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Am J Physiol Gastrointest Liver Physiol 290: G120-G128, 2006. First published September 8, 2005; doi:10.1152/ajpgi.00350.2004
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LIVER AND BILIARY TRACT

Thrombopoietin stimulates migration and activates multiple signaling pathways in hepatoblastoma cells

Roberto G. Romanelli,1 Ilaria Petrai,1 Gaia Robino,2 Eva Efsen,1 Erica Novo,2 Andrea Bonacchi,1 Gabriella Pagliai,3 Alberto Grossi,3 Maurizio Parola,2 Nadia Navari,1 Wanda Delogu,1 Francesco Vizzutti,1 Krista Rombouts,1 Paolo Gentilini,1 Giacomo Laffi,1 and Fabio Marra1,4

1Dipartimento di Medicina Interna, University of Florence, Florence, Italy; 2Dipartimento di Medicina e Oncologia Sperimentale, University of Turin, Torino, Italy; 3Division of Hematology, Azienda Ospedaliera Careggi, Florence, Italy; and 4Center for Research, Transfer and High Education "Denothe," University of Florence, Florence, Italy

Submitted 3 August 2004 ; accepted in final form 24 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Thrombopoietin (TPO), a cytokine that participates in the differentiation and maturation of megakaryocytes, is produced in the liver, but only limited information is available on the biological response of liver-derived cells to TPO. In this study, we investigated whether HepG2 cells express c-Mpl, the receptor for TPO, and whether TPO elicits biological responses and intracellular signaling in this cell type. Specific transcripts for c-Mpl were detected in HepG2 cells by RT-PCR, and expression of the protein was demonstrated by Western blot analysis and immunofluorescence. Exposure of HepG2 cells to TPO was associated with a dose-dependent increase in cell migration and chemoinvasion through Matrigel-coated filters. A checkerboard analysis showed that the effects of TPO on cell migration were dependent on both chemotaxis and chemokinesis. Exposure of HepG2 cells to TPO resulted in the activation of different members of the MAPK family, including ERK and JNK, as assessed using phosphorylation-specific antibodies and immune complex kinase assays. TPO also activated phosphatidylinositol 3-kinase (PI3K) and the downstream kinase Akt in a time-dependent manner. Finally, activation of c-Mpl was associated with increased activation of nuclear factor-{kappa}B. With the use of specific inhibitors, tyrosine phosphorylation and activation of PI3K were found to be required for the induction of migration in response to TPO. We conclude that TPO exerts biological actions on cultured hepatoblastoma cells via activation of c-Mpl and its downstream signaling.

Mpl; invasion; mitogen-activated protein kinases; phosphatidylinositol 3-kinase; nuclear factor-{kappa}B; apoptosis

THROMBOPOIETIN (TPO) is the primary regulator of platelet production by megakaryocytes (24, 30). TPO's actions are mediated by expression on the cell surface of the product of the protooncogene c-mpl, originally isolated as the cellular homolog of the transforming oncogene v-mpl of the myeloproliferative leukemia virus (5, 52). Mpl belongs to the type I group of the cytokine receptor superfamily and shares structural features with the receptors for IL-3, IL-5, and granulocyte colony-stimulating factor (19). Despite the fact that c-Mpl does not possess intrinsic enzymatic activity, receptor dimerization is associated with activation of a number of intracellular signaling pathways, including Jak tyrosine kinases and molecules of the signal transducers and activators of transcription (Stat) family (19). The majority of information concerning Mpl signaling derives from studies performed in megakaryocytes using both receptor mutants or pharmacological inhibition of signaling molecules.

Studies conducted in different areas have indicated that Mpl expression is not limited to megakaryocytes. In fact, hematopoietic cell lines of various origins have been shown to respond to TPO, including the erythroid, granulocytic, and macrophage compartments, (8, 21, 31, 34, 56), and Mpl has been shown to mediate proliferation of hematopoietic stem cells (32). Moreover, TPO has been described to be biologically active in endothelial cells, where it is responsible for the increased production of platelet-activating factor (11). The liver has been identified as a major site of TPO production, with hepatocytes being the most important cellular source of this growth factor (27). In fact, low levels of TPO have been reported in patients with fulminant hepatitis or advanced chronic liver disease, possibly contributing to the occurrence of thrombocytopenia in these clinical conditions (23, 39, 42, 55).

