Vol. 180, No. 2, 1991 October 31, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 765-773
Scatter Factor from Human Embryonic Lung Fibroblasts is Probably Identical to Hepatocyte Growth Factor T e t s u m i Konishi 1, Toyohiro T a k e h a r a 1, T a k a s h i Tsuji 1, Keiichi O h s a t o 2, Kunio M a t s u m o t o I and Toshikazu N a k a m u r a I § 1DepartmentofBiology, Facul~ ofScience, Kyushu Un~ersity, F u k u o k a 812, J a p a n 2First D e p a r t m e n t of Surgery, School of Medicine, University of O c c u p a t i o n a l and Environmental Health, K i t a k y u s h u 807, J a p a n Received September i0, 1991
SUMMARY: H u m a n e m b r y o n i c l u n g f i b r o b l a s t s (MRC5) p r o d u c e d s c a t t e r factor w h i c h e n h a n c e d motility of M a d i n - D a r b y c a n i n e k i d n e y (MDCK) epithelial cells a n d a factor w h i c h s t i m u l a t e s DNA s y n t h e s i s of a d u l t r a t h e p a t o c y t e s in p r i m a r y c u l t u r e . T h e s e activities w e r e b o t h completely neutralized by antibody against human hepatocyte growth factor (HGF). H u m a n r e c o m b i n a n t HGF i n d u c e d a m a r k e d s c a t t e r i n g of MDCK cells. Moreover, MRC5 cells highly e x p r e s s e d 6 k b mRNA w h i c h h y b r i d i z e d with H G F cDNA p r o b e a n d s c a t t e r factor cDNA cloned from the MRC5 cDNA library h a d the s a m e s e q u e n c e as that of H G F cDNA from h u m a n l e u k o c y t e s . T h e s e r e s u l t s indicate t h a t H G F p o s s e s s e s s c a t t e r f a c t o r activity a n d t h e s c a t t e r factor derived from t h e MRC5 cells is p r o b a b l y identical to HGF. ®1 9 9 1 A c a d e m i c P ..... I n c .
E n h a n c e m e n t of cell g r o w t h a n d cell motility is closely r e l a t e d to v a r i o u s biological p h e n o m e n a , i n c l u d i n g e m b r y o g e n e s i s , a n g i o g e n e s i s , t u m o r g e n e s i s a n d t i s s u e repair. C y t o k i n e s w h i c h e n h a n c e cell motility h a v e r e c e n t l y b e e n identified a n d c h a r a c t e r i z e d (1). Among these c o m p o u n d s , s c a t t e r factor, a p r o t e i n p r o d u c e d b y v a r i o u s f i b r o b l a s t cell lines including t r a n s f o r m e d cells e n h a n c e s motility a n d t r a n s l o c a t i o n of cohesive epithelial colonies a n d finally i n d u c e s s c a t t e r i n g of the colonies (2). T h r e e g r o u p s i n d e p e n d e n t l y purified s c a t t e r factor to h o m o g e n e i t y from t h e c o n d i t i o n e d m e d i a of MRC5 cells (3) a n d from r a s - t r a n s f o r m e d NIH3T3 f i b r o b l a s t s (4). S c a t t e r factor is c o m p o s e d of two s u b u n i t s of Mr 5 7 , 0 0 0 - 6 2 , 0 0 0 a n d Mr 3 0 , 0 0 0 - 3 4 , 0 0 0 linked b y disulfide b o n d (3, 4). A l t h o u g h several g r o w t h factors have b e e n e x a m i n e d b y m e a s u r i n g their § To w h o m c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d .
765
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s t i m u l a t o r y effects on cell growth, s o m e of t h e s e g r o w t h factors were s e e n to p o s s e s s s t i m u l a t o r y effects on cell motility. H e p a t o c y t e g r o w t h f a c t o r (HGF), a p o t e n t m i t o g e n for p r i m a r y c u l t u r e d r a t h e p a t o c y t e s , w a s first identified in t h e s e r a of p a r t i a l l y h e p a t e c t o m i z e d r a t s (5) a n d w a s p u r i f i e d f r o m r a t p l a t e l e t s to h o m o g e n e i t y (6, 7). T h e r e a f t e r , HGF w a s also p u r i f i e d f r o m h u m a n p l a s m a (8, 9). HGF is a h e t e r o d i m e r molecule c o m p o s e d of the 69kD as u b u n i t a n d the 34kD ~ - s u b u n i t .
Molecular cloning a n d s e q u e n c i n g of
h u m a n a n d r a t HGF cDNAs revealed HGF is to be derived from a singlec h a i n p r e c u r s o r of 728 a m i n o acid r e s i d u e s b y proteolytic processing a n d it h a s four kringle d o m a i n s in the m-chain (10-13). As d e d u c e d from m a r k e d i n c r e a s e s of HGF mRNA a n d HGF activity in the injured liver a n d p l a s m a a f t e r v a r i o u s liver i n s u l t s ( 1 4 - 1 8 ) , HGF s e e m s to a c t as a h e p a t o t r o p h i c factor for liver regeneration. D e t e r m i n a t i o n of partial a m i n o acid s e q u e n c e s of purified s c a t t e r factor revealed t h a t this factor a n d HGF were either the s a m e or h i g h l y r e l a t e d p r o t e i n s (3, 19). In t h i s work, to clarify w h e t h e r t h e s e two f a c t o r s were t h e s a m e p r o t e i n s or not, we have n o w c o m p a r e d t h e biological a n d i m m u n o c h e m i c a l properties of HGF a n d s c a t t e r f a c t o r p r o d u c e d by MRC5 cells.
