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[Cancer Research 60, 1394-1402, March 1, 2000]
© 2000 American Association for Cancer Research

Tumor Biology

Autocrine Stimulatory Mechanism by Transforming Growth Factor ß in Human Hepatocellular Carcinoma1

Koichi Matsuzaki2, Masataka Date, Fukiko Furukawa, Yoshiya Tahashi, Masanori Matsushita, Kazushige Sakitani, Noriyo Yamashiki, Toshihito Seki, Hidetsugu Saito, Mikio Nishizawa, Junichi Fujisawa and Kyoichi Inoue

Third Department of Internal Medicine [K. M., M. D., F. F., Y. T., M. M., K. S., N. Y., T. S., K. I.], Department of Medical Chemistry [M. N.], and Department of Microbiology [J. F.], Kansai Medical University, Osaka 570-8507, and Department of Internal Medicine, School of Medicine, Keio University, Tokyo 160-8582 [H. S.], Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The serum concentration of transforming growth factor ß (TGF-ß) is elevated as tumors progress in hepatocellular carcinoma (HCC) patients. In this study, we examined whether modulation of tumor-derived TGF-ß signal transduction contributes to malignant progression. We investigated the production of TGF-ß1, the biological effects of TGF-ß and neutralizing antibody on HCC cells, activation of Smad 2, Smad 3, and Smad 4, induction of antagonistic Smads (Smad 6 and Smad 7), and promoter activities of two target genes, plasminogen activator inhibitor type 1 (PAI-1) and p15INK4B. In human cell lines HCC-M and HCC-T, TGF-ß accelerates their proliferation. Smad 2 was activated constitutively by an autocrine mechanism, because in the absence of exogenous TGF-ß, a high level of Smad 2 phosphorylation, induction of PAI-1 transcripts, and nuclear localization of Smad 2 were observed. This constitutive activation of Smad 2 was, at least in part, attributable to the lack of induction of antagonistic Smads by TGF-ß. However, Smads activated by tumor-derived TGF-ß constantly suppressed p15INK4B expression. In addition, 3 of 10 human HCC tissues showed nuclear localization of Smad 2 and low mRNA levels of p15INK4B and antagonistic Smads but a high level of PAI-1. Our observations suggest that this constant suppression of the p15INK4B gene could be involved in the malignant progression of HCC.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TGF3 -ß is a potent growth inhibitor for most epithelial cells. The TGF-ß signal is transduced by heteromeric complexes of its type I and type II transmembrane Ser/Thr kinase receptors (1) . The receptor complex is activated when the type II receptor kinase transphosphorylates the GS domain of the type I kinase (2) . This activates the type I kinase and transiently associates with and phosphorylates R-Smads, such as Smad 2 and Smad 3 (3 , 4) . The phosphorylation of R-Smads results in formation of a heteromeric complex with another common mediator Smad, Smad 4 (5) , and translocates into the nucleus (3) . The activated Smad complex binds to target promoters in association with DNA-binding cofactors including FAST-1 (6) , FAST-2 (7) , and Fos/Jun (8) , and recruits coactivators or corepressors in activating or inhibiting transcription, respectively (9 , 10) . Target genes include PAI-1 gene and one of the CDK inhibitors, the p15INK4B gene, which is a potential effector of TGF-ß-mediated cell cycle arrest (11) . In contrast to R-Smads and Smad 4, the antagonistic Smads Smad 6 and Smad 7 appear to block the ligand-dependent signaling (12 , 13) .

In hepatocytes of normal liver, TGF-ß1 is not detected by immunohistochemical analysis and in situ hybridization, whereas human HCC cells display significant intracellular expression of TGF-ß1 (14) . Moreover, TGF-ß concentration in the plasma of HCC patients is increased (15) . In light of the well-known fact that TGF-ß regulates hepatocyte growth negatively, there is a discrepancy between high serum TGF-ß level and the high proliferating rate in HCC cells. This implies that the change of growth response to TGF-ß at the cellular level is the more important step in malignant progression; therefore, we hypothesize that HCC cells show a different regulatory mechanism for TGF-ß signal transduction than normal hepatocytes. We investigated the biological effects of TGF-ß on HCC cells, activation of signaling molecules, and promoter activities of two target genes, PAI-1 and p15INK4B.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Cell Separation Procedure
HCC-M and HCC-T cell lines were grown as subconfluent monolayer cultures in DMEM supplemented with 10% FCS, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were kept under 95% air and 5% CO2 at 37°C. All experiments were carried out in the log phase of growth after the cells had been plated for 24 h. Hepatocytes were isolated from normal rat liver as described (16) .

Northern Blot Hybridization Analysis
mRNAs were isolated and hybridized with labeled cDNAs probes for TGF-ß1, Smad 6, Smad 7, PAI-1, p15INK4B, and human GAPDH as described (16) . Total RNA was isolated from cultured cells or frozen tissues by extraction in guanidinium isothiocyanate/phenol/chloroform, and poly(A)-rich RNA was selected using oligo(dT)-cellulose (New England Biolabs Inc., Beverly, MA). Aliquots (2 µg) of poly(A)-rich RNA were denatured with 2.2 M formaldehyde/50% formamide, electrophoresed through 1% agarose gels containing 2.2 M formaldehyde, and transferred onto nylon membranes (Hybond-N+ RPN303B; Amersham International, Buckinghamshire, United Kingdom). cDNAs were labeled with [{alpha}-32P]dCTP (3000 Ci/mmol; Amersham International) by the random primer labeling method. The filters were hybridized with the cDNA probe in solution containing 40% formamide, 5x Denhardt’s solution, 5x saline-sodium phosphate-EDTA, 0.5% SDS, and 100 µg/ml sonicated salmon testis DNA for 16 h at 42°C. After washing with 2x SSC containing 0.1% SDS at 55°C, the filters were exposed to an RX film (Fuji Photo Film Co., Kanagawa, Japan).

