LDH Assay

After starvation for 8h, cells were treated with TGF-β for 72h. For HCC-M and HuH7, starvation medium was supplemented with 1% heat inactivated FCS. LDH content in supernatant and adherent cells (disrupted with 1% Triton X-100 in HBSS) was detected using Cytotoxicity Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany). Cell death was calculated as percentage of LDH in the medium as compared to total LDH levels.

MTT Assay

Cells were cultured in medium containing 0.25% heat inactivated FCS with 5ng/ml TGF-β for 2 or 6 days (addition of new FCS and TGF-β after 3 days). 4h after adding MTT reagent (final concentration 500µg/ml), reduced dye in viable cells was resolved in acidified DMSO solution (10% SDS, 0.6% glacial acid in DMSO). Absorbance was measured at 570nm (reference 630nm). Values of treated samples were normalized to untreated controls.

RNA Extraction, Reverse Transcription, Real Time PCR Analysis

HCC cell lines were treated with TGF-β for 2 or 24h. RNA was isolated using RNeasy Mini Kit, integrity ensured by agarose gel electrophoreses and reverse transcribed using QuantiTect Reverse Transcription Kit (QIAGEN, Hilden, Germany). mRNA levels of Smad7, TGF-β1, TβRI, TβRII and 18S-rRNA were detected using TaqMan® probes and TaqMan® Universal PCR Master Mix, No AmpErase® UNG; PRAJA, ELF, Bim, PAI-1 and 18S-RNA using Power SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Primer pairs used for SYBR green Real time PCR were as followed: PRAJA (For: 5’-TCGCCATTTTCCACTACTCGT-3’; Rev: 5’-GTTCCCGAACTCTCGCTGT-3’), ELF (For: 5’-AGCTGGAAGGCAGATTCAAG-3’; Rev 5’-CGTCCATCTCGAAGGTCAGT-3’), Bim (For: 5’-TAAGTTCTGAGTGTGACCGAGA-3’; Rev 5’-GCTCTGTCTGTAGGGAGGTAGG-3’), PAI-1 (For: 5’-CACAAATCAGACGGCAGCACT-3’; Rev 5’-CATCGGGCGTGGTGAACTC-3’), Smad4 (For: 5’-GCTGCTGGAATTGGTGTTGATG-3’; Rev 5’-AGGTGTTTCTTTGATGCTCTGTCT-3’) and 18S rRNA (For: 5’- AAACGGCTACCACATCCAAG-3’; Rev 5’-CCTCCAATGGATCCTCGTTA-3’). Following assays were obtained from Applied Biosystems and used for TaqMan, Real time PCR: Smad (Hs00178696_m1), TGF-β1 (Hs00171257_m1), TGFBR1 (TβRI, Hs00610318_m1), TGFBR2 (TβRII, Hs00559661_m1), Smad2 (Hs00998181_gH), Smad3 (Hs00969205_g1) and 18S rRNA (Hs03003631_g1). Relative expression levels were determined using the ΔΔCt method (reference 18S-rRNA level). As 18S-rRNA expression values are very stable (see Figure S1) only one reference gene was used according to literature [13].

Immunoblot Analysis

Blotted proteins were detected using antibodies against pSmad2C (Ser465/467), Smad2 (D43B4), Smad3, Akt, pAKT (Ser473) (587F11), pc-JUN (Ser63), pP38 MAPK (Thr180/Tyr182) (12F8), cleaved Caspase-3 (Asp175) (5A1E), PARP, BCL-2 (50E3) and BCL-XL (54H6) (Cell Signaling Technology, Danvers, MA, USA), Smad4 (B-8), PCNA (F-2), c-MYC (2Q329), ERK1/2 (MK1) and pERK (E-4) (Santa Cruz Biotechology, Santa Cruz, California, USA.), P21WAF1/Cip1 (CP74) (Sigma Aldrich, St. Louis, Missouri, USA) and pSmad3L (pS423/425) (Epitomics, Burlingame, California, USA). Secondary antibodies were goat anti-rabbit IgG-HRP or goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, California, USA). Antibodies’ sources, dilutions and buffer conditions are given in table S1. All experiments were performed multiple times, number of independent experiments and densitometric analysis are given in figures S2, S3, S4, S5 and S6.

