Partial purification and characterization of ‘injurin-like’ factor which stimulates production of hepatocyte growth factor

Biochimica et Biophysica Acta, 1220 (1994) 291-298

291

© 1994 Elsevier Science B.V. All rights reserved 0167-4889/94/$07.00

BBAMCR 13512

Partial purification and characterization of 'injurin-like' factor which stimulates production of hepatocyte growth factor Hiroko Okazaki a,1 Kunio Matsumoto a,b and Toshikazu Nakamura a,, a Division of Biochemistry, Biomedical Research Center, Osaka University School of Medicine, Suita, Osaka 565 (Japan) and b 'Cell and Information' Group, Presto, Research Development Corporation of Japan (JRDC) Seika, Sohra-Gun, Kyoto 619-02 (Japan)

(Received 15 July 1993)

Key words: Hepatocyte growth factor; Injurin; Liver regeneration; MRC-5; (Human) We have previously reported the evidence for presence of a humoral factor 'injurin', which induces expression of the hepatocyte growth factor (HGF) gene in MRC-5 human embryonic lung fibroblasts. We have now purified a factor from porcine liver which stimulates HGF production but differs from injurin. When injurin activity was measured as a stimulatory effect on HGF production by MRC-5 cells, this activity was found in various acid extracts from porcine tissues, including liver, kidney, brain, and lung, and acid extracts from the liver was used for purification. When the acid extract was applied to Q-Sepharose anion-exchange chromatography, 50-60% of the total injurin activity was adsorbed to the column and the remaining activity was detected in the flow through fractions. Injurin activity was eluted from the Q-Sepharose column by NaCI concentration gradient with four peaks at 0.5-0.6 M, 0.7-0.8 M, 0.9-1.2 M. 1.5-2.0 M NaCI, thereby suggesting that the factor exists in heterogenous or various forms in tissues. The major active fractions were combined and applied to Mono-Q FPLC anion-exchange chromatography. Injurin activity eluted with a single peak at 0.9-1.5 M NaC1 and this activity was 4286 fold purified from the starting extract. Addition of this fraction to MRC-5 cells increased the amount of HGF pulse-labeled with [35S]methionine to a 3-4-fold higher level than that seen in control cells, whereas it had no significant effect on HGF mRNA levels. Therefore, this factor seems to stimulate HGF synthesis affecting translational processes and is distinct from the previously characterized injurin which stimulates HGF gene expression. Chemical treatments and SDS-polyacrylamide gel electrophoresis of this injurin-like factor indicated that injurin-like factor is a acid- and heat-stable non-proteinous factor with an apparent M r of 8-15 kDa. Since the injurin activity of the factor was decreased by heparinase treatment, the factor may be a polysulfated glycosaminoglycan related to heparin or to heparan sulfate. These results suggest that HGF production may be regulated by this non-proteinous injurin-like factor and that this factor may also play an important role in the regeneration of organs, through translationally enhancing HGF production.

Introduction Hepatocyte growth factor ( H G F ) was originally purified to homogeneity from rat platelets, as a potent mitogen for mature hepatocytes, [1,2]. H G F was thereafter purified from h u m a n plasma [3], rabbit plasma [4], liver of rats with CCl4-induced hepatitis [5] and conditioned m e d i u m of h u m a n fibroblasts [6]. H G F is a heterodimer composed of a 69 k D a a-subunit and a 34 kDa of /3-subunit and it has four kringle domains in the ot-subunit [7-11]. T h e high-affinity cellular receptor for H G F is the c-met protooncogene product which has a cytoplasmic tyrosine kinase domain [12-14].

* Corresponding author. Fax: + 81 6 8757419. 1 Present address: Central Research Laboratory, Ajinomoto Co., Totsuka, Yokohama 244, Japan. SSDI 0167-4889(93)E0161-R

Recent studies have shown that H G F is a pleiotropic factor. In addition to its mitogenic activity for various epithelial and endothelial cells [6,10,11,15], H G F has unique morphoregulatory functions for several types of cells, acting as epithelial m o r p h o g e n [16-18]. H G F induces a blanching tubule structure in M D C K renal epithelial cells [16] and hepatic bile duct epithelial cells grown in a collagen gel matrix [17], and a lumen-like structure is induced in colon carcinoma cells in monolayer culture [18]. Moreover, the coincidence of c D N A sequences of scatter factor and tumor cytotoxic factor with H G F indicate that H G F has motogenic activity (enhancement of cell motility) for various epithelial cells [19,20], acting as motogen, while it inhibits growth of various tumor cells in vitro and in vivo [21-23]. H G F m R N A and H G F protein increase markedly and rapidly in the liver after various injuries and diseases [10,11,24-27], and thus H G F is considered to be a hepatotropic factor for liver regeneration. This hepa-

