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(American Journal of Pathology. 2000;157:1693-1701.)
© 2000 American Society for Investigative Pathology

Regular Articles

The Mitogenic Activity of Hepatocyte Growth Factor on Rat Hepatocytes Is Dependent upon Endogenous Transforming Growth Factor-{alpha}

Tomoaki Tomiya*, Itsuro Ogata*, Miho Yamaoka{dagger}, Mikio Yanase*, Yukiko Inoue* and Kenji Fujiwara{dagger}

From the First Department of Internal Medicine,*
Faculty of Medicine, University of Tokyo, Tokyo; and the Third Department of Internal Medicine,{dagger}
Saitama Medical School, Saitama, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Both transforming growth factor-{alpha} (TGF-{alpha}) and hepatocyte growth factor (HGF) induce DNA synthesis in hepatocytes in vitro and in vivo. Hepatic and circulating levels of HGF have been reported to increase before an increase in TGF-{alpha} levels in several rat models of liver regeneration. In addition, serum TGF-{alpha} levels increase after an increase in serum HGF levels in patients with either partial hepatectomy or acute hepatitis. In this study, we investigate the significance of TGF-{alpha} in hepatocyte proliferation. TGF-{alpha} contents and DNA synthesis in cultured rat hepatocytes increased in response to HGF addition to the culture medium in a dose-related manner. These increases were suppressed by the addition of anti-sense TGF-{alpha} mRNA oligonucleotide. Furthermore, the addition of anti-TGF-{alpha} rabbit IgG suppressed the increase in DNA synthesis. When the anti-TGF-{alpha} antibody was administered to rats after partial hepatectomy, the number of mitotic hepatocytes was reduced in comparison to rats treated with normal rabbit IgG. These results were observed even though hepatic HGF levels were increased equally in rats given either anti-TGF-{alpha} antibody or normal rabbit IgG. Our results suggest that HGF stimulates TGF-{alpha} production in rat hepatocytes, and that the mitogenic activity of HGF depends on endogenous TGF-{alpha} activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Several mitogens have been identified for hepatocytes in culture.1-3 Among them, transforming growth factor-{alpha} (TGF-{alpha}) and hepatocyte growth factor (HGF) have been reported to stimulate hepatocyte proliferation in vivo.4-9 Hepatocytes express both TGF-{alpha} mRNA and mRNA encoding the TGF-{alpha}/epidermal growth factor (EGF) receptor.1,10-16 In contrast, whereas hepatocytes express mRNA encoding the receptor for HGF, the c-met proto-oncogene gene product, only nonparenchymal cells of the liver produce HGF.17-24 Therefore, TGF-{alpha} and HGF are thought to act in an autocrine and paracrine manner, respectively.1,3

Previous reports have demonstrated that HGF mRNA and the circulating and hepatic levels of HGF are increased in rats either after partial hepatectomy or after acute liver injury induced by carbon tetrachloride intoxication. These increases occur before an increase in endogenous TGF-{alpha} and TGF-{alpha} mRNA.13,25-28 In humans, serum HGF levels have been shown to increase before an increase of serum TGF-{alpha} levels after partial hepatectomy or in patients with acute hepatitis.29-33 These observations raise questions as to whether TGF-{alpha} is required in addition to HGF for the proliferation of hepatocytes.

The present study investigates the significance of TGF-{alpha} in hepatocyte proliferation even in the presence of HGF. We examined the effect of the inhibition of the synthesis and activity of TGF-{alpha}, through the use of anti-sense TGF-{alpha} mRNA oligonucleotide and anti-TGF-{alpha} antibodies, respectively, on the DNA synthesis by rat hepatocytes cultured in the presence of HGF. Furthermore, we examined the effect of anti-TGF-{alpha} rabbit IgG administration on hepatocyte proliferation in rats after partial hepatectomy. Our results suggest that HGF stimulates TGF-{alpha} production in rat hepatocytes, and that the mitogenic activity of HGF depends on endogenous TGF-{alpha} activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Sources

Type I collagen-coated tissue culture dishes were purchased from Iwaki Glass (Corning, Tokyo, Japan); the 96-well assay plate was from A/S Nunc (Roskilde, Denmark); 20% neutral buffered formalin solution was from Muto Pure Chemicals Ltd. (Tokyo, Japan); phenylmethyl-sulfonyl fluoride was from Sigma Chemical Company (St. Louis, MO); Williams’ medium E (WE) from ICN Biochemicals Inc. (Costa Mesa, CA); fetal calf serum (FCS) from Life Technologies Inc. (Grand Island, NY); insulin from Novo Industri A/S (Copenhagen, Denmark); dexamethasone from Takeda Medical Industries Ltd. (Osaka, Japan); blocking agents derived from skim milk, ie, Block Ace (BA) from Snow Brand Milk Products Co., Ltd. (Sapporo, Japan); peroxidase-conjugated anti-rabbit IgG solution from Otsuka Assay Laboratories (Tokushima, Japan); free and peroxidase-conjugated anti-rabbit IgG antibodies preabsorbed with rat serum from Chemicon International Inc. (Temecula, CA); normal rabbit IgG from InterCell Technologies Inc. (Hopewell, NJ); rat TGF-{alpha} from Bachem Feinchemikalien AG (Bubendorf, Switzerland); rat EGF from Otsuka Assay Laboratories (Tokushima, Japan); heparin-binding EGF-like growth factor (HB-EGF) from R&D Systems Inc. (Minneapolis, MN); 2,2'-azino-di-[3-ethylbenzthiazoline sulfonate6 ] from Boe-hringer Mannheim (Mannheim, Germany); and dye reagent concentrate of Bio-Rad Protein Assay from Bio-Rad Laboratories (Hercules, CA). Recombinant HGF, a 5-amino acid-deleted form of HGF (dHGF), and anti-TGF-{alpha} rabbit IgG were generously provided from Snow Brand Milk Products Co., Ltd. and Kyowa Medex Co., Ltd., (Tokyo, Japan) respectively.34 Rat HGF enzyme-linked immunosorbent assay (ELISA) kit, 5-bromo-2'-deoxy-uridine (BrdU) labeling and detection kits II and III, and rat interlukin-6 (IL-6) ELISA kits were obtained from the Institute of Immunology (Tokyo, Japan), Boehringer Mannheim, and Immuno-Biological Laboratories (Fujioka, Japan), respectively.35 Other reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan).