Immunofluorescent Staining of Linker Phosphorylated Smad3

Cells were seeded with a density of 300.000 cells per well in 6-well plates without further treatment. After 1 day, cells were washed twice with PBS pH7.4, fixed in 4% PFA/PBS for 10 minutes, permeabilized with 0.3% Triton X-100 v/v in PBS pH 7.4 for 5 min, fixed a second time with 2% PFA/PBS for 5 min and washed 3 times in PBS. Fixed and permeabilized cells were blocked in 1% (w/v) BSA/PBS pH 7.4 for 1h, then incubated with rabbit-anti Smad3L antibody (Immunobiological Laboratories No. 28029) over night at 4°C, washed 3 times in PBS and incubated with Alexa-555-goat anti–rabbit IgG (Molecular Probes/Invitrogen) for 50 min at room temperature. Nuclei were stained with DRAQ5 (Biostatus Ltd, Shepshed Leicestershire, UK) in PBS for 15 min. Fixed samples were washed 3 times with PBS pH 7.4 and mounted on glass slides using DakoCytomation Fluorescent Mounting Medium (DakoCytomation, Hamburg, Germany). Confocal images were obtained with a Leica laser scanning spectral confocal microscope, model DM IRE2, with an HCX PL Apo 40x/1.32 numeric aperture oil objective (Leica Microsystems, Wetzlar, Germany). Excitation was performed with an argon laser emitting at 488 nm, a krypton laser emitting at 568 nm, and a helium/neon laser emitting at 633 nm. Images were acquired with a TCS SP2 scanner and Leica Confocal software, version 2.5 (Leica Microsystems).

Cell Culture

HCC-M, HCC-T, HepG2, Hep3B, HuH7, PLC/PRF/5 (PLC, Alexander), HLE, HLF, FLC-4 and HuH6 cells were cultured in DMEM (Lonza Group Ltd., Cologne, Germany) supplemented with 2mM glutamine (PAA Laboratories GmbH, Cölbe, Germany) and 10% FCS (Invitrogen, Darmstadt, Germany) at 37°C and 5% CO₂ in a humidified incubator. Experiments were conducted between passage numbers 2 and 12 without full confluency throughout experiments. Absence of mycoplasma contamination was confirmed by Venor®GeMtest (Minerva Biolabs GmbH, Berlin, Germany). Prior to experiments, medium was changed to starvation medium without FCS for at least 7h, if not indicated otherwise. 5ng/ml TGF-β (PeproTech GmbH, Hamburg, Germany) were used. Cell line characteristics are given in table S2 in the supplementary data.

Smad3 Transcriptional Activity

Adherent cells were infected for 2h with adenovirus carrying a Smad3 reporter construct, (CAGA) ₉MLP-Luc [14] or control adenovirus with β-Galactosidase [15]. After overnight starvation, TGF-β was added for 9h. Luciferase and β-Galactosidase activities were analyzed using Luciferase Assay Reagent and β-Galactosidase Enzyme Assay (Promega, Madison, WI, USA). After normalizing luciferase activity to β-Gal expression, TGF-β dependent induction of Smad3 transcriptional activity was normalized to the untreated control sample. Each condition was analyzed in triplicates.

Smad2 transcriptional activity

Smad2 transcription factor complexes recognize activin response elements (ARE). Smad2 is unable to bind DNA and needs assistance by, e.g. Fast-1, as cofactor. Hence, a luciferase gene under the control of ARE was co-transfected with a Fast-1 expression plasmid to evaluate Smad2/Smad4 transcriptional activity. For this, HCC cell lines were cultured at a confluency of 70-80%. ARE-Luc, Fast-1 and a β-Galactosidase control vector (pCR3lacZ; Invitrogen) at a ratio of 6:2:1 were introduced into the cell using Lipofectamine 2000 (Invitrogen, Darmstadt, Germany) according to the manufacturer’s protocol. After transfection, cells were starved over night until treatment with TGF-β for 9h. β-Galactosidase and Luciferase assay was performed as described above.

