in Growth Factor Research. Vol. 3. pp. 67-85. 1991 Printed in Great Britain. All rights reserved.

Progrm

STRUCTURE HEPATOCYTE

OY55-2235191 $O.OO+ .5i1 I991 Pergamon Press plc

AND FUNCTION OF GROWTH FACTOR

Toshikazu Department

t

Nakamura

of Biology, Faculty Kyushu University Fukuoka 8 12. Japan

of Science

Hepatocytegrowthfactor (HGF), a potent mitogenfor mature hepatocytes in primary culture, was first found in sera of partial hepatectomized rats and seems to be a hepatotrophic factor for liver regeneration which has not been pur@ed over the past 30 years. HGF is composed of the 69 kDa a-subunit and the 34 kDa P-subunit. Molecular cloning reveals that HGF is derived from a single chain precursor of 728 amino acid residues and it contains 4 kringle domains in the u-subunit. HGFgene spans about 70kb and consists of 18 exons and 17 introns. HGF is now thought to be a pleiotropicfactor itq?uencing a cell growth and cell motility for various epithelial cells. HGF receptor with K, = X1-30~~ is widely distributed in various epithefial cells including hepatocytes. HGF mRNA and HGF activity increase markedly in liver after various fiver injuries and in kidney,fXowing unilateral nephectomy or acute renal injury. Moreover. HGF mRNA is induced even in the intact lung in response to liver and kidney injury. In situ hybridization reveals that HGF-producing ceils are mesenchymal cells such as Kupfler cells and sinusoidal endothelial cells in liver, fenestrated endothelial cells in kidne?,. and macrophages and endothelial cells in lung. Thus, HGF may play an important role as a puracrine or endocrine mediator through an epithelial-mesenchymal interaction in wound-healing, tissue or organ regeneration, morphogenesis and carcinogenesis.

Hepatocyte growth factor, hepatocyte proliferation, hepatotropic factor, liver regeneration,HGF gene,HGF receptor, renotropic factor. renal regeneration. cell surfacemodulator. Keywords:

INTRODUCTION The liver plays important and diverse roles in the metabolism of the body. It also regenerates actively after injury such as partial hepatectomy and hepatitis. Liver regeneration is one of the most dramatic examples of tissue repair in animals. Investigators have studied the mechanisms of liver regeneration and many attempts have been made to demonstrate a hepatotrophic factor that acts as a trigger for liver regeneration after partial hepatectomy, since it was suggested about 30 years ago that

Ach-no~~/edg~~menIs-This work was supported by a research grant for Science and Cancer from the Ministry of Education, Science and Culture of Japan and by research grants from the Princess Takamatsu Cancer Research Fund. the Cell Science Research Foundation. and the Terumo Life Science Foundation.

67

T. Nakamura

68

liver regeneration is mediated by a humoral factor by using liver transplantation and cross-circulation experiments with parabiotic rats [l-6]. However, no such humoral factor has been purified and characterized, because a simple, reproducible and sensitive in vitro method is not available for its assay. In the past decade, however several studies, including our own [7-121, have demonstrated that adult rat hepatocytes in primary culture, which retain many liver specific functions and respond to various hormones like their in vivo counterparts [13-l 51, can proliferate at low cell density when insulin and EGF are added to the culture medium. In 1984, using this in vitro assay system we first identified a putative hepatotrophic factor in the serum of partially hepatectomized rats. It strongly stimulated DNA synthesis and growth of adult rat hepatocytes in primary culture. We partially purified it and named it, hepatocyte growth factor (HGF), or hepatotropin [ 161. Subsequently, HGF was also found in rat platelets [ 17,181, in normal rat serum (19), in the sera of patients after sugical removal of hepatoma (201 and in the plasma of patients with fulminant hepatic failure [21]. In 1986, we purified HGF to homogeneity from platelets of 2,000 rats, and demonstrated that it is a new growth factor [22,23]. Finally, we recently succeeded in cloning HGF cDNA and determined the complete amino acid sequence of rat and human HGF from the nucleotide sequences of their cDNAs [24,25]. Here I describe recent studies on the structure and function of HGF and discuss its possible roles in tissue or organ repair as well as liver regeneration. CHEMICAL