Measurement of TGF-ß1 by ELISA
Cells were plated in 60-mm dishes in regular growth medium. When they were 50–75% confluent, the monolayers were washed twice with PBS and replaced by serum-free DMEM. The conditioned media were collected 60 h later. To determine the concentration of TGF-ß1 in the media, we used an enzyme-linked immunoassay kit for human TGF-ß1 (R&D System, Minneapolis, MN).

Cell Proliferation Experiments
Cells were plated at a density of 2 x 104 in 60-mm dishes. Two hundred pM TGF-ß1, 5 µg/ml anti- TGF-ß antibody (R&D system), or nonimmune rabbit IgG in 0.2% FCS/DMEM was added at the time of inoculation and on day 3. Cells were trypsinized on day 5, and the cell number was counted as described (17) .

For measurement of DNA synthesis, 3 x 104 cells/well were plated in 12-well plates and changed to serum-free medium 24 h later. TGF-ß1 or the antibodies were added the following day. Twenty h later, DNA synthesis was measured by the incorporation of 2 µCi/well [3H]thymidine (25 Ci/mmol; Amersham International) into 5% trichloroacetic acid-precipitable material after a 3-h pulse as described (18) .

Binding and Covalent Affinity Cross-Linking of Ligand
Carrier-free TGF-ß1 was iodinated to a specific activity of 1 x 105 cpm/ng (25 µCi/ng) by the chloramine-T method as described (19) . The cells were seeded at 3 x 105 cells/60-mm dish. Receptors were cross-linked to bound ligand with disuccinimidyl suberate and solubilized with buffer containing Triton X-100. Cell extracts were clarified by centrifugation and then subjected to 7% sodium SDS-PAGE and autoradiography.

Constructs and Reagents
Mammalian expression vectors with an NH2- or COOH-terminal tag (Myc, Flag, or HA) were constructed by inserting oligonucleotides encoding for epitope tag sequences into pcDNA3 (Invitrogen, San Diego, CA). The coding regions of TGFßRI, Smad 2, Smad 3, Smad 4, Smad 6, or Smad 7 were amplified by PCR and subcloned into Myc-pcDNA3, Flag-pcDNA3, or HA-pcDNA3. Constitutively active (T204D) or kinase-inactive (K232R) forms of TßRI and the mutants Smad 2 or Smad 3 (3S-A) were produced by PCR-based mutagenesis. The integrity of the constructs was confirmed by sequencing.

Transfection, Metabolic Labeling, and Immunoprecipitation
Conditions for cell culture and transfection have been described (20) . HCC-M cells were seeded at 3 x 105 cells/60-mm dish. The cells were subjected to the transfection with LipofectAMINE and 1 µg of the indicated constructs 24 h after seeding and incubated for 4 h. After a wash with the medium, the cells were then incubated for 20 h in serum-free DMEM in the absence or presence of 5 µg/ml anti-TGF-ß antibody (R&D System). The cells were preincubated with phosphate-free DMEM (Life Technologies, Inc., Rockville, MD) for 1 h. The cells were then incubated with the same phosphate-free medium containing 500 µCi/ml [32P]phosphate for 2 h at 37°C and stimulated with 200 pM TGF-ß1 for 30 min. To determine TßRI and Smad protein levels, HCC-M cells were incubated with methionine- and cysteine- free DMEM (Life Technologies) containing 50 µCi/ml [35S]methionine and [35S]cysteine mixture (Pro-mix cell labeling mix; Amersham International) for 2 h. Subsequently, the metabolically labeled cells were solubilized in 1 ml of lysis buffer [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP40, 1 mM EDTA, 10 µg/ml aprotinin, and 0.3 mM phenylmethylsulfonyl fluoride] at 4°C for 20 min. Insoluble debris was removed by centrifugation at 10,000 rpm for 5 min. Myc-tagged receptors or Flag- or HA-tagged Smads were then immunoprecipitated from the supernatant for 1 h at 4°C with 2 µg of anti- Myc 9E10 antibody (BAbCO, Richmond, CA), anti-Flag M2 antibody (Kodak, New Haven, CT), or anti-HA 12CA5 antibody (Boehringer-Mannheim, Indianapolis, IN), followed by absorption to protein A-Sepharose (Pharmacia, Uppsala, Sweden). Beads were washed six times with lysis buffer, and bound protein was eluted by heating in SDS-PAGE sample buffer containing DTT.

Immunofluorescence Study
Subcellular localization of Smad 2 was determined as described previously (21) . Cells grown in LAB TEK chambers (Nunc, Naperville, IL) were transfected with WT or mutant (3S-A) Flag-Smad 2 alone or with TßRI (T204D)-Myc or HA-Smad 7. After fixation with 4% paraformaldehyde, slides were incubated with 3 µg/ml anti-Flag antibody at 4°C for 16 h. Then, 1 µg/ml FluoroLink Cy2-labeled goat antimouse IgG (H&L; Amersham Life Science, Arlington Heights, IL) was added. After mounting the slides with Perma Fluor Aqueous Mounting Medium (Shandon Lipshaw, Pittsburgh, PA), the cells were observed with a fluorescence microscope.