Smad7 Promoter Activity

β-Galactosidase control vector (pCR3lacZ; Invitrogen) and Smad7 promoter deletion mutant, p(-625 SacI)-Smad7prom-Luc, constructed from the 1,321-bp rat Smad7 promoter region (-1276 to -41) [16] were transiently transfected using Lipofectamine 2000 (Invitrogen, Darmstadt, Germany). Immediately post-transfection, cells were starved for 24h until treatment with TGF-β for 6h. β-Galactosidase and Luciferase assays were performed as described above.

Knockdown of Smad2 and Smad3 with siRNA

Hep3B, HuH7 and PLC/PRF/5 cells were cultured at medium density to perform siRNA knockdown experiments. Smad2 and Smad3 or unspecific siRNA (Order No SI02757496, SI00082495 and 1027281; Qiagen, Hilden, Germany) was introduced into the cells using Lipofectamine RNAiMax (Invitrogen, Darmstadt, Germany) according to the manufacturer’s protocol but with 2 µl RNAiMax per ml medium. Final siRNA concentrations were 10 µM for Hep3B and PLC/PRF/5 and 20 µM for HuH7 cells. Knockdown was allowed to establish for 48 h in medium supplemented with 1% heat inactivated FCS. Cells were treated with starvation medium supplemented with 5 ng/ml TGF-β for 1 h (proof of knockdown; Western blot analysis) or 72 h (LDH assay). Medium for HuH7 cells additionally contained 1% heat inactivated FCS. LDH and Western blot analysis was performed as described above. Additionally, content of LDH in adherent cells was used to evaluate proliferation.

Clustering Analysis and Statistics

Clustering analysis was performed by using Cluster 3.0 software and the data were further visualized with TreeView 1.6 [17]. Results are shown as means +/- standard error of 3-5 (MTT Assay), 3-4 (LDH Assay), 3-4 (Real Time PCR, basal expression), 2-3 (Real Time PCR, induced expression), 3 (Smad3 transcriptional und Smad7 promotor activity), 2-3 (Smad2 transcriptional activity) independent experiments. Significant differences were determined by two-tailed unpaired Student’s t test.

Cytostatic TGF-β response is maintained in Hep3B, HuH7 and PLC HCC cell lines

Sensitivity to TGF-β induced cytostasis was evaluated in 10 HCC cell lines. 2 days TGF-β treatment significantly inhibited cell proliferation in Hep3B, HuH7 and PLC, as determined by MTT-assay (Figure 1A, right panel), HepG2 and HuH6 displayed no or a weak response after 2 days, respectively. However, they show a strong response 6 days upon TGF-β addition. Proliferation of HCC-M, HCC-T, HLE, FLC-4 and HLF was not reduced. HCC-T even displayed an increased proliferation after 6 days treatment. In line with MTT-assay data, expression of proliferation associated marker P21 showed the strongest induction comparing before and after TGF-β treatment in those cell lines with TGF-β dependent proliferation inhibition. Consistently, expression of proliferation facilitating protein c-MYC was specifically down regulated in cell lines sensitive to TGF-β induced proliferation control, whereas it was even induced late in HCC-T cells (Figure 1A, left panel, Figure S2). Western Blot analysis was not sensitive enough to confirm weak cell death (+7%) of HepG2 upon TGF-β treatment on protein level.

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Figure 1. TGF-β induces cell death and/or inhibits proliferation in PLC, HepG2, HuH6, Hep3B and HuH7.

