Proc. Nati. Acad. Sci. USA Vol. 89, pp. 3800-3804, May 1992 Medical Sciences

Identification and characterization of "injurin," an inducer of expression of the gene for hepatocyte growth factor (organ regeneration/liver regeneration)

KUNIO MATSUMOTO, HISAO TAJIMA, MASAHIRO HAMANOUE*, SEIHO KOHNO, TAISEI KINOSHITA, AND TOSHIKAZU NAKAMURAt Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812, Japan

Communicated by Takashi Sugimura, December 30, 1991 (received for review December 6, 1991)

ABSTRACT The marked and rapid increase of hepatocyte growth factor (HGF) mRNA in the intact lung of rats after partial hepatectomy or unilateral nephrectomy suggests the existence of a humoral factor mediating a signal of injury to distal organs and may induce the expression of HGF gene in these organs. We have now identified a proteinous factor in the sera of rats with injury of liver or kidney that increases HGF mRNA in the intact lung. When the serum of rats with liver insult caused by partial hepatectomy or ischemic treatment was injected i.p. into normal noninjured rats, it induced a marked HGF mRNA expression in the lung of the recipient rats. The addition of serum from rats with various hepatic or renal injuries to MRC-5 human embryonic lung fibroblasts in culture also led to the induction of HGF mRNA expression, so that the production of HGF by MRC-5 cells after treatment with the sera was remarkably increased in the culture medium. However, serum from the normal intact rat induced no HGF production and no HGF mRNA in the lung in vivo and lung fibroblasts in vitro. This factor, which increases HGF production, was purified >200-fold from sera of CCI4-treated rats. The factor proved to be an acid- and heat-stable protein with an apparent molecular mass of 10-20 kDa in SDS/PAGE. Its activity markedly increased within 3-6 hr in the plasma of rats after various treatments that injured the liver or kidney. These results suggest that the factor specifically appears in the blood of rats with organ injury and may be involved in organ regeneration through the potential to increase the synthesis of HGF. Since the factor seems to mediate various organ injuries, we named it "injurin."

induced in the kidney after renal injury (26). More recently, we found that HGF mRNA was markedly and rapidly increased even in noninjured organs such as lung and spleen after partial hepatectomy and unilateral nephrectomy (27). Thus, it seems logical to assume that a humoral factor that induces HGF mRNA in distal organs is present in the blood after liver or renal injuries. Here we report evidence for the existence of a proteinous factor that induces HGF mRNA in distal noninjured organs. We partially purified and characterized this factor, which we call "injurin."

MATERIALS AND METHODS Operation of Animals and Preparation of Sera. Adult male Wistar rats (130-150 g) were used in the following experiments. After the animals were anesthetized with ether, the abdomen was incised, and the medial and left hepatic lobes were removed. For unilateral nephrectomy, after a right lumbodorsal incision, the right kidney of a separate group of rats was removed. Sera were obtained from intact rats and those with hepatic or renal injuries at various times after 70% partial hepatectomy, CCL4 administration (2 mg of CC14 per gram of body weight), ischemia caused by partial ligation of the portal vein, unilateral nephrectomy, HgCl2 administration (2 mg of HgCl2 per gram of body weight), or kanamycin administration (0.3 mg of kanamycin per gram of body weight). RNA (Northern) Blot Hybridization. Total RNA was extracted from the lung or MRC-5 cells (human embryonic lung fibroblasts) by the acid-guanidine-phenol-chloroform method (28). Poly(A)+ RNAs were purified by using Oligotex dT-30 (Roche Pharmaceuticals). Northern blot hybridization was carried out as described (6) with the EcoRI fragment of RBC-1 (6) or BamHI-Sal I fragment of pBS-7 (5) as a probe. The EcoRI fragment of RBC-1 included the fourth kringle domain of the a chain, the entire P3 chain, and part of the 3' noncoding region of rat HGF cDNA. The BamHI-Sal I fragment of clone pBS-7 included the full-length open reading frame of human HGF cDNA. Assay for HGF-Inducing Activity (Injurin Activity) in Vivo. Two milliliters of sera obtained from intact rats or those with injury of the liver were injected i.p. into other normal intact rats. Six hours later the lung was removed, and HGF mRNA expression in the lung was analyzed by Northern hybridization.