Despite the considerable amount of information concerning the role of the liver in the production of TPO, little is known regarding the possible effects of this growth factor on liver cells. The present study was undertaken to explore whether cultured hepatoblastoma cells express functional TPO receptors and to characterize the related biological actions and signal transduction mechanisms. We provide evidence that binding of Mpl is associated with activation of multiple signaling pathways and with the induction of cell migration and chemoinvasion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. Polyclonal antibodies against the phosphorylated forms of ERK, JNK, I{kappa}-B{alpha}, and Akt (Ser473) were from Cell Signaling Technology (Beverly, MA). Polyclonal anti-Mpl antibodies used for immunofluorescence, anti-ERK antibodies used for Western blot analysis, and polyclonal anti-Akt antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-Mpl antibodies used for immunoprecipitation and immunoblotting were kindly provided by Dr. Luigi Pegoraro (University of Turin, Torino, Italy). Human recombinant TPO, hepatocyte growth factor (HGF), and epidermal growth factor (EGF) were purchased from Peprotech (Rocky Hill, NJ). PD-98059, genistein, and LY-294002 were from Calbiochem (San Diego, CA). Radionuclides were purchased from MP Biochemicals (Costa Mesa, CA). All other reagents were of analytical grade.

Cell culture. HepG2 cells, derived from a human hepatoblastoma expressing wild-type p53, were maintained in DMEM (Sigma Chemical; St. Louis, MO) and supplemented with 10% fetal bovine serum, 5 mM sodium pyruvate, 5 mM nonessential amino acids, and antibiotic/antimicotic solution at 37°C in a humidified incubator containing 5% CO2. All experiments were performed in subconfluent cells after they were incubated overnight in serum-free medium.

B1647 cells and human hepatic stellate cells were cultured as previously described (6, 45).

RT-PCR. Analysis of c-Mpl expression was performed as previously described (37), with minor modifications. Reverse transcription of 1 µg of total RNA was allowed to proceed at 42°C for 30 min in a final volume of 20 µl of 10 mM Tris (pH 8.3), 5 mM MgCl2, 1 unit RNAse inhibitor, 2.5 units cloned Moloney murine leukemia virus reverse transcriptase, and 2.5 µM random hexamers (all from Perkin-Elmer; Boston, MA). Amplification of reverse-transcripted cDNA (5 µl) was performed in a final volume of 50 µl of 10 mM Tris (pH 8.3), 2 mM MgCl2, 200 µM each dNTP, and 1 unit Ampli-Taq DNA polymerase. Each tube contained 100 nmol of both sense and antisense primers for c-Mpl (sense primer 5'-TGGAGATGCAGTGGCACTTG-3'; antisense primer 5'-AGAACTGTGGGGGTCTGTAGT-3'). PCR conditions were as follows: 60 s at 94°C, 60 s at 58°C, and 60 s at 72°C for a total of 40 cycles in a Gene Amp 2400 system (Perkin-Elmer). Positive (RNA from the B1647 cell line) (45) and negative (tubes containing no cDNA) controls were always included in the reaction. Aliquots (20 µl) of the PCR mix were finally electrophoresed on a 2% agarose gel and directly visualized by ethidium bromide staining.

Cell migration and invasion assay. Cell migration assays were performed essentially as previously described using Boyden chambers equipped with 8-µm porosity polyvinylpyrrolidone-free polycarbonate filters (14). Polycarbonate filters were coated on the lower surface with 20 µg/ml human type I collagen (Collaborative Biomedical Products; Bedford, MA) for 30 min at 37°C and placed between the lower chamber and upper chamber. The lower chamber was filled with DMEM (200 µl) containing TPO or EGF. Serum-deprived HepG2 cells were washed, trypsinized, resuspended in serum-free medium containing 1% albumin at a concentration of 3 x 105 cells/ml, and placed in the upper chamber. The Boyden apparatus was assembled by interposing the filter between the upper and lower chambers, and cells were incubated at 37°C in a cell-culture incubator for 6 h. In selected experiments, TPO was added also to the upper chamber to modify the gradient across the filter. Migration was quantitated by counting the cells that migrated to the lower surface of the polycarbonate filters using a Zeiss microscope (Oberkochen, Germany) equipped with bright-field optics (x40 magnification). For each filter, the number of cells in six randomly chosen fields was counted, and the counts were averaged (means ± SD). Results are expressed as the number of migrated cells per high-power field. When inhibitors were used, cultured cells were incubated with the drugs to be tested or with their vehicle for 15 min before trypsinization, and equal concentrations were added to both chambers of the Boyden apparatus.