292 totropic role of HGF was clearly evidenced by the finding that intravenously injected recombinant HGF into mice markedly enhanced liver regeneration in vivo [28], and liver regeneration was completed during a shorter period in transgenic mice in which HGF was persistently expressed in the liver [29]. Moreover, because the expression of HGF rapidly increases in the kidney following various injuries and because intravenously injected HGF markedly enhanced renal regeneration in experimental animals [30-32], HGF is now considered to function as the long-sought after renotropic factor for renal regeneration. On the basis of the 'trophic function' of HGF to enhance regeneration of organs and tissues following onset of injuries and diseases, we asked how the expression of HGF is regulated during injury. We found that acid- and heat-stable proteinous factor 'injurin', which enhances H G F gene expression and HGF production in MRC-5 human embryonic lung fibroblasts, increases in the plasma of rats after various injuries of the liver or kidney [33]. In the present study, we purified and characterized one of the 'injurin-like factors' from porcine liver. The factor stimulates H G F production through a post-transcriptional mechanism, and it is a acid- and heat-stable non-proteinous molecule, presumably related to heparin or heparan sulfate, with an apparent M r of 8-15 kDa.

tial medium (MEM) supplemented with 10% FCS, HL-60 cells were in RPMI1640 medium supplemented with 10% FCS, MRC-9 cells were in Eagle's MEM supplemented with 10% FCS, and non-essential amino acids composed of 8.9 mg/1 L-alanine, 10.0 mg/l Lasparagine, 13.3 mg/1 L-asparagic acid, 14.7 mg/l Lglutamic acid, 11.5 mg/1 L-proline, 10.5 mg/1 L-serine, and 7.5 mg/1 L-glycine. Normal human skin samples were obtained during plastic surgery and normal human keratinocytes were cultured as described elsewhere [35].

Measurement of injurin activity Cells were seeded on 24-well or 48-well plates (Costar) at a density of 5.104 cells/cm 2, and then cultured for 24 h. After medium was replaced with fresh DME medium with 1% FCS, samples were added to each well, and the preparation was further cultured for 24 h. H G F in each conditioned medium was measured by enzyme-linked immunosorvent assay (ELISAI as described [33]. One unit of injurin activity was defined as the activity which gives a half-maximal stimulatory effect on the production of HGF when acid extract from porcine lung (see below) was added. When HL-60 cells were used as responders, the cells were cultured in the presence of 0.1 n g / m l TPA.

Preparation of tissue extracts Materials and Methods

Materials Human recombinant HGF was purified from the culture medium of CHO ceils or C-127 cells transfected with expression vector containing human H G F cDNA [7,34]. 12-O-Tetradecanoylphorbol-13-acetate (TPA), Q-Sepharose and L-[35S]methionine (1100-1300 Ci/mmol) were purchased from Sigma (St. Louis, MO), Pharmacia and Du Pont-New England Nuclear (Boston), respectively.

5 ml of 1 M acetate buffer (pH 3.5) was added tc one g of tissue and the preparation was homogenizec with Polytron (Dipergier-und Mischtechnik, Switzer. land) for 2 min at 0°C. After stirring the homogenat~ for 3 h on ice, it was centrifuged at 100000 × g for 1 t at 4°C. The supernatant was neutralized to pH 7.0 centrifuged at 10000 × g for 20 min, then was dialyzec against phosphate-buffered saline (PBS), passec through a 0.22 mm pore-size filter (Millex GV, Milli pore), and used for assay of injurin activity.

Cell cultures

Partial purification of injurin-like factor from porcin, liver

MRC-5 (human embryonic lung fibroblasts, CCL171), WI-38 (human lung fibroblasts, JCRB0517), A172 (human, glioblastoma, JCRB0228), IMR-90 (human embryonic lung fibroblasts, JCRB0516), HL-60 (human acute promyelocytic leukemia, JCRB0085), MRC-9 (human fetal lung fibroblasts, CCL212) were obtained from the Japanese Cancer Research Resources Bank. HepG2 (human hepatoeellular carcinoma) was a kind gift from Genentech (San Francisco, CA). MRC-5, WI-38, Balb/c 3T3 (clone A31), A172, HepG2, A431, and normal human skin fihroblasts were cultured in Dulbecco's modified Eagle's (DME) medium supplemented with 10% fetal calf serum (FCS). IMR-90 cells were cultured in Eagle's minimum essen-

