Lung may have an endocrine function producing hepatocyte growth factor in response to injury of distal organs

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31, 1992

Lung May Have an Endocrine

Function

Producing

802-809

Hepatocyte

Growth Factor in Response to Injury of Distal Organs Kimihiko Yanagita1.2, Mika Nagaikel, Hiromi Ishibashiz, Yoshiyuki Niho2, Kunio Matsumoto l, and Toshikazu NakamuraL* 1 Department

2 First

of Biology, Faculty of Science, Fukuoka 8 12, Japan

Department Kyushu

Received

December

19,

of Internal University,

Medicine, Fukuoka

Kyushu

Faculty

University,

of Medicine,

812, Japan

1991

SUM-Y: Hepatocyte growth factor (HGF) is a potent growth factor for various epithelial cells including mature hepatocytes and renal tubular cells. When 70% of the rat liver was excised, HGF mRNA in the intact lung markedly increased at 6 h later, then decrease to normal levels at 24 h. A similar marked increase of HGF mRNA was found in the lung of rats with hepatitis induced by CC14. Moreover HGF mRNA in the intact lung also increased to about a 5 times higher level than the normal, within 12 h after unilateral nephrectomy. Isolated alveolar macrophages significantly expressed HGF mRNA, yet the amount remained unchanged after injury of the liver. The marked increase of HGF mRNA in lungs of partially hepatectomized rats remained even after removal of alveolar macrophages. In situ hybridization showed a marked increase of HGF mRNA signal found in endothelial cells in the lung after partial hepatectomy. We postulate that endothelial cells in the lung recognize damage of distal organs through a mediator and that lungderived HGF may contribute to tissue repair or regeneration of injured organs, through endocrine-related mechanisms. 0 1992Academic Press,Inc.

Hepatocyte regeneration, was is a heterodimer and contains four

growth factor (HGF), a putative hepatotropic factor for liver first purified to homogeneity from rat platelets (l-3). HGF molecule composed of 69 kD o-chain and 34 kD P-chain, kringle domains in the o-chain (4-7). In addition to mature

hepatocytes, HGF has been shown to stimulate growth of various epithelial cells including renal tubular cells (8). epidermal melanocytes (9), and keratinocytes (10, 11). On the other hand, in situ hybridization revealed that HGF-producing and endothelial *To whom

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cells are mesenchymal cells but not epithelial requests

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should Inc. reserved.

cells such as cells (12).

be addressed.

802

fibroblasts,

Kupffer

cells,

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When hepatitis was induced in rats by the administration of carbontetrachloride (CC14). HGF mRNA in the liver increased markedly 5 h later (5, 13). and HGF activity in the liver increased to 20-fold higher value than that of normal value at 36 h (14). Similar marked increases in HGF mRNA were also found in the liver by ischemia or crush insults (15). Thus, in the presence of hepatitis, liver ischemia, and liver crush, HGF produced by Kupffer cells and sinusoidal endothelial cells may act as a hepatotropic factor for liver regeneration, in a paracrine both HGF mRNA and HGF activity

fashion. On the other hand, although increased in the remnant liver after

removal of 70% rat liver by partial hepatectomy (16), these increases were much lower and later than those in the liver of rats with hepatitis. However, more

than

within

a 5-fold

6 h after

liver regeneration from extra-hepatic tissues contribute

increase

partial

in HGF activity

hepatectomy

(16,

was noted

17). Therefore,

in the blood we considered

plasma that

may also depend on an endocrine mechanism supplied organs. So we asked whether extra-hepatic organs or to this endocrine mechanism for liver regeneration by

HGF. We describe markedly

in the

here rat

to the

evidence

intact

lung

hypothesis

that

expression

following

findings

lead

that

induction the lung

of HGF in the lung through has an endocrine function

injuries

injuries

of HGF

mRNA

of distal

of other

organs

increases

organs.

These

may

trigger

a mediator via blood circulation and that producing HGF to facilitate regeneration

of an organ.