Animals

Male Sprague-Dawley rats, 5 to 6 weeks old, were obtained from Japan SLC (Shizuoka, Japan). They were housed in cages at 22°C under a 12-hour light-dark cycle and fed a commercial diet and water ad libitum. All animal study protocols conformed to the guideline of Faculty of Medicine, University of Tokyo for humane care.

Preparation of Oligonucleotides

Oligonucleotides were synthesized with an automated DNA synthesizer (Model 391 PCR-Mate DNA Synthesizer; Applied Biosystems, Foster City, CA) using the phosphoramidite protocol provided by the manufacturer.36,37 The pentadecadeoxynucleotide (5'-GGCCGCG-GGGACCAT-3'), complementary to the initiation codon and the next four codons of rat TGF-{alpha} mRNA,38 was used as an anti-sense TGF-{alpha} mRNA oligonucleotide. A nonsense pentadecadeoxynucleotide, 5'-CGCGAGGCTAGGCGC-3', was prepared containing the same numbers of G, C, A, and T as the anti-sense oligonucleotide.

Determination of BrdU Incorporation, TGF-{alpha} Content, Mitochondria Function, and Total Protein Content of Cultured Hepatocytes

Fixation of hepatocytes and BrdU detection were performed according to the manufacturer’s instructions for the BrdU labeling and detection kits. Incorporated BrdU was determined by ELISA using BrdU labeling and detection kit III. In addition, immunohistological detection of BrdU was performed using BrdU labeling and detection kit II, and the number of labeled nuclei were counted in >1,000 hepatocytes in each well for calculations of BrdU nuclear labeling index. The TGF-{alpha} content in fixed hepatocytes was determined using an immunoblotting method as previously described39,40 with some modifications. Briefly, 200 µL of methanol containing 0.5% H2O2 was added to each culture well, incubated for 30 minutes at 4°C, and removed by aspiration. After five washes with 350 µl of 10 mmol/L phosphate-buffered saline (PBS), pH 7.2, the wells were filled with 350 µl of BA, incubated for 5 hours at 4°C, and washed three times with 350 µl of 10% BA in 10 mmol/L PBS. Fifty µl of anti-TGF-{alpha} rabbit IgG diluted to 5 µg/ml in 10 mmol/L PBS containing 10% BA, 10 mmol/L ethylenediaminetetraacetic acid, and 0.25% polyoxyethelene-10-octyphenyl ether (BA buffer) was added to each well, and incubated overnight at 4°C. The well was washed six times with 350 µl of BA buffer and 50 µl of peroxidase-conjugated anti-rabbit IgG solution diluted 50-fold in BA buffer was added to each well. The wells were incubated for 1 hour at room temperature, followed by six washings. To each well, 100 µl of o-phenylenediamine solution was added, incubated at room temperature for 20 minutes, followed by the addition of 100 µl of 0.5 N oxalic acid solution. The optical density of the solution in each well was determined at 490 nm in an automated microplate reader (MR 700; Dynatech Laboratories, Inc., Chantilly, VA).

For the measurement of the total cellular protein by Bradford’s method, the cells were solubilized in 100 µl of 1 N NaOH at 65°C for 15 minutes and neutralized with the same amount of 1 N HCl.41 Mitochondria function of cultured hepatocytes was determined using a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assay as previously described.42

Assay for HGF and TGF-{alpha} in the Liver

Liver extracts were prepared according to the manufacturer’s instructions of a HGF ELISA kit. Extracts were applied to HGF ELISA as previously described.35 Hepatic TGF-{alpha} levels were determined by ELISA as previously reported.25

Assay for Serum Rabbit IgG and IL-6 Levels in Rats

Serum rabbit IgG levels in rats dosed with rabbit IgG after partial hepatectomy were measured by sandwich ELISA as follows. In each well of a 96-well assay plate, 50 µl of 5 µg/ml anti-rabbit IgG preabsorbed with rat serum in 50 mmol/L of PBS (pH 7.4) was added, incubated overnight at 4°C, and removed by aspiration. After washing three times with 400 µl of 50 mmol/L PBS (pH 7.2), the well was filled with 400 µl of 70% (v/v) BA in 50 mmol/L PBS (pH 7.2), incubated overnight at 4°C, and removed by aspiration. The well was washed three times with 400 µl of 10% BA in 50 mmol/L of PBS (pH 7.2). To each well, 100 µl of assay samples diluted with 10% BA containing 10 mmol/L of ethylenediaminetetraacetic acid in 50 mmol/L of PBS (pH 7.2) was added, and incubated overnight at 4°C. The wells were washed eight times with 400 µl of 0.05% polyoxyethylene-20-sorbitan monolaurate and 10% BA in 50 mmol/L PBS (pH 7.2) followed by the addition of 50 µl of 1 µg/ml peroxidase conjugated anti-rabbit IgG in the 10% BA to each well. The plate was incubated overnight at 4°C, followed by similar washings. To each well, 100 µl of 2,2'-azino-di-[3-ethylbenzthiazoline sulfonate(6)] solution was added, incubated at room temperature for 20 minutes, followed by the addition of 50 µl of 0.25 mol/L oxalic acid solution. The optical density of the solution in each well was determined at 405 nm on an automated microplate reader. The lower limit of this assay was 160 pg/ml. Serum IL-6 levels in rats after partial hepatectomy were measured using a rat IL-6 ELISA according to the manufacturer’s instructions.

Determination of the Reactivity of Anti-TGF-{alpha} Rabbit IgG

The reactivity of anti-TGF-{alpha} rabbit IgG to TGF-{alpha}, EGF, and HB-EGF was determined by an immunoblotting method. Various concentrations of growth factors in 50 mmol/L of PBS (pH 7.4) were distributed in each well of a 96-well assay plate, incubated overnight at 4°C, and removed by aspiration. Subsequent procedures were similar to those used to detect TGF-{alpha} in cultured hepatocytes as described above.

Histological Examination

Liver specimens were fixed in formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The mitotic index of hepatocytes was determined by counting more than 3,000 hepatocytes in each liver specimen.

Experiments with Cultured Hepatocytes

Hepatocytes were isolated from rat livers according to Seglen’s method.43 They were plated on collagen-coated dishes at 5 x 104 cells/cm2 in WE containing 5% (v/v) FCS, 1 mmol/L insulin, and 1 mmol/L dexamethasone. After 2 hours, the culture medium was replaced with WE supplemented with 5% FCS. After a further 24 hours of incubation, the medium was changed to WE containing 5% FCS and various concentrations of dHGF. After another 24 hours incubation, the medium was replaced with media supplemented with 2% FCS containing 1 mmol/L BrdU, instead of the 5% FCS. The cells were harvested 16 hours later for the determinations of cellular TGF-{alpha} contents and BrdU incorporation into cellular DNA.