For partial purification of injurin-like factor fron porcine liver, the acid extract (50 mg protein/ml, 20q ml) in 20 mM sodium phosphate buffer (pH 7.2) wa loaded on a column of Q-Sepharose (20 cm 2 × 10 cm equilibrated with 20 mM sodium phosphate buffer, pt 7.2). After washing with 20 mM phosphate buffer con taining 0.5 M NaCI, the column was eluted with linear gradient of 0.5-2 M NaC1 in phosphate buffel Each fraction dialyzed against distilled water wa lyophilized and the lyophilized materials were di~, solved in PBS and used for injurin activity assay. I~ jurin active fractions were dialyzed against 20 ml~ sodium phosphate buffer (pH 7.2) and applied t Mono-Q anion-exchanger column equilibrated with th same buffer. The column was washed with 20 mb

293 phosphate buffer containing 0.5 M of NaCl and then eluted at a flow rate of 0.5 m l / m i n with a linear gradient of 0.5-2 M NaCl. Injurin activity in each fraction was measured as described above.

Northern blot hybridization RNA was purified from MRC-5 cells using the acid-guanidium thiocyanate-phenol-chloroform method [36]. 10 ~g of total RNA were electrophoresed in a 1% agarose, 0.7% formaldehyde gel and transferred to a Hybond-N nylon membrane. The BamHI-SalI fragment of clone pBS-7, including the full-length open reading frame eDNA for human H G F [7], was labeled with [a-32p]dCTP, using the multiprime labeling system, according to the manufacturer's instruction. The membrane was hybridized with radiolabeled eDNA at 42°C for 18 h in solution composed of 50% (v/v) formamide, 5 x SSPE, 4 × Denhardt's, 0.5% SDS, and 1 0 0 / z g / m l salmon sperm DNA. The filter was washed with 0.2 x SSPE containing 0.1% SDS for 15 min at 65°C, then was dried and autoradiographed.

Protein synthesis and immunoprecipitation MRC-5 cells were seeded on 6-well plates (Costar) at a density of 3" 104 cells/cm 2 and cultured for 24 h, then the partially purified injurin-like factor from porcine liver was added and cells were cultured for 12 h. The medium was changed to 1 ml of methionine-free medium supplemented with 20 nM [35S]methionine (27 /zCi/ml) and cultured for 2 h. Conditioned media were collected and the remaining cells were solubilized in lysis buffer composed of 150 mM NaCl., 50 mM Tris-HCl (pH 7.5), 0.05% SDS, 1% (v/v) Nonidet P-40, 1 mM PMSF, and 1 m g / m l soybean trypsin inhibitor. The conditioned medium and cell lysate were separately incubated with preimmune rabbit serum and subsequently with proteinA-agarose. After centrifugation at 1000 x g for 10 min, the supernatants were further incubated with anti-human H G F rabbit IgG or preimmune rabbit IgG, and subsequently with proteinA-agarose. After centrifugation, the precipitated proteinA-agarose was dissolved in sample buffer for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and centrifuged at 10000 Xg for 10 min. The supernatant was subjected to SDS-PAGE using 10-20% gradient gel according to the methods of Laemmli [37]. The gel was dried and autographed using Imaging plate BAS-III (Fiji Photo Film, Tokyo) for 24-48 h and analyzed with BIO-IMAGE analyzer (Fuji Photo Film).

SDS-PAGE and extraction of injurin-like factor SDS-PAGE was carried out by the method of Laemmli [37] under nonreducing conditions. Extraction of injurin-like factor from SDS-polyacrylamide gel was done as described elsewhere [33].

Chemical treatment of injurin-like factor Injurin-like factor after Mono-Q anion-exchanger FPLC was subjected to various treatments, as follows: The injurin active fraction was acidified to pH 3.5 with acetic acid. For protease treatments, the injurin active fraction containing 5 / z g protein was incubated in the presence of 50 ng of lysylendopeptiase, Vs protease, trypsin, chymotrypsin and proteinase K per ml at 37°C for 3 h, respectively, and the enzyme digestion was terminated by the addition of 1 m g / m l bovine serum albumin and rapid freezing at -40°C. When the injurin fraction was treated with heparinase, the fraction containing 5/zg protein was treated with 1.5/~g heparinase (Seikagaku Kogyo, Tokyo) at 37°C for 20 h. Protein concentration was measured by microBCA protein assay kit (Pierce Chemical), using bovine serum albumin as the standard. Results

Tissue distribution of injurin activity To examine the tissue distribution of injurin, we prepared acid extracts from various porcine tissues and measured injurin activity using MRC-5 cells. Except for platelet extract, the addition of acid extracts prepared from various tissues stimulated H G F production in the conditioned medium of MRC-5 cells (Table I). Relatively high injurin activities were found in extracts of the lung, cerebrum, cerebellum, spleen, and liver.