Transcriptional Response Assay
The cells were seeded at 1 x 105 cells/well into six-well clusters. The cells were subjected to the transfection with LipofectAMINE and 0.4 µg of reporter plasmid and indicated constructs or with an empty vector alone 24 h after seeding and incubated for 4 h. After a wash with the medium, cells were then incubated for an additional 20 h in serum-free DMEM in the absence or presence of 200 pM TGF-ß1 or 5 µg/ml anti-TGF-ß antibody. Cells were lysed, and the luciferase activities of cell extracts were measured as relative light units by a luminometer (Berthold, Bad Wildbad, Germany) using the Dual-Luciferase Reporter Assay System (Promega Corp., Madison, WI). The luciferase activities were normalized on the basis of the Renilla luciferase activity.

Immunohistochemistry
Immunohistochemical staining was performed on frozen tissues as described (21) . Frozen tissue sections (4-µm thick) were air dried, fixed with acetone at 4°C for 10 min, and treated with PBS containing 0.3% H2O2 for 10 min at room temperature. After preincubation with PBS containing normal goat serum for 30 min at room temperature to block nonspecific binding, sections were incubated with affinity-purified rabbit polyclonal anti-Smad 2 antibody (Ref. 22 ; a generous gift from Dr. ten Dijke, Ludwig Institute for Cancer Research, Melbourne, Australia) in a humidified chamber at 4°C overnight. Next, tissue sections were washed thoroughly with PBS and incubated with biotinylated goat antirabbit IgG (Vector Laboratories, Burlingame, CA) at room temperature for 40 min and then with biotin-avidin-peroxidase reagent (Vector Laboratories) at room temperature for 30 min. The bound immunocomplex was visualized by incubation with 0.02% 3,3'-deaminobenzidine tetrahydrochloride in PBS containing 0.006% H2O2 for several minutes. Finally, the sections were counterstained lightly with Mayer hematoxylin (Sigma Chemical Co., St. Louis, MO).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TGF-ß1 Accelerates HCC-M and HCC-T Cell Growth in an Autocrine Fashion
Initially, we examined the TGF-ß1 expression and secretion into the conditioned medium by Northern blot hybridization and ELISA techniques using HCC-M and HCC-T cell lines, which were derived from HCC patients carrying hepatitis B virus and non-A/non-B virus, respectively (23 , 24) . These results demonstrate that HCC-M and HCC-T cells displayed significant expression of TGF-ß1 at both protein and mRNA levels (Fig. 1)Citation . Next, we studied the TGF-ß effect on cellular proliferation by measuring cell growth and [3H]thymidine incorporation into DNA of HCC-M and HCC-T cells. For long-term cell growth assay, TGF-ß1 stimulated cell growth (Fig. 2A)Citation . After inoculation of 2 x 104 cells, HCC-M cell numbers on day 5 were 9.7 x 105 and 6.4 x 105 with and without 200 pM TGF-ß1, respectively. HCC-T cell numbers were also 3.1 x 105 and 2.5 x 105 with and without TGF-ß, respectively. Thus, HCC-M and HCC-T cell numbers on day 5 in the presence of TGF-ß1 showed a 51 and 24% increase, respectively, to those in the absence of TGF-ß1. Moreover, 200 pM TGF-ß1 showed slight, but significant, stimulatory effects on DNA synthesis (Fig. 2B)Citation . By contrast, primary rat hepatocytes are potently inhibited by TGF-ß1 with an ID50 of ~0.4 pM (data not shown). If the secreted TGF-ß1 functions as a positive autocrine growth regulator in these cells, antibody-induced blockage of the endogenous TGF-ß may indirectly inhibit growth. In fact, HCC-M and HCC-T cell numbers on day 5 in the presence of the antibody showed 34 and 38% reduction, respectively, to those in the absence of the antibody (Fig. 2A)Citation . Furthermore, the addition of anti-TGF-ß antibody to HCC-M and HCC-T cells in the absence of serum caused 10 and 13% reduction, respectively, in DNA synthesis 24 h after antibody treatment (Fig. 2B)Citation . These results indicate that TGF-ß, which is produced and secreted by HCC-M and HCC-T cells, stimulates their growth in an autocrine fashion.



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  		Fig. 1. Levels of TGF-ß1 mRNA and protein in human cultured HCC cells. A, Northern blot hybridization was performed with poly(A)+ RNAs (2 µg/lane) extracted from HCC-M and HCC-T cells. Blots were hybridized with 32P-labeled random-primed cDNAs for TGF-ß1 and GAPDH. Lower panel, GAPDH mRNA expression as an internal control. B, TGF-ß1 concentration in the conditioned media. Samples were collected from conditioned media in which these cells were cultured for 3 days and measured by enzyme-linked immunoassay, as described in the text. The data are from one representative experiment with each point determined in duplicate; bars, SD.

 


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  		Fig. 2. Effects of TGF-ß1 and anti-TGF-ß antibody on cell growth (A) and DNA synthesis (B) in HCC-M and HCC-T cells. The cells were plated at a density of 2 x 104 in 60-mm dishes in 0.2% FCS/DMEM (A). Two hundred pM TGF-ß1, 5 µg/ml anti-TGF-ß antibody, or nonimmune rabbit IgG was added at the time of inoculation and on day 3. After 5 days, the cells were counted using a hemocytometer. The cells were subjected to a [3H]thymidine incorporation assay in the absence or presence of TGF-ß1 or the antibody in serum-free DMEM (B). The percentage of DNA synthesis was calculated by measuring relative to [3 H]thymidine without exogenous reagents. Means for triplicate samples are shown; bars, SD.