To examine the effects of anti-sense oligonucleotides, hepatocytes cultured for 26 hours as described above were used. The medium was changed to WE containing 5% FCS and 10 ng/ml of dHGF. After 24 hours, the medium was replaced with WE containing 2% FCS, 10 ng/ml of dHGF, and 1 mmol/L of BrdU, along with various concentrations of either anti-sense TGF-{alpha} mRNA oligonucleotide or nonsense oligonucleotide. The cells were harvested 16 hours later for the determinations of cellular TGF-{alpha} contents and BrdU incorporation into cellular DNA.

To examine the effects of antibodies against TGF-{alpha}, hepatocytes cultured for 50 hours as described above were used. The medium was replaced with WE containing 5% FCS, 10 ng/ml of dHGF, and 1 mmol/L of BrdU, along with various concentrations of either anti-TGF-{alpha} rabbit IgG or normal rabbit IgG. After 16 hours, the cells were harvested for the determination of BrdU incorporation into cellular DNA.

In addition to these experiments, hepatocytes were cultured in serum-free WE containing 0.5 µg/ml of aprotinin along with either anti-sense TGF-{alpha} mRNA oligonucleotides or antibodies for TGF-{alpha} to avoid potential effects of growth factors and cytokines in the serum.

Animal Experiments

Rats were subjected to a two-thirds resection of the liver under diethyl ether anesthesia. The rats were injected intraperitoneally with normal rabbit IgG (0.5 mg in 2.5 ml saline/kg body weight) at 0 and 12 hours after surgery. Rats were anesthetized with diethyl ether, and blood was collected through the inferior caval vein using an empty plastic syringe to prepare serum for the determination of serum rabbit IgG levels.

Similarly operated rats were injected intraperitoneally with either anti-TGF-{alpha} rabbit IgG or normal rabbit IgG (0.5 mg in 2.5 ml saline/kg body weight) at 0 and 12 hours after the operation. Twenty-four hours later, blood was collected through the inferior caval vein to prepare serum for the determination of serum IL-6 levels. After collecting blood, the liver was perfused with 25 ml of saline at 37°C through the portal vein after near-total exsanguination, and excised. A portion of the liver was immersed in 20% neutral-buffered formalin solution for histological examination. The remaining liver was frozen in liquid nitrogen, and stored at -80°C for use in the HGF assay.

Statistical Analyses

The differences between two unpaired samples were defined as significant when P values by both Student’s t-test and Mann-Whitney U test were <0.05. Spearman’s correlation test was used for analysis of correlations. The dose-related effects were tested by one-way analysis of variance followed by Spearman’s correlation test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TGF-{alpha} Content and BrdU Incorporation in Cultured Rat Hepatocytes in the Presence of HGF

As shown in Figure 1 , when dHGF was added to the medium at increasing concentrations, cultured rat hepatocytes increased their contents of TGF-{alpha} in a dose-related manner up to 10 ng/ml dHGF. The addition of dHGF at concentrations of 20 ng/ml or more resulted in a reduction of TGF-{alpha} content in hepatocytes compared to 10 ng/ml of dHGF addition. A similar dose-related response was observed in the BrdU incorporation into hepatocytes treated with dHGF.



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  		Figure 1. Effect of HGF on cellular TGF-{alpha} content and BrdU incorporation in cultured rat hepatocytes. A: Cellular TGF-{alpha} in the presence of increasing concentrations of HGF (expressed as percent of levels in the absence of HGF). Hepatocytes were plated on collagen-coated dishes in WE containing 5% FCS, 1 mmol/L insulin, and 1 mmol/L dexamethasone. After 2 hours, the culture medium was replaced with WE supplemented with 5% FCS. After a further 24 hours of incubation, the medium was changed to WE containing 5% FCS and dHGF. After another 24 hours incubation, the medium was replaced with media supplemented with HGF and 2% FCS instead of the 5% FCS. The cells were harvested 16 hours later. B: Same cultures as in A except that the medium supplemented with 2% FCS was containing 1 mmol/L BrdU, examined for BrdU incorporation. Data are mean ± SEM of six dishes. *, P < 0.05; **, P < 0.01 compared with the values in the absence of HGF.

 
Effect of Anti-Sense TGF-{alpha} mRNA Oligonucleotide on TGF-{alpha} Content and BrdU Incorporation in Cultured Rat Hepatocytes in the Presence of HGF

In the presence of 10 ng/ml of dHGF, anti-sense TGF-{alpha} mRNA oligonucleotide in the culture medium reduced TGF-{alpha} content in hepatocytes in a dose-related manner by up to 52% of the levels observed with the addition of control nonsense oligonucleotide (F = 8.9, P < 0.01; r = -0.75, P < 0.01) (Figure 2A) . The addition of anti-sense oligonucleotide resulted in a similar decrease in BrdU incorporation, as determined by ELISA (F = 76.7, P < 0.01; r = 0.87, P < 0.01) (Figure 2, B-1) . The anti-sense was effective in reducing BrdU incorporation even when hepatocytes were cultured in serum-free medium (Figure 2, B-2) . The BrdU nuclear labeling index of hepatocytes was also reduced significantly by the addition of 80 µmol/L of the anti-sense oligonucleotide, when compared to the addition of nonsense oligonucleotide (mean ± SD: 36.5 ± 1.4% versus 51.3 ± 0.5%, P < 0.01). The total cellular protein content and the mitochondrial function of the cells, as determined by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assay, were not affected by the addition of either oligonucleotide (data not shown).