AND BIOLOGICAL

PROPERTIES

OF HGF

Chemical Properties

HGF was first purified to homogeneity, from rat platelets, by a four step procedure including heparin affinity chromatography [22,23]. In 1984, we noticed that HGF has strong affinity to heparin-Sepharose CL-6B during a survey of good ligands for affinity chromatography of HGF [16]. This affinity to heparin greatly facilitated purification of HGF and, consequently, HGF could be purified from rat platelets to homogeneity. resulting in high yield by only four steps. Thereafter, other investigators also used this heparin-affinity chromatography for purification of HGF from the plasma of patients with fulminant hepatitis [26] and human serum [27]. HGF is also a glycoprotein which has strong affinity to concanavalin A. HGF has a molecular weight of 82-85 kDa on SDS-polyacrylamide gel electrophoresis [23]. Under reducing conditions, it gives two bands with apparent molecular weights of 69 kDa and 34 kDa, respectively. Thus, HGF is a heterodimeric molecule composed of an cc-subunit of 69 kDa and a p-subunit of 34 kDa, linked by a disulfide bond. HGF is a heat-labile protein; it loses activity appreciably on heating to 56°C for 30 min or completely on boiling for 1.5 min. Activity is also lost partially on treatment with acetic acid and completely by trypsin digestion or reduction with dithiothreitol. Chemical and biological properties of HGF are summarized in Table 1. In 1988, we determined 19 and 27 amino acid residues of the N-terminal portions of a- and P-subunit, respectively. These amino acid sequences had no homology to known growth factors. Biological Properties

HGF alone markedly

stimulated

DNA

synthesis of adult rat hepatocytes. The

Hepatocyte

Growth

Factor

TABLE 1. Chemical and biological properties of HGF.

(1)

Chemical properties Molecular weight N-terminal

amino

Treatments

causing

Affinity Affinity

(2)

(SDS-PAGE)

82kDa (nonreduced) 69kDa and 34kDa (reduced) z-chain-Pyro-Gln @hain-Val -heating (100°C. 3 min) -acid (1 M acetic acid) -trypsin digestion --dithiothreitol reduction high high

acids inactivation

to heparin to Concanavalin

A

Biological properties Receptor high affinity low affinity Concentration for maximum Additivity of effect Species specificity Target cells Growth stimulation

Growth

Stimulation

inhibition

of motility

activity

Kd : 2&30 PM sites/cell : 20&1000 Kd : 300-500 PM sites/cell : 1-2 x IO4 5-10 rig/ml (60-90~~) with EGF and insulin no hepatocytes renal tubular epithelial cells epidermal keratinocytes epidermal melanocytes Mv ILu (mink lung epithelial cells) Balb/MK (mouse keratinocytes) B6/F 1 (mouse melanoma I KB (human squamous carcinoma cells) HepG2 (human hepatoma) Epidermal keratinocytes MDCK (canine kidney epithelial cells) Lu99 (human lung epithelin cells) A431 (human epidermoid carcinoma cells) HepG2.

purified rat HGF was markedly effective at a concentration as low as 1 rig/ml and was maximally effective at 8 rig/ml (about 90pM). The maximum activity is 2 or 3 times higher than that of EGF. At this maximum dose of HGF, about 40% of rat hepatocytes entered the S-phase,judging from the labeling index. The effect of HGF is additive with those of insulin and EGF or TGF-ar. After treatment with HGF, EGF and insulin, over 80% of adult rat hepatocytes entered the S phase. HGF doesnot exhibit speciesspecificity in activity. For example, rat HGF markedly stimulates DNA synthesis of human hepatocytes in primary culture prepared from liver biopsy specimens.In addition, rat HGF also stimulates DNA synthesis of dog, pig, and mousehepatocytes in primary culture. Conversely, human HGF hasthe same potency on stimulation of DNA synthesis of primary cultured rat hepatocytes as that of rat HGF. However, HGF does not stimulate DNA synthesis and growth of nonparenchymal liver cellsand many fibroblasts suchasSwiss3T3 cellsand NRK-49F cells. It was recently found that the effect of HGF is not restricted to hepatocytes. It markedly stimulated DNA synthesisand growth of normal rabbit renal epithelial cells

70

T. Nakamura

in secondary cultures [28], normal human epidermal melanocytes [29] and keratinocytes [30]. In addition to stimulating cell growth. HGF markedly enchanced the motility of many epithelial cells suchascanine kidney epithelial cells[31,32] and human keratinocytes [30]. Moreover, HGF strongly inhibited the growth of several tumor cells such as B6/Fl melanoma [33] and HepG2 hepatoma cells [33,34]. Recently, scatter factor, which enhanced the motility of eplithelial cells causing colonies to scatter, was found to be identical to HGF [35,36]. Similarly, a fibroblastderived tumor cytotoxic factor was also found to be the same as HGF [37]. HGF is therefore a multifunctional factor for many epithelial cells and may be involved in various important biological processes, such as wound-healing, tissue or organ regeneration, and organogenesisduring embryogenesis. MOLECULAR