Invasion assays were performed essentially as described by Albini et al. (2). A thin layer of Matrigel solution (50 µg/ml) was overlaid on the upper surface of the same polycarbonate filter used for the migration assays. The Matrigel was allowed to gel by incubating the filters for 30 min at 37°C. Subsequently, the cell ability to invade this substratum was tested by using TPO. Serum-deprived HepG2 cells were trypsinized and resuspended at a concentration of 106 cells/ml; 105 cells were added to the top well of the Boyden apparatus assembled as described above and then incubated for 24 h at 37°C. They were processed and fixed as described for the migration assays. The number of cells that invaded the Matrigel layer was quantitated following the same protocol used for the migration assays.

Indirect immunofluorescence. HepG2 or hepatic stellate cells were cultured on glass coverslips at a low cell density and deprived of serum for 16 h. Cells were fixed with acetone-methanol (1:1) for 10 min at 4°C and stained for indirect immunofluorescence using polyclonal anti-Mpl (dilution 1:100) or anti-smooth muscle {alpha}-actin (1:100) as primary antibodies and anti-rabbit Cy3-conjugated antibodies (Amersham) as secondary antibodies (1:1,000). Nuclear DNA was detected by DAPI fluorescence staining. Some slides were stained solely with the secondary antibody as a negative control.

Preparation of nuclear extracts and gel mobility shift assay. Nuclear extracts were prepared according to Andrew and Faller (3). Gel mobility shift analysis was performed using NF-{kappa}B consensus oligonucleotides (Promega; Madison, WI) as described elsewhere (6). Briefly, 5 µg of nuclear extracts were incubated for 30 min at room temperature in a buffer containing 35mM HEPES (pH 7.8), 0.5 mM EDTA, 0.5 mM DTT, 10% glycerol, 10 µg/ml poly-dI-dC, 0.28 mM spermidine, and 100,000 counts/min 32P-labeled oligonucleotide probe. The DNA-protein complexes were separated by polyacrylamide gel electrophoresis in 0.5x Tris-borate-EDTA. At the end of the run, the gel was dried and autoradiographed. Some nuclear extracts were preincubated with polyclonal antibodies against the p65 subunit of NF-{kappa}B before the binding reaction was started.

Preparation of cell lysates and Western blot analysis. HepG2 cells were treated under the appropriate conditions, quickly placed on ice, and washed with ice-cold PBS. The monolayer was lysed in RIPA buffer [20 mM Tris·HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 0.05% (wt/vol) aprotinin]. Insoluble proteins were discarded by high-speed centrifugation at 4°C. The protein concentration in the supernatant was measured in triplicate using a commercially available assay (Pierce; Rockford, IL). Equal amounts of total cellular proteins were separated by SDS-PAGE and analyzed by Western blot analysis as previously described (7).

Immune complex kinase assays. The JNK assay was performed as described in detail elsewhere (43). Briefly, proteins were immunoprecipitated with polyclonal anti-JNK antibodies (Santa Cruz Biotechnology). After being washed, immunobeads were incubated in reaction buffer [20 mM HEPES (pH 7.6), 20 mM MgCl2, 2 mM DTT, 25 mM {beta}-glycerophosphate, and 0.1 mM sodium orthovanadate] containing 20 mM ATP, 5 µCi [{gamma}-32P]ATP, and 1 µg recombinant activating transcription factor 2 (ATF-2) (Santa Cruz Biotechnology) or GST-Jun for 30 min at 37°C. The reaction was stopped by the addition of Laemmli buffer, and samples were then subjected to 10% SDS-PAGE. The p38 MAPK assay was carried out as described for the JNK assay except for the use of anti-p38 polyclonal antibodies for immunoprecipitation and the constant use of ATF-2 as a substrate.

The phosphatidylinositol 3-kinase (PI3K) assay was performed after immunoprecipitation with anti-phosphotyrosine antibodies, as described elsewhere (38). Radioactive lipids were separated by thin-layer chromatography using chloroform-methanol-30% ammonium hydroxide- water (46:41:5:8). After being dried, plates were autoradiographed. The radioactive spots were then scraped and counted in a {beta}-counter.