MATERIALS

AND METHODS

Partial

hepatectomy and Unilateral nephrectomy: Adult male Wistar rats (150-200 g of body weight) were anesthetized with ether. The abdomen was incised, and then the medial and left hepatic lobes were removed according to the method of Higgins and Anderson (18). For unilateral nephrectomy, following a right lumbodorsal incision, the right kidney was removed. Isolation of alveolar macrophages: Rats anesthetized with Nembutal were tracheotomized to cannulate and the lungs were lavaged with 50 ml of prewarmed phosphate bufferd saline (pH 7.0). Alveolar macrophages were precipitated by centrifugation of the lavaged fluid at 200 x g for 10 min. Northern-blot analysis: Total RNA was isolated using the acid-guanidium thiocyanate-phenol-chloroform (AGPC) method (19). Ten pg of total RNA were applied to gel-electrophoresis on 1.0% agarose/formaldehyde gels and transferred to a Hybond-N membrane filter. The EcoRl fragment (1.4 kb) of rat HGF cDNA (REX-1 clone, ref 5) was labeled with the random primer DNA labeling kit (TaKaRa) according to the manufacturer’s instruction. RBC-1 encodes the 5’-portion including the fourth kringle domain of a-chain, the entire P-chain of HGF, and a part of 3’-noncoding region. Hybridization was performed at 42’C for 20 h in solution composed of 50% (v/v) formamide, 5 x SSPE (1 x SSPE consists of 0.15 M of NaCl, 10 mM of sodium phosphate buffer (pH 7.4). and 1 mM of EDTA), 4 x Denhardt’s solution, 0.5% SDS, and 150 pg/ml salmon sperm DNA. The filter was washed with 0.2 x SSPE-0.1% 803

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SDS for 15 min at 65°C. then was dried and autoradiographed on Fuji X-ray film. In situ hybridization: In situ hybridization was performed as described elsewhere (12). Briefly, the lung was fixed with 4% paraformaldehyde and paraffin-embedded. Serial sections (4 Km thickness) were collected on slide glass coated with poly-L-lysin and were treated with 5 pg/ml proteinase K at 37°C for 15 min. Sections were hybridized with a [35S]-labeled probe synthesized from RBC-1 clone with a Megaprime DNA labelling system (Amersham). After washing, the slide glass was dipped into emulsion (l/4diluted Kodak NTB2). and exposed in the dark for two days. The sections were developed with Kodak D-19, then stained with hematoxylin and eosin. RESULTS HGF mRNA expression 70% found HGF,

liver or renal

injury

in the remnant kidney after unilateral nephrectomy newly synthesized in the respective injured organs,

participate lung,

in lung after

We reported that HGF mRNA increases in the remnant liver following partial hepatectomy (16). Similarly, the induction of HGF mRNA was

in regeneration

of each

HGF mRNA increased

markedly

organ.

Surprisingly

and rapidly

(20). Therefore was thought to

even in

after partial

the intact

hepatectomy

or

unilateral nephrectomy, as shown in Fig. 1. HGF mRNA in the lung increased markedly only 6 h after partial hepatectomy: the value is more than 5 times higher than the normal. Within 24 h of the surgery, normal levels were regained.

A similar

marked

and rapid PH -r-----Y

increase UN

6 kb -

Fig. 1. Northern blots unilateral nephrectomy.

of HGF mRNA in the lung was

HGF mRNA

-

of HGF mRNA in rat lung and CClq-administration.

after

partial

hepatectomy,

RNA was extracted and purified from the lung at various times after the treatment with AGPC method. Ten Fg of total RNA were separated on 1.0% agarose/formaldehyde gel electrophoresis, transferred to Hybond-N nylon membrane filter, and hybridized with EcoRI fragment of rat HGF cDNA as a probe. N. normal: PH, partial hepatectomy: UN, unilateral nephrectomy; CC14, 12 h after CC14administration. 804

