natl. Acad. Sci. USA 87, 1252-1256. cleavage modify its activity. 34. Kinoshita, T., Tashiro, K. & Nakamura, T. (1989) Biochem. Biophys. Res. Commun. 165, 1229-1234. Note. After submission of this manuscript, reports appeared describ- 35. Seno, M., Sasada, R., Kurokawa, T. & Igarashi, K. (1990) Eur. J. ing a cytotoxic activity ofHGF on certain tumor cell lines (39) as well Biochem. 188, 239-245. as structural similarity if not identity of HGF to scatter factor, a 36. Ruoslahti, E. & Pierschbacher, M. D. (1987) Science 238, 491-497. molecule previously shown to stimulate the dissociation and scat- 37. Dan0, K., Andreasen, P. A., Gr0ndahl-Hansen, J., Kristensen, P., Nielsen, L. S. & Skriver, L. (1985) Adv. Cancer Res. 44, 139-266. tering of epithelial cells (40). 38. Saksela, 0. & Rifkin, D. B. (1988) Annu. Rev. Cell Biol. 4, 93-126. 39. Higashio, K., Shima, N., Goto, M., Itagaki, Y., Nagao, M., Yasuda, H. We thank R. Halaban for performing thymidine incorporation & Morinaga, T. (1990) Biochem. Biophys. Res. Commun. 170, 397-404. assays on human melanocytes; M. Stampfer for providing the 40. Gherardi, E. & Stoker, M. (1990) Nature (London) 346, 228. Downloaded by guest on September 26, 2021