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  		Figure 2. Effect of anti-sense TGF-{alpha} mRNA oligonucleotide on cellular TGF-{alpha} levels and BrdU incorporation in rat hepatocytes cultured in the presence of HGF. A: Hepatocytes cultured for 26 hours as described in the legend of Figure 1 were used. The medium was changed to WE containing 5% FCS and 10 ng/ml HGF. After 24 hours, the medium was replaced with WE containing 2% FCS, 10 ng/ml of dHGF along with either anti-sense TGF-{alpha} mRNA oligonucleotide or nonsense oligonucleotide. The cells were harvested 16 hours later for the determination of cellular TGF-{alpha} contents of hepatocytes. B: Similar cultures and conditions as in A, examined for BrdU incorporation. B-1: Hepatocytes were cultured in the same medium as in A except that the medium supplemented with 2% FCS was containing 1 mmol/L BrdU. B-2: Similar cultures as in B-1. However, the medium contained 0.5 µg/ml of aprotinin instead of FCS. Data are mean ± SEM of six dishes. *, P < 0.05; **, P < 0.01 compared with the values in the presence of nonsense oligonucleotide at same concentrations.

 
Effect of Anti-TGF-{alpha} Rabbit IgG on BrdU Incorporation in Cultured Rat Hepatocytes in the Presence of HGF

The anti-TGF-{alpha} rabbit IgG used was specific for TGF-{alpha} and did not recognize up to 500 ng of either EGF or HB-EGF by immunoblotting assays (Figure 3) . BrdU incorporation by cultured hepatocytes in the presence of 10 ng/ml dHGF was decreased by 1 µg/ml and 10 µg/ml of anti-TGF-{alpha} rabbit IgG addition, as determined by ELISA (F = 50.2, P < 0.01; r = -0.80, P < 0.01) (Figure 4) . A similar dose-related decrease in BrdU incorporation by addition of the antibody was observed even when hepatocytes were cultured in serum-free medium (Figure 4) . The addition of 10 µg/ml of anti-TGF-{alpha} rabbit IgG suppressed the BrdU nuclear labeling index of hepatocytes, when compared to that in the presence of normal rabbit IgG (mean ± SD: 34.9 ± 1.9% versus 50.7 ± 1.0%, P < 0.01). The total cellular protein content and the mitochondrial function by hepatocytes were not affected by the addition of the rabbit IgGs (data not shown).



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  		Figure 3. Reactivity of anti-TGF-{alpha} rabbit IgG to growth factors. Closed circles, open circles, and squares indicate the reactivity of anti-TGF-{alpha} rabbit IgG with TGF-{alpha}, EGF, and HB-EGF, respectively.

 


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  		Figure 4. Effect of anti-TGF-{alpha} rabbit IgG on BrdU incorporation in rat hepatocytes cultured in the presence of HGF. Hepatocytes cultured for 26 hours as described in the legend of Figure 1 were used. The medium was changed to WE containing 10 ng/ml of dHGF along with either 5% FCS or 0.5 µg/ml of aprotinin. After another 24 hours incubation, the medium was replaced with WE containing either 5% FCS or 0.5 µg/ml of aprotinin, 10 ng/ml of dHGF, and 1 mmol/L of BrdU, along with either anti-TGF-{alpha} rabbit IgG or normal rabbit IgG. After 16 hours, the cells were harvested for the determination of BrdU incorporation into cellular DNA. Closed lines indicate hepatocytes were cultured in medium containing 5% of FCS. Dotted lines indicate hepatocytes were cultured in medium without FCS. Closed circles denote rat hepatocytes cultured in the presence of 10 ng/ml dHGF with the addition of increasing concentrations of anti-TGF-{alpha} rabbit IgG. Open circles denote experiments using the control normal rabbit IgG. Data are mean ± SEM of six dishes. **, P < 0.01 compared with the values in the presence of normal rabbit IgG at the same concentrations.

 
Effect of Anti-TGF-{alpha} Rabbit IgG on the Mitotic Index of Hepatocytes after Partial Hepatectomy in Rats

As shown in Figure 5 , rats dosed with normal rabbit IgG after partial hepatectomy possessed serum rabbit IgG levels in excess of 1 µg/ml at 2 hours after the surgery. These rats attained a plateau of ~10 µg/ml at 12 hours. In addition, rats given either normal rabbit IgG or anti-TGF-{alpha} rabbit IgG after partial hepatectomy increased hepatic HGF levels comparable to that obtained when rats were dosed with the vehicle alone (Figure 6) . However, the increased mitotic index of hepatocytes 24 hours after partial hepatectomy was suppressed by the administration of anti-TGF-{alpha} rabbit IgG to 20% of the mitotic index of rats given normal rabbit IgG (Figure 6) . Hepatic TGF-{alpha} levels were significantly decreased in rats treated with anti-TGF-{alpha} IgG compared to controls (Figure 6) . Serum IL-6 levels were not affected by administration of either IgG (data not shown).



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  		Figure 5. Changes of serum rabbit IgG levels in rats after partial hepatectomy and dosed with rabbit IgG. Data are mean ± SEM of four rats.

 


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  		Figure 6. Effect of anti-TGF-{alpha} rabbit IgG on the mitotic index of hepatocytes and hepatic HGF and TGF-{alpha} levels 24 hours after partial hepatectomy in rats. Data are mean ± SEM of four rats. "Before" means the levels of HGF or TGF-{alpha} and the mitotic index of hepatocytes before the operation; "Ve" indicates levels in rats dosed with vehicle alone; "IgG" labels the levels in rats dosed with normal rabbit IgG; and "Ab" indicates levels in rats dosed with anti-TGF-{alpha} rabbit IgG. Left: Shaded and open columns indicate hepatic levels of HGF and TGF-{alpha}, respectively. **, P < 0.01 compared with the levels before operation. #, P < 0.05 compared with the levels in rats dosed with vehicle alone or normal rabbit IgG. Right: Circles indicate mitotic index of hepatocytes. **, P < 0.01 compared with the levels in rats dosed with normal rabbit IgG.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results indicate that the TGF-{alpha} production and DNA synthesis of rat hepatocytes in primary culture were enhanced in a dose-related manner by the addition of HGF to the medium. These enhancements were attenuated by the addition of anti-sense TGF-{alpha} mRNA oligonucleotide. Furthermore, anti-TGF-{alpha} rabbit IgG reduced DNA synthesis in hepatocytes in vitro and in vivo, even in the presence of HGF.