CLONING

AND PRIMARY

STRUCTURE OF HGF

To isolate the cDNA encoding rat HGF, we synthesized various oligonucleotides designedto have a sequencecorresponding to the N-terminus of the rat-HGF P-chain asa hybridization probe and constructed cDNA library in hgt 10 from poly (A)‘ RNA extracted from livers of Ccl, treated rats, becausethe livers of rats treated the Ccl, contain large amounts of HGF, as described later. The screening of 2 x lOh recombinant phagesyielded one positive clone. This positive clone had the 1.4kb insert which contained a open reading frame coding a 404 amino acid residue protein. In the middle of this open reading frame, we confirmed the 27 amino acid sequenceof Nterminal portion of the p-subunit. Using this rat HGF cDNA. the size of HGF mRNA was investigated. Northern blot hybridization showed that the size of HGF mRNA was about 6 kb. Therefore, both rat and human HGF cDNA. which are sufficient to encode the entire mRNA of 6kb. were screenedfrom human or rat liver cDNA library using rat HGFcDNA as a probe. Finally, both full size cDNAs of rat and human were cloned in 1989 [24,25]. The nucleotide sequenceand deduced amino acid sequenceof human HGF are shown in Fig. 1. The sequence comprises a single open reading frame of 2,184 nucleotides and 3,580 nucleotides of the 3 noncoding regions, which contain many A’U-rich sequences.The A’ U-rich sequenceis known to be a recognition signal for an mRNA processing pathway for degradation of the mRNA, and mRNAs possessing this A’U-rich sequenceare thought to be expressedtransiently. In the open reading frame, we also confirmed the 19 amino acid residuesof N-terminus of the cc-subunitof rat HGF. Thus, both a- and p-subunits of HGF are encoded in a single mRNA. A polyeptide of 728 amino acids is encoded by human HGF cDNA. The first 31 amino acid residues,beginning with methionine are hydrophobic, which is typical of a signal sequence.The C-terminus of the m-subunitis followed directly by the N-terminus of the /I- subunit. The sequenceat the cleavage site between the a and p subunits is Arg-Val (residues 495 and 496), and this sequencemay be cleaved by a trypsin-like protease. Thus, HGF is synthesized as a pre-pro HGF precursor of 728 amino acids and then mature HGF is formed by proteolytic cleavage (Fig. 2). Thecr- and P-chains of human HGF consist of 463 and 234 amino acids, respectively. The predicted molecular weight of the a-chain and B-chain are 54,180 and 26,089, respectively. Each subunit contains two N-linked glycosylation sites, which are located at positions 294 and 402 of the cychain and at positions 566 and 653 of the p-chain. The differences between the

1. Nucleotide sequence of human HGF and its deduced amino acid sequence. Nucleotide numbers are shown at the right, and amino acid numbers above the sequence. The amino acid sequences of the HGF & and /3chains determined experimentally are underlined. Dotted underlining indicates kringle domains. The proteolytic cleavage site is boxed. Putative Nglycosylation sites are marked by wavy lines. The glutamate at position 534 and the tyrosine at 676 (circled) correspond to the hi&dine and serine, respectively in plasmin, which form the active center for the protease. The cysteine residues that form an interchain disulfide bond between the a- and pchains are indicated by asterisks.

T. Nakamura

72

P-subunit

(234 a.a.)

1

processing

FIGURE 2. Representation of hmnaa pm-pro HGF and schematic closed box represents the signal peptide. The solid arrow indicates

structare of the predicted the processing site.

mature

form. The

molecular weights determined by SDS-PAGE and those calculated from the cDNAs may be due to the glycosylation of each subunit and over-estimation of the molecular weight caused by destruction of disulfide bonds. Interestingly, HGF has considerable homology (38%) with plasminogen, although it has no homology with any known growth factors. As shown in the predicted primary structure of human HGF (Fig. 2), the or-chain of HGF contains four kringle structures, like the A-chain of plasmin which has five kringle domains. Kringle structures are also found in several proteins such as tissue plasminogen activator, urokinase, prothrombin, and coagulation factor XII [38]. The kringle domain is thought to play a role in the interaction between macromolecules. On the other hand, the P-chain of the HGF has significant homology with various serine proteases, including the B-chain of plasmin. However, the histidine and serine residues of the active sites of the serine proteases are replaced in the a-chain of HGF by glutamine and tyrosine, respectively. So HGF should not have proteolytic activity. This has recently been confirmed using a large amount of purified human recombinant HGF. Neither plasminogen nor plasmin has HGF activity which stimulates DNA synthesis of adult rat hepatocytes in primary culture. In mature HGF, Cys 487 in the a-chain and Cys 604 in the P-chain seem to form an interchain bridge as shown in Fig. 2. Human HGF cDNA was also cloned by Miyazawe et al. (39)