The immune complex kinase assay of Akt activity was performed as described elsewhere (7). Briefly, 100 mg of protein were immunoprecipitated using anti-Akt antibodies and protein G-agarose. The immunobeads were washed three times with washing buffer [20 mM HEPES (pH 7.5), 40 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 0.5% Nonidet P-40, 20 mM {beta}-glycerophosphate, 0.5 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 10 µg/ml aprotinin]. The assay was performed by resuspending the beads in kinase buffer [50 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 10 mM MnCl2, 10 mM glycerophosphate, and 0.5 mM sodium orthovanadate] in the presence of 1 µM protein kinase A inhibitor peptide, 50 µM unlabeled ATP, and 6 µCi [{gamma}-32P]ATP using exogenous histone 2B (1.5 µg/assay tube) as the substrate and an incubation for 20 min at room temperature. The proteins in the samples were resolved by 12% SDS-PAGE, and the gel was stained with Coomassie blue and subjected to autoradiography.

Statistical analysis. The effects of TPO on cell proliferation and migration were analyzed on at least three independent experiments. The effects on intracellular signaling pathways were assessed using densitometry scans from three or more independent experiments and comparing the baseline values with peak activation. Data were considered significant for P values of ≤0.05 (by Student's t-test).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hepatoblastoma cells express Mpl. We first investigated whether the hepatoblastoma cell line HepG2 expresses transcripts for the protooncogene c-mpl, which codes for the TPO receptor. With the use of RT-PCR, a single amplification product of the expected size was detected using RNA isolated from HepG2 cells as well as from the megakaryocytic cell line B1647, which was used as a positive control (Fig. 1A). To confirm the expression of Mpl at the protein level, cell lysates from HepG2 cells were immunoprecipitated with polyclonal anti-Mpl antibodies and analyzed by immunoblotting using the same antibodies (Fig. 1B). Cell lysates from B1647 cells were used as a positive control. Analysis of lysates from HepG2 cells showed a band corresponding to the known size of Mpl and comigrating with one of identical size present in B1647. In contrast, cell lysates from human hepatic stellate cells, which do not express Mpl, did not show this band when subjected to the same protocol used for HepG2 cells (Fig. 1B).



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  		Fig. 1. HepG2 cells express c-Mpl. A: total RNA isolated from HepG2 cells (lane 1) or from the megakaryocytic cell line E1647 (lane 2, positive control) was subjected to RT-PCR using specific primers for c-mpl. A 206-bp amplification product was observed in both cell types. Lane 3, negative control. B: 500 µg of total protein isolated from HepG2 cells (lane 2) or from human hepatic stellate cells (HSCs; lane 3, negative control) were subjected to immunoprecipitation with anti-Mpl antibodies, extensively washed, and analyzed by Western blot using the same antibody. In lane 1, 25 µg of total cell lysate from B1647 cells were run as a positive control. Migration of the molecular mass marker is indicated on the left.

 
To confirm the expression of Mpl with an alternative technique, HepG2 cells were analyzed by immunofluorescence using antibodies against Mpl (Fig. 2, A and C). Evident membrane staining was observed in all cells, whereas the addition of the fluorochrome-conjugated secondary antibody, in the absence of the primary antibody, did not yield any signal (Fig. 2E). To provide additional evidence for the specificity of the immunofluorescence signals, we also analyzed human hepatic stellate cells, which do not express Mpl (see Fig. 1B). As expected, no staining was observed in this cell type (Fig. 2, I and K), which was clearly stained by smooth muscle {alpha}-actin antibodies (Fig. 2O). These data indicate that in HepG2 cells, mRNA transcripts for Mpl are associated with expression at the protein level.



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  		Fig. 2. Analysis of Mpl expression by immunofluorescence. HepG2 cells (A–H) or HSCs (I–P) were deprived of serum for 16 h and analyzed for expression of Mpl (A, C, I, and K) or smooth muscle {alpha}-actin (G and O) using indirect immunofluorescence (red). In E and M, the primary antibody was omitted, and cells were exposed only to the secondary conjugated antibody. Cells were also stained with the fluorescent dye DAPI (blue) to provide the correspondent visualization of nuclei for any acquired field of immunofluorescence (B, D, F, H, J, L, N, and P). Original magnification: x100 in A, B, and G–N and x400 in C–F, O, and P.