 
HCC-M and HCC-T Cells Display TGF-ß Receptors
The TGF-ß effects can be modulated by various mechanisms, including regulators of ligand binding, receptor activity, Smad activation, nuclear translocation, or the available repertoire of DNA-binding partner and modulator molecules, such as coactivators and corepressors. To clarify the stimulatory mechanisms of TGF-ß in the cells, we first analyzed binding and covalent affinity cross-linking of ligand to its receptors. 125I-Labeled TGF-ß1 bound to two different proteins in HCC-M and HCC-T cells with molecular weights of Mr ~65,000 and ~85,000–110,000, which disappeared with the addition of an excess of unlabeled TGF-ß1. On the basis of the molecular weights, the two proteins appear to represent TßRI and TßRII, respectively (Fig. 3)Citation . Northern blot hybridization also confirmed the expressions of TßRI and TßRII mRNAs (data not shown).



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  		Fig. 3. HCC-M and HCC-T cells display TGF-ß receptors. Cells were affinity labeled with 50 pM 125I-labeled TGF-ß1 in the absence or presence of 5 nM unlabeled TGF-ß1. Labeled proteins were separated by SDS-PAGE and visualized by autoradiography.

 
TGF-ß Receptor Constitutively Activated by Endogenous TGF-ß1 Highly Phosphorylates Smad 2, Translocates It to the Nucleus, and Induces PAI-1 Transcripts in HCC-M Cells
We further studied the mechanisms of R-Smad activation in HCC-M cells. Ligand-dependent phosphorylations of Smad 2 and Smad 3 occur on serine residues within the conserved SSXS motif at the COOH terminus of these proteins (3) . To determine whether the TGF-ß signaling pathway stimulates phosphorylation of Smad 2 and Smad 3, NH2-terminal Flag-tagged Smad 2 and Smad 3 proteins were transiently expressed in HCC-M cells, and phosphorylation levels for Smad 2WT and Smad 3WT were compared with those for mutant Smad 2 (3S-A) and Smad 3 (3S-A), in which the three COOH-terminal conserved serine residues were changed to alanine. The cells were metabolically labeled with [32P]phosphate and subsequently incubated with or without exogenous TGF-ß. Surprisingly, as shown in Fig. 4ACitation , upper panel, the phosphorylation level of Smad 2WT without exogenous TGF-ß was almost equal to that with exogenous ligand, whereas the phosphorylation level of Smad 2 (3S-A) in the absence of TGF-ß was insignificant and did not change upon the addition of TGF-ß, suggesting that the SSXS sites for Smad 2 are constitutively phosphorylated. On the other hand, the basal level for Smad 3WT phosphorylation was identical to those of its derivatives and did not change in the presence of exogenous TGF-ß. The results suggest that ligand-independent sites of Smad 3 are phosphorylated in HCC-M cells. However, Smad 4 was not phosphorylated in response to TGF-ß. As a control, using Mv1Lu cells in the same conditions, dramatic exogenous ligand-dependent phosphorylation of Smad 2 was observed (Fig. 4B)Citation . The phosphorylation of Smad 2 in HCC-M cells was increased by cotransfection with constitutively active TßRI (T204D) (Ref. 25 ; 30% gain, as normalized for the 35S-labeled band; Fig. 4CCitation , left panel). By contrast, the basal level for Smad 2 phosphorylation was dramatically reduced by the addition of neutralizing antibody against TGF-ß (Fig. 4CCitation , middle panel). The high level of phosphorylation appeared to be mediated by TßRI, because the phosphorylation of Smad 2 was diminished by cotransfection with dominant-negative TßRI (K232R) (Ref. 2 ; Fig. 4CCitation , right panel). Taken together, these data indicate that the constitutive phosphorylation of Smad 2 observed in HCC-M cells occurs by an autocrine mechanism.



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  		Fig. 4. TGF-ß receptor constitutively activated by endogenous TGF-ß1 highly phosphorylates Smad 2. A, the ligand- dependent phosphorylation of Smad 2 is submaximal, even without an addition of exogenous TGF-ß in HCC-M cells. The cells transiently transfected with the WT or mutant (3S-A) Flag-Smad 2 or Smad 3, or Flag-Smad 4 WT, were [32P]phosphate labeled and incubated with (+) or without (-) 200 pM TGF-ß1 for 30 min prior to lysis. Smads were subsequently purified by immunoprecipitation using an anti-Flag M2 monoclonal antibody and analyzed by SDS-PAGE and autoradiography (top). The migration of phosphorylated Smads are indicated (right), and the positions of the molecular mass markers (in kDa) are shown on the left. Expression levels of Smads were monitored by labeling the cells with [35S]methionine/cysteine (bottom). B, ligand-dependent phosphorylation of Smad 2 in Mv1Lu cells. The cells transiently transfected with the WT or mutant (3S-A) Flag-Smad 2 were labeled with [32P]phosphate and purified as described above. C, phosphorylation of Smad 2 by the constitutively active type I receptors in HCC-M cells (left panel). The cells transfected with constitutively active TßRI (T204D)-Myc together with Flag-Smad 2WT were labeled with [32P]phosphate and purified as described above. The expression level of TßRI (T204D) was monitored by purification with anti-Myc antibody (bottom). The ligand dependent-phosphorylation of Smad 2 is diminished by the addition of the neutralizing TGF-ß antibody in HCC-M cells (middle panel). The cells transfected with Flag-Smad 2WT were labeled with [32P]phosphate and incubated with (+) or without (-) 5 µg/ml TGF-ß antibody overnight prior to lysis. Experimental conditions were the same as above, and the expression level of Smad 2 was monitored (bottom). Right panel, dominant-negative type I receptor interferes the phosphorylation of Smad 2 in HCC-M cells. The cells transfected with the dominant-negative TßRI (K232R)-Myc together with Flag-Smad 2WT were labeled with [32P]phosphate and purified as described above. The expression level of TßRI (K232R) was monitored by purification with anti-Myc antibody (bottom).