73

Heputocyte Growth Factor

To demonstrate that HGF cDNA clone may produce a functional protein, human HGF precursor cDNA was introduced into an expression vector with the SV40 early promoter and then transfected into COS-1 cells by the DEAE-dextran method. The conditioned medium from the cells transfected with the plasmid carrying the HGF cDNA revealed the dose-dependent stimulation of DNA synthesis of adult rat hepatocytes in primary culture [24]. Human recombinant HGF had a similar molecular weight, and composition of a- and P-chains to the mature, native HGF, as estimated by SDS-PAGE. rat: human:

MMJ-GlXLLPVLLLQHVLLHLLLLPVTIPYm --"-----A--------------IA----H--H------I------~---

a

I-

QKKRRNTLHEFKKSAKTT

50

rat: human:

LTKEDPLVKIKTKKVNSADECANRCIRNKGFPFTCKAFVFDKSRKRCYWY

rat : huma":

PFNSMSSGVKKGFGHEFDLYENKDYIRNCIIGKGGSYKGTVSITKS(~IKC -----------E----------------------R-----------.----

150

rat: human:

QPWNSMIPHEHSFLPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYE ---S------------------------------------------.----

200

rat: human:

VCDIPQCSEVECMTCNGESYRGPMDHTESGKTCQRWDQQTPHRHKFLPER ----------------------~--------1-----"--------.----

250

rat: human:

YPDKGFDDNYCRNPDGKPRPWCYTLDPDTPWEYCAI~CAHSAVNETDVP

300

rat: human:

METTECIKGQGEGYRGTTNTIWNGIPCQRWDSQYPHKHDITPENFK(:KDL

rat: human:

RENYCRNPDGAESPWCFTTDPNIRVGYCSQIPKCDVSSGQDCYRGN[;KNY ----------S---------------------N--M-N--------~---

400

rat: human:

MGNLSKTRSGLTCSMWDKNMEDLHRHIFWEPDASKLTKNYCRNPDDI)AHG -----Q----------N-------------------NE--------~---

450

rat: numa*:

PWCYTGNPLVPWDYCPISRCEGDTTPTIVNLDHPVISCAKTKQL ---------I-----------------------------------

rat: huma":

PTQTTVGWMVSLKYRNKHICGGSLIKESWVLTARQCFPARNKDLKDYEAW --R-N----I--R-------------------------S----..---

550

rat: human:

LGIHDVHERGEEKRKQILNISQLVYGPEGSDLVLLKLARPAILDNFVSTI -------G--------V--V--------------M------V--D--N--

600

rat: human:

DLPSYGCTIPEKTTCSIYGWGYTGLINADGLLRVAHLYIMGNEKCSQHHQ ---N---------S--V----------y-------------------.--R

650

rat: human:

GKVTLNESELCAGAEKIGSGPCEGDYGGPLICEQHKMRMVLGVIVP(;RGC ---------I--------------------V----------------.---

700

rat: huma,,:

AIPNRPGIFVRVAYYAKWIHKVILTYKL

728

100

-I-I--AL--------T--Q-----T--N-L-----------A--Q.-L-F

----------------Q----------H-R-------T--DNT--D~---

350

-------Q---------A---------------------M------~---

P GI F __---

500

---------------------I-----VPQS

FIGURE 3. Alignment of amino acid sequences of rat and human HGF. Amino acid numbers right hand side. Identical amino acids are indicated by dashes in the sequence of human HGF.

are given at the

Figure 3 shows a comparable alignment of amino acid sequences of human and rat HGF. The amino acid sequence of human HGF is about 90% identical to that of rat HGF, hence, HGF seems to have no species specificity for its biological activity. There are however some insertions and deletions in the amino acid sequence of the P-subunit

74

T. Nakamura

of human and rat. Therefore, the length of the /?-subunit of human and rat HGF is 234 and 233 amino acid residues, respectively. The C-terminus of human P-subunit is serine whereas that of rat is leucine. Another minor difference is found in the length of the signal sequence of HGF; 3 1 amino acid residues in human HGF and 32 amino acid residues in rat HGF. Recently. cDNA for a variant of HGF was cloned from human leukocytes [40] and human embryonic lung fibroblasts [41]. The sequence showed that it had an in-frame deletion of 15 base pairs within the first kringle region. A transient expression using COS- 1 cells revealed that deletion of 5 amino acid residues caused by the 15 base pair deletion did not affect the biological activiity of HGF in vitro. HUMAN