 
TPO stimulates chemotaxis and chemoinvasion of hepatoblastoma cells. The ability to move according to chemotactic cues is a characteristic of malignant cells and may be associated with a more aggressive tumor phenotype. We tested whether TPO behaves as a chemotactic stimulus for HepG2 cells using a migration assay in modified Boyden chambers. The addition of TPO resulted in a marked and significant stimulation of cell migration through collagen-coated filters (Fig. 3A). This effect was already evident at low concentrations of TPO and peaked at 100 ng/ml, when a fourfold induction of cell migration was observed. To establish to what extent the induction of cell migration was dependent on chemotactic, i.e., gradient-dependent, or chemokinetic actions, we performed checkerboard analyses using different concentrations in the upper and lower chambers (Table 1). The ability of TPO to induce migration of HepG2 cells was only partially abolished when the chemotactic gradient was reduced or neutralized by the addition of increasing amounts of cytokine in the upper chamber. These data indicate that the induction of HepG2 cells migration by TPO is the result of both chemotaxis and chemokinesis. We also evaluated whether TPO was able to modify HepG2 cell proliferation, as assessed by measurement of DNA synthesis. However, no increase in cell proliferation was observed when serum-deprived cells were incubated with 100 ng/ml TPO (data not shown).



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  		Fig. 3. Thrombopoietin (TPO) stimulates chemotaxis and invasion of HepG2 cells. A: HepG2 cell migration was analyzed in modified Boyden chambers as described in MATERIALS AND METHODS by adding TPO at the indicated concentrations in the lower chamber. After 6 h, cells migrating through the filter were counted. The bar graph shows means ± SD of 4 independent experiments. *P < 0.005 vs. control. B: the ability of HepG2 cells to migrate through a filter covered with basement membrane-like matrix in response to TPO was measured as described in MATERIALS AND METHODS. The bar graph shows means ± SD of 3 independent experiments. *P < 0.05 vs. control.

 

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  		Table 1. Checkerboard analysis of the effects of TPO on HepG2 cell migration

 
Degradation of basement membrane-like matrix facilitates tumor invasiveness and formation of metastasis. This characteristic of invading cells may be evaluated in vitro by measuring the ability of cells to migrate through a filter covered with Matrigel in response to soluble mediators. When chemoinvasion was measured in the presence of TPO in the lower chamber of the Boyden apparatus, a significant dose-dependent induction was observed (Fig. 3B).

TPO activates multiple MAPK cascades in hepatoblastoma cells. To obtain information on signal transduction pathways activated by Mpl in this cell type, HepG2 cells were exposed to TPO for different periods of time, and the activation of intracellular signaling was assessed. We first explored MAPK cascades that transduce signals generated by different soluble mediators in several systems, focusing on the three major pathways, represented by ERK, JNK, and p38 MAPK. These molecules are activated through a phosphorylation cascade that involves the sequential activation of small G proteins of the Ras superfamily, a serine/threonine kinase, and a dual-specificity kinase that ultimately leads to phosphorylation of MAPK (28). Exposure of HepG2 cells to TPO (Fig. 4A) resulted in a two- to fourfold increase in ERK phosphorylation, as an index of activation, that was statistically significant when several independent experiments were considered (5.98 ± 0.83 vs. 13.73 ± 4.84 densitometry units, P = 0.02, n = 4). In addition, increased phosphorylation of JNK was evident, particularly for the 54-kDa isoform, whereas the faster migrating form was only modestly affected (Fig. 4B). To confirm the activation of JNK, we conducted immune complex kinase assays and analyzed the enzymatic activity of JNK immunoprecipitates. TPO led to a rapid and sustained twofold increase in JNK activity over unstimulated control cells, thus confirming the induction of the JNK pathway (Fig. 4C). Also, in this case, the effects of TPO on JNK activity were statistically significant (33.33 ± 8.50 vs. 63.67 ± 6.03 densitometry units, P = 0.007, n = 3). Activation of p38 MAPK's enzymatic activity was also stimulated by TPO, although the effects were less evident and did not reach statistical significance (data not shown).



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  		Fig. 4. TPO activates different MAPK pathways. Serum-deprived HepG2 cells were incubated with 100 ng/ml TPO for the indicated time points or with epidermal growth factor (EGF; 200 ng/ml) or IL-6 (100 ng/ml) for 15 min. At the end of the experiments, total protein lysates were prepared and used as follows. A: 25 µg of protein were analyzed by Western blot analysis using antibodies directed against phosphorylated (p)ERK (top) or total ERK (bottom). Migration of the molecular mass marker is indicated on the left. B: the experiment was carried out as described in A by analyzing the blots with antibodies against pJNK (top) or total JNK (bottom). C: 25 µg of protein were immunoprecipitated with antibodies against JNK, and JNK activity was assayed in the presence of [32P]ATP using recombinant ATF-2 as a substrate. At the end of the kinase reaction, an aliquot was analyzed by SDS-PAGE, and the gel was dried and autoradiographed.