Partial purification of injurin-like factor from porcine liver Acid extract from the porcine liver was dissolved in 20 mM phosphate buffer (pH 7.2) and was first subjected to anion-exchange chromatography using a QTABLE I Injurin activityin acid extracts fromvarious porcine tissues Tissue Spleen Lung Liver Kidney(cortex) Kidney(medulla) Adrenal gland Heart Aorta Platelet Cerebellum Cerebrum

Specificactivity mU/mg 0.53 1.08 0.50 0.29 0.31 0.23 0.28 0.37 0.019 0.99 1.53

U/g wet wt. 3.71 9.50 3.87 2.58 2.85 1.70 1.81 2.31 0.30 2.40 2.39

MRC-5 cells were seeded on 48-well plate at a density of 5" 104 cells/cm2. After adding the extracts prepared under acidic conditions, cells were cultured for 24 h, and HGF in conditionedmedium was measured by ELISA. Each value represents the mean of triplicate measurements.

294

Sepharose column (Fig. 1A). In this condition, 50-60% of total injurin activity loaded onto the column was adsorbed to Q-Sepharose, and the remaining 40-50% of the activity was detected in the flow through fractions. Injurin activity adsorbed to Q-Sepharose was eluted at 0.5-0.6 M, 0.7-0.8 M, 0.9-1.2 M, and 1.5-2.0 M of NaCI, and 25.8% of the total injurin activity applied to the column was eluted at 0.9-1.2 M NaC1. Injurin active fractions (peak III) eluted at 0.9-1.2 M of NaCl from Q-Sepharose column were combined and further purified on a Mono-Q FPLC anion-exchange chromatography (Fig. 1B). Most of the injurin activity was eluted as a single peak at 0.9-1.5 M NaC1. Fig. 2 shows the dose-response curve of HGF synthesis when starting acid extract, or when the injurin active fraction after Mono-Q FPLC was added to MRC-5 cells. In the case of starting material, HGF synthesis was maximilly stimulated at 500 ~ g / m l °f

A

100

"~

I

I

0

100

I

I

I

50

0

I

2OO 3OO Protein (l~g/ml)

4O0

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t

I

alk

A

30

2s

j

i

10

-2.0

).2

-1.5

i

!

"~ 50

"1.0

).1

T

O_t

0

-0.S

0

) Flow through (290ml)

0

20

40

60

0.S 75

Injurln active fraction

I

I

-2.0

i

,'""

- 0.4

-1 .G

?

o.2T -1.0

| ~

I

1.5

Fig. 2. Dose-responsive stimulation of HGF production in MRC-5 cells by the addition of crude acid extract and partially purified injurin-like factor. (A) Crude acid extract of porcine liver; (13) injurin-like factor purified by two step column ehromatographies.

-0

80

Fraction number (4.S nd In each)

B

I t 0.5 1.0 Protein (p.g~t)

2S

...f -0.S

0~

protein (Fig. 2A). On the other hand, the stimulatory effect of this fraction on H G F synthesis was detected even at 0.25 /zg/ml and the maximal stimulation by 4.6-fold was seen at 0.3 /~g/ml of protein (Fig. 2B). The results of a typical purification are summarized in Table II. The recovery of injurin activity in this fraction was 14.4% with a 4286-fold increase in the specific activity over that in the crude acid extract.

0 0

1S

30

45

60

Fraction number (1 ml in each)

Fig. 1. Elution profiles of injurin-like factor on column chromatography. Crude acid extract prepared from ,porcine liver was applied on a Q-Sepharose column (A). Injurin active fractions were combined and applied on a Mono-Q FPLC column (B). Injurin activity was determined by measuring stimulatory effects on HGF production in MRC-5 cells, as described in Materials and Methods, and one unit of injurin activity was defined as the activity which gives a half-maximal stimulatory effect on the production of HGF when acid extract prepared from porcine lung was added. Fractions indicated by bars w e r e pooled and used for the following purification or for analysis of biological and chemical properties of injurin-like factor.