HGF-ENCODING

GENE

Recently the human HGF gene was cloned and characterized [42]. Figure 4 shows restriction map and the location of exons of human HGF gene. This gene spans approximately 70kb and consists of 18 exons, interrupted by 17 introns. The first exon contains the S-untranslated region and the signal peptide. The next ten exons encode the a-chain which contains four kringle structures. Each kringle domain is encoded by two exons as observed in other kringle-containing proteins. The cleavage site between the IY- and P-chain encoded in the thirteenth exon. The remaining six exons constitute the p-chain. The last, eighteenth, exon contains the C-terminal part of the pchain and a long 3’-noncoding region. The organization of the human HGF gene is highly homologous to that of human plasminogen (43) suggesting that the HGF gene is evolutionally related to plasminogen genes. Signal(29 aa.)

a

(463a.a.)

wk-

CM2

LEADER

H

I

II A

R

R

IUN B

R

RR

C RR

V

H

UvlI

D

RR

4, I ’ 1’ I11 I I III1 0

5

10

15

20

25

R

RR

I 30

I RR

I I II

35

40

I

XXI Ii

G

CATALYTICDOMAIN

H

VIIIK

F

E R

I

*xlu%%m J R RR

I I 45

--c

CHO

KRINGLE4

KRINUE 3

KRINGLE 2

H

p (234 a.a.) CHO

K

MNOP RR R

Q

III IIIIII I

Ill 50

L RR

55

60

65

70 I0

FIGURE 4. Exon-intron structure of the human HGF gene. Positions of the exons are shown with narrow black boxes and numbered with Roman numerals. The introns are indicated with capital letters. R indicates the restriction site of Eco RI.

We also found that there are many kinds of c&elements-regulatory motifs upstream from the TATA box on HGF genes of humans and rats. More recently, the locus of HGF gene was analyzed in chromosomes of mouse and woodchuck and it is located in the q22 of the chromosome 3 in woodchuck, and the proximal region of the chromosome 5 in mouse. We also determined in humans that it is located in the chromosome 7.

Hepatocyte Growth Factor

7.5

PHYSIOLOGICAL

ROLES OF HGF IN LIVER REGENERATION

Induction of Gene Expression of HGF in Rat Liver Physiological studies on HGF in regenerating livers have shown that HGF has a central role in liver regeneration as a hepatotrophic factor after liver injuries such as partial hepatectomy and hepatitis. Hepatitis can be induced in rats by administration of hepatotoxin such as Ccl, and D-galactosamine. HGF activity markedly increasedin the sera of rats with induced hepatitis in a dosedependent fashion after treatment with various concentrations of Ccl,. HGF activity in the seracorrelated well with the degreeof liver damage, determined by the increaseof serum GPT and histological observations. HGF activity in the livers of rats with hepatitis also increasesmarkedly 12h after Ccl, administration and reached over 20fold higher than that of the normal level after 30h [44]. This finding indicates that liver itself can produce HGF when injured and suggeststhat the marked increaseof HGF in sera of rats with hepatitis is derived in part from the injured tissue. 1 .o

I

I

0

IO

I

I

+ E a E % 3

0.5

s! : E i

1

0 Time

after

I 20

adminlstration

I 30 of CC14

40 (hr)

FIGURE 5. Time courses of changes in HGF mRNA level and HGF activity in Poly(A)RNAs were extracted from the livers at the indicated times after treatment Northern hybridization. HGF mRNA amounts (0) were determined by scanning activity (0) was assayed by [‘*“I] iododeoxyuridine incorporation into DNA in adult culture and expressed as units/liver.

the liver of rats with Ccl,. with Ccl, and analyzed by the autoradiograms. HGF rat hepatocytes in primary

Northern blot analysis of HGF mRNA confirmed that regenerating liver itself produces HGF. As shown in Fig. 5, HGF mRNA level is negligible or undetectable in normal rat liver, but it increased time-dependently in the liver of rats treated with Ccl,, being detectable at 5h after administration and at a maximum level at 10h [25,45]. Dgalactosamine, which causessevere hepatitis, also induces HGF mRNA in rat liver with a good correlation between its concentration and the degree of liver damage. Similarly, a marked increase in HGF mRNA was found in the liver of a patient with hepatitis. A high level of HGF protein was also observed in the plasma of a patient with

T. Nakamura

76

fulminant hepatic failure [46]. An increasein HGF activity wasalso found in the ascites and plasma from patients with liver cirrhosis, but not in those from patients without cirrhosis [47]. More recently, we found that HGF mRNA also increased markedly in the liver of the rat after partial ligation of portal vein (ischemic damage) or physical damage (liver crush). Thusthe liver is one of the major HGF producing organs and HGF production is induced before the start of liver regeneration, following liver injury. HGF Producing

Cells in Liver

To determine which type of liver cell produces HGF, we first examined, using Northern blot analysis, whether parenchymal or nonparenchymal liver cells express HGF mRNA. Northern blot hybridization, after isolation of parenchymal hepatocytes and nonparenchymal liver cells, revealed that HGF mRNA was present only in the nonparenchymal liver cells and that their HGF mRNA level increased markedly after administration of Ccl,, as shown in Fig. 6.