 
The subcellular localization of Smad 2 was also investigated in unstimulated cultures. HCC-M cells were transfected with either Flag-Smad 2WT or Smad 2 (3S-A) in the presence or absence of the dominant-negative TßRI (K232R). The Flag-Smad 2 localization was determined by immunofluorescence and confocal microscopy using a mouse anti-Flag antibody. Smad 2WT was localized in the nucleus in the absence of exogenous TGF-ß, and inactivation of Smad 2 resulted in its cytosolic distribution. Furthermore, in the presence of dominant-negative TßRI (K232R), the nuclear translocation of Smad 2 in response to endogenous TGF-ß signals was blocked (Fig. 5)Citation . The nuclear localization of Smad 2WT protein was observed in 80% of transfectants. Together, these data suggest that the activated TGF-ß receptor always translocates Smad 2 protein to the nucleus in HCC-M cells.



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  		Fig. 5. Smad 2 is localized in the nucleus because of the activation of TGF-ß receptor by endogenous TGF-ß in HCC-M cells. The cells were transfected with Flag-Smad 2 alone or together with dominant-negative TßRI (K232R)-Myc. Flag-Smad 2 was detected by immunofluorescence using anti-Flag M2 antibody and FITC-conjugated secondary antibody and analyzed by confocal microscopy. Smad 2WT is localized in the nucleus, even without exogenous addition of TGF-ß in HCC-M cells, and inactivation of Smad 2 resulted in its cytosolic distribution. Coexpression with dominant-negative TßRI (K232R) prevents this nuclear accumulation, resulting in a cytosolic staining pattern.

 
To study the specific gene response in the HCC-M cells by endogenous TGF-ß1, we investigated whether PAI-1 expression was regulated by endogenous TGF-ß. Northern blot analysis of PAI-1 on mRNA from TGF-ß-stimulated HCC-M cells revealed a slight increase in PAI-1 mRNA (2.2 and 3.2 kb) in response to TGF-ß stimulation, although exogenous TGF-ß prominently stimulated PAI-1 expression of Mv1Lu cells in the same conditions (data not shown). However, an addition of the neutralizing anti-TGF-ß antibody dramatically reduced the mRNA (data not shown). To obtain direct evidence that the constitutive activation of Smad 2 and TßR-I by endogenous TGF-ß is involved in TGF-ß signaling, we further investigated the effects of the TGF-ß antibody and dominant-negative TßRI and Smad 2 on the PAI-1 promoter activity. We used p3TP-Lux (generously provided by Joan Massagué, Memorial Sloan-Kettering Cancer Center, New York, NY), which contains ~100 bp of the PAI-1 promoter linked to a luciferase reporter (26) . HCC-M cells were transfected with p3TP-Lux, either alone or together with TßRI or Smad 2. Cells were incubated overnight in the absence or presence of TGF-ß1 or neutralizing antibody, and the relative luciferase activity was measured in cell lysates. Transfection of p3TP-Lux alone into HCC-M cells resulted in high basal levels of transcription in untreated cells, which was weakly induced by stimulation with exogenous TGF-ß1 (Fig. 6)Citation . However, we observed a strong suppression of the 3TP promoter by the addition of neutralizing antibody. The TGF-ß signaling could be propagated through TßRI and Smad 2, because transfection of dominant-negative TßRI (K232R) or Smad 2 (3S-A) led to strong suppression of the 3TP promoter that was stimulated by coexpression of the constitutively active TßRI (T204D). Together, these data suggest that Smad 2 is constitutively phosphorylated by TßRI upon endogenous ligand binding and constantly stimulates the 3TP promoter.



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  		Fig. 6. Endogenous TGF-ß can constantly stimulate the 3TP promoter in HCC-M cells. The cells were transfected with p3TP-Lux alone or with TßRI or Smad 2. Cells were incubated overnight in the absence or presence of 200 pM TGF-ß1 or neutralizing TGF-ß antibody, and the relative luciferase activity was measured in cell lysates. The luciferase activity was normalized to the Renilla luciferase activity and is expressed as the means (n = 3) from a representative experiment; bars, SD.