Effect on 35S-labeled H G F synthesis To determine whether the increase of HGF concentration in conditioned medium is due to enhancement of the net protein synthesis of H G F or merely to enhancement of protein secretion, proteins synthesized by MRC-5 cells were pulse-labeled for 2 h with [3SS]methionine and subjected to subsequent immunoprecipitation of HGF, using anti-HGF antibodies (Fig. 3). Autoradiograms of immuno-preeipitated proteins from conditioned medium indicated that asS-labeled H G F with a molecular mass of 82-84 kDa was specifi-

295 TABLE II

Oh

Summary of partial purification of a factor with injurin activity from porcine liver Protein (mg)

0.50 0.57 293.2 2,143

1 -

100 40.7

586 4,286

25.8 14.4

Injurin activity of each fraction was assayed using MRC-5 cells. 1 U was defined as the activity with a half-maximal stimulating effect on the synthesis of HGF when acid extract from porcine lung was added. For further details see Materials and Methods.

cally detected when using anti-HGF antibody but not with preimmune IgG. When the injurin active fraction was added to MRC-5 cells, the intensity of the band corresponding to H G F increased 3-4-fold higher level than in control one (Fig. 3). However, the addition of the injurin active fraction did not enhance the total protein synthesis as determined by incorporation of [35S]methionine into TCA insoluble proteins (not shown). These results indicate that the increase in H G F concentrations in the conditioned medium of MRC-5 cells by the addition of injurin active fraction was due to a stimulatory effect on the net protein synthesis of HGF. Effect on H G F mRNA levels We next examined changes in H G F mRNA levels in MRC-5 ceils after addition of the injurin active frac-

injurin-Iike factor anti-HGF preimmune

IgG IgG

HGF

--

//H

Total Specific Purification Recovery activity activity (-fold) (%) (U) (U/mg)

starting extract 10,000 4,950 flow through 3,507 2,016 Q-Sepharose (peak III) 4.4 1,290 Mono-Q 0.34 720

+

.

+

-

+

+

+ +

--

12h I

/

/

HGF

(6

Fig. 4. Northern blot analysis of HGF mRNA in MRC-5 cells treated with injurin-like factor. MRC-5 cells were cultured in the absence or presence of injurin-like factor for 12 h and RNA was extracted from the cells. Northern blots were hybridized with 32p-labeled human HGF eDNA probe. Ribosomal RNAs stained with ethidium bromide are shown in the lower photograph, to indicate the amount of RNAs loaded onto the gel.

tion (Fig. 4). 12 h after the addition, RNA was extracted and HGF mRNA levels were analyzed by Northern hybridization. Although this factor stimulated H G F synthesis, H G F mRNA levels in MRC-5 cells were not changed. Therefore, this factor stimulates H G F synthesis affecting translational processes but not by stimulating HGF mRNA expression. This factor is thus distinct from our previously characterized injurin [33] which stimulates H G F synthesis by enhancing H G F gene expression. We then characterized this factor as injurin-like factor.

84 kD

Fig. 3. Increase in the amount of [35S]methionine-labeled HGF. MRC-5 cells were seeded on 6-well plates at a density of 3" 104 cells/cm 2 and cultured for 20 h. Injurin-like factor was added and cultured for 12 h. Cells were then pulse-labeled with 20 nM [3SS]methionine for 2 h in the medium depleted with methionine. Immunoprecipitated proteins from conditioned medium using preimmune or anti-HGF IgG were respectively dissolved and subjected to SDS-PAGE. Standard proteins used for determination of molecular weight were phosphorylase b (97 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), soybean trypsin inhibitor (21 kDa), and lysozyme (14 kDa).

Stimulation of H G F synthesis in various cells To investigate which type of cells synthesize HGF and whether their H G F synthesis is regulated by injurin-like factor, we measured the stimulatory effect of injurin-like factor on HGF synthesis (Table III). HGF synthesis was detected in mesenchymal cells, including fibroblasts such as MRC-5, IMR-90, WI-38 cells, normal human skin fibroblasts, and Balb/c 3T3 and HL-60 hematopoietic cells. The addition of injurin-like factor increased H G F synthesis 3-4 fold in the fibroblasts. In contrast, HepG2, A172, A431 and normal human keratinocytes did not produce HGF, either in the absence or presence of the injurin-like factor.