FIGURE 6. HGF mRNA levels in parenchymsl and nonparenchymal liver cells of rats with or without Ccl,. Liver cells were isolated by iosituperfusion with cokgenase, and parenchymal and noqarenchymal cells were separated by differential centrifugation. Their RNAs were extracted and analyzed by Northern hybridization. P, parenchymal hepatocytes; N, nonparenchymal liver cells.

Hepatoeyte Growth Factor

77

To identify the nonparenchymal liver cells responsible for HGF production. we carried out in situ hybridization on serial sections of rat livers treated with Ccl,. As shown in Fig. 7, HGF mRNA localized in Kupffer cells and sinusoidal endothelial cells, whereas no expression of HGF mRNA is found in any parenchymal hepatocytes [48]. We also confirmed this finding using Northern blot analysis of Kupffer cells and sinusoidal endothelial cells isolated by counter-current elutriation method. Therefore, the growth of hepatocytes during liver regeneration after hepatitis may be mainly regulated by HGF produced by Kupffer cells and sinusoidal endothelial cells, in a paracrine fashion.

FIGURE 7. Localization Kupffer cells.

E.utrahepatic

of HGF

Production

mRNA

in liver of rat treated

qf HGF

After Partial Nephrectomy

with CCI,.

E, sinusoidal

Hepatectomy

endothelinl

cells; K,

and Unilateral

It is of particular interest that both HGF mRNA and HGF activity in remnant liver after 70% partial hepatectomy increased, but the increase is not so marked and rapid as that found in the case of hepatitis induced by Ccl, treatment. Since the initial peak of DNA synthesis in remnant liver has been shown to appear about 24h after the partial hepatectomy, the initial mitogenic signal should act within 12h. Therefore, HGF newly synthesized in the remnant liver is unlikely to trigger the initial prominent DNA synthesis of hepatocytes, but it may function to sustain subsequent hepatocyte growth and functional maturation. However, HGF activity in the blood plasma markedly increased within 3h after 70% partial hepatectomy, as shown in Fig. 8, suggesting that the initiation of the liver regeneration after the operation depends on HGF supplied from extrahepatic organs.

78

T. Nakamura

02

14 Q0 0

, 6

Hours

FIGURE 8. Time course of the increase in HGF 0; partial hepatectomy, 0; sham operation.

L,

12

after

18

partial

activity

~. ~

24

hepaiectomy

in the blood plasma of rats after partial

hepatectomy.

Previously we have shown that HGF mRNA is present not only in the liver, but also in various other tissuesof the rat [25]. For example, rat platelets contain a relatively large amount of HGF and it was secreted from platelets after the aggregation [17, 18, 221. We recently confirmed a wide tissue distribution of HGF using an immunohistochemical technique. Thus, it is conceivable that the HGF-producing organs responding to liver injuries are not restricted to the liver. In fact. we recently found that HGF mRNA rapidly increased in intact lung, spleen and kidney after partial hepatectomy or Ccl, administration. Similar marked increasesin HGF mRNA were also found not only in the remaining kidney [49] but also in intact lung after unilateral nephrectomy. In situ hybridization revealed that the cells expressing HGF mRNA seemto be fenestrated endothelial cells in the kidney and endothelial cells in the lung [48,49]. Therefore, in addition to paracrine mechanisms,the endocrine mechanismof HGF supplied from other tissuesor organs may be involved in liver regeneration. In fact, initiation of liver regeneration after partial hepatectomy may depend on HGF from endocrine pathway rather than the paracrine system. In addition to the role of HGF produced from other organs in liver regeneration, HGF may play an important role in the repair of these organs themselves. We are interested in the possible role of HGF as a putative renal trophic factor in compensatory growth and regeneration of the kidney after nephrectomy or the injury. This is in view of the following data: HGF exhibits potent mitogenic activity for renal tubular epithelial cells; HGF mRNA expression in the kidney increasesafter unilateral nephrectomy and various treatments with nephrotoxins; and a high affinity receptor for HGF in kidney and renal tubular cells has been identified. HGF RECEPTOR A specific receptor for HGF was recently identified, using “sl-labeled human recombinant HGF asa ligand, on rat hepatocytes [50]. Scatchard analysisrevealed that HGF receptor had a Kd of 20-3Op~, a value in good accord with half maximum dose