 
The PI3K/Akt pathway is a target of activated Mpl in HepG2 cells. Activation of the PI3K pathway is critical to link surface receptor activation with cell motility. For this reason, we investigated whether TPO is able to induce this signaling pathway in HepG2 cells. Analysis of PI3K activity, as assessed by the ability of phosphotyrosine immunoprecipitates to phosphorylate phosphatidylinositol in vitro, was increased by two- to threefold at different time points after exposure of HepG2 cells to TPO (Fig. 5A). Generation of 3-phosphorylated inositol lipids by PI3K is associated with downstream activation of the serine/threonine kinase Akt. To confirm activation of this pathway, we analyzed Akt using antibodies that recognized the phosphorylation status of Ser473, which is critical for activation of this molecule. TPO significantly increased Akt Ser473 phosphorylation (23.33 ± 1.53 vs. 69.67 ± 18.18 densitometry units, P = 0.011, n = 3), with a peak at 10–15 min after the addition of the cytokine (Fig. 5B). Increased phosphorylation was associated with a twofold increase in Akt enzymatic activity in cells exposed to TPO, as indicated by the ability to phosphorylate histone 2B in vitro (Fig. 5C). Thus the PI3K/Akt pathway is an additional target of Mpl activation in HepG2 cells.



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  		Fig. 5. TPO activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in HepG2 cells. Serum-deprived HepG2 cells were incubated with 100 ng/ml TPO for the indicated time points or with hepatocyte growth factor (HGF; 100 ng/ml) or IL-6 (100 ng/ml) for 15 min. At the end of the experiments, total protein lysates were prepared and used as follows. A: 100 µg of protein were immunoprecipitated with monoclonal anti-phosphotyrosine antibodies, and PI3K activity was assayed as described in detail in MATERIALS AND METHODS. Migration of phosphatidylinositol phosphate (PIP) after thin-layer chromatography and autoradiography is shown by the arrow. B: 25 µg of protein were analyzed by Western blot analysis using antibodies directed against pAkt (top) or total Akt (bottom). Migration of the molecular mass marker is indicated on the left. C: 50 µg of protein were immunoprecipitated with anti-Akt antibodies, and Akt activity was assayed in the presence of [32P]ATP using recombinant histone 2B as a substrate. At the end of the kinase reaction, an aliquot was analyzed by SDS-PAGE, and the gel was dried and autoradiographed.

 
Chemotactic effect of TPO is differentially regulated by postreceptor pathways. Induction of biological activities such as cell migration is dependent on cytokine interaction with cognate receptors and downstream activation of multiple signaling pathways. These pathways are both cell and agonist specific, as demonstrated by experiments conducted in different cell types, including liver resident cells (6). On the basis of the activation pattern resulting from TPO stimulation in HepG2 cells, we explored the requirement for specific signaling intermediates in the induction of cell migration. When HepG2 cells were pretreated with PD-98059, an inhibitor of MEK that effectively limits the activation of ERK (18), only a 35% inhibition of cell migration in response to TPO was observed (Fig. 6A). In contrast, the pan-specific tyrosine kinase inhibitor genistein more effectively blocked the action of TPO on cell migration, resulting in a significant 76% inhibition. We also tested the requirement for PI3K activation using LY-294002, which competitively blocks activation of all PI3K isoforms. In these conditions, inhibition of the chemotactic effects of TPO was virtually complete. These data indicate that the action of TPO on motility of HepG2 cells is critically dependent on tyrosine kinase activity and on PI3K. To rule out the possibility that the lack of effect of PD-98059 on cell migration was due to ineffective inhibition of ERK activation, we evaluated the effects of different inhibitors on TPO-stimulated ERK activation (Fig. 6B). PD-98059 completely prevented ERK activation, whereas the PI3K inhibitor LY-294002 had no effect. Conversely, this compound effectively inhibited the PI3K/Akt pathway (Fig. 6C). As expected, the tyrosine kinase inhibitor genistein prevented both ERK and PI3K/Akt activation (Fig. 6B and data not shown).