MATERIALS AND METHODS Materials: Protein A S e p h a r o s e CL-4B was p u r c h a s e d from P h a r m a c i a LKB B i o t e c h n o l o g y (Uppsala). T h e m u l t i p r i m e l a b e l l i n g s y s t e m , m e m b r a n e filter H y b o n d - N , [~-32p]dCTP (10 m C i / m l ) a n d [1251]d e o x y u r i d i n e (1 m C i / m l ) were from A m e r s h a m . X-ray film (RX-5) w a s from Fuji Photo Film (Tokyo). Oligotex-dT30 w a s from Daiichi Pure C h e m i c a l s (Tokyo). Cell cultures: MRC5 h u m a n embryonic lung fibroblasts a n d Madin-Darby c a n i n e k i d n e y (MDCK) epithelial cells were o b t a i n e d f r o m J a p a n e s e Cancer Research Resources Bank. MRC5 cells were c u l t u r e d in D u l b e c c o ' s modified Eagle's m e d i u m (DMEM) c o n t a i n i n g 10% fetal calf s e r u m (FCS), a n d MDCK cells were c u l t u r e d in DMEM c o n t a i n i n g 5% FCS. HGF and anti-HGF antibody: H u m a n r e c o m b i n a n t HGF was purified from c u l t u r e m e d i u m of C127 m o u s e f i b r o b l a s t s t r a n s f e c t e d w i t h p l a s m i d c o n t a i n i n g h u m a n leukocyte HGF cDNA (13). IgG fractions from sera of a p r e - i m m u n e rabbit and the rabbit after i m m u n i z a t i o n with h u m a n r e c o m b i n a n t HGF were respectively purified u s i n g Protein A S e p h a r o s e column. A s s a y for s c a t t e r factor a c t i v i t y and HGF activity: Scatter factor activity w a s m e a s u r e d as described elsewhere (4). Briefly, MDCK cells s u s p e n d e d in DMEM c o n t a i n i n g 10% FCS were m i x e d w i t h a n e q u a l v o l u m e of serially diluted conditioned m e d i u m (CM) from MRC5, a n d plated onto a 96-well plate at 3000 cells/well. After a 24 h c u l t u r e period, the cells were fixed, s t a i n e d a n d p h o t o g r a p h e d u n d e r a light microscope. HGF activity was d e t e r m i n e d by m e a s u r i n g the s t i m u l a t o r y effect on DNA s y n t h e s i s of a d u l t r a t h e p a t o c y t e s as p r e v i o u s l y d e s c r i b e d (20). 766
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Briefly, h e p a t o c y t e s were c u l t u r e d on a 24-well p l a t e in Williams' E m e d i u m s u p p l e m e n t e d with 10 -9 M d e x a m e t h a s o n e , 10 -9 M insulin, a n d 5 U / m l a p r o t i n i n . After a d d i n g CM to e a c h c u l t u r e , t h e ceils were c u l t u r e d for 22 h t h e n were pulse-labelled w i t h [12SI]-deoxyuridine (0.3 m C i / m l ) for 6 h. After w a s h i n g the cells t h r e e t i m e s with PBS a n d t h e n twice w i t h ice-cold TCA, t h e cells were d i s s o l v e d w i t h 1 M NaOH, followed b y d e t e r m i n a t i o n of r a d i o a c t i v i t y u s i n g a T-counter. When n e u t r a l i z a t i o n of s c a t t e r factor or HGF activity b y a n t i b o d y w a s examined, CM w a s i n c u b a t e d with control IgG or anti-HGF IgG for 2 h at 37 °C a n d a s s a y e d as described above. Northern blot hybridization: Total RNA was extracted from MRC5 cells b y t h e a c i d - g u a n i d i n i u m - t h i o c y a n a t e - p h e n o l - c h l o r o f o r m m e t h o d (21), a n d poly(A)+RNA w a s p u r i f i e d u s i n g O l i g o t e x - d T 3 0 . One tlg of poly(A)+RNA was electrophoresed on a 1% agarose gel c o n t a i n i n g 0.66 M f o r m a l d e h y d e a n d t r a n s f e r r e d to a Hybond-N n y l o n m e m b r a n e filter. The BamHI-SalI f r a g m e n t of clone pBS-7, w h i c h i n c l u d e d the full-length open r e a d i n g frame cDNA for h u m a n leukocyte HGF (13), was labelled w i t h [(~32p]dCTP u s i n g t h e m u l t i p r i m e l a b e l l i n g s y s t e m , a c c o r d i n g to t h e m a n u f a c t u r e r ' s i n s t r u c t i o n . The m e m b r a n e w a s h y b r i d i z e d w i t h radiolabelled cDNA probe at 42°C for 24 h in s o l u t i o n c o m p o s e d of 750 mM NaC1, 75 mM s o d i u m citrate, 50% f o r m a m i d e , 2.5 x D e n h a r d t ' s , 0.5% (w/v) BSA, 50 mM s o d i u m p h o s p h a t e (pH 6.8), 0.5% (w/v) SDS a n d 200 I~g/ml s a l m o n s p e r m DNA. The filter w a s w a s h e d twice w i t h b u f f e r c o m p o s e d of 30 mM NaC1, 3 m M citrate (pH 6.8), 0.1% SDS for 30 rain a t 65 °C, t h e n e x p o s e d on X - r a y film for 36 h at -70°C, u s i n g a n i n t e n s i f y i n g screen.
RESULTS W h e n conditioned m e d i u m (CM) from MRC5 cells was a d d e d to the c u l t u r e s of MDCK cells, it m a r k e d l y e n h a n c e d motility of the MDCK cells a n d t h e r e s u l t was cell scattering (Fig. 1). In u n t r e a t e d c u l t u r e s of MDCK cells, m o s t of t h e cells were t i g h t l y l i n k e d as s h o w n in Fig. 1A. S c a t t e r i n g of t h e cells by CM could be d e t e c t e d even w h e n 32-fold d i l u t e d CM w a s a d d e d a n d m a x i m a l cell s c a t t e r i n g o c c u r r e d w i t h 8-fold d i l u t e d CM (Fig. 1B, C a n d D). T h u s , MRC5 cells p r o d u c e a n d secrete s c a t t e r factor w h i c h e n h a n c e s the motility of MDCK cells. To f u r t h e r c h a r a c t e r i z e t h e s c a t t e r i n g activity in t h e CM, we e x a m i n e d w h e t h e r s c a t t e r factor activity p r o d u c e d by MRC5 cells could be n e u t r a l i z e d b y polyclonal a n t i b o d y a g a i n s t h u m a n HGF. The CM from MRC5 cells w a s p r e i n c u b a t e d w i t h r a b b i t a n t i - H G F a n t i b o d y a n d t h e n a d d e d to t h e c u l t u r e . A l t h o u g h p r e - i m m u n e control IgG h a d no effect on s c a t t e r factor activity in the CM (Fig. 1E), the a n t i - H G F a n t i b o d y completely n e u t r a l i z e d s c a t t e r factor activity (Fig. IF). On the o t h e r h a n d , w h e n the CM of MRC5 cells was a d d e d to a d u l t r a t h e p a t o c y t e s in p r i m a r y c u l t u r e , the CM s t i m u l a t e d DNA s y n t h e s i s of m a t u r e hepatocytes, in a d o s e - d e p e n d e n t m a n n e r as s h o w n in Fig. 2A. To d e t e r m i n e w h e t h e r t h e s t i m u l a t o r y effect of t h e CM on DNA s y n t h e s i s 767
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S c a t t e r factor a c t i v i t y in CM o f MRC5 c e l l s and i n h i b i t i o n by antiHGF a n t i b o d y . MDCK cells were incubated for 24 h without (11) or with
32-fold (B), 16-fold (C), or 8-fold (D) diluted CM of MRC5 ceils cultured for 72 h. MDCK cells were also cultured for 24 h in the p r e s e n c e of 8fold diluted CM which w a s p r e i n c u b a t e d with 4 ~ g / m l p r e - i m m u n e control IgG (E) or 4 ~ g / m l anti-HGF IgG (F). To e x a m i n e the effect of antibody, 8-fold diluted CM was treated with control IgG or anti-HGF IgG at 37 °C for 2 h t h e n the treated CM was added to the culture of MDCK cells. After fixation with f o r m a l d e h y d e , the ceils were s t a i n e d with methylene blue. The b a r r e p r e s e n t s 200 ~m.