(A) Left side: HCC cell lines were treated with 5 ng/ml TGF-β for indicated time points. Changes in c-MYC and P21 expression were detected using Western blot analysis with GAPDH as loading control. As HCC-T cells show a delayed TGF-β response, c-MMYC was induced after 72 h of TGF-β treatment. Right side: After TGF-β treatment for 0 (grey line), 2 (white bars) or 6 days (black bars) cell proliferation was evaluated by MTT assays. Treated samples were normalized to the corresponding control. (B) Left side: Western blot analysis of PARP and Caspase 3 cleavage using GAPDH expression as loading control after 0 and 48 h TGF-β treatment. In the case of HuH7 cells, the control sample was treated with TGF-β for 3 h. Right side: Cell death induced by 5 ng/ml TGF-β over 72 h (filled bars) was quantified by detecting LDH release normalized to total amount of LDH. Untreated samples were defined as 0 (grey line). Significant differences are indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001 (Student’s t test).

https://doi.org/10.1371/journal.pone.0072252.g001

Besides controlling proliferation, TGF-β is a prominent modulator of hepatocyte apoptosis. We analyzed apoptosis of TGF-β treated cells by LDH release to the medium and cleavage of PARP and Caspase3 (Figure 1B, Figure S2). Again, Hep3B, HuH7 and PLC cells showed increased cell death after 72h TGF-β stimulation, whereas HepG2 and HuH6 exhibited very low TGF-β induced cell death rates. Increased cytotoxicity was accompanied by elevated levels of PARP and Caspase3 cleavage (Figure 1B). No significant pro apoptotic effect of TGF-β was found in the other cell lines. Our results revealed heterogeneous cytostatic TGF-β effects in HCC cell lines. Sensitivity for cell death induction and proliferation inhibition generally occur in common (Hep3B, HuH7, PLC; to a lower extent HepG2, HuH6). HCC-M, HCC-T, HLE, HLF and FLC-4 are completely resistant to cytostatic TGF-β effects.

Upregulated TGF-β production and high expression of inhibitory Smad7 correlate with loss of cytostatic response in HCC cell lines

We next tried to correlate (loss of) cytostatic response with disposability of TGF-β signaling components and dynamics of its downstream signaling. One feature of progressed cancer cells is TGF-β production and autocrine stimulation. Therefore, we analyzed endogenous TGF-β expression by qRT-PCR. A broad spectrum of expression levels was found in the cell lines (1-10 fold) (Figure 2A). Interestingly, intrinsic levels of inhibitory Smad7 similarly varied and relative expression strongly correlated with that of TGF-β1 (Pearson correlation, r = 0.87, p = 0.0011) (Figure 2A) increasing in the following order: HCC-M, PLC, HCC-T, HepG2, Hep3B, HuH7, HLE, HLF, FLC-4 and HuH6. Cytostatic TGF-β effects could be correlated to low endogenous TGF-β/Smad7 expression levels (HCC-M, HCC-T, PLC, HepG2, HuH7, exception Hep3B). The special position taken by HCC-M and HCC-T has to be pointed out. Both cell lines express rather low TGF-β and Smad7 levels, but do not respond to TGF-β mediated cytostasis. This issue will be discussed further below.

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Figure 2. Expression levels of TGF-β/Smad signaling components in HCC cell lines.

Relative TGF-β1 and Smad7 (A), TGF-β receptor I (TβRI) and II (TβRII) (B) and Smad2, Smad3, and Smad4 (C) expression levels were detected using real time PCR. Expression of 18s rRNA was used as reference and results were analyzed with the ΔΔCt method. A strong correlation of Smad7 and TGF-β1 expression was identified by calculating the Pearson coefficient (r = 0.87; p = 0.0011) (A). Further, TβRII expression (filled bars) was generally increased compared to the expression of TβRI (grey line) in the same cell line (B, right). Additionally, total and phosphorylated Smad2 and Smad3 as well as basal Smad4 protein levels were evaluated by Western Blot using GAPDH as loading control (C).

https://doi.org/10.1371/journal.pone.0072252.g002

Endogenous levels of TGF-β receptor I (TβRI, ALK-5) were relatively even within the different cell lines, although HCC-M displayed especially high levels (Figure 2B). TGF-β receptor II (TβRII) expression was upregulated in cell lines with an asserved cytostatic TGF-β response, showing high (HepG2 and PLC) or medium (Hep3B and HuH7) TβRII mRNA levels (Figure 2B). HuH6 with delayed proliferation inhibition upon TGF-β treatment expressed low amounts of TβRII, whereas HLE cells lacking TGF-β dependent cytostasis displayed intermediate expression levels.