We cultured hepatocytes in the medium containing FCS. Furthermore, we performed the experiments using serum-free medium to confirm the results. Serum deprivation, which causes apoptosis of many types of cultured cells, is liable to induce death of hepatocytes in culture, although the addition of aprotinin to the medium would prevent it.44-48 On the other hand, HGF has been reported to have a cytoprotective effect as well as a mitogenic effect on hepatocytes.49-51 Recently, it was reported that HGF may prevent apoptosis of hepatocytes.49,52 To minimize the possibility that HGF may act on hepatocytes as a cytoprotective factor, and not to cloud interpretation of the results, we performed experiments using the medium containing FCS as well as serum-free medium supplemented with aprotinin in hepatocyte culture. In addition, we used dHGF (a 5-amino acid deleted form of HGF) in vitro experiments. dHGF is known to be more potent than HGF in the stimulation of DNA synthesis in hepatocytes.53

To examine DNA synthesis by cultured hepatocytes in the presence of the anti-sense oligonucleotides, we added BrdU to the culture medium. We used a BrdU concentration >10 times higher than that of the added oligonucleotides to avoid a reduced uptake of BrdU because of the presence of degraded oligonucleotides in the medium. Furthermore, a nonsense oligonucleotide containing the same numbers of G, C, A, and T as the anti-sense oligonucleotide was used as a control. The addition of oligonucleotides did not affect the mitochondrial function of the hepatocytes, as well as cellular protein content, suggesting that the addition of oligonucleotides did not influence the viability of hepatocytes. Suppressed TGF-{alpha} production by anti-sense TGF-{alpha} mRNA oligonucleotide in cultured hepatocytes resulted in a reduced DNA synthesis by these cells. These observations are consistent with a previous report describing the inhibition of tumor growth in liver epithelial cells transfected with a TGF-{alpha} anti-sense gene.54

Currently, there are six known ligands for the EGF/TGF-{alpha} receptor. At least three of them, EGF, TGF-{alpha}, and HB-EGF, have been found to stimulate DNA synthesis in cultured rat hepatocytes.1,55 Of these, hepatocytes produce only TGF-{alpha} in substantial quantities,1,10-16 although a possible transient production of a small amount of EGF by hepatocytes has been reported.1,56 Our results demonstrate that the addition of highly specific anti-TGF-{alpha} rabbit IgG to the culture medium inhibited DNA synthesis by hepatocytes, even though the antibody did not influence hepatocyte viability. These results suggest that the anti-TGF-{alpha} rabbit IgG may inhibit the action of TGF-{alpha} produced by hepatocytes, leading to a suppression of DNA synthesis by hepatocytes. Previously, several reports have described the suppression of TGF-{alpha} activity of various kinds of TGF-{alpha}-producing cells by the addition of monoclonal or polyclonal anti-TGF-{alpha} antibodies to the culture medium.57-61 In those studies, the effective concentration of the antibodies in the medium varied from 0.5 to 50 µg/ml depending on the antibodies and cells used.57-61 In the present study, hepatocyte DNA synthesis was suppressed by the addition of 1 µg/ml or more of anti-TGF-{alpha} IgG to the medium, consistent with the previous reports concerning antibody concentration.

To examine the activity of anti-TGF-{alpha} rabbit IgG on hepatocytes in vivo, we administered 0.5 mg/kg body weight of the anti-TGF-{alpha} rabbit IgG intraperitoneally to rats at 0 and 12 hours after partial hepatectomy. When we treated rats in a similar manner with normal rabbit IgG, serum rabbit IgG levels exceeded 1 µg/ml at 2 hours, and were maintained at ~10 µg/ml at 12 hours or more after the operation. Previously, it has been reported that, in rats subjected to partial hepatectomy, the hepatic and serum TGF-{alpha} levels are significantly increased at 12 hours after the operation when compared to the preoperative levels.1,14,25 The TGF-{alpha} reaches maximal levels at 24 hours, coinciding with the maximum number of mitotic hepatocytes. These observations confirm that the rise in serum and hepatic levels of TGF-{alpha} in rats after partial hepatectomy can be accompanied by a rise in serum levels of anti-TGF-{alpha} rabbit IgG in rats dosed with the antibody. The concentration of antibody in these rats during this time should be comparable to the concentration of anti-TGF-{alpha} rabbit IgG that was effective in the suppression of DNA synthesis in cultured hepatocytes. Indeed, our results demonstrate that the antibody administration suppressed hepatocyte proliferation 24 hours after partial hepatectomy, even when the increase of HGF was not affected. In addition, serum levels of IL-6, which may play a critical role in liver regeneration, were not influenced by the antibody administration.62-68 As for hepatic TGF-{alpha} levels, the moderate but significant decrease was observed in rats treated with anti-TGF-{alpha} IgG compared to controls. The antibody may not suppress the synthesis of TGF-{alpha} by hepatocytes, whereas TGF-{alpha} trapped by the antibody may not be internalized into hepatocytes. The reduction of hepatic TGF-{alpha} contents might reflect these situations. Recently, Russell et al69 reported no significant difference in hepatocyte proliferation after partial hepatectomy in TGF-{alpha} knockout mice and wild-type mice. As mentioned above, there are other ligands for the TGF-{alpha}/EGF receptor, such as EGF, which is abundantly produced by salivary glands in mice.1,15 The effects of the loss of TGF-{alpha}, in knockout mice, might be obviated by the opportunistic compensation of such ligands.69 Furthermore, it has been demonstrated that the targeting of a gene can increase the expression of functionally related proteins.70 Therefore, other proteins that have the potential to act as ligands to the TGF-{alpha}/EGF receptor may compensate for the loss of TGF-{alpha}. To clarify this point, studies using TGF-{alpha}/EGF receptor knockout mice will be required. In addition, it would be important to learn whether neutralizing antibodies to HGF would prevent the subsequent increase of TGF-{alpha} during liver regeneration to clarify the relationship between HGF and TGF-{alpha} in vivo.

The communication between HGF, TGF-{alpha}, and other growth factors has been a topic of study for several years. TGF-{alpha} secretion by cultured rat hepatocytes has been shown to be enhanced by EGF, TGF-{alpha}, or HGF.14,26 In addition, TGF-{alpha} mRNA expression is stimulated by TGF-{alpha} in human colon carcinoma cells and by HGF in human pancreatic cancer cells.71,72 The mitogenic effects of HGF and TGF-{alpha} have been shown to be additive in cultured fetal and adult hepatocytes.26,73,74 Recently, a communication between HGF and TGF-{alpha}/EGF receptor systems was reported in rat liver epithelial cells.75,76 The TGF-{alpha}/EGF receptor and c-met, the HGF receptor, are tyrosine kinases that autophosphorylate, as well as phosphorylate, to activate other enzymes. The src-homology regions of activated c-met and the TGF-{alpha}/EGF receptor are reported to bind different downstream signal transduction elements, suggesting that HGF and TGF-{alpha}, via their receptors, might stimulate unique targets.77,78 These observations, together with our results, highlight the significance of the communication between growth factors and underlie the need for further research.