w a s l i n k e d to H G F o r to o t h e r g r o w t h f a c t o r s , t h e C M w a s p r e i n c u b a t e d w i t h a n t i - H G F a n t i b o d y a n d a d d e d to c u l t u r e d h e p a t o c y t e s (Fig. 2B).
The
s t i m u l a t i o n o f D N A s y n t h e s i s of h e p a t o c y t e s b y t h e C M w a s c o m p l e t e l y i n h i b i t e d a t a c o n c e n t r a t i o n of 2 0 B g / m l of t h e a n t i b o d y , w h e r e a s c o n t r o l IgG from the pre-immune
s e r u m w a s w i t h o u t effect.
Both scatter factor
a c t i v i t y a n d H G F a c t i v i t y in t h e CM w e r e c o m p l e t e l y l o s t b y h e a t i n g (100 768
V o l . 1 80, N o . 2, 1 991
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
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Fig.2. Stlmulatory effect of CM from MRC5 cells o n t h e DNA s y n t h e s i s of adult rat h e p a t o c y t e s in primary culture and its i n h i b i t i o n b y anti-HGF a n t i b o d y . (A), Effect of CM on DNA synthesis of adult rat hepatocytes;
(B), Effect of CM preincubated with pre-immune control IgG (lightly shaded bars) or anti-HGF IgG (solid bars). Hatched bar shows the effect of non-treated CM. MRC5 cells were cultured at confluent in serum free medium for 72 h and the CM were added to primary cultured adult rat hepatocytes. When effect of antibody was examined, 2-fold diluted CM of MRC5 cells was preincubated with serial concentration of anti-HGF IgG or the same concentration of pre-immune control IgG at 37 °C for 2 h. DNA synthesis was determined by measuring incorporation of [125I]deoxyuridine. Each value is the mean of triplicate measurements. °C, 3 rain), r e d u c t i o n w i t h d i t h i o t h r e i t o l , or b y t r y p s i n digestion.
The
activities were m a r k e d l y d e c r e a s e d b y t r e a t m e n t w i t h 1 N a c e t i c acid a n d b o t h a c t i v i t i e s in t h e CM h a d a s t r o n g affinity for h e p a r i n ( d a t a n o t shown). T h u s , s c a t t e r i n g a c t i v i t y for MDCK a n d H G F a c t i v i t y for h e p a t o c y t e in t he CM h a d the s a m e c h e m i c a l p r o p e r t i e s as H G F (6). H u m a n r e c o m b i n a n t H G F h a d a p o t e n t s c a t t e r f a c t o r act i vi t y for MDCK cells, as s h o w n in Fig. 3.
H G F s t r o n g l y s t i m u l a t e d motility of t h e
MDCK cells to be followed b y s c a t t e r i n g .
T h e s t i m u l a t i o n of cell m ot i l i t y
w a s o b s e r v e d with 0.5 n g / m l H G F a n d t h e m a x i m a l s t i m u l a t i o n w a s s e e n w i t h a c o n c e n t r a t i o n of 2 n g / m l (Fig. 3B a n d C). activity which
occurred
in t h e
presence
This scatter factor
of r e c o m b i n a n t
HGF was
c o m p l e t e l y n e u t r a l i z e d b y a n t i - H G F a n t i b o d y (Fig. 3D). All r e s u l t s s h o w t h a t HGF h a s s c a t t e r f a c t o r activity a n d t h a t s c a t t e r facto r a n d H G F are hi ghl y h o m o l o g o u s m o l e c u l e s . T herefore, we a n a l y z e d t h e mRNA s p e c i e s in MRC5 ceils w h i c h h y b r i d i z e s w i t h t he h u m a n H G F cDNA p r o b e . Fig. 4 s h o w s t h a t MRC5 cells e x p r e s s a hi gh level of 6 k b mRNA, t h e size of w h i c h m a t c h e d H G F mRNA. We s c r e e n e d a cDNA l i b r a r y c o n s t r u c t e d w i t h poly(A)+RNA of MRC5 cells u s i n g h u m a n H G F cDNA as a p r o b e .