Receptor-Smad2 and co-Smad4, like TβRI, were equally expressed in the different HCC cell lines, whereas Smad3 exhibited strikingly high mRNA and protein expression in HLE and HLF (Figure 2C, Figure S3). However, no significant correlation between Smad3 expression levels and the HCC cells cytostatic response could be concluded.

TGF-β effects on expression of its signaling components in HCC cell lines

In order to mimic the response of hepatocytes to TGF-β secreted by other cell types, we investigated the impact of TGF-β stimulation on expression of TGF-β signaling components. Smad2 and Smad4 (Figure 3A, 3B, Figure S4) levels did not vary upon 24h TGF-β treatment, whereas Smad3 (Figure 3A, 3B, Figure S4) and Smad7 (Figure 4A) expression was significantly induced mostly in cytostasis responsive cell lines (including HuH6), 24h and 2h after TGF-β treatment. TGF-β-induced expression of Smad7 (Figure 4A) was inversely correlated with intrinsic Smad7 expression, excluding HCC-M and HCC-T (Figure 2.A).

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Figure 3. Induction of Smad3 and TβRI expression correlates with cytostatic responsiveness upon TGF-β treatment.

Cells were cultured with or without 5 ng/ml TGF-β for 24 h. Changes in Smad2, Smad3 and Smad4 levels after TGF-β treatment were evaluated by (A) Real Time PCR and (B) Western Blot analysis using 18S rRNA or GAPDH as reference genes. ^◊ and ^{◊ ◊} indicate which GAPDH belongs to Smad2 and Smad3 or Smad4, respectively. (C) TGF-β dependent expression levels of TGF-β Receptor I (TβRI) and II (TβRII) were detected and correlated against untreated samples using real time PCR with 18S rRNA as reference gene. Untreated samples are shown as grey lines, while filled bars display TGF-β treated samples. Significant differences are indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001 (Student’s t test).

https://doi.org/10.1371/journal.pone.0072252.g003

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Figure 4. Induction of Smad7 expression by TGF-β correlates with cytostatic responsiveness.

(A) Left side: Cells were treated with or without TGF-β for 2 h. Smad7 expression and 18S rRNA levels as reference gene were detected by real time PCR. Right side: For evaluation of transcriptional activity, HCC cell lines were transfected with a construct containing a luciferase gene under control of the Smad7-promotor and treated with TGF-β for 6 h. Treated samples were correlated to untreated controls. (B) Changes in expression levels of Smad3 target genes Bim and PAI-1 were detected after 2 h (PAI-1) or 24 h (Bim) using real time PCR analysis with rS18 as reference gene. The tables highlight cell lines with highest CAGA activity (black fields). Significant differences are indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001 (Student’s t test).

https://doi.org/10.1371/journal.pone.0072252.g004

While TβRII levels did not vary upon TGF-β stimulation, TβRI expression was strongly induced in cytostatic responsive cell lines (PLC, Hep3B, and HuH7) (Figure 3C), suggesting a regulatory role for TβRI in driving the effects of TGF-β dependent cytostasis.

Prolonged vs short term Smad2 signaling in HCC cell lines

As altered expression levels of signaling components may not necessarily reflect activated signal transduction, we investigated the phosphorylation i.e. activation of Smad2 (Figure 5A, Figure S5). Some cell lines (HCC-M, HCC-T, PLC, Hep3B, HuH7) exhibited a prolonged pSmad2 signal after continuous stimulation with TGF-β up to 48h, whereas others (HepG2, HLE, HLF) faded out after 1-7h of TGF-β treatment. In FLC-4 und HuH6 cells pSmad2 signal peaks after 1 hour and then stabilizes on a lower level. This let us sort the cell lines into transiently and long term activated responders with regard to Smad2 phosphorylation.

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Figure 5. While Smad2 phosphorylation duration correlates to TGF-β and Smad7 expression, cytostatic responsiveness relates to CAGA-reporter-activation.