 
Endogenous TGF-ß Cannot Induce Smad 6 and Smad 7 Expression in HCC-M Cells, Although Overexpression of Exogenous Smad 6 and Smad 7 Blocks Smad 2 Activation in HCC-M Cells
In contrast with the constant activation of Smad 2 by endogenous TGF-ß in HCC-M cells, the activation of R-Smads are tightly restricted in other cells, such as Mv1Lu cells. On the other hand, antagonistic Smads participate in negative feedback loops that may regulate the intensity or duration of TGF-ß responses. Thus, TGF-ß usually induces antagonistic Smads, the production of which result in interference with receptor binding and phosphorylation of R-Smads (12 , 13 , 27) . Therefore, we examined the involvement of antagonistic Smads in HCC-M cells and investigated whether the expression of Smad 6 and Smad 7 was regulated by TGF-ß. Northern blot analyses of Smad 6 and Smad 7 on RNA prepared from Mv1lu cells stimulated with TGF-ß revealed that Smad 6 mRNA (3.0 kb) and Smad 7 mRNA (4.4 kb) were rapidly induced by TGF-ß. By contrast, the mRNA expression was marginal, if any, and did not change in response to TGF-ß stimulation and the addition of the antibody in HCC-M cells (Fig. 7A)Citation . However, Smad 6 and Smad 7 could exert a negative role in TGF-ß signaling by interfering with Smad 2 activation in HCC-M cells, when they were expressed by the transfection of expression plasmid. Smad 6 and Smad 7 inhibited the endogenous TGF-ß-induced phosphorylation of Smad 2 (Fig. 7B)Citation . In addition, cotransfection of Smad 6 or Smad 7 with p3TP-Lux resulted in the decrease of luciferase activities by 45 and 28%, respectively, when compared with control cells transfected with p3TP-Lux alone (Fig. 7C)Citation . Furthermore, in the presence of excess Smad 7, the nuclear translocation of Smad 2 in response to endogenous TGF-ß signal was blocked (Fig. 7D)Citation . Excess Smad 6 also retained Smad 2 protein in the cytosol (data not shown). Together, these results suggest that the constitutive activation is, at least in part, attributable to the lack of induction of antagonistic Smads by TGF-ß.



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  		Fig. 7. Endogenous TGF-ß cannot induce Smad 6 and Smad 7 expression in HCC-M cells, although the overexpression of exogenous Smad 6 and Smad 7 blocks the Smad 2 activation in HCC-M cells. A, endogenous TGF-ß1 does not induce Smad 6 and Smad 7 transcripts in HCC-M cells. Northern blot hybridization was performed with poly(A)+ RNAs (2 µg/lane) from HCC-M cells cultured in the presence of 200 pM TGF-ß1 or 5 µg/ml anti-TGF-ß antibody. Blots were hybridized with 32P-labeled random-primed cDNAs for Smad 6, Smad 7, and GAPDH. B, Smad 6 and Smad 7 can prevent the Smad 2 phosphorylation by activated TGF-ß receptor in HCC-M cells. HCC-M cells were transfected with the indicated combinations of Flag-Smad 2, HA-Smad 6, or Smad 7. Cells were labeled with [32P]phosphate, and Flag-Smad 2 was immunoprecipitated with anti-Flag M2 antibody and analyzed by SDS-PAGE and autoradiography. The expressions of Flag-Smad 2, HA-Smad 6, and Smad 7 were monitored by purification with anti-Flag antibody (upper) or anti-HA antibody (bottom). Right, migration of each protein. C, Smad 6 and Smad 7 inhibit transcriptional activation of the 3TP promoter induced by endogenous TGF-ß in HCC-M cells. HCC-M cells were transiently transfected with p3TP-Lux alone or with Smad 6 and Smad 7. Cells were cultured overnight without exogenous TGF-ß, and the relative luciferase activities were measured in cell lysates. The luciferase activity was normalized to the Renilla luciferase activity and is expressed as the means (n = 3) from a representative experiment; bars, SD. D, Smad 2 protein translocates from the nucleus to the cytosol by the overexpression of Smad 7 in HCC-M cells. HCC-M cells were transfected with Flag-Smad 2 together with HA-Smad 7. Coexpression of Flag-Smad 2 with Smad 7 blocks this nuclear accumulation, resulting in a cytosolic staining pattern.

 
Activated Smad Complex Inhibits p15INK4BTranscription in HCC-M Cells
The discovery that endogenous TGF-ß constitutively activates Smads, despite promoting cell growth of HCC-M cells, raises questions regarding the distinct role of activated Smads in cell cycle control. To address these questions, we focused on p15INK4B, because p15INK4B is one of the CDK inhibitors whose rapid induction in response to TGF-ß mediates cell cycle arrest (11) . Northern blot analysis of RNA from TGF-ß-treated HCC-M cells revealed that p15INK4B mRNA (2.2 kb) was insignificant and did not change in response to TGF-ß stimulation (data not shown). On the contrary, the mRNA showed 2-fold increase by the addition of the neutralizing anti-TGF-ß antibody (data not shown). The expression level of p21CIP1, which is another TGF-ß-inducible CDK inhibitor, did not show any change after treatment with TGF-ß and the antibody (data not shown). To obtain further proof that the inactivation of Smad 2 and TßRI results in the enhancement of p15INK4B transcript, we investigated the effects of the neutralizing TGF-ß antibody, dominant-negative Smad 2 (3S-A), and antagonistic Smads on the p15INK4B promoter activities. We used plasmid p15P113-Luc (generously provided by Xiao-Fan Wang, Duke University, NC), which contains 113 bp of the p15INK4B promoter linked to a luciferase reporter (28) . Cells were transfected with p15P113-Luc either alone or together with Smad 2WT, Smad 2 (3S-A), Smad 6, or Smad 7, and then luciferase assays were performed with cells untreated or treated with neutralizing TGF-ß antibody for 20 h. In HCC-M cells transfected with p15P113-Luc alone, the blockage of TGF-ß signal by the neutralizing anti-TGF-ß antibody led to 1.3-fold increase in the luciferase activity, and exogenous TGF-ß suppressed it (Fig. 8)Citation . As a control, using HepG2 cells under the same conditions, a 5-fold increase in the activity was observed by the addition of TGF-ß (data not shown). Cotransfection of constitutively active TßRI or Smad 2WT with p15P113-Luc also showed a minimal response in the p15INK4B promoter to TGF-ß. In contrast, when dominant-negative TßRI, Smad 2 (3S-A), Smad 6, and Smad 7 were used in the assays, 1.35, 2.0, 2.1, and 1.8-fold increases were observed in the luciferase activities, respectively. These are precisely opposite results to those that indicated that Smad 2 activated by the TßRI upon endogenous ligand binding constantly promotes PAI-1 transcription (Fig. 6)Citation . Furthermore, the data indicate that p15INK4B transcription is suppressed by the activation of Smad 2 and TßRI in HCC-M cells. Therefore, the constant suppression of p15INK4B transcription by endogenous TGF-ß in HCC-M cells may lead to the acceleration of their growth and involvement in the malignant progression of the cells.