   Footnotes
 
Address reprint requests to Tomoaki Tomiya, M.D., Ph.D., First Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: tomiya-1im{at}h.u-tokyo.ac.jp

Supported in part by a grant for scientific research from the Ministry of Education, Science and Culture of Japan.

Accepted for publication August 3, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Fausto N: Hepatic regeneration. Zakim D Boyer TD eds. Hepatology. 1996, :pp 32-57 W. B. Saunders, Philadelphia
  2. Michalopoulos G: Liver regeneration: molecular mechanisms of growth control. FASEB J 1990, 4:176-187[Abstract]
  3. Zarnegar R, DeFrances MC, Michalopoulos GK: Hepatocyte growth factor. Its role in hepatic growth and pathology. Liver: Biology and Pathology. Edited by IM Arias, JL Boyer, N Fausto, WB Jakoby, DA Schachter, DA Shafritz. New York, Raven Press, 1994, pp 1047–1057
  4. Jhappan C, Stahle C, Harkins RN, Fausto N, Merlino GT: TGF{alpha} overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. Cell 1990, 61:1137-1146[Medline]
  5. Shiota G, Wang TC, Nakamura T, Schmidt EV: Hepatocyte growth factor in transgenic mice: effects on hepatocyte growth, liver regeneration and gene expression. Hepatology 1994, 19:962-972[Medline]
  6. Ishiki Y, Ohnishi H, Muto Y, Matsumoto K, Nakamura T: Direct evidence that hepatocyte growth factor is a hepatotropic factor for liver regeneration and has a potent antihepatitis effect in vivo. Hepatology 1992, 16:1227-1235[Medline]
  7. Fujiwara K, Nagoshi S, Ohno A, Hirata K, Ohta Y, Mochida S, Tomiya T, Higashio K, Kurokawa K: Stimulation of liver growth by exogenous human hepatocyte growth factor in normal and partially hepatectomized rats. Hepatology 1993, 18:1443-1449[Medline]
  8. Webber EM, Godowski PJ, Fausto N: In vivo response of hepatocytes to growth factors requires an initial priming stimulus. Hepatology 1994, 19:489-497[Medline]
  9. Liu ML, Mars WM, Zarnegar R, Michalopoulos GK: Collagenase pretreatment and mitogenic effects of hepatocyte growth factor and transforming growth factor-{alpha} in adult rat liver. Hepatology 1994, 19:1521-1527[Medline]
  10. Todaro GJ, Fryling C, deLarco JE: Transforming growth factors produced by certain human tumor cells: polypeptides that interact with epidermal growth factor receptors. Proc Natl Acad Sci USA 1980, 77:5258-5262[Abstract/Free Full Text]
  11. Massague J: Epidermal growth factor-like transforming growth factor. II. Interaction with epidermal growth factor receptors on human placenta membrane and A431 cells. J Biol Chem 1983, 258:13614-13620[Abstract/Free Full Text]
  12. Derynck R: Transforming growth {alpha}. Cell 1988, 54:593-595[Medline]
  13. Johnson AC, Garfield SH, Merlino GT, Pastan I: Expression of epidermal growth factor receptor proto-oncogene mRNA in regenerating rat liver. Biochem Biophys Res Commun 1988, 150:412-418[Medline]
  14. Mead JE, Fausto N: Transforming growth factor {alpha} may be a physiological regulator of liver regeneration by means of an autocrine mechanism. Proc Natl Acad Sci USA 1989, 86:1558-1562[Abstract/Free Full Text]
  15. Fisher DA, Lakshmanan J: Metabolism and effects of epidermal growth factor and related growth factors in mammals. Endocr Rev 1990, 11:418-442[Abstract]
  16. Fausto N, Webber EM: Liver regeneration. Arias IM Boyer JL Fausto N Jakoby WB Schachter DA Shafritz DA eds. The Liver: Biology and Pathology. 1994, :pp 1059-1084 Raven Press, New York
  17. Kinoshita T, Tashiro K, Nakamura T: Marked increase of HGF mRNA in non-parenchymal liver cells of rats treated with hepatotoxins. Biochem Biophys Res Commun 1989, 165:1229-1234[Medline]
  18. Noji S, Tashiro K, Koyama E, Nohno T, Ohyama K, Taniguchi S, Nakamura T: Expression of hepatocyte growth factor gene in endothelial and Kupffer cells of damaged rat livers, as revealed by in situ hybridization. Biochem Biophys Res Commun 1990, 173:42-47[Medline]
  19. Zarnegar R, DeFrances MC, Oliver L, Michalopoulos G: Identification and partial characterization of receptor binding sites for HGF on rat hepatocytes. Biochem Biophys Res Commun 1990, 173:1179-1185[Medline]
  20. Naldini L, Vigna E, Narsimhan RP, Gaudino G, Zarnegar R, Michalopoulos GK, Comoglio PM: Hepatocyte growth factor (HGF) stimulates the tyrosine kinase activity of the receptor encoded by the proto-oncogene c-MET. Oncogene 1991, 6:501-504[Medline]
  21. Bottaro DP, Rubin JS, Faletto DL, Chan AM-L, Kmiecik TE, Vande Woude GF, Aaronson SA: Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 1991, 251:802-804[Abstract/Free Full Text]
  22. Higuchi O, Nakamura T: Identification and change in the receptor for hepatocyte growth factor in rat liver after partial hepatectomy or induced hepatitis. Biochem Biophys Res Commun 1991, 176:599-607[Medline]
  23. Schirmacher P, Geerts A, Pietrangelo A, Dienes HP, Rogler CE: Hepatocyte growth factor/hepatopoietin A is expressed in fat-storing cells from rat liver but not myofibroblast-like cells derived from fat-storing cells. Hepatology 1992, 15:5-11[Medline]
  24. Hu Z, Evarts RP, Fujio K, Marsden ER, Thorgeirsson SS: Expression of hepatocyte growth factor and c-met genes during hepatic differentiation and liver development in the rat. Am J Pathol 1993, 142:1823-1830[Abstract]
  25. Tomiya T, Ogata I, Fujiwara K: Transforming growth factor {alpha} levels in liver and blood correlate better than hepatocyte growth factor with hepatocyte proliferation during liver regeneration. Am J Pathol 1998, 153:955-961[Abstract/Free Full Text]