A l t h o u g h t h e s e q u e n c e of t h e cDNA c l o n e w a s n o t 769
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Fig.3. Scatter factor a c t i v i t y of HGF. MDCK cells were incubated for 24 h without (A), or with 0.5 n g / m l (B), 2 n g / m l (C) of HGF or 2 n g / m l HGF plus 4 ~tg/ml anti-HGF IgG {D). For neutralization of HGF, 2 n g / m l of HGF was p r e i n c u b a t e d with anti-HGF IgG at 37 °C for 2 h and added to cultures of MDCK cells. After fixation with formaldehyde, the cells were stained with methylene blue. The bar represents 200 Ixm. s h o w n , we c o n f i r m e d t h a t t h e e n t i r e s e q u e n c e o f all t h r e e i n d e p e n d e n t cDNA clones
of scatter
factor
obtained
from
MRC5
cells
matched
c o m p l e t e l y t h a t o f h u m a n l e u k o c y t e H G F cDNA (13). iiii:~i,~iiiiii¸ i~iiii~iiiiiiil iiii~i!i!i!ii!ii i!i!i!i!i~!~ii~ii!iiii ~iiF?!iiiiiiiiiil
ii i"28s ---- iii!iiiiii!iiiii~iii 18S
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liiiiiiiiiiiiiiiiiiil~ ii!ii!iii!ii?~lii!i~ ~il?ii~iiiiiii~i!i i?~,ili!i!!ii!iiiiiil,i iiiiii!!iiiiiii~!i
Fig.4. Northern blot analysis of mRNA from MRC5 cultured cells. One ~g of poly(A)+RNA was electrophoresed into a 1% agarose gel containing 0.66 M f o r m a l d e h y d e and blotted onto a Hybond-N nylon m e m b r a n e filter. The filter was then hybridized with a 32p-labelled h u m a n HGF cDNA probe. Arrows are directed to the positions of 28S and 18S ribosomal RNA markers. 770
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DISCUSSION In t h i s s t u d y we o b t a i n e d evidence t h a t s c a t t e r factor p r o d u c e d b y MRC5 fibroblasts is identical to HGF, b a s e d on t h e following: (a) MRC5 cells p r o d u c e d a n d secreted not only scattering activity of MDCK cells b u t also s t i m u l a t o r y a c t i v i t y of DNA s y n t h e s i s in a d u l t r a t c u l t u r e d h e p a t o c y t e s ; (b) T h e s e activities were acid- a n d h e a t - labile a n d were i n d i s t i n g u i s h a b l e biologically a n d i m m u n o c h e m i c a l l y from HGF; (c) H u m a n r e c o m b i n a n t HGF h a d a p o t e n t s c a t t e r i n g activity; (d) The mRNA species in MRC5 cells w h i c h hybridized with HGF cDNA probe w a s t h e s a m e size with HGF mRNA a n d the entire s e q u e n c e of the c o r r e s p o n d i n g s c a t t e r factor cDNA clone m a t c h e d completely t h a t of h u m a n HGF cDNA. F u r l o n g et al. also f o u n d t h a t m u r i n e s c a t t e r factor purified from r a s - t r a n s f o r m e d NIH3T3 cells s t i m u l a t e d DNA s y n t h e s i s of p r i m a r y c u l t u r e d r a t h e p a t o c y t e s a n d t h a t t h i s HGF a c t i v i t y w a s c o m p l e t e l y i n h i b i t e d b y a n t i b o d y a g a i n s t m u r i n e s c a t t e r f a c t o r (22). F r o m t h e s e findings, t h e y suggested t h a t s c a t t e r factor a n d HGF are the s a m e protein. On the o t h e r h a n d , R o s e n et al. f o u n d t h a t s c a t t e r factor derived from h u m a n iliac a r t e r y s m o o t h m u s c l e h a d cell s c a t t e r i n g a c t i v i t y w h i c h differed from m u r i n e s c a t t e r factor: t h e former s c a t t e r e d a n d s t i m u l a t e d migration of several lines of h u m a n s q u a m o u s c a r c i n o m a cells, while t h e l a t t e r l a c k e d t h i s activity (23). The p a r t i a l a m i n o acid s e q u e n c e of s c a t t e r f a c t o r purified from h u m a n t e r m p l a c e n t a s h a r e d no s i m i l a r i t y w i t h HGF (24). T a k e n t o g e t h e r with o u r p r e s e n t s t u d y , t h e r e m a y be multiple s c a t t e r factors a n d different ones m a y be purified from different s o u r c e s , however, s c a t t e r factor p r o d u c e d b y MRC5 cells s e e m s to be identical with t h e HGF molecule. HGF h a s s c a t t e r factor activity w h i c h e n h a n c e s the motility of various epithelial cells s u c h as k e r a t i n o c y t e s (25) a n d h e p a t o m a cells (data n o t shown) as well as MDCK cells. R e c e n t s t u d i e s have s h o w n t h a t HGF s t i m u l a t e s growth of v a r i o u s e p i t h e l i a l cells, i n c l u d i n g r e n a l t u b u l a r cells (26), h u m a n e p i d e r m a l k e r a t i n o c y t e s (25) a n d m e l a n o c y t e s (27), a n d several epithelial cell lines (28), o t h e r t h a n m a t u r e h e p a t o c y t e s . On t h e c o n t r a r y , H G F s t r o n g l y i n h i b i t s t h e g r o w t h of epithelial t u m o r cells (29, 30). T h u s , HGF is a m u l t i f u n c t i o n a l growth factor w h i c h r e g u l a t e s b o t h cell g r o w t h a n d cell motility. T h e s e multiple biological a c t i o n s m a y be d u e to differences in properties of t h e HGF receptor. However, all t h e s e activities are exerted at 2-10 n g / m l HGF (16-80 pM HGF) w h i c h are c o n s i s t e n t w i t h 2 x Kd v a l u e s of HGF receptor. We f o u n d no significant differences in Kd v a l u e s a n d the n u m b e r of HGF receptor in n o r m a l a n d t u m o r cells (26, 27, 30, 31). T h e HGF r e c e p t o r w a s f o u n d to be t h e c - m e t p r o t o - o n c o g e n e p r o d u c t w h i c h h a s intraceUular t y r o s i n e k i n a s e activity (32). Therefore, 771
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the multiple actions of HGF seem to be related to signal t r a n s d u c t i o n pathways down-stream from the primary tyrosine kinase reaction. Since HGF acts b o t h as a mitogen and a motogen which enhances cell motility of epithelial cells a n d HGF is p r o d u c e d b y cells of m e s e n c h y m a l origin, HGF m a y play an important role in morphogenesis, embryogenesis, and tissue repair as well as in regeneration of organs s u c h as liver and kidney through epithelial-mesenchymal interaction.
Acknowledgments. We t h a n k M. Ohara for helpful comments. This work was s u p p o r t e d by a Research Grant for Studies on Science and Cancer from the Ministry of Education, Science and Culture of Japan. REFERENCES i.