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-

k “Pi4

,HGF -

mRNA

HGF mRNA

FW. 2. Expression of HGF mRNA in alveolar macrophages and alveolar macrophage-deprived lung after partial hepatectomy (A) or CC14administration (B). Bronchoalveolar lavage (BAL) was performed on normal, partially hepatectomized. and CCla-administrated rats to obtain alveolar macrophages. N, normal: macrophage: treatment.

also than after seen

(A) Lane 1, 2, alveolar PH. 6 h after partial lane 3, 4, lung after

macrophage; lane 3, 4, lung after BAL . hepatectomy. (B) Lane 1, 2, alveolar BAL. N, normal: CC14. 12 h after CC14-

noted after unilateral nephrectomy: the value is about 5 times higher the normal at 6-12 h. The level decreased rapidly to normal values 24 h nephrectomy. A similar dramatic increase in HGF mRNA in the lung was 12 h after inducing hepatitis with 2 g/kg body weight of CC14.

HGF mRNA in alveolar macrophages and other lung cells Since cells which synthesize HGF in the CCl4-treated rat liver are Kupffer and sinusoidal endothelial cells (12). we assumed that alveolar macrophages might be one of the HGF-producing cells in the lung. We isolated alveolar macrophages from normal and partial hepatectomized rats by bronchoalveolar lavage and analyzed the HGF mRNA expression in these macrophages and in the lungs deprived of alveolar macrophages (Fig. 2A). Since a significant amount of HGF mRNA was expressed in alveolar macrophages isolated even from the lung of normal rats, alveolar macrophages are one of cells producing HGF in the lung. However, the HGF mRNA level in alveolar macrophages did not increase after partial hepatectomy. Rather, HGF mRNA was still expressed at a high level in the lavaged lung and the level increased to about 5 fold after partial hepatectomy. The increase in HGF mRNA in the lavaged lung was also found in rats with hepatitis induced by CC14 (Fig. 2B). The HGF mRNA level in the lung increased to about 5 times that in the intact rat, and a similar increase was observed in the lavaged lung. The HGF mRNA did not increase in alveolar macrophages from lung of rats with hepatitis. Thus, the induction of HGF 805

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mRNA in the lung of rats with liver injuries is presumably in HGF mR.NA in cells other than alveolar macrophages. HGF-producing cells in the lung To identify cells which produce localization of HGF mRNA in the lung

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due to the increase

HGF in the lung, we next examined of normal and partial hepatectomized

rats, using in situ hybridization. Distribution of dense silver grains indicating the presence of HGF mRNA was more marked in the lung of rat after partial hepatectomy consistent

than with

that

that

of normal

rat (Fig. 3, A, B, D, and E), the result

of northern

in the cells with characteristics (Fig. 3, C and F): One type

hybridization.

Dense

grains

of alveolar macrophages of the cells has a large

is

were distributed

and endothelial round nucleus

cells and

protruded into alveolar space, thus seems to be alveolar macrophage. The other type has a small and flat nucleus and faced to capillary vessel, which is typical of endothelial cell. The accumulation of grains in alveolar macrophages showed

no significant

hepatectomized grains

increased

cells apparently

difference

rats,

between

whereas,

remarkably produce

lungs

the number after

partial

from

normal

of endothelial

hepatectomy.

HGF in the lung following

and

cells

Therefore,

partially

with

dense

endothelial

liver injury.