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  		Fig. 6. Contribution of different signaling pathways to TPO-induced cell migration. A: serum-deprived HepG2 cells were preincubated with 50 µM PD-98059, 10 µM genistein, 10 µM LY-294002, or vehicle (DMSO) for 30 min. HepG2 cell migration was subsequently analyzed by adding TPO (100 ng/ml) or EGF (200 ng/ml, positive control) in the lower chamber of the Boyden system. The bar graph shows means ± SD of 3 independent experiments. *P < 0.005 vs. control;°P < 0.01 vs. TPO + vehicle. B: serum-deprived HepG2 cells were preincubated with the indicated inhibitors at the same concentrations indicated in A for 30 min, followed by the addition of TPO (100 ng/ml) for 15 min. Twenty-five micrograms of protein were analyzed by Western blot analysis using antibodies directed against pERK (top) or total ERK (bottom). Migration of the molecular mass marker is indicated on the left. C: HepG2 cells were preincubated with 10 µM LY-294002 or its vehicle for 30 min, followed by 100 ng/ml TPO for 15 min, as indicated. Twenty-five micrograms of protein were analyzed by Western blot analysis using antibodies directed against pAkt (top) or {beta}-actin (bottom). Migration of the molecular mass marker is indicated on the left.

 
NF-{kappa}B as a target of TPO in HepG2 cells. The transcription factor NF-{kappa}B is a critical modulator of different biological activities in liver cells and contributes to hepatocyte survival and expression of proinflammatory cytokines. To investigate whether NF-{kappa}B transduces signals downstream of Mpl, we first analyzed the ability of HepG2 nuclear extracts to bind consensus NF-{kappa}B oligonucleotides in gel mobility shift assays. Compared with unstimulated cells, HepG2 cells exposed to TPO showed a more than twofold increase in NF-{kappa}B binding to labeled oligonucleotides (Fig. 7A). NF-{kappa}B activation was evident at early time points and persisted for as long as 1 h after stimulation. To be active, NF-{kappa}B must dissociate from the inhibitory protein I{kappa}-B{alpha} and migrate to the nucleus (15). Because phosphorylation of I{kappa}B{alpha} leads to dissociation from NF-{kappa}B and degradation through the ubiquitine-proteasome pathway, we analyzed changes in the phosphorylation status of I{kappa}B{alpha} to obtain additional evidence for the ability of TPO to induce NF-{kappa}B. Indeed, in cells incubated with TPO, a marked increase in I{kappa}B{alpha} phosphorylation was observed, with a time course and an extent of induction similar to those observed for the appearance of NF-{kappa}B activity in the nucleus (Fig. 7B). The effects of TPO on this pathway were statistically significant (8.00 ± 0.44 vs. 17.74 ± 8.74 densitometry units, P = 0.027, n = 3). Finally, we tested the specificity of the gel mobility shift signals by preincubating nuclear extracts with antibodies directed against the p65 subunit of the NF-{kappa}B complex. Compared with control extracts, anti-p65 antibodies resulted in a clear reduction of the intensity of the shifted band. These data confirm the specificity of gel mobility shift signals and indicate that p65 is a major component of the NF-{kappa}B complex in these cells together with other Rel proteins responsible for the band(s) not affected by this antibody (Fig. 7C). All NF-{kappa}B complexes were no longer present when an excess of cold oligonucleotide was used as a competitor (Fig. 7C).



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  		Fig. 7. TPO activates NF-{kappa}B in HepG2 cells. A: serum-deprived HepG2 cells were incubated with 100 ng/ml TPO for the indicated time points or with IL-6 (100 ng/ml) for 30 min, as indicated. Nuclear extracts were analyzed by electrophoretic mobility shift assay as described in MATERIALS AND METHODS using a radiolabeled consensus NF-{kappa}B oligonucleotide as a probe. Migration of the specific NF-{kappa}B complexes is indicated by an arrow. NS, nonspecific bands. B: serum-deprived HepG2 cells were incubated with 100 ng/ml TPO for the indicated time points. Twenty-five micrograms of protein were analyzed by Western blot analysis using antibodies directed against pI{kappa}B{alpha} (top) or total I{kappa}B{alpha} (bottom). In lanes 7 and 8, 10 µg of lysates from HeLa cells cultured in the absence or presence of TNF-{alpha} were run as negative or positive controls, respectively, for I{kappa}B{alpha} phosphorylation. Migration of the molecular mass marker is indicated on the left. C: serum-deprived HepG2 cells were incubated with 100 ng/ml TPO for the indicated time points or with IL-6 (100 ng/ml) for 30 min, as indicated. The electrophoretic mobility shift assay was carried out as in A, but nuclear extracts were preincubated with antibodies against the p65 subunit of NF-{kappa}B (lanes 5–8) or with a 50-fold excess of unlabeled oligonucleotide (lanes 9–12) before the binding reaction was started.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The relationships between hematopoietic growth factors and the liver are manyfold. It has been shown that the liver is a major site of production of TPO in the adult (48), and modulation of TPO expression takes place in different pathophysiological conditions. TPO is a target molecule during the acute phase, and IL-6, a critical cytokine for induction of the acute phase response, stimulates TPO expression in cultured liver cells (22, 29, 54). On the other hand, low levels of TPO have been suggested to contribute to thrombocytopenia in advanced chronic liver disease or during fulminant hepatitis (23, 39, 42, 55). In patients with liver tumors such as hepatocellular carcinoma or hepatoblastoma, it has been proposed that the increase in platelet count may be related to increased TPO production, and HepG2 hepatoblastoma cells have been used as a model system to explore the regulation of this cytokine in vitro (26, 29, 35, 36, 55). Despite the tight links between TPO and the liver, only limited information is available on the biological response of liver-derived cells to TPO. In this study, we report for the first time that TPO is a cytokine that targets hepatoblastoma cells, as indicated by three lines of evidence. First, HepG2 cells express Mpl, the cognate receptor for TPO. Second, exposure of HepG2 cells to TPO is associated with an induction of cell migration and chemoinvasion. Finally, binding of Mpl with recombinant TPO activates multiple intracellular signaling pathways. Taken together, these data identify a novel soluble mediator capable of modifying the biology of HepG2 cells.