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Asami, O., Ihara, I., Shimidzu, N., Shimizu, S., Tomita, Y., Ichihara, A. and Nakamura, T. (1991) J. Biochem. 109, 8-13 Kinoshita, T., Hirao, S., Matsumoto, K. and Nakamura, T. (1991) Biochem. Biophys. Res. Commun. 177, 330-335 Gherardi, E. and Stoker, M. (1990) Nature 346, 228 Nakamura, T., Arakaki, R. and Ichihara, A. (1988) Exp. Cell Res. 179, 488-497 Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156159 Furlong, R.A., Takehara, T., Taylor, W.G., Nakamura, T. and Rubin, J.S. (1991) J. Cell Sci., in press Rosen, E.M., Meromsky, L., Setter, E., Vinter, D.W. and Goldberg, I.D. (1990) Invasion Metastasis I 0 , 49-64 Rosen, E.M., Meromsky, L., Romero, R., Setter, E. and Goldberg, I.D. (1990) Biochem. Biophys. Res. Commun. 168, 1082-1088 Matsumoto, K., Hashimoto, K., Yoshikawa, K. and Nakamura, T. (1991) Exp. Cell Res., in press Igawa, T., Kanda, S., Kanetake, H., Saitoh, Y., Ichihara, A., Tomita, Y. and Nakamura, T. (1991) Biochem. Biophys. Res. Commun. 174, 831-838 Matsumoto, K., Tajima, H. and Nakamura, T. (1991) Biochem. Biophys. Res. Commun. 176, 45-51 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 Higashio, K., Shima, N., Goto, M., Itagaki, Y., Nagao, M., Yasuda, H. and Morinaga, T. (1990) Biochem. Biophys. Res. Commun. 170, 397-404 Tajima, H., Matsumoto, K. and Nakamura, T. (1991) FEBS Lett., in press Higuchi, O. and Nakamura, T. (1991) Biochem. Biophys. Res. Commun. 178, 599-607 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, 8028O4
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Scatter Factor from Human Embryonic Lung Fibroblasts is Probably Identical to Hepatocyte Growth Factor T e t s u m i Konishi 1, Toyohiro T a k e h a r a 1, T a k a s h i Tsuji 1, Keiichi O h s a t o 2, Kunio M a t s u m o t o I and Toshikazu N a k a m u r a I § 1DepartmentofBiology, Facul~ ofScience, Kyushu Un~ersity, F u k u o k a 812, J a p a n 2First D e p a r t m e n t of Surgery, School of Medicine, University of O c c u p a t i o n a l and Environmental Health, K i t a k y u s h u 807, J a p a n Received September i0, 1991
SUMMARY: H u m a n e m b r y o n i c l u n g f i b r o b l a s t s (MRC5) p r o d u c e d s c a t t e r factor w h i c h e n h a n c e d motility of M a d i n - D a r b y c a n i n e k i d n e y (MDCK) epithelial cells a n d a factor w h i c h s t i m u l a t e s DNA s y n t h e s i s of a d u l t r a t h e p a t o c y t e s in p r i m a r y c u l t u r e . T h e s e activities w e r e b o t h completely neutralized by antibody against human hepatocyte growth factor (HGF). H u m a n r e c o m b i n a n t HGF i n d u c e d a m a r k e d s c a t t e r i n g of MDCK cells. Moreover, MRC5 cells highly e x p r e s s e d 6 k b mRNA w h i c h h y b r i d i z e d with H G F cDNA p r o b e a n d s c a t t e r factor cDNA cloned from the MRC5 cDNA library h a d the s a m e s e q u e n c e as that of H G F cDNA from h u m a n l e u k o c y t e s . T h e s e r e s u l t s indicate t h a t H G F p o s s e s s e s s c a t t e r f a c t o r activity a n d t h e s c a t t e r factor derived from t h e MRC5 cells is p r o b a b l y identical to HGF. ®1 9 9 1 A c a d e m i c P ..... I n c .
E n h a n c e m e n t of cell g r o w t h a n d cell motility is closely r e l a t e d to v a r i o u s biological p h e n o m e n a , i n c l u d i n g e m b r y o g e n e s i s , a n g i o g e n e s i s , t u m o r g e n e s i s a n d t i s s u e repair. C y t o k i n e s w h i c h e n h a n c e cell motility h a v e r e c e n t l y b e e n identified a n d c h a r a c t e r i z e d (1). Among these c o m p o u n d s , s c a t t e r factor, a p r o t e i n p r o d u c e d b y v a r i o u s f i b r o b l a s t cell lines including t r a n s f o r m e d cells e n h a n c e s motility a n d t r a n s l o c a t i o n of cohesive epithelial colonies a n d finally i n d u c e s s c a t t e r i n g of the colonies (2). T h r e e g r o u p s i n d e p e n d e n t l y purified s c a t t e r factor to h o m o g e n e i t y from t h e c o n d i t i o n e d m e d i a of MRC5 cells (3) a n d from r a s - t r a n s f o r m e d NIH3T3 f i b r o b l a s t s (4). S c a t t e r factor is c o m p o s e d of two s u b u n i t s of Mr 5 7 , 0 0 0 - 6 2 , 0 0 0 a n d Mr 3 0 , 0 0 0 - 3 4 , 0 0 0 linked b y disulfide b o n d (3, 4). A l t h o u g h several g r o w t h factors have b e e n e x a m i n e d b y m e a s u r i n g their § To w h o m c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d .
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s t i m u l a t o r y effects on cell growth, s o m e of t h e s e g r o w t h factors were s e e n to p o s s e s s s t i m u l a t o r y effects on cell motility. H e p a t o c y t e g r o w t h f a c t o r (HGF), a p o t e n t m i t o g e n for p r i m a r y c u l t u r e d r a t h e p a t o c y t e s , w a s first identified in t h e s e r a of p a r t i a l l y h e p a t e c t o m i z e d r a t s (5) a n d w a s p u r i f i e d f r o m r a t p l a t e l e t s to h o m o g e n e i t y (6, 7). T h e r e a f t e r , HGF w a s also p u r i f i e d f r o m h u m a n p l a s m a (8, 9). HGF is a h e t e r o d i m e r molecule c o m p o s e d of the 69kD as u b u n i t a n d the 34kD ~ - s u b u n i t .
Molecular cloning a n d s e q u e n c i n g of
h u m a n a n d r a t HGF cDNAs revealed HGF is to be derived from a singlec h a i n p r e c u r s o r of 728 a m i n o acid r e s i d u e s b y proteolytic processing a n d it h a s four kringle d o m a i n s in the m-chain (10-13). As d e d u c e d from m a r k e d i n c r e a s e s of HGF mRNA a n d HGF activity in the injured liver a n d p l a s m a a f t e r v a r i o u s liver i n s u l t s ( 1 4 - 1 8 ) , HGF s e e m s to a c t as a h e p a t o t r o p h i c factor for liver regeneration. D e t e r m i n a t i o n of partial a m i n o acid s e q u e n c e s of purified s c a t t e r factor revealed t h a t this factor a n d HGF were either the s a m e or h i g h l y r e l a t e d p r o t e i n s (3, 19). In t h i s work, to clarify w h e t h e r t h e s e two f a c t o r s were t h e s a m e p r o t e i n s or not, we have n o w c o m p a r e d t h e biological a n d i m m u n o c h e m i c a l properties of HGF a n d s c a t t e r f a c t o r p r o d u c e d by MRC5 cells.