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  		Fig. 8. Activated Smad complex inhibits p15INK4B transcription in HCC-M cells. The cells were transfected with p15P113-Luc alone or with TßRI, Smad 2, Smad 6, or Smad 7. The cells were incubated overnight in the absence or presence of TGF-ß1 or neutralizing TGF-ß antibody, and the relative luciferase activities were measured. The luciferase activity was normalized to the Renilla luciferase activity and is expressed as the means (n = 3) from a representative experiment; bars, SD.

 
Smad 2 Proteins Are Located in Nuclei of Human HCC Tissue, Which Expressed Low Levels of p15INK4B, Smad 6, and Smad 7 mRNAs But a High Level of PAI-1 mRNA
To further confirm whether TGF-ß propagates its signal in a similar manner in vivo, we investigated the subcellular localization of Smad 2 in human HCC tissues and transcriptional levels of p15INK4B, PAI-1, Smad 6, and Smad 7 as target genes in HCC using Northern blot hybridization.

(a) To clarify the cell type-specific differences in mRNA expressions of p15INK4B, PAI-1, Smad 6, and Smad 7 between noncancerous tissues and HCC, mRNA levels from noncancerous tissues were compared with those for HCC (Fig. 9A)Citation . mRNAs for p15INK4B, Smad 6, and Smad 7 species were clearly detected in adjacent liver tissue but exhibited much lower expression levels in the HCC cells. In contrast, poly(A) RNA from the cancer cells showed the same levels of PAI-1 mRNA as adjacent liver tissues did. Quantitation of the p15INK4B, PAI-1, Smad 6, and Smad 7 mRNAs by scanning densitometry revealed that the levels of expression of p15INK4B, Smad 6, and Smad 7 mRNAs in the cancer cells were 50, 25, and 30% of the adjacent tissues, respectively, whereas PAI-1 mRNA remained at the same level. Furthermore, Northern blot analyses with Smad-specific probes revealed Smad 2, Smad 3, and Smad 4 mRNA expressions, and no mutations of these Smads were observed (data not shown).



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  		Fig. 9. Smad 2 proteins are located in the nuclei of human HCC tissue, which expressed low levels of p15INK4B, Smad 6, and Smad 7 mRNAs but a high level of PAI-1 mRNA. A, mRNA levels of p15INK4B, PAI-1, Smad 6, and Smad 7 in human HCC tissue. Northern blot hybridization was performed with poly(A) RNAs (2 µg/lane) extracted from human HCC and adjacent liver tissue. Blots were hybridized with 32P-labeled random-primed cDNA probes for p15INK4B, PAI-1, Smad 6, Smad 7, and GAPDH. Immunohistochemical staining with Smad 2 antibody in human hepatocytes (B) and HCC tissue (C) is shown. High-power microscopic views of a cancerous area demonstrate a moderate signal for Smad 2 in the nucleus of human HCC (arrow). Bar, 50 µm.

 
(b) The localization was detected by immunofluorescence using specific antisera. Moderate immunoreactivities with the anti-Smad 2 antibody were observed in the cytoplasm of normal hepatocytes (Fig. 9B)Citation . However, Smad 2 proteins were dominantly localized in the nuclei of the human HCC cells (Fig. 9C)Citation . Control rabbit IgG did not react with the antigen in the cells. The nuclear localization of Smad 2 protein and the expression patterns of these target genes were observed in 3 of 10 human HCC patients.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have shown that HCC-M and HCC-T cells displayed significant expression of TGF-ß1 at both protein and mRNA levels. The cells expressed cell surface receptors that could bind to TGF-ß. Furthermore, exogenous TGF-ß stimulated cell growth, and the neutralizing antibody abolished the positive growth effects of endogenous TGF-ß, resulting in an inhibition of cell growth. These results indicate that TGF-ß acts as an autocrine positive growth regulator in HCC-M and HCC-T cells.

The effects of growth factor are modulated by various mechanisms, such as alterations in postreceptor pathways. In contrast to the tight restriction of Smad 2 activation in other cells, such as Mv1Lu cells, Smad 2 was constitutively activated in HCC-M cells by an autocrine mechanism. At a high level of Smad 2 phosphorylation, the induction of PAI-1 transcript and nuclear localization of Smad 2 by endogenous TGF-ß were observed in the cells. We examined the involvement of antagonistic Smads for the loose restriction of Smad 2 activation in HCC-M cells, because the expressions of antagonistic Smads were rapidly elevated in response to exogenous TGFß in Mv1Lu cells, and they could inhibit R-Smads activation. Thus, we investigated whether the expression of antagonistic Smads were regulated by endogenous TGF-ß. Accordingly, our present data demonstrate that endogenous TGF-ß1 cannot induce antagonistic Smads. Therefore, this constitutive activation of Smad 2 can be attributed to the lack of induction of antagonistic Smads by endogenous TGF-ß.