HCC cell lines were treated with 5 ng/ml TGF-β for 0, 1 (3),, 7, 24 and 48 h. (A) Left side: Western blot analysis of Smad2 phosphorylation and GAPDH expression. Right side: To evaluate TGF-β dependent transcriptional activity of Smad2, cells were transfected with plasmids carrying a luciferase gene under control of the activin response element (ARE) and additionally with a FAST-1 expression construct. Luciferase activity of cells treated with 5 ng/ml TGF-β for 9 h was correlated to untreated control samples. (B) Left side: Western blot analysis of Smad1/3 phosphorylation (1: upper/3: lower band); loading control GAPDH. Right side: TGF-β dependent transcriptional activity of Smad3 was evaluated 9 h after TGF-β treatment in cells infected with an adenovirus carrying a luciferase gene controlled by a CAGA response element. Treated samples were correlated to the corresponding untreated control. In the table, cell lines marked as black, show TGF-β induced cell death and growth inhibition whereas cells marked as grey mainly react with the latter one. The gradient from white to black displays increasing basal Smad7 expression levels. (C) Immunofluorescent detection of linker phosphorylated Smad3 in uninduced HCC-T cells. Red fluorescence indicates nuclear localization of pSmad3L. Blue staining indicates nuclei stained with DRAQ5. Significant differences are indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001 (Student’s t test).

https://doi.org/10.1371/journal.pone.0072252.g005

Smad7 is a potent inhibitor of TGF-β signaling. In line, our data suggest that Smad7 expression is critical in controlling the duration of Smad2 activation. We found a negative correlation (Pearson coefficient r=-0.54) between endogenous (not induced) Smad7 expression levels and the stability of TGF-β induced pSmad2 signals (Figure 2A, Figure 5A). Except HepG2, all cell lines with low Smad7 expression levels (HCC-M, HCC-T, PLC, Hep3B) exhibited a prolonged induction of pSmad2, while cell lines with high Smad7 levels, displayed more transient Smad2 phosphorylation. However, the latter do not show significant Smad7 induction by TGF-β, indicating that the TGF-β dependent negative feedback function of Smad7 is not controlling the duration of Smad2 phosphorylation. In contrast, induction of Smad7 correlates with CAGA reporter system activation by TGF-β which is low in HCC-M and HCC-T but high in PLC and Hep3B (see below) but not with Smad2 phosphorylation duration. We conclude, that basal Smad7 levels predetermine the cells sensitivity towards TGF-β induced cytostasis. Inducibility of Smad7 itself, however, is strictly dependent of Smad3 transcriptional activity and therefore high in cytostatic responsive cell lines. But this induced Smad7 is not abrogating or negatively controlling the cytostatic program of TGF-β.

Interestingly, Smad2 dependent ARE reporter activation was not correlated with Smad2 phosphorylation duration, but with cytostatic sensitivity, as reflected by strong activation of luciferase in PLC, HepG2 and HuH7 cells after TGF-β treatment (Figure 5A). Significant, but less activation is seen in Hep3B.

Smad3/4 dependent CAGA-Luc reporter activation and target gene transcription correlate with cytostatic TGF-β effects

All cell lines display immediate induction of Smad3 phosphorylation upon 1h of TGF-β treatment. The induction is stable up to 48 hours in Hep3B, HCC-M, HLE, HuH7, HCC-T and PLC cells, while it is only transient in HepG2, HLF, HuH6, and FLC4 cells (Figure 5B, Figure S5). Surprisingly, Smad3 phosphorylation events were, in contrast to Smad2, not correlated to Smad7 expression. Also, no correlation to Smad7 induction by TGF-β (Figure 4A) was detectable. Because R-Smad phosphorylation only represents the first step in TGF-β signaling, it may not solely explain differences in the cytostatic TGF-β response among cell lines. Therefore, we additionally investigated induction of TGF-β/pSmad3 dependent transcription by measuring the relative CAGA reporter activity after 9h TGF-β treatment. HepG2, Hep3B, PLC and HuH7 cells exhibited relatively high CAGA-Luc-activity upon TGF-β treatment, while the other cell lines did not (Figure 5B). We found that the responsive cell lines have relatively little endogenous TGF-β and Smad7 (compare Figure 2A, Figure 5B). Most obviously, we demonstrated that CAGA reporter activation by TGF-β strongly correlated with sensitivity of the cell lines towards cytostatic TGF-β effects. Very intriguing, HCC-M and HCC-T display strong and prolonged TGF-β induced Smad3 activation, however very low if any CAGA reporter activation. In HCC-T cells, using immunofluorescence, we identified strong nuclear staining with an antibody that detects serine 208/213 phosphorylation at the linker region of Smad3 (Figure 5C). Such intrinsic linker phosphorylation was previously described to inhibit signaling of C-terminally phosphorylated Smad3 [18] and thus, may explain the controversy between Smad3 activation and transcriptional activity. Such mechanism however remains to be functionally proven.