296 TABLE III

TABLE IV

Effect of injurin-like factor on HGF synthesis in various species of cells

Effect of various treatment on injurin-like factor

Cell line

HGF synthesis (ng/ml)

MRC-5 IMR-90 MRC-9 WI-38 HL-60 Balb/c 3T3 Skin fibroblast A172 HepG2 A431 Keratinoeyte

None

Partially purified injurin

14.4 14.4 12.6 6.0 6.4 0.9 0.2 0 0 0 0

65.2 48.8 25.9 25.5 14.8 1.6 1.0 0 0 0 0

4.5 3.4 2.1 4.3 2.3 1.7 5.0 -

Each value represents the mean of triplicate measurements. Standard deviations of each value were within 10% of the mean value.

Chemical properties of injurin-like factor To analyze the chemical properties of injurin-like factor, the preparation was subjected to various treatments and its stimulatory effect on HGF synthesis in MRC-5 cells was examined (Table IV). Injurin-like factor was stable against acid-treatment (1 M acetic acid, 1 h), heat-treatment (100°C, 3 min), reduction with dithiothreitol (50 mM), and protease digestion with lysylendopeptidase, V8 protease, trypsin, chymotrypsin, or proteinase K. I

I

I

I

I/

5e

~

ae

4

i" •

1t1

0

e

-

Treatment

Injurin activity (% of control)

Untreated Heat (100°C, 3 min) Acid (1 M acetic acid, 1 h) Dithiotheitol (50 mM) Protease digestion (end Lys-C) (Va protease) (trypsin) (chymotrypsin) (proteinase K) Heparinase treatment

100 120 86 82 113 99 104 95 91 44

-fold

i;aa Imm NImlN¢ Fig. 5. Erectruphoretic separation of i~jurin-like factor by SDSPAGE. The iajurin active fraction conteining 25 mg of protein from the Mono-Q column was subjected to SDS-PAGE, under nonreducins conditions. After SDS-PAGE, proteins were extracted from the polyacrylamide gel and then precipitated with ethanol to exclude SDS before measurement of injurin activity. Standards for estimation of molecular mass were poephorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), soybean trypsin inhibitor (21 kDa), and lysozyme (14 kDa).

Since injurin-like factor seems to be non-proteinous factor and possess numerous negative charged groups in its molecule, we asked whether injurin activity of the factor would be affected by heparinase treatment. Indeed, injurin activity of this factor was decreased after treatment with heparinase. SDS-PAGE of injurin-like factor and subsequent measurements of injurin activity in extracts from gel slices indicated that injurin-like factor has an apparent M, of 8-15 kDa (Fig. 5). Discussion

In the present study, we obtained evidence that (1) injurin-like factor, which stimulates HGF synthesis in MRC-5 cells is distributed in a wide variety of tissues, (2) injurin-lLke factor was purified 4286-fold by acidic extraction, Q-Sepharose anion-exchanger chromatography, and Mono-Q FPLC anion-exchange chromatography, (3) injurin-like factor stimulates HGF synthesis in distinct types of cells by regulating translational mechanisms, and (4) this factor is an acid- and heatstable non-proteinous molecule with apparent M r of 8-15 kDa and may be a heparin-like molecule. We reported that a humoral factor, 'injurin,' which increases HGF mRNA in the intact lung and in MRC-5 cells in culture, was detected in the plasma of rats with liver injury, and we partially purified it from the plasma of rats administrated with CCI 4 by monitoring the stimulatory effect on HGF synthesis by MRC-5 cells [33]. Although attempts to define the chemical structure of injurin have yet been unsuccessful, injurin partially purified from the rat plasma proved to be an acid- and heat,stable, proteinous factor. Therefore, iajurin-like factor derived from the porcine liver is distinct from h~urin, in both biochemical and chemical properties. Since injurin-like factor was eluted from Q-Sepharose and Mono,Q FPLC columns at relatively higher NaCI concentrations, this factor seems to possess numerous negative charged groups in its molecule.