  26. Webber EM, FitzGerald MJ, Brown PI, Bartlett MH, Fausto N: Transforming growth factor-{alpha} expression during liver regeneration after partial hepatectomy and toxic injury, and potential interactions between transforming growth factor-{alpha} and hepatocyte growth factor. Hepatology 1993, 18:1422-1431[Medline]
  27. Kinoshita T, Hirao S, Matsumoto K, Nakamura T: Possible endocrine control of liver regeneration after partial hepatectomy. Biochem Biophys Res Commun 1991, 177:330-335[Medline]
  28. Zarnegar R, DeFrances MC, Kost DP, Lindroos P, Michalopoulos G: Expression of hepatocyte growth factor mRNA in regenerating rat liver after partial hepatectomy. Biochem Biophys Res Commun 1991, 177:559-565[Medline]
  29. Tomiya T, Fujiwara K: Serum levels of transforming growth factor-{alpha} in patients after partial hepatectomy as determined with an enzyme-linked immunosorbent assay. Hepatology 1993, 18:304-308[Medline]
  30. Tomiya T, Tani M, Yamada S, Hayashi S, Umeda N, Fujiwara K: Serum hepatocyte growth factor levels in hepatectomized and nonhepatectomized surgical patients. Gastroenterology 1992, 103:1621-1624[Medline]
  31. Tomiya T, Hayashi S, Yanase M, Umeda N, Tani M, Yamada S, Masaki N, Ogata I, Fujiwara K: Serum transforming growth factor {alpha} level can be a parameter for evaluating liver regeneration after partial hepatectomy in patients with liver cancer. Semin Oncol 1997, 24(Suppl 6):S14-S17
  32. Tomiya T, Fujiwara K: Liver regeneration in fulminant hepatitis as evaluated by serum transforming growth factor {alpha} levels. Hepatology 1996, 23:253-257[Medline]
  33. Tomiya T, Nagoshi S, Fujiwara K: Significance of serum human hepatocyte growth factor levels in patients with hepatic failure. Hepatology 1992, 15:1-4[Medline]
  34. Shima N, Nagao M, Ogaki F, Tsuda E, Murakami A, Higashio K: Tumor cytotoxic factor/hepatocyte growth factor from human fibroblasts: cloning of its cDNA, purification and characterization of recombinant protein. Biochem Biophys Res Commun 1991, 180:1151-1158[Medline]
  35. Yamada A, Matsumoto K, Iwanari H, Sekiguchi K, Kawata S, Matsuzawa Y, Nakamura T: Rapid and sensitive enzyme-linked immunosorbent assay for measurement of HGF in rat and human tissues. Biomed Res 1995, 16:105-114
  36. Helene C, Toulme JJ: Specific regulation by antisense, sense and antigene nucleic acids. Biochim Biophys Acta 1990, 1049:99-125[Medline]
  37. Goodchild J, Agrawal S, Civeira MP, Sarin PS, Sun D, Zamecnik PC: Inhibition of human immunodeficiency virus replication by antisense oligonucleotides. Proc Natl Acad Sci USA 1988, 85:5507-5511[Abstract/Free Full Text]
  38. Lee DC, Rose TM, Webb NR, Todaro GI: Cloning and sequence analysis of a cDNA for rat transforming growth factor-{alpha}. Nature 1985, 313:489-491[Medline]
  39. Inagaki H, Katoh M, Kurokawa-Ohsawa K, Tanaka S: A new sandwich enzyme-linked immunosorbent assay (ELISA) for transforming growth factor {alpha} (TGF{alpha}) based upon conformational modification by antibody binding. J Immunol Methods 1990, 128:27-37[Medline]
  40. Katoh M, Inagaki H, Kurosawa-Ohsawa K, Katsuura M, Tanaka S: Detection of transforming growth factor alpha in human urine and plasma. Biochem Biophys Res Commun 1990, 167:1065-1072[Medline]
  41. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 1976, 72:248-254[Medline]
  42. Mosmann T: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983, 65:55-63[Medline]
  43. Seglen PO: Preparation of isolated rat liver cells. Methods Cell Biol 1976, 13:29-83[Medline]
  44. Pandey S, Wang E: Cells en route to apoptosis are characterized by the upregulation of c-fos, c-myc, c-jun, cdc2, and RB phosphorylation, resembling events of early cell-cycle traverse. J Cell Biochem 1995, 58:135-150[Medline]
  45. Chiao C, Zhang Y, Kaufman DG, Kaufmann WK: Phenobarbital modulates the type of cell death by rat hepatocytes during deprivation of serum in vitro. Hepatology 1995, 22:297-303[Medline]
  46. Hoffman B, Liebermann DA: The proto-oncogene c-myc and apoptosis. Oncogene 1998, 17:3351-3357[Medline]
  47. Nakamura T, Asami O, Tanaka K, Ichihara A: Increased survival of rat hepatocytes in serum-free medium by inhibition of a trypsin-like protease associated with their plasma membranes. Exp Cell Res 1984, 155:81-91[Medline]
  48. Asami O, Nakamura T, Mura T, Ichihara A: Identification of trypsin inhibitor in bovine pituitary extracts as a survival factor for adult rat hepatocytes in primary culture. J Biochem 1984, 95:299-309[Abstract/Free Full Text]
  49. Tsubouchi H: Hepatocyte growth factor for liver disease. Hepatology 1999, 30:333-334[Medline]
  50. Okano J, Shiota G, Kawasaki H: Protective action of hepatocyte growth factor for acute liver injury caused by D-galactosamine in transgenic mice. Hepatology 1997, 26:1241-1249[Medline]
  51. Yoshikawa A, Kaido T, Seto S, Yamaoka S, Sato M, Ishii T, Imamura M: Hepatocyte growth factor promotes liver regeneration with prompt improvement of hyperbilirubinemia in hepatectomized cholestatic rats. J Surg Res 1998, 78:54-59[Medline]
  52. Kosai K, Matsumoto K, Funakoshi H, Nakamura T: Hepatocyte growth factor prevents endotoxin-induced lethal hepatic failure in mice. Hepatology 1999, 30:151-159[Medline]
  53. Shima N, Tsuda E, Goto M, Yano K, Hayasaka H, Ueda M, Higashio K: Hepatocyte growth factor and its variant with a deletion of five amino acids are distinguishable in their biological activity and tertiary structure. Biochem Biophys Res Commun 1994, 200:808-815[Medline]