MATERIALS AND METHODS Materials: Protein A S e p h a r o s e CL-4B was p u r c h a s e d from P h a r m a c i a LKB B i o t e c h n o l o g y (Uppsala). T h e m u l t i p r i m e l a b e l l i n g s y s t e m , m e m b r a n e filter H y b o n d - N , [~-32p]dCTP (10 m C i / m l ) a n d [1251]d e o x y u r i d i n e (1 m C i / m l ) were from A m e r s h a m . X-ray film (RX-5) w a s from Fuji Photo Film (Tokyo). Oligotex-dT30 w a s from Daiichi Pure C h e m i c a l s (Tokyo). Cell cultures: MRC5 h u m a n embryonic lung fibroblasts a n d Madin-Darby c a n i n e k i d n e y (MDCK) epithelial cells were o b t a i n e d f r o m J a p a n e s e Cancer Research Resources Bank. MRC5 cells were c u l t u r e d in D u l b e c c o ' s modified Eagle's m e d i u m (DMEM) c o n t a i n i n g 10% fetal calf s e r u m (FCS), a n d MDCK cells were c u l t u r e d in DMEM c o n t a i n i n g 5% FCS. HGF and anti-HGF antibody: H u m a n r e c o m b i n a n t HGF was purified from c u l t u r e m e d i u m of C127 m o u s e f i b r o b l a s t s t r a n s f e c t e d w i t h p l a s m i d c o n t a i n i n g h u m a n leukocyte HGF cDNA (13). IgG fractions from sera of a p r e - i m m u n e rabbit and the rabbit after i m m u n i z a t i o n with h u m a n r e c o m b i n a n t HGF were respectively purified u s i n g Protein A S e p h a r o s e column. A s s a y for s c a t t e r factor a c t i v i t y and HGF activity: Scatter factor activity w a s m e a s u r e d as described elsewhere (4). Briefly, MDCK cells s u s p e n d e d in DMEM c o n t a i n i n g 10% FCS were m i x e d w i t h a n e q u a l v o l u m e of serially diluted conditioned m e d i u m (CM) from MRC5, a n d plated onto a 96-well plate at 3000 cells/well. After a 24 h c u l t u r e period, the cells were fixed, s t a i n e d a n d p h o t o g r a p h e d u n d e r a light microscope. HGF activity was d e t e r m i n e d by m e a s u r i n g the s t i m u l a t o r y effect on DNA s y n t h e s i s of a d u l t r a t h e p a t o c y t e s as p r e v i o u s l y d e s c r i b e d (20). 766
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Briefly, h e p a t o c y t e s were c u l t u r e d on a 24-well p l a t e in Williams' E m e d i u m s u p p l e m e n t e d with 10 -9 M d e x a m e t h a s o n e , 10 -9 M insulin, a n d 5 U / m l a p r o t i n i n . After a d d i n g CM to e a c h c u l t u r e , t h e ceils were c u l t u r e d for 22 h t h e n were pulse-labelled w i t h [12SI]-deoxyuridine (0.3 m C i / m l ) for 6 h. After w a s h i n g the cells t h r e e t i m e s with PBS a n d t h e n twice w i t h ice-cold TCA, t h e cells were d i s s o l v e d w i t h 1 M NaOH, followed b y d e t e r m i n a t i o n of r a d i o a c t i v i t y u s i n g a T-counter. When n e u t r a l i z a t i o n of s c a t t e r factor or HGF activity b y a n t i b o d y w a s examined, CM w a s i n c u b a t e d with control IgG or anti-HGF IgG for 2 h at 37 °C a n d a s s a y e d as described above. Northern blot hybridization: Total RNA was extracted from MRC5 cells b y t h e a c i d - g u a n i d i n i u m - t h i o c y a n a t e - p h e n o l - c h l o r o f o r m m e t h o d (21), a n d poly(A)+RNA w a s p u r i f i e d u s i n g O l i g o t e x - d T 3 0 . One tlg of poly(A)+RNA was electrophoresed on a 1% agarose gel c o n t a i n i n g 0.66 M f o r m a l d e h y d e a n d t r a n s f e r r e d to a Hybond-N n y l o n m e m b r a n e filter. The BamHI-SalI f r a g m e n t of clone pBS-7, w h i c h i n c l u d e d the full-length open r e a d i n g frame cDNA for h u m a n leukocyte HGF (13), was labelled w i t h [(~32p]dCTP u s i n g t h e m u l t i p r i m e l a b e l l i n g s y s t e m , a c c o r d i n g to t h e m a n u f a c t u r e r ' s i n s t r u c t i o n . The m e m b r a n e w a s h y b r i d i z e d w i t h radiolabelled cDNA probe at 42°C for 24 h in s o l u t i o n c o m p o s e d of 750 mM NaC1, 75 mM s o d i u m citrate, 50% f o r m a m i d e , 2.5 x D e n h a r d t ' s , 0.5% (w/v) BSA, 50 mM s o d i u m p h o s p h a t e (pH 6.8), 0.5% (w/v) SDS a n d 200 I~g/ml s a l m o n s p e r m DNA. The filter w a s w a s h e d twice w i t h b u f f e r c o m p o s e d of 30 mM NaC1, 3 m M citrate (pH 6.8), 0.1% SDS for 30 rain a t 65 °C, t h e n e x p o s e d on X - r a y film for 36 h at -70°C, u s i n g a n i n t e n s i f y i n g screen.