DISCUSSION In addition to endocrine organs, heart, kidney, produce various bioactive peptides involved in endocrine metabolic

regulation,

vasodynamics,

the heart

predominantly

functions

hematopoiesis, as a circulatory

role in the regulation of blood volume organ, through its potential to produce (21, 22). The kidney (23) and

the

liver

produces produces

organ,

and liver including

cell growth. it no doubt

While has a

and blood pressure, as an endocrine and secrete atrial natriuretic peptide

and secretes insulin-like

and

intestine, functions,

erythropoietin growth

factor

for hematopoiesis (Somatomedins)

in

response to growth hormone (24). Therefore, various organs may well have endocrine function in addition to their own predominant functions. Here, we obtained evidence that HGF mRNA in the lung markedly increased following partial hepatectomy and unilateral nephrectomy. Because these insults do not cause any direct injury in the lung and the lung is intact, HGF newly produced in the lung is thought to function as a hepatotropic or renotropic factors already know that partial hepatectomy

for regeneration of liver and kidney, respectively. We the increase of HGF mRNA in the remnant liver after was much lower and later than that after CC14-

administration (lG).However, a marked increase of HGF activity was found in the blood of partially hepatectomized rats within 6 h after treatment, and elevated levels of plasma HGF activity persisted for up to 24 h after the

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Fig. 3. Localization of HGF mRNA in lung from intact and partially hepatectomized rats. Lungs were prepared from intact rat (A, B, C) and partially hepatectomized rat (D. E, F) 6 h after the treatment, and fured by perfusing with 4% paraformaldehyde solution. HGF mRNA on sections was hybridized with [35S]-labelled cDNA probe: A, C, D. and F, bright-field views for histological identification: B and D, the corresponding dark-field views showing autoradiography signal (white grains). Note that the dense grains are sparse (A, B), and mainly located on alveolar macrophages (C) in the intact rat lung. The amount of the dense accumulation of grains increased in the lung after partial hepatectomy (D, E), and the increase seemed to be predominantly related to the increase of dense grains on endothelial cells (F). Scale bar in A, B, D. and E, 200pm: C and F, 25pm.

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treatment. It is unlikely that the increase in HCF activity in the plasma relates to an increased production in the liver. We postulate that the elevated levels of HGF in the plasma may be derived from some organs and tissues other than the liver. In addition, a marked down regulation of HGF receptor on plasma membranes of the liver or kidney occurred within 12 h after partial hepatectomy (25) or unilateral nephrectomy (20), respectively. Downregulation of the HGF receptor may follow internalization of the receptor after binding and signaling of HGF, so that HGF may stimulate proliferation of parenchymal epithelial cells in the injured organ. An important point is that down-regulation occurs specifically in the injured organ and not in intact organs (26). Down-regulation of the HGF receptor was rare in the lung after partial hepatectomy or unilateral nephrectomy, despite the marked increase of HGF mRNA in the lung. HGF newly synthesized in the lung responding to injuries of other organs probably does not exert biological actions within the lung itself. Taken together, it is conceivable that the increase of HGF activity in the plasma of partially hepatectomized rats is due to a rapid production and secretion at least from the intact lung, and HGF newly produced in the lung, in response to injuries of other organs may act as a mitogen for epithelial cells of a distal injured organ. Thus, the lung may have an endocrine function producing HGF for regeneration of injured tissues or organs. We assume that a mediator which induces HGF mRNA in the lung might be involved. Indeed, we have confirmed the existence of such a factor in the blood of rats with injury of liver or kidney, the factor induces an increase of HGF mRNA expression and production of HGF in the intact lung in vivo and also in cultured lung fibroblasts in vitro. Various known cytokines did not have such activities. The factor seems to be an acid- and heat-stable protein which we named “injurin” because of its possible role to mediate organ injuries (27). Purification, characterization, and molecular cloning of injurin are underway and important information is expected to be forthcoming on molecular mechanisms of organogenesis and organ-interactions involved in the homeostasis of organ mass, as well as for eluciating organ regeneration. Acknowledgment: This study was supported by a Research Grant for Studies on Science and Cancer from the Ministry of Education Science, and Culture of Japan. REFERENCES 1. Nakamura. T., Nawa, K., and Ichihara. A. (1984) Biochem. Biophys. Res. Commun. 122, 1450-1459 2. Nakamura, T., Teramoto, H, and Ichihara, A. (1986) Proc. Natl. Acad. Sci. USA 83, 6489-6493 808

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