297

Moreover, injurin activity of the factor was decreased by heparinase, but not by protease treatments. Taken together, we assume that this factor may be a molecule(s) related to polysulfated glycosaminoglycans such as heparin and heparan sulfate, and we are now testing whether various types of glycosaminoglycans have biological activity to stimulate HGF synthesis. Because a significant amount (up to 40-50%) of injurin activity in the starting extract of porcine liver did not adsorb to the Q-Sepharose column, injurin which stimulates H G F gene expression may be present in other fractions and be distinct from injurin-like factor. Although HGF has been identified, purified, and molecularly cloned as a potent mitogen for mature hepatocytes in primary culture and its hepatotrophic function for liver regeneration has been well characterized, this is now considered to be a pleiotropic factor which possesses unique motogenic functions as a scatter factor [19,20] and epithelial morphogenic function [16-18]. Because the epithelial morphoregulatory function of HGF has not been duplicated with any other defined cytokines, and the mitogenic, motogenic, and morphogenic activities are all essential for the construction of normal tissue structure, H G F is thought to play important roles in embryogenesis, organogenesis and organ regeneration. Indeed, the expression of H G F mRNA and H G F protein increases markedly in response to the onset of injury and disease of the kidney and lung, as well as liver. Thus HGF seems to act as a 'trophic factor' which enhances regeneration of organs [30-32,38]. The manner in which H G F synthesis is regulated by overriding factors need to be addressed. We have recently shown that several chemically defined molecules regulate H G F gene expression. Interleukin-1 (IL-1) and tumor necrosis factor-a stimulate H G F synthesis by inducing H G F mRNA expression [39], whereas transforming growth factor-/31 and glucocorticoid suppress HGF mRNA, thereby inhibiting H G F production [40]. Though chemical structures of injurin and injurin-like factor have yet to be defined, since their chemical properties differ from the above well-definded cytokines, injurin and injurin-like factor are likely to be previously unknown molecules or a known molecule(s) with hitherto unsuspected injurin activity. Finally, because injurin activity, i.e., stimulation of H G F synthesis, was detected in a wide variety of tissues, injurin and injurin-like factor seem to distribute in various cells and tissues and may also exert 'trophic function' for regeneration of various organs, through biological activity to stimulate H G F synthesis. Furthermore, based on unique biological activities of H G F as mitogen, motogen, and morphogen, ongoing purification and elucidation of the chemical structure of injurin and injurin-like factor appear to be important for elucidating molecular mechanisms not only for mor-

phogenic tissue formation, i.e., embryogenesis, organogenesis, and organ regeneration, but also for pathogenesis of various organ diseases.

Acknowledgements We thank M. Ohara for helpful comments and to H. Tajima, T. Kinoshita, and S. Kohno for technical supports. This work was funded by a Research Grant for Studies on Science and Cancer from the Ministry of Education, Science and Culture of Japan, Nagase Science and Technology Foundation, Mochida Memorial Foundation for Medical and Pharmacological Research, and Terumo Life Science Foundation.

References 1 Nakamura, T., Teramoto, H. and Ichihara, A. (1986) Proc. Natl. Acad. Sci. USA 83, 6489-6493. 2 Nakamura, T., Nawa, K., Kaise, A. and Nishino, T. (1987) FEBS Lett. 224, 311-318. 3 Gohda, E., Tsubouchi, H., Nakayama, H., Hirono, S., Sakiyama, O., Takahashi, K., Miyazaki, H., Hashimoto, S. and Daikuhara, Y. (1988) J. Clin. Invest. 81, 414-419. 4 Zarnegar, R. and Michalopoulos, G.K. (1989) Cancer Res. 49, 3314-3320. 5 Asami, O., lhara, I., Shimidzu, I., Shimizu, S., Tomita, Y., Ichihara, A. and Nakamura, T. (1991) J. Biochem. 109, 8-13. 6 Rubin, J.S., Chan, A.M.-L., Bottaro, D.P., Burgess, W.H., Taylor, W.G., Cech, A.C., Hirschfield, D.W., Wong, J., Miki, T., Finch, P.W. and Aaronson, S.A. (1991) Proc. Natl. Acad. Sci. USA 88, 415-419. 7 Nakamura, T., Nishizawa, T., Hagiya, M., Seki, T., Shimonishi, M., Sugiyama, A., Tashiro, K. and Shimizu, S. (1989) Nature 342, 440-443. 8 Miyazawa, K., Tsubouchi, H., Naka, D., Takahashi, ~ , Okigaki, M., Arakaki, N., Nakayama, Y., Hirono, S., Sakiyama, O., Takahashi, K., Gohda, E., Daikuhara, Y. and Kitamura, N. (1989) Biochem. Biophys. Res. Commun. 163, 967-973. 9 Tashiro, K., Hagiya, M., Nishizawa, T., Seki, T., Shimonishi, M., Shimizu, S. and Nakamura, T. (1990) Proc. Natl. Acad. Sci. USA 87, 3200-3204. 10 Nakamura, T. (1991) Prog. Growth Factor Res. 3, 67-85. 11 Matsumoto, K. and Nakamura, T. (1992) Crit. Rev. Oncogenesis 3, 27-54. 12 Bottaro, D.P., Rubin, J.S., Faletto, D.L., Chan, A.M. -L., Kmiecik, T.E., Vande Woude, G.F. and Aaronson, S.A. (1991) Science 251, 802-804. 13 Naldini, L., Vigna, E., Narsimhan, R.P., Gaudino, G., Zarneger, R., Michalopoulos, G.K. and Comoglio, P.M. (1991) Oncogene 6, 501-504. 14 Higuchi, O., Mizuno, K., Vande woude, G.F. and Nakamura, T. (1992) FEBS Lett. 301, 282-286. 15 Igawa, T., Kanda, S., Kanetake, H., Saitoh, Y., Ichihara, A., Tomita, Y. and Nakamura, T. (1991) Biochem. Biophys. Res. Commun. 174, 831-838. 16 Montesano, R., Matsumoto, K., Nakamura, T. and Orci, L. (1991) Cell 67, 901-908. 17 Johnson, M.E., Matsumoto, K., Nakamura, T. and Iyer, A. (1993) Hepatology 17, 1052-1061. 18 Tsarfaty, I., Resau, J.H., Rulong, S., Keydar, I., Falleto, D.L. and Vande Woude, G.F. (1992) Science 257, 1258-1261. 19 Gherardi, E. and Stoker, M. (1991) Cancer Cells 3, 227-232.