Hepatocyte growth factor (HGF) was first purified from rat platelets as a potent mitogen for mature hepatocytes (1, 2). HGF is an unusual cytokine composed of the 69-kDa a subunit and the 34-kDa P3 subunit. Molecular cloning of both human (3-5) and rat (6) HGF cDNAs revealed that HGF is derived from a single prepropeptide of 728 amino acid residues by proteolytic cleavage and that it contains four kringle domains in the a chain. Until recently, HGF was considered to have a narrow target-cell specificity; however, recent findings suggest that it is a pleiotropic factor that is produced by mesenchymal cells and acts on various epithelial cells (7, 8). HGF strongly enhances the growth (9-12) or motility (13-16) of various cells, induces epithelial tubule formation as an epithelial morphogen (17), and yet inhibits growth of several tumor cell lines (18-20). HGF has been shown to act as a hepatotropic factor for liver regeneration, and recently as a renotropic factor for renal regeneration, from the findings that HGF mRNA and HGF activities were markedly induced in the liver or plasma of rats after various liver insults (21-25), and they were also

Abbreviations: HGF, hepatocyte growth factor; IL-la, IL-1f, IL-2, IL-6, interleukins la, 1f3, 2, and 6; aFGF and bFGF, acidic and basic fibroblast growth factors; TGF-a and TGF-P, transforming growth factors a and (8. *Present address: The First Department of Surgery, Faculty of Medicine, Kagoshima University, Kagoshima 890, Japan. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Assay for HGF-Inducing Activity (Injurin Activity) in Vitro. MRC-5 cells were cultured in 24-well plates in Dulbecco's modified Eagle's medium (DMEM) containing 10%o (vol/vol) fetal calf serum and antibiotics (100 units of penicillin and 100 ,ug of streptomycin per ml). After reaching subconfluency, the medium was replaced with fresh DMEM lacking fetal calf serum, various sera were added to each well, and the cells were cultured for 24 hr. HGF in conditioned medium of MRC-5 cells was measured by an ELISA. Rabbit anti-human HGF antibodies were coated on a 96-well plate at 370C for 15 hr. After blocking with bovine serum albumin solution, conditioned medium was added to each well, and the preparation was incubated for 2 hr at 370C. Wells were washed three times with PBS-Tween [phosphate-buffered saline (PBS) containing 0.025% Tween 20], biotinylated polyclonal anti-human HGF IgG was added, and incubation was allowed to proceed for 2 hr at 370C. Wells were washed three times with PBS-Tween, incubated with the horseradish peroxidase-conjugated streptoavidin-biotin complex in PBS-Tween at 370C for 1 hr, and then washed three times with PBS-Tween. The enzyme reaction was initiated by adding substrate solution composed of 50 mM citric acid, 100 mM sodium phosphate, 2.5 mg o-phenylenediamine per ml, and 0.015% H202. The enzyme reaction was halted by adding 1 M H2SO4, and absorbance at 490 nm was measured. One unit of injurin activity was defined as that with a half-maximal stimulatory effect on the production of HGF when CC14-administrated rat serum was added at the maximal dose. Partial Purification of Injurin. CCL4-treated rat sera were obtained 15 hr after the i.p. injection of CCL4. Serum was acidified to pH 3.5 with formic acid, stirred at 4°C for 2 hr, and passed through a 0.22-,m pore-sized filter (Millipore). The acidified serum (20 ml) was loaded onto a Bio-Gel P-60 (Bio-Rad) column (19.6 cm2 x 60 cm) equilibrated with 100 mM ammonium formate (pH 3.5). To assay injurin activity, each fraction was extensively Iyophilized, dissolved in 50 mM Hepes-NaOH (pH 7.4), and sterilized by filtration. Injurin fractions from Bio-Gel P-60 were dissolved in 10 mM Hepes-NaOH buffer (pH 7.2) containing 0.2 M NaCI and loaded onto a Sephadex G-150 (Pharmacia) column (5.3 cm2 x 90 cm) equilibrated with the same buffer. An aliquot of each fraction was added to cultures of MRC-5 cells for the assay of injurin activity. SDS/PAGE and Extraction of Injurin from Polyacrylamide Gel. SDS/PAGE was carried out by the method of Laemmli (29) under nonreducing conditions. After SDS/PAGE, the polyacrylamide gel was cut into small pieces (3-mm width) and crushed with a Teflon homogenizer. Phosphate-buffered saline was added to each tube, and incubation was carried out at 4°C for 15 hr with shaking. After centrifugation at 1000 x g for 20 min, bovine serum albumin was added to the supernatant to a final concentration of 25 ,g/ml. The extract was dialyzed against H20 at 4°C for 12 hr, Iyophilized, and dissolved in 10 mM Hepes-NaOH buffer (pH 7.2) containing 0.15 M NaCl. Then bovine serum albumin was added as a carrier to a final concentration of 4 mg/ml. Cold ethanol (-20°C) was added, and the preparation was incubated on ice for 30 min. After centrifugation at 15,000 x g for 10 min, the precipitate was dissolved in 10 mM ammonium acetate and Iyophilized; then the material was dissolved in 10 mM Hepes-NaOH (pH 7.2) containing 0.15 M NaCI and used for the measurement of injurin activity. Analysis of Chemical Properties. Active fractions of injurin after molecular-sieve chromatography on a Sephadex G-150 column were subjected to various treatments as follows. The injurin fraction was acidified to pH 5.0 or 3.5 with acetic acid and to 1.0 with HCl. For trypsin treatment, the injurin fraction (470 ,ug of protein per ml) was incubated in the presence of 100 Ag of trypsin (type III; Sigma) per ml at 370C