As endogenous Smad7 levels are relatively low in TGF-β responsive cell lines and Smad7 per se is a TGF-β target gene of the early signature group, we compared the ability of TGF-β to induce the Smad7 promotor and Smad7 mRNA expression (Figure 4). We observed that TGF-β mediated Smad7 promoter activation perfectly reflected its mRNA induction, indicating transcriptional regulation of the expression. Furthermore, the level of Smad7 induction paralleled that of TGF-β induced CAGA reporter activation (PLC, HepG2, Hep3B, HuH7).

Other Smad3 dependent TGF-β target genes (Bim, PAI-1) were also investigated (Figure 4B). In line with its apoptotic function, Bim expression was upregulated upon TGF-β treatment in PLC, Hep3B and HuH 7 cell lines showing a cytostatic TGF-β response. PAI-1 expression also correlated to TGF-β sensitivity and was induced in PLC, Hep3B and HepG2.

No coherent survival signaling status regulates cytostatic TGF-β effects

As balance between survival and cytostatic pathways plays an important role in determining the fate of hepatocytes and chronic liver disease progression, we asked whether the activity of classical survival pathways may explain differences in cytostatic responsiveness of HCC cell lines. Expression of BCL-2, BCL-XL, P21 and AKT as well as phosphorylation of P38, c-JUN, ERK and AKT do not exhibit any general link to TGF-β dependent cytostasis in the tested HCC cell lines (Figure 6A, Figure S6). However, induction of P21 expression (and to some extent downregulation of c-MYC) by TGF-β is a significant marker for TGF-β dependent cytostasis (see Figure 1A).

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Figure 6. Endogenous expression of survival factors and Smad3 signaling modulators PRAJA and ELF in HCC cells.

HCC cell lines were cultured in starvation medium for 24 h. (A) Western blot analysis was performed to evaluate expression levels of Akt, Bcl-2, Bcl-XL, p21 and GAPDH as loading control. Additionally, phosphorylation levels of ERK, c-Jun, Akt (lower band) and p38 were detected. (B) Basal PRAJA and ELF mRNA levels were quantified by Real time PCR analysis using rS18 as reference gene. Tables in (A) and (B) highlight cell lines responding to TGF-β with cell death and growth arrest in black. Cells which mainly show an inhibition of proliferation are marked as grey.

https://doi.org/10.1371/journal.pone.0072252.g006

Potential regulation of Smad3 signaling by ELF and PRAJA

It was reported that adaptor protein ELF is essential for Smad3 dependent TGF-β signaling and its down-regulation, e.g. by PRAJA mediated proteasomal degradation, can decrease Smad3 signaling and cause loss of the cytostatic response [8,19]. Thereby, loss of ELF could support gastric cancer and HCC development [20–22]. Testing this mechanism comparatively in HCC cell lines, we observed heterogeneous expression of both proteins. However, cytostatically responsive Hep3B and HuH7 cells expressed high levels of ELF and rather low levels of PRAJA (Figure 6B). In HepG2 and Huh6 showing a weaker cytostatic TGF-β response, ELF and PRAJA expression is intermediate. HCC-M, HCC-T, HLE and HLF do not show cytostasis upon TGF-β treatment and express relatively low ELF, but high PRAJA. Generally spoken, relative ELF/PRAJA ratios are higher in cytostatically responsive cell lines than in insensitive ones.