  54. Laird AD, Brown PI, Fausto N: Inhibition of tumor growth in liver epithelial cells transfected with a transforming growth factor alpha antisense gene. Cancer Res 1994, 54:4224-4232[Abstract/Free Full Text]
  55. Russell WE, Carver RS: A diversity of EGF-like ligands and receptors in liver. Fleig WE eds. Normal and Malignant Liver Cell Growth. 1999, :pp 33-46 Kluwer Academic Publishers/The Netherlands, Dordrecht
  56. Mulhaupt B, Feren A, Fodor E, Jones A: Liver expression of epidermal growth factor RNA. : Rapid increase in immediate-early phase of liver regeneration. J Biol Chem 1994, 269:19667-19670[Abstract/Free Full Text]
  57. Machida T, Taga M, Minaguchi H: Effects of epidermal growth factor and transforming growth factor alpha on the mouse trophoblast outgrowth in vitro. Eur J Endocrinol 1995, 133:741-746[Abstract]
  58. Fang K: An enhanced and sensitive autocrine stimulation by transforming growth factor-{alpha} is acquired in the brain metastatic variant of a human non-small-cell lung cancer cell line. Br J Cancer 1996, 74:1776-1782[Medline]
  59. Asano K, Nakamura H, Lilly CM, Klagsbrun M, Drazen JM: Interferon {gamma} induces prostaglandin G/H synthase-2 through an autocrine loop via the epidermal growth factor receptor in human bronchial epithelial cells. J Clin Invest 1997, 99:1057-1063[Medline]
  60. Seki S, Sakai Y, Kitada T, Kawakita N, Yanai A, Tsutsui H, Sakaguchi H, Kuroki T, Monna T: Induction of apoptosis in a human hepatocellular carcinoma cell line by a neutralizing antibody to transforming growth factor-{alpha}. Virchows Arch 1997, 430:29-35[Medline]
  61. Murthy S, Mathur SN, Bishop WP, Field FJ: Inhibition of apolipoprotein B secretion by IL-6 is mediated by EGF or an EGF-like molecule in CaCo-2 cells. J Lipid Res 1997, 38:206-216[Abstract]
  62. Cressman DE, Greenbaum LE, DeAngelis RA, Ciliberto G, Furth EE, Poli V, Taub R: Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice. Science 1996, 274:1379-1383[Abstract/Free Full Text]
  63. Yamada Y, Kirillova I, Peschon JJ, Fausto N: Initiation of liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor. Proc Natl Acad Sci USA 1997, 94:1441-1446[Abstract/Free Full Text]
  64. Yamada Y, Webber EM, Kirillova I, Peschon JJ, Fausto N: Analysis of liver regeneration in mice lacking type 1 or type 2 tumor necrosis factor receptor: requirement for type 1 but not type 2 receptor. Hepatology 1998, 28:959-970[Medline]
  65. Yamada Y, Fausto N: Deficient liver regeneration after carbon tetrachloride injury in mice lacking type 1 but not type 2 tumor necrosis factor receptor. Am J Pathol 1998, 152:1577-1589[Abstract]
  66. Maione D, Di Carlo E, Li W, Musiani P, Modesti A, Peters M, Rose-John S, Della Rocca C, Tripodi M, Lazzaro D, Taub R, Savino R, Ciliberto G: Coexpression of IL-6 and soluble IL-6R causes nodular regenerative hyperplasia and adenomas of the liver. EMBO J 1998, 17:5588-5597[Medline]
  67. Webber EM, Bruix J, Pierce RH, Fausto N: Tumor necrosis factor primes hepatocytes for DNA replication in the rat. Hepatology 1998, 28:1226-1234[Medline]
  68. Taub R, Greenbaum LE, Peng Y: Transcriptional regulatory signals define cytokine-dependent and -independent pathways in liver regeneration. Semin Liver Dis 1999, 19:117-127[Medline]
  69. Russell WE, Kaufmann WK, Sitaric S, Luetteke NC, Lee DC: Liver regeneration and hepatocarcinogenesis in transforming growth factor-{alpha}-targeted mice. Mol Carcinog 1996, 15:183-189[Medline]
  70. Zhang W, Behringer RR, Olson EN: Inactivation of the myogenic bHLH gene MRF4 results in up-regulation of myogenin and rib anomalies. Genes Dev 1995, 9:1388-1399[Abstract/Free Full Text]
  71. Jiang D, Liang J, Humphrey LE, Yang H, Brattain MG: Expression of TGF{alpha} autocrine activity in human colon carcinoma CBS cells is autoregulated and independent of exogeneous epidermal growth factor. J Cell Physiol 1998, 175:174-183[Medline]
  72. Ohba N, Funatomi H, Seki T, Makino R, Mitamura K: Hepatocyte growth factor stimulates cell growth and enhances the expression of transforming growth factor a mRNA in AsPC-1 human pancreatic cancer cells. J Gastroenterol 1999, 34:498-504[Medline]
  73. Hernandez J, Zarnegar R, Micalopoulos GK: Characterization of the effects of human placental HGF on rat hepatocytes. J Cell Physiol 1992, 150:116-121[Medline]
  74. Fabregat I, de Juan C, Nakamura T, Benito M: Growth stimulation of rat fetal hepatocytes in response to hepatocyte growth factor: modulation of c-myc and c-fos expression. Biochem Biophys Res Commun 1992, 189:684-690[Medline]
  75. Stolz DB, Michalopoulos GK: Comparative effects of hepatocyte growth factor and epidermal growth factor on motility, morphology, mitogenesis and signal transduction of primary rat hepatocytes. J Cell Biochem 1994, 55:445-464[Medline]
  76. Presnell SC, Stolz DB, Mars WM, Jo M, Michalopoulos GK, Strom SC: Modifications of the hepatocyte growth factor/c-met pathway by constitutive expression of transforming growth factor-{alpha} in rat liver epithelial cells. Mol Carcinog 1997, 18:244-255[Medline]
  77. Cantley LC, Auger KR, Carpenter C, Duckworth B, Graziani A, Kapeller R, Soltoff S: Oncogenes and signal transduction. Cell 1991, 64:281-302[Medline]
  78. Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Powson T, Haser WG, King F, Roberts T, Ratnofsky S, Lechleider RJ: SH2 domains recognize specific phosphopeptide sequence. Cell 1993, 72:767-778[Medline]




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