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for 3 hr, and the enzymatic digestion was terminated by addition of 200 gg of soybean trypsin inhibitor (type I-S; Sigma) per ml. The injurin fraction (470 ,ug of protein per ml) was incubated in the presence of 0.31 milliunits of heparinase per ml at 370C for 1 hr, and the reaction was terminated by the addition of 1 M HCL.

RESULTS Injurin Activity in Sera After Rat Organ Injuries. Demonstration of injurin activity in vivo. To identify a factor that induces HGF mRNA in the intact lung after partial hepatectomy, we first obtained sera from rats subjected to partial hepatectomy or liver ischemia and injected the serum i.p. into normal intact rats. Six hours after the injection, HGF mRNA expression in the lung was analyzed by Northern hybridization (Fig. 1). In the intact lung, 6-kilobase (kb) HGF mRNA was expressed, and injection of sera from rats with liver injuries increased the HGF mRNA level in the lung. The induction of HGF mRNA was found with sera obtained at 6, 12, and 18 hr after partial hepatectomy and at 12 hr after liver ischemia. The injection of serum from the normal intact rat produced no change. These results suggest the presence of a factor inducing HGF mRNA expression in the sera of rats with liver injuries and that this factor is responsible for the increase of HGF mRNA in the lung after partial hepatectomy and unilateral nephrectomy. Since the factor seems to mediate the initial regenerative processes responding to organ injuries through its potential to induce HGF mRNA, we tentatively named it injurin. To characterize injurin, rat serum obtained after partial hepatectomy was treated with acid (pH 3.5) or with acid and heat (100°C for 5 min) or with ultrafiltration, and HGF mRNA in the lung was analyzed 6 hr after the injection of serum into normal intact rats (Fig. 2). HGF mRNA in the lung remained increased even when the serum was treated with acid or with acid and heat. With ultrafiltration treatment, the increase of HGF mRNA was also found in the fraction of molecular mass > 10 kDa but not in the flow-through fraction < 10 kDa. Therefore, injurin seems to be an acid- and heat-stable factor with an apparent molecular mass of >10 kDa. Injurin activity in vitro. Injurin activity in the sera of rats was also found in vitro when MRC-5 fibroblasts derived from the human embryonic lung were used as responder cells. When sera from rats given CCl4 was added to cultures of MRC-5 cells, HGF mRNA in the MRC-5 cells was markedly

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FIG. 1. Expression of HGF mRNA in the rat lung following the i.p. injection of serum obtained from rats at various hours (h) after liver insult induced by 70%o partial hepatectomy (lanes P.H.) or ischemia. Two milliliters of sera was injected i.p. into normal rats, and their lungs were resected 6 hr later. Poly(A)+ RNAs (2 'g) were electrophoresed, and the expression of HGF mRNA was analyzed by Northern hybridization, with the EcoRI fragment of RBC-1 as probe.

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i 0 FIG. 2. Expression of HGF mRNA (arrows) in the rat lung after i.p. injection of serum from the 70% partially hepatectomized (lanes P.H.) rat. Acid and acid with heat (Left) or ultrafiltration (Right) were used for treatment. Sera (2 ml) treated with acid (pH 3.5), with acid and heat (100TC for 5 min) or with both ultrafiltrated and unfiltrated fractions derived from 2 ml of rat sera were injected into normal rats, and the lung was resected 6 hr later. Poly(A)+ RNAs (2 jug) were electrophoresed, and expression of HGF mRNA was analyzed by Northern hybridization with the EcoRI fragment of RBC-1 as probe. kD, kDa.