RNAi with Smad3 conflicts TGF-β dependent cytostasis in TGF-β sensitive HCC cells

Hierarchical clustering summarized our biochemical results and confirmed three HCC cell line groups (Figure 7): (1) HepG2, Hep3B, HuH7, PLC; (2) HLE, HLF, FLC-4, HuH6 as well as (3) HCC-M plus HCC-T. The clusters differ in their sensitivity towards TGF-β induced cytostasis, which is correlated to different Smad7 and TGF-β expression levels, duration of induced Smad2 phosphorylation, Smad3 and Smad2 transcriptional activity, TβRII expression and inducibility of TβRI mRNA.

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Figure 7. Hierarchical clustering analysis of HCC cell lines based on TGF-β/Smad signaling and cytostatic outcome.

TGF-β related observations on the 10 HCC-derived cell lines (as summarized in Figure 9) were converted into an all-or-none fashion (black/white boxes) to produce a matrix which was organized by hierarchical clustering algorithm. Observations were divided into 5 broad categories (A-E, left side). (A) TGF-β dependent cytostasis (black boxes: cell death >5%; proliferation inhibition >50%), (B) basal TGF-β signaling (black boxes: 2^-ΔΔCt > 2.5 for all, but >4 for Smad3), (C) induced TGF-β signaling (black boxes rel. induction of expression 2^-ΔΔCt > 2, induction of CAGA and Smad7 promoter and expression > 2.8, induction of ARE reporter > 5 ; prolonged duration), (D) survival signaling (black boxes: >3-fold increased for pAKT total AKT, 6-fold increased for pERK; > 40% inhibition of c-MYC), (E) basal ELF and PRAJA expression (black boxes: 2^-ΔΔCt >2 and 2^-ΔΔCt >1.4 respectively).

Based on the integrated above observations, clustering analysis unambiguously clustered the 10 cell lines. Beside HCC-M and HCC-T cell lines, 2 main groups were identified: group 1 included HepG2, PLC, Hep3B, HuH7 and group 2 included HLE, HLF, HuH6, FLC-4.

https://doi.org/10.1371/journal.pone.0072252.g007

Taken together, the clusters demonstrate that disrupted Smad3 downstream signaling is required for loss of cytostatic TGF-β effects in liver cancer. Additionally, TGF-β strongly enhanced Smad3 expression and its transcriptional activity in cell lines with retained TGF-β mediated cytostasis. For functional proof of the critical role of Smad3 in TGF-β mediated cytostasis, we knocked down Smad3 or Smad2 in Hep3B, HuH7 and PLC, and investigated the resulting TGF-β effects on apoptosis and proliferation inhibition (Figure 8). In line with our hypothesis, we find that Smad3 knock down diminishes TGF-β induced cytostasis, while the effect of Smad2 knock down is comparably little. The fact that siRNA against Smad2 also reduces Smad3 expression to some extent may even direct the observed Smad2 knock down effects towards Smad3 function.

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Figure 8. TGF-β induced cell death is Smad3 dependent.

RNA interference technology was used to downregulate Smad2 (S2) and Smad3 (S3) in PLC/PRF/5 (upper panel), Hep3B (middle panel) and HuH7 (lower panel) cells. An unspecific siRNA sequence (co) was used as control. Knockdown was allowed to take place for 48 h. Afterwards, each setup was treated with or without 5 ng/ml TGF-β for 1 (Immunoblot) or 3 days (cell death and proliferation) (Left). Western blot analysis against phosphorylated and total Smad2 and Smad1/3 was performed to confirm successful knockdown. GAPDH was used as loading control (Middle). After 3 days with or without TGF-β, cell death rates were evaluated using an LDH assay. Untreated cells for each siRNA were defined as 0 (grey line). Filled bars show TGF-β treated samples (Right). LDH content of viable (adherent) cells was used to determine proliferation rates. TGF-β treated samples (filled bars) were related to untreated siRNA samples (grey line).

https://doi.org/10.1371/journal.pone.0072252.g008

These results functionally confirm the predominant role of Smad3 in cytostatic outcome of TGF-β on liver parenchymal cells and indicate loss of Smad3 mediated downstream effects as critical for carcinogenic transdifferentiation.