70

Hepatocyte Growth Factor

for HGF activity, and a receptor density of 500-600 sites/cell. More recently, Zarnega c’t al. also reported that the Kd values of the HGF receptor and the receptor number per hepatocyte were 3.5nM and 120,000 respectively [51]. Their K,, value and receptor density were about IOO-fold higher than ours. This difference probably relates to the different ligands used for the receptor assay: they used HGF purified from rabbit serum. a preparation which contains a contaminant with MW of about 30kDa. Affinit) cross-linking of the receptor with ‘*‘I-HGF showed that the HGF receptor in the plasma membranes from rat liver was a polypeptide of MW of approximately 220 kDa. Interestingly, the number of HGF receptors in liver plasma membranes decreased rapidly without change in Kd value after partial hepatectomy or the administration of CCI,, as shown in Fig. 9. This rapid down-regulation of the HGF receptor before the initiation of liver regeneration suggests that HGF is immediately supplied to the injured liver and is internalized after binding to its receptor and signaling to hepatocyte. After one week when liver regeneration was complete, the receptor number was restored to the level in normal rat liver. These findings further suggest that HGF supplied within 3-6h from both hepatic and extrahepatic tissues after liver injury acts as a trigger for liver regeneration. I

I

I

II

0 0

0.1

‘261.ffiF fmoU50

0.2

bound, @g protel”

" 2

1 Time

after

treatment

II ”

F 7

(days)

FIGURE 9. Change in the number of HGF receptor on plasma membranes from rat livers after partial hepatectomy and hepatitis induced by CC&. Plasma membranes were purified from the residual liver of hepatectomized rats (0) or the liver from Ccl, treated rats (a) at the indicated time. The inset panel shows a Scatchard plot:(A), normal liver: 3h (W) and t2h (0) after partial hepatectomy; 3h (0) and 24h (A) after the treatment with Ccl,.

The HGF receptor is also present in rat lung, kidney, spleen, many endocrine organs, and in various epithelial cell lines. However, the HGF receptor was not observed in nonparenchymal liver cells and in many cell lines of mesenchymal origin including blood cells and fibroblasts. Alternatively, mesenchymal-derived cells such as Kupffer cells and endothelial cells synthesize and secrete HGF. Therefore, HGF is likely to be a factor influencing epithelial cell growth or cell motility through an epitheliaC

80

T. Nakamura

mesenchymal interaction. Moreover, the tissue distribution of HGF receptor correlates with that of HGF mRNA. Also HGF strongly stimulated DNA synthesis and cell growth of many epithelial cells such as rabbit renal tubular cells [28], human melanocytes [29], and epidermal keratinocytes [30]. Thus, HGF may play an important role not only in liver regeneration, but also in repair of other tissues or in homeostatic turnover. To elucidate the molecular mechanism of HGF action, purification, characterization and molecular cloning of the HGF receptor are required. Structure, function, and expression of HGF receptor may elucidate the roles of HGF in embryogenesis, wound-healing or tissue repair, and carcinogenesis. Recently, Aaronson’s group reported that the HGF receptor was identified as the c-met protooncogene product using its antibody [52]. The c-met proto-oncogene product is a receptor-like tyrosine kinase composed of disulfide-linked subunits of 5OkDa and 145 kDa [53,54]. We are now confirming whether the transfected c-met gene actually encodes a high affinity HGF receptor with Kd of 20-30 PM. MOLECULAR