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increased 3 hr later; however, normal rat serum did not increase HGF mRNA level in these cells (Fig. 3). Moreover, the addition of rat serum obtained after CC14 administration or partial hepatectomy to MRC-5 cells increased the production of HGF by 3- to 4-fold in a dose-dependent manner, whereas addition of normal rat serum did not increase HGF-production (data not shown). Therefore, measurement of HGF production by MRC-5 cells provides a useful assay system for the purification and characterization of injurin. Changes of injurin activity after various injuries of rat liver and kidney. After CC14 treatment, injurin activity in the serum reached a maximal level as early as 3 hr later, and this level persisted for at least 24 hr (Fig. 4 Upper). In the case of partial hepatectomy, injurin activity reached a maximal level in 3-6 hr. In the case of liver ischemia, injurin activity increased 3 hr after the treatment, reached a maximal level at 12 hr, and then decreased. Therefore, changes in injurin activity in sera of rats after partial hepatectomy or liver ischemia as determined in vitro were consistent with the

FIG. 4. Injurin activity of sera from rats with liver or renal injuries. (Upper) Injurin activity in rat sera after hepatic injuries caused by CC!4 administration (e), partial hepatectomy (o), or liver ischemia (A). (Lower) Injurin activity in rat sera after renal injury caused by unilateral nephrectomy (A), HgCl2 administration (I), or kanamycin administration (a). Injurin activity was determined by measuring the increase in HGF production in MRC-5 cells with the addition of rat serum. Various sera were added to cultures of MRC-5 cells, and HGF in conditioned medium was measured by ELISA.

FIG. 3. Expression of HGF mRNA in MRC-5 cells at various hours (h) after addition of serum from normal or CC14 administrated rats. Total RNA (10 .&g) from MRC-5 cells was electrophoresed and analyzed by Northern hybridization with the BamHI-Sal I fragment of pBS-7 as probe.

results obtained in vivo. Increases in injurin activity were also noted in sera of rats with renal injury (Fig. 4 Lower). Increase of injurin activities in sera of rats after unilateral nephrectomy, HgCl2 administration, or kanamycin administration initiated 3 hr after the treatment, reaching a maximal level during hours 3-6 after treatment. Partial Purification and Characterization of Injurin. Serum obtained from CC4-treated rats was subjected to molecularsieve chromatography on a Bio-Gel P-60 column under acidic conditions (Fig. 5 Upper). The major injurin activity was eluted from fractions 45-60 and was clearly separated from the major protein peak. Minor peaks associated with injurin activity were detected in other fractions; however, these peaks did not consistently appear in repeated experiments. After molecular-sieve chromatography on a Bio-Gel P-60 column, injurin was purified to -100-fold of the starting serum, and this fraction was further purified on a Sephadex G-150 column (Fig. 5 Lower). Injurin activity was eluted from fractions 56-64 but showed no sharp single peak. Judging from the positions of marker proteins, the molecular mass of injurin seems to be 10-30 kDa, given that the nonspecific interaction between injurin and the gel was negligible. After the chromatography, injurin was purified > 200-fold from the

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ity was detected at positions corresponding to a molecular mass of 10-20 kDa as a major peak and of about 50 kDa as a second small peak as shown in Fig. 6. Protein staining of the corresponding polyacrylamide gel indicates that the injurin active fraction from the Sephadex G-150 column still contained several proteins (Fig. 6). Two relatively diffuse bands were visible between 10 and 20 kDa, although it is unclear at present whether one of these bands truly represents injurin. To assess whether well-characterized cytokines might mimic the stimulatory effect of injurin on HGF production by MRC-5 cells, MRC-5 cells were incubated with y interferon; tumor necrosis factor a; interleukins la, 1(3, 2, and 6 (IL-la, IL-13, IL-2, and IL-6); acidic and basic fibroblast growth factors (aFGF and bFGF; also called heparin-binding growth factor types 1 and 2); and transforming growth factors a and P (TGF-a and TGF-P3). None of these cytokines at either 1 ng/ml or 10 ng/ml increased the production of HGF by MRC-5 cells.