RESULTS W h e n conditioned m e d i u m (CM) from MRC5 cells was a d d e d to the c u l t u r e s of MDCK cells, it m a r k e d l y e n h a n c e d motility of the MDCK cells a n d t h e r e s u l t was cell scattering (Fig. 1). In u n t r e a t e d c u l t u r e s of MDCK cells, m o s t of t h e cells were t i g h t l y l i n k e d as s h o w n in Fig. 1A. S c a t t e r i n g of t h e cells by CM could be d e t e c t e d even w h e n 32-fold d i l u t e d CM w a s a d d e d a n d m a x i m a l cell s c a t t e r i n g o c c u r r e d w i t h 8-fold d i l u t e d CM (Fig. 1B, C a n d D). T h u s , MRC5 cells p r o d u c e a n d secrete s c a t t e r factor w h i c h e n h a n c e s the motility of MDCK cells. To f u r t h e r c h a r a c t e r i z e t h e s c a t t e r i n g activity in t h e CM, we e x a m i n e d w h e t h e r s c a t t e r factor activity p r o d u c e d by MRC5 cells could be n e u t r a l i z e d b y polyclonal a n t i b o d y a g a i n s t h u m a n HGF. The CM from MRC5 cells w a s p r e i n c u b a t e d w i t h r a b b i t a n t i - H G F a n t i b o d y a n d t h e n a d d e d to t h e c u l t u r e . A l t h o u g h p r e - i m m u n e control IgG h a d no effect on s c a t t e r factor activity in the CM (Fig. 1E), the a n t i - H G F a n t i b o d y completely n e u t r a l i z e d s c a t t e r factor activity (Fig. IF). On the o t h e r h a n d , w h e n the CM of MRC5 cells was a d d e d to a d u l t r a t h e p a t o c y t e s in p r i m a r y c u l t u r e , the CM s t i m u l a t e d DNA s y n t h e s i s of m a t u r e hepatocytes, in a d o s e - d e p e n d e n t m a n n e r as s h o w n in Fig. 2A. To d e t e r m i n e w h e t h e r t h e s t i m u l a t o r y effect of t h e CM on DNA s y n t h e s i s 767
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S c a t t e r factor a c t i v i t y in CM o f MRC5 c e l l s and i n h i b i t i o n by antiHGF a n t i b o d y . MDCK cells were incubated for 24 h without (11) or with
32-fold (B), 16-fold (C), or 8-fold (D) diluted CM of MRC5 ceils cultured for 72 h. MDCK cells were also cultured for 24 h in the p r e s e n c e of 8fold diluted CM which w a s p r e i n c u b a t e d with 4 ~ g / m l p r e - i m m u n e control IgG (E) or 4 ~ g / m l anti-HGF IgG (F). To e x a m i n e the effect of antibody, 8-fold diluted CM was treated with control IgG or anti-HGF IgG at 37 °C for 2 h t h e n the treated CM was added to the culture of MDCK cells. After fixation with f o r m a l d e h y d e , the ceils were s t a i n e d with methylene blue. The b a r r e p r e s e n t s 200 ~m.
w a s l i n k e d to H G F o r to o t h e r g r o w t h f a c t o r s , t h e C M w a s p r e i n c u b a t e d w i t h a n t i - H G F a n t i b o d y a n d a d d e d to c u l t u r e d h e p a t o c y t e s (Fig. 2B).
The
s t i m u l a t i o n o f D N A s y n t h e s i s of h e p a t o c y t e s b y t h e C M w a s c o m p l e t e l y i n h i b i t e d a t a c o n c e n t r a t i o n of 2 0 B g / m l of t h e a n t i b o d y , w h e r e a s c o n t r o l IgG from the pre-immune
s e r u m w a s w i t h o u t effect.
Both scatter factor
a c t i v i t y a n d H G F a c t i v i t y in t h e CM w e r e c o m p l e t e l y l o s t b y h e a t i n g (100 768
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BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
15 A I
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Fig.2. Stlmulatory effect of CM from MRC5 cells o n t h e DNA s y n t h e s i s of adult rat h e p a t o c y t e s in primary culture and its i n h i b i t i o n b y anti-HGF a n t i b o d y . (A), Effect of CM on DNA synthesis of adult rat hepatocytes;
(B), Effect of CM preincubated with pre-immune control IgG (lightly shaded bars) or anti-HGF IgG (solid bars). Hatched bar shows the effect of non-treated CM. MRC5 cells were cultured at confluent in serum free medium for 72 h and the CM were added to primary cultured adult rat hepatocytes. When effect of antibody was examined, 2-fold diluted CM of MRC5 cells was preincubated with serial concentration of anti-HGF IgG or the same concentration of pre-immune control IgG at 37 °C for 2 h. DNA synthesis was determined by measuring incorporation of [125I]deoxyuridine. Each value is the mean of triplicate measurements. °C, 3 rain), r e d u c t i o n w i t h d i t h i o t h r e i t o l , or b y t r y p s i n digestion.
The
activities were m a r k e d l y d e c r e a s e d b y t r e a t m e n t w i t h 1 N a c e t i c acid a n d b o t h a c t i v i t i e s in t h e CM h a d a s t r o n g affinity for h e p a r i n ( d a t a n o t shown). T h u s , s c a t t e r i n g a c t i v i t y for MDCK a n d H G F a c t i v i t y for h e p a t o c y t e in t he CM h a d the s a m e c h e m i c a l p r o p e r t i e s as H G F (6). H u m a n r e c o m b i n a n t H G F h a d a p o t e n t s c a t t e r f a c t o r act i vi t y for MDCK cells, as s h o w n in Fig. 3.
H G F s t r o n g l y s t i m u l a t e d motility of t h e
MDCK cells to be followed b y s c a t t e r i n g .
T h e s t i m u l a t i o n of cell m ot i l i t y
w a s o b s e r v e d with 0.5 n g / m l H G F a n d t h e m a x i m a l s t i m u l a t i o n w a s s e e n w i t h a c o n c e n t r a t i o n of 2 n g / m l (Fig. 3B a n d C). activity which
occurred
in t h e
presence
This scatter factor
of r e c o m b i n a n t
HGF was
c o m p l e t e l y n e u t r a l i z e d b y a n t i - H G F a n t i b o d y (Fig. 3D). All r e s u l t s s h o w t h a t HGF h a s s c a t t e r f a c t o r activity a n d t h a t s c a t t e r facto r a n d H G F are hi ghl y h o m o l o g o u s m o l e c u l e s . T herefore, we a n a l y z e d t h e mRNA s p e c i e s in MRC5 ceils w h i c h h y b r i d i z e s w i t h t he h u m a n H G F cDNA p r o b e . Fig. 4 s h o w s t h a t MRC5 cells e x p r e s s a hi gh level of 6 k b mRNA, t h e size of w h i c h m a t c h e d H G F mRNA. We s c r e e n e d a cDNA l i b r a r y c o n s t r u c t e d w i t h poly(A)+RNA of MRC5 cells u s i n g h u m a n H G F cDNA as a p r o b e .