It is currently unclear whether R-Smads have distinct actions or whether they function similarly for specific target genes. Several reports indicate that TGF-ß propagates its negative signal through R-Smads, and TGF-ß simultaneously induces CDK inhibitors including p15INK4B and p21CIP1 as well as PAI-1 (29 , 30) . These results are in contrast to our current findings that constitutive activation of Smad 2 rather suppresses p15INK4B promoter activity in HCC-M cells, whereas it leads to the constant activation of PAI-1 promoter. Thus, this reverse response may explain the growth-stimulatory effect of TGF-ß1 in the cells (Fig. 10)Citation . The discrepancy between the two apparent opposite effects of activated Smad 2 in the cells is most likely attributable to the differences in associating cofactors, including DNA binding partners or putative inhibitors of Smad 2 and/or modulator molecules, such as coactivators and corepressors on two specific genes (16 , 17) . Furthermore, the levels of p21CIP1 expression were not altered by the addition of TGF-ß1 and its antibody in HCC-M cells. Therefore, it is possible that loss of the responsiveness of p21CIP1 to TGF-ß1 is also an important step for HCC development.



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  		Fig. 10. Autocrine stimulatory mechanism by TGF-ß in Human HCC. Human HCC cells produce TGF-ß and activate their TGF-ß receptors in an autocrine fashion. The TGF-ß receptors can constantly phosphorylate Smad 2 under low levels of antagonistic Smads, and the activated Smad 2 protein is localized in the nucleus, even without exogenous TGF-ß in HCC-M and HCC-T. Furthermore, it accelerates cell growth partly because the activated Smads complex inhibits the transcription of p15INK4B. The cellular response may be determined by combinations of DNA-binding partners. Thus, specific inhibitors may interfere with the binding of the activated Smads complex to their responsible elements in the p15INK4B gene.

 
TGF-ß negatively regulates hepatocyte proliferation. By contrast, several reports indicate that elevated levels of TGF-ß mRNA and protein in HCC tissues are associated with cancer progression (14) . Although most cancer cells secrete large quantities of latent TGF-ß, both HCC-M and HCC-T cells secreted biologically active TGF-ß1 into conditioned media, which inhibited the growth of primary rat hepatocytes. This growth inhibition was blocked by anti-TGFß antibody, whereas nonimmune rabbit IgG had no effect (data not shown). The discrepancy between the high level of TGF-ß expression and the negative growth response to TGF-ß leads to the possibility that alteration of the TGF-ß signaling pathway may be involved in malignant progression of this human cancer. Concerning this mechanism, numerous reports have described the loss of the responsiveness to TGF-ß in human tumor-derived cell lines. Disruption of TGF-ß signaling could therefore be an important cause of cancer. Moreover, several mutations of signaling molecules, including TGF-ß family members, their receptors, or Smad genes, have been reported as direct evidence for disrupted TGF-ß signaling in many cancers, such as colon and pancreatic cancer (31 , 32) . We therefore sought the mutations that cause the inactivation of TGF-ß signal transduction in 10 human HCC tissues and seven cultured cell lines. However, no mutations in the genes could be found among them. This is almost consistent with the recent reports that the mutations are distinctly uncommon in human HCC (33) . In addition, disruption of TGF-ß signaling was not observed in all cultured HCC cells.

Our current results demonstrate the autocrine stimulatory mechanism by TGF-ß in some HCC cells. However, the endogenous TGF-ß signal acts on cell growth negatively in other HCC cells, such as HuH-7 cells, because neutralization of TGF-ß in the medium or the blockage of the signal transduction pathway by the induction of dominant-negative Smad 2/3 results in stimulation of cell growth.4 Therefore, it will also be important to investigate regulatory mechanisms for TGF-ß as an autocrine inhibitor.


   ACKNOWLEDGMENTS
 
We thank Dr. S. W. Qian (National Cancer Institute, Bethesda, MD), Dr. R. Derynck (University of California at San Francisco, San Francisco, CA), Drs. K. Miyazono and M. Kawabata (The Cancer Institute), Dr. P. ten Dijke (Ludwig Institute for Cancer Research), Dr. J. Massagué (Memorial Sloan-Kettering Cancer Center, New York, NY), and Dr. X-F. Wang (Duke University, NC) for providing us with cDNAs of rat TGF-ß1, human Smad 2, Smad 3, Smad 4, mouse Smad 6, human TGF-ßRI, mouse Smad 7, and anti-Smad 2 serum, p3TP-Lux vector, and p15P113-Luc vector, respectively, and surgeons of the First Department of Surgery (Kansai Medical University, Japan) for cooperation in this study. We also thank Dr. K. Miyazono for helpful discussion.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture, Japan. Back

2 To whom requests for reprints should be addressed, at Third Department of Internal Medicine, Kansai Medical University, 10-15 Fumizonocho, Moriguchi, Osaka 570-8507, Japan. Phone: 81-6-6992-1001, extension 3221; Fax: 81-6-6996-4874; E-mail: matsuzak{at}takii.kmu.ac.jp Back

3 The abbreviations used are: TGF, transforming growth factor; TßRI, TGF-ß type I receptor; R-Smad, receptor-regulated Smad; CDK, cyclin-dependent kinase; PAI-1, plasminogen activator inhibitor type 1; HCC, hepatocellular carcinoma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WT, wild type; HA, hemagglutinin. Back

4 Unpublished observations. Back

Received 7/16/99. Accepted 12/20/99.


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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