MECHANISM OF THE INITIATION REGENERATION

OF LIVER

In normal liver lobules, hepatocytes are arranged radially around central veins and the cells are in tight contact. In these conditions hepatocytes do not grow but express specific functions fully, being in the quiescent G, phase. As previously reported [55-591, the regulation of growth and differentiated functions of hepatocytes through cell-cell contact was elucidated in in vitro studies using adult rat hepatocytes in primary culture. In cultures at high cell density, the hepatocytes form tight cellLcel1 contact and remain in the quiescent G, phase, as in normal liver lobules; they do not proliferate even in the presence of excess amounts of growth factors, but they fully express liver specific functions. On the contrary, in cultures at low cell density, the cells do not form tight cell contact and so move to the G, phase, in which they can respond to growth factors and their cell cycle progresses from G, to the S phase. This G,-G, cell cycle transition in hepatocytes through cell-cell contact was shown to be regulated by a plasma membrane protein named ‘cell surface modulator’ (CSM) [56]. This mechanism may also operate in viva in regulating the growth and differentiation of hepatocytes in liver regeneration: in intact lobules, CSM on hepatocyte cell membranes gives a signal to suppress their growth and stimulate their expression of differentiated functions. However, when the liver is injured such as by hepatitis or partial hepatectomy, cell-cell contact of the hepatocytes is loosened. The growth suppression of hepatocytes by CSM may be released and the cells may progress from the G, to the G, phase. The existence of this mechanism is supported by the finding that cellcell contact between hepatocytes is loosened in regenerating liver in viva [60-621, and that the c-nzvc gene is expressed at an early stage after partial hepatectomy [63]. Hepatocytes in the G, phase respond to HGF supplied by paracrine and endocrine system and start to grow. The initiation mechanism of liver regeneration is summarized in Fig. 10. HGF is induced in response to various types of injury. These findings suggest that there must exist an HGF inducer which responds to various injuries and mediates the induction of HGF mRNA in HGF-producing cells. We are now interested in the

Hepatocyte

Growth

HI

Factor

Intact liver Lung’ Kidney Spleen

Go w

0

CSM

FIGURE

10. Possible

mechanisms

Kupffer

( Endotheiial

a -

cells cells 1

Paramine

Endocrine

G1

u

No>arenchymal liver cells

1 (FEF)

Liver Regeneration involved

in the initiation

of liver

regeneration

after

the injury.

identification of this HGF inducer. The purification and the molecular cloning of the HGF inducer should provide information on the molecular mechanism of not only tissue repair and liver regeneration, but also the homeostasis of tissue organization. CONCLUSION Hepatocyte growth factor (HGF) is the most potent mitogen for mature hepatocytes in primary culture and seems to be a hepatotrophic factor which has not been purified during the past 30 years. HGF is a heterodimeric molecule composed of a 69kDa LXsubunit and a 34KDa psubunit. cDNA cloning revealed that the mature heterodimer, HGF, is derived from a single chain precursor of 728 amino acid residues. HGF has no amino acid sequence homology with other known growth factors, but a 38% homology with plasmin. The a-subunit of HGF contains four kringle structures and the HGF Psubunit has a 37% homology with a serine protease domain of plasmin. Both a- and Pchain have two IV-glycosylation sites in each subunit. The homology between rat and human HGF is about 90%. Biologically active recombinant HGF was expressed from COS-1 cells and CHO cells transfected with the cloned cDNA. cDNA for a variant of HGF was also cloned from human leukocytes and embryonic lung fibroblasts. The variant HGF has a deletion of 5 amino acid residues within the first kringle domain. The human HGF gene spans about 70kb and consists of 18 exons and 17 introns. The organization of HGF gene is highly homologous to that of plasminogen. HGF activity and the HGF mRNA level are markedly increased in the liver following injury such as hepatitis caused by the administration of hepatotoxins. ischemia, physical damage (liver crush), and paritial hepatectomy. In situ hybridization and cell fractionation revealed that HGF-producing cells in liver are nonparenchymal cells, presumably Kupffer cells and sinusoidal endothelial cells. After liver injury, HGF mRNA is also rapidly induced in the lung, spleen and kidney. Therefore, HGF from neighboring cells in liver and from extrahepatic organs may function as a trigger for liver regeneration by both paracrine and endocrine mechanisms. This was supported

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by the finding that a high affinity HGF receptor with Kd of 20-30 PM on liver plasma membranes was rapidly down-regulated within 6h of partial hepatectomy and Ccl,administration. Affinity cross-linking experiments revealed that the molecular weight of the HGF receptor is approximately 220kDa. Also HGF may function as a renotrophic factor as well as hepatotrophic factor, because HGF mRNA is markedly induced in the remaining kidney after unilateral nephrectomy. HGF has mitogenic activity for renal tubular epithelial cells, epidermal melanocytes and keratinocytes as well as mature hepatocytes, whereas it strongly inhibits growth of various carcinoma cells. Moreover, HGF has the potential to promote cell motility for some epithelial cell types. The HGF receptor is also detected in many epithelial cells as well as in various tissues. Therefore, HGF is likely to be a multifunctional factor derived from mesenchymal cells influencing epithelial cell growth and cell motility through an epithelial-mesenchymal interaction. HGF may have important roles in wound-healing, tissue regeneration, morphogenesisand carcinogenesis. REFERENCES I. 2.

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Structure and function of hepatocyte growth factor.

Hepatocyte growth factor (HGF), a potent mitogen for mature hepatocytes in primary culture, was first found in sera of partial hepatectomized rats and...
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