A l t h o u g h t h e s e q u e n c e of t h e cDNA c l o n e w a s n o t 769
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Fig.3. Scatter factor a c t i v i t y of HGF. MDCK cells were incubated for 24 h without (A), or with 0.5 n g / m l (B), 2 n g / m l (C) of HGF or 2 n g / m l HGF plus 4 ~tg/ml anti-HGF IgG {D). For neutralization of HGF, 2 n g / m l of HGF was p r e i n c u b a t e d with anti-HGF IgG at 37 °C for 2 h and added to cultures of MDCK cells. After fixation with formaldehyde, the cells were stained with methylene blue. The bar represents 200 Ixm. s h o w n , we c o n f i r m e d t h a t t h e e n t i r e s e q u e n c e o f all t h r e e i n d e p e n d e n t cDNA clones
of scatter
factor
obtained
from
MRC5
cells
matched
c o m p l e t e l y t h a t o f h u m a n l e u k o c y t e H G F cDNA (13). iiii:~i,~iiiiii¸ i~iiii~iiiiiiil iiii~i!i!i!ii!ii i!i!i!i!i~!~ii~ii!iiii ~iiF?!iiiiiiiiiil
ii i"28s ---- iii!iiiiii!iiiii~iii 18S
~
6kb
liiiiiiiiiiiiiiiiiiil~ ii!ii!iii!ii?~lii!i~ ~il?ii~iiiiiii~i!i i?~,ili!i!!ii!iiiiiil,i iiiiii!!iiiiiii~!i
Fig.4. Northern blot analysis of mRNA from MRC5 cultured cells. One ~g of poly(A)+RNA was electrophoresed into a 1% agarose gel containing 0.66 M f o r m a l d e h y d e and blotted onto a Hybond-N nylon m e m b r a n e filter. The filter was then hybridized with a 32p-labelled h u m a n HGF cDNA probe. Arrows are directed to the positions of 28S and 18S ribosomal RNA markers. 770
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DISCUSSION In t h i s s t u d y we o b t a i n e d evidence t h a t s c a t t e r factor p r o d u c e d b y MRC5 fibroblasts is identical to HGF, b a s e d on t h e following: (a) MRC5 cells p r o d u c e d a n d secreted not only scattering activity of MDCK cells b u t also s t i m u l a t o r y a c t i v i t y of DNA s y n t h e s i s in a d u l t r a t c u l t u r e d h e p a t o c y t e s ; (b) T h e s e activities were acid- a n d h e a t - labile a n d were i n d i s t i n g u i s h a b l e biologically a n d i m m u n o c h e m i c a l l y from HGF; (c) H u m a n r e c o m b i n a n t HGF h a d a p o t e n t s c a t t e r i n g activity; (d) The mRNA species in MRC5 cells w h i c h hybridized with HGF cDNA probe w a s t h e s a m e size with HGF mRNA a n d the entire s e q u e n c e of the c o r r e s p o n d i n g s c a t t e r factor cDNA clone m a t c h e d completely t h a t of h u m a n HGF cDNA. F u r l o n g et al. also f o u n d t h a t m u r i n e s c a t t e r factor purified from r a s - t r a n s f o r m e d NIH3T3 cells s t i m u l a t e d DNA s y n t h e s i s of p r i m a r y c u l t u r e d r a t h e p a t o c y t e s a n d t h a t t h i s HGF a c t i v i t y w a s c o m p l e t e l y i n h i b i t e d b y a n t i b o d y a g a i n s t m u r i n e s c a t t e r f a c t o r (22). F r o m t h e s e findings, t h e y suggested t h a t s c a t t e r factor a n d HGF are the s a m e protein. On the o t h e r h a n d , R o s e n et al. f o u n d t h a t s c a t t e r factor derived from h u m a n iliac a r t e r y s m o o t h m u s c l e h a d cell s c a t t e r i n g a c t i v i t y w h i c h differed from m u r i n e s c a t t e r factor: t h e former s c a t t e r e d a n d s t i m u l a t e d migration of several lines of h u m a n s q u a m o u s c a r c i n o m a cells, while t h e l a t t e r l a c k e d t h i s activity (23). The p a r t i a l a m i n o acid s e q u e n c e of s c a t t e r f a c t o r purified from h u m a n t e r m p l a c e n t a s h a r e d no s i m i l a r i t y w i t h HGF (24). T a k e n t o g e t h e r with o u r p r e s e n t s t u d y , t h e r e m a y be multiple s c a t t e r factors a n d different ones m a y be purified from different s o u r c e s , however, s c a t t e r factor p r o d u c e d b y MRC5 cells s e e m s to be identical with t h e HGF molecule. HGF h a s s c a t t e r factor activity w h i c h e n h a n c e s the motility of various epithelial cells s u c h as k e r a t i n o c y t e s (25) a n d h e p a t o m a cells (data n o t shown) as well as MDCK cells. R e c e n t s t u d i e s have s h o w n t h a t HGF s t i m u l a t e s growth of v a r i o u s e p i t h e l i a l cells, i n c l u d i n g r e n a l t u b u l a r cells (26), h u m a n e p i d e r m a l k e r a t i n o c y t e s (25) a n d m e l a n o c y t e s (27), a n d several epithelial cell lines (28), o t h e r t h a n m a t u r e h e p a t o c y t e s . On t h e c o n t r a r y , H G F s t r o n g l y i n h i b i t s t h e g r o w t h of epithelial t u m o r cells (29, 30). T h u s , HGF is a m u l t i f u n c t i o n a l growth factor w h i c h r e g u l a t e s b o t h cell g r o w t h a n d cell motility. T h e s e multiple biological a c t i o n s m a y be d u e to differences in properties of t h e HGF receptor. However, all t h e s e activities are exerted at 2-10 n g / m l HGF (16-80 pM HGF) w h i c h are c o n s i s t e n t w i t h 2 x Kd v a l u e s of HGF receptor. We f o u n d no significant differences in Kd v a l u e s a n d the n u m b e r of HGF receptor in n o r m a l a n d t u m o r cells (26, 27, 30, 31). T h e HGF r e c e p t o r w a s f o u n d to be t h e c - m e t p r o t o - o n c o g e n e p r o d u c t w h i c h h a s intraceUular t y r o s i n e k i n a s e activity (32). Therefore, 771
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the multiple actions of HGF seem to be related to signal t r a n s d u c t i o n pathways down-stream from the primary tyrosine kinase reaction. Since HGF acts b o t h as a mitogen and a motogen which enhances cell motility of epithelial cells a n d HGF is p r o d u c e d b y cells of m e s e n c h y m a l origin, HGF m a y play an important role in morphogenesis, embryogenesis, and tissue repair as well as in regeneration of organs s u c h as liver and kidney through epithelial-mesenchymal interaction.
Acknowledgments. We t h a n k M. Ohara for helpful comments. This work was s u p p o r t e d by a Research Grant for Studies on Science and Cancer from the Ministry of Education, Science and Culture of Japan. REFERENCES i.
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