298 20 Tajima, H., Matsumoto, K. and Nakamura, T. (1992) Exp. Cell Res. 202, 423-431. 21 Higashio, K., Shima, N, Goto, M., Itagaki, Y., Nagao, M., Yasuda, H. and Morinaga, T. (1990) Biochem. Biophys. Res. Commun. 170, 397-404. 22 Tajima, H., Matsumoto, K. and Nakamura, T. (1991) FEBS Lett. 291,229-232. 23 Shiota, G., Rhoads, D.P., Wang, T.C., Nakamura, T. and Schmidt, E.V. (1992) Proc. Natl. Acad. Sci. USA 89, 373-377. 24 Kinoshita, T., Tashiro, K. and Nakamura, T. (1989) Bioehem. Biophys. Res. Commun. 165, 1229-1234. 25 Selden, C., Jones, M., Wade, D. and Hodgson, H. (1990) FEBS Lett. 270, 81-84. 26 Hamanoue, M., Kawaida, K., Takao, S., Shimizu, H., Noji, S., Matsumoto, K. and Nakamura, T. (1992) Hepatology 16, 14851492. 27 Zarnegar, R., DeFrances, M.C., Kost, D.P., Lindroos, P., and Michalopoulos, G.K. (1991) Biochem. Biophys. Res. Commun. 177, 559-565. 28 Ishiki, Y., Ohnishi, H., Muto, Y., Matsumoto, K. and Nakamura, T. (1992) Hepatology 16, 1227-1235. 29 Shiota, G., Wang, T.C., dela Monte, S., Nakamura, T. and Schmidt, E.V. (1993) J. Clin. Invest., in press. 30 Nagaike, M., Hirao, S., Tajima, H., Noji, S., Taniguchi, S., Mat-

31 32 33

34

35 36 37 38 39 40

sumoto, K. and Nakamura, T. (1991) J. Biol. Chem. 266, 2278122784. Igawa, T., Kanda, S., Kanetake, H., Saitoh, Y., Matsumoto, K. and Nakamura, T. (1993) Am. J. Physiol. 265, F61-F69. Kawaida, K., Shimazu, H., Matsumoto, K. and Nakamura, T (1993) Proc. Natl. Acad. Sci. USA in press. Matsumoto, K., Tajima, H., Hamanoue, M., Kohno, S., Kinoshita, T. and Nakamura, T (1992) Proc. Natl. Acad. Sci. USA 89, 3800-3804. Seki, T., Ihara, I., Sugimura, A., Shimonishi, M., Nishizawa, T., Asami, O., Hagiya, M., Nakamura, T., amd Shimizu, S. (1990) Biochem. Biophys. Res. Commun. 172, 321-327. Matsumoto, K., Hashimoto, K., Yoshikawa, K. and Nakamura, T. (1991) Exp. Cell Res. 196, 114-120. Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156-159. Laemmli, U.K. (1970) Nature 227, 680-685. Yanagita, K., Matsumoto, K., Sekiguchi, K., Ishibashi, H., Niho, Y. and Nakamura, T. (1993) J. Biol. Chem. 268, 21212-21217. Matsumoto, K., Okazaki, H. and Nakamura, T. (1992) Bioehem. Biophys. Res. Commun. 188, 235-243. Matsumoto, K, Tajima, H, Okazaki H. and Nakamura, T. (1992) J. Biol. Chem. 267, 24917-24920.