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FIG. 5. Chromatographic separation of injurin from senim of rats treated with CC14. (Upper) Molecular-sieve chromatograpi&y of sera from CC14-treated rats on Bio-Gel P-60 column under acicdic conditions. (Lower) Molecular-sieve chromatography on a !Sephadex G-150 column under neutral conditions of the injurin frac.tion from the Bio-Gel P-60 column. Injurin activity was determinedd by measuring the increase of HGF production in MRC-5 cells with the addition of each fraction. Standard proteins for the estirmation of molecular mass indicated by arrows were aldolase (158 kDaa), bovine serum albumin (67 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (14 kDa).

starting material, and this fraction was used as matterial for the characterization of injurin in the following expe riments. The effects of various treatments on injurin activ ity were examined (Table 1). Injurin activity was diminish ed after trypsin digestion but remained active after treatme nts with heat (60-100°C), acid (pH 1.0-5.0), reduction by dit]lhiothreitol, or digestion with heparinase. To estimate the mLolecular mass of injurin, the injurin fraction was subjected Ito SDS/ PAGE and extracted from polyacrylamide gel. Injurin activ-

Injuries of organs or tissues have been known to induce the expression of growth factors such as aFGF (30), TGF-a (31), TGF-,B (32), and platelet-derived growth factor (33) as well as HGF (7, 8). The production of some of these growth factors increases in autoregulation mechanisms in vitro (34, 35); however, it is important to elucidate regulatory mechanisms functioning in vivo and to search for a molecule with a predominant role in regulating production of one or more growth factors. In the present study we obtained evidence that injurin, which enhances the production of HGF in the noninjured organ, indeed is present in the sera of rats with organ injuries. Supportive evidence is as follows. (i) Injection of sera from rats with various organ injury into normal, intact rats induced a rapid increase of HGF mRNA in the intact lung, and the sera also increased HGF production by human lung fibroblasts (MRC-5 cells) in vitro. (ii) The appearance of injurin in the blood preceded the increase of HGF mRNA in the lung-i.e., injurin activity in the serum reached an almost maximal level as early as 3 hr after either partial hepatectomy or unilateral nephrectomy, and the increase in HGF mRNA levels in the lung started 6 hr after these treatments (27). 50 0

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Gel slice number FIG. 6. Electrophoretic separation of injurin by SDS/PAGE. The injurin fraction containing 300 g.g of protein from the Sephadex G-150 column was subjected to SDS/PAGE under nonreducing conditions. After SDS/PAGE, proteins were extracted from the polyacrylamide gel and precipitated by ethanol to exclude SDS before measurement of injurin activity. Standards for the estimation of molecular mass were phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), soybean trypsin inhibitor (21 kDa), and lysozyme (14 kDa). Proteins were visualized by silver staining.

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Chemical treatments of injurin fractions suggest that injurin is a protein(s) with a molecular mass of 10-20 kDa, is destroyed by trypsin, but is resistant to heat, acid, dithiothreitol, or heparinase treatment. Additionally, it seems to have no species specificity in biological activity, as sera of rats with organ injury increased both HGF mRNA expression and HGF production in MRC-5 fibroblasts derived from the human lung. It is unlikely that injurin represents hormones such as insulin, glucagon, and vasopressin because the molecular masses greatly differ. Moreover, the stimulatory effect on the production of HGF by MRC-5 cells was not duplicated by known cytokines, including y interferon, tumor necrosis factor a, IL-la, IL-1/3, IL-2, IL-6, aFGF, bFGF, TGF-a, and TGF-13. Taken together with its chemical and physiological properties, injurin is likely to be a previously unknown molecule(s) or a known molecule(s) with hitherto unsuspected injurin activity. Injurin is widely distributed in liver, kidney, lung, spleen, brain, and leukocytes (unpublished data), and it also increases the production of HGF by other cells, including HL-60 human promyelocytic leukemia cells and normal human skin fibroblasts. Based on considerations that (i) HGF functions as a mitogen, a motogen, and a morphogen for various epithelial cells derived from various tissues and (ii) its expression is regulated by injurin, the injurin/HGF system seems to be involved not only in organ regeneration but also in homeostasis of the organ mass and tissues. Injurin may be a unique molecule functioning in an unidentified selfdefensive system through its potential to regulate expression of the HGF gene and probably other genes involved in tissue organization. We thank M. Ohara for helpful comments. This study was supported by a Research Grant for Studies on Science and Cancer from the Ministry of Education, Science, and Culture of Japan.

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Identification and characterization of "injurin," an inducer of expression of the gene for hepatocyte growth factor.

The marked and rapid increase of hepatocyte growth factor (HGF) mRNA in the intact lung of rats after partial hepatectomy or unilateral nephrectomy su...
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