The ability of TPO to exert biological activities in nonhematopoietic cells is supported by studies demonstrating Mpl expression in vascular endothelial cells, where an interaction with TPO activates a proinflammatory cascade involving the lipid mediator platelet-activating factor (11). Interestingly, despite the fact that the large majority of primary tumors or cancer cell lines did not express c-Mpl transcripts, fetal liver tissue and the hepatocarcinoma cell line Hep3B have been shown to express Mpl by RT-PCR analysis (17). On the other hand, it should be noted that in that study, HepG2 cells were not found to be positive for c-mpl expression. Although the reason for this discrepancy with our data is uncertain, it may be related to the low abundance of c-Mpl transcript in HepG2 cells and to differences in the RT-PCR protocol. Expression of Mpl has also been described in liver sinusoidal endothelial cells, where TPO stimulates both growth and production of different proinflammatory cytokines (12, 13), as well as in surgical specimens collected from patients with hepatoblastoma (35). Taken together, these data indicate that under particular conditions, such as development and cancer, liver tissue may be a site of Mpl expression. Along these lines, it has been shown that liver precursor cells share the expression of several surface molecules with hematopoietic progenitors, including CD34, or c-kit (47), and regenerating liver cells and HepG2 cells express a chemokine receptor, CX3CR1, which is also expressed in cells of hematopoietic lineage (20).

An important finding of the present study is the observation that Mpl expression on hepatoblastoma cells is associated with the induction of biological activities that could be relevant for this tumor. In particular, exposure of HepG2 cells to recombinant TPO induced a marked increase in cell migration and chemoinvasion. The ability of tumor cells to migrate in response to chemotactic stimuli is critical for the local spread of the tumor and/or for the formation of metastases. In particular, in the case of hepatoblastoma, it should be kept in mind that the local concentration of the cognate ligand, i.e., TPO, is extremely elevated due to the high expression by resident liver cells. In this condition, it could be hypothesized that the TPO-Mpl axis may represent an additional factor favoring the in vivo diffusion of hepatoblastoma within liver parenchyma. This possibility is supported by the study of Komura et al. (35), which demonstrated that Mpl is expressed in five of eight hepatoblastomas analyzed by PCR after surgery, an observation that led the authors to hypothesize the occurrence of a paracrine/autocrine action of TPO in hepatoblastoma. The data reported herein are the first biological demonstration of the possible occurrence of this event.

The results of the present study provide additional information on signal transduction pathways activated by the receptor for TPO in nonhematopoietic cells and the relevance of the different signals for the induction of biological activities. Activation of the Ras/ERK pathway by TPO has been extensively demonstrated in megakaryocytic cell lines and normal megakaryocytes, where it is necessary for endomitosis (44). Similarly, activation of the PI3K/Akt pathway has been implicated in mediating the proliferation signals of TPO in UT7 cells (10). Signals generated by Ras/ERK and PI3K/Akt have been shown to modulate several aspects of the biology of HepG2 cells, including migration, proliferation, and metabolic pathways (9, 16, 46, 49). In cells exposed to TPO, an inhibitor of MEK exerted minor effects on cell migration, indicating that the ERK pathway is not critical for the motogenic action of TPO in this cell type. In contrast, inhibition of PI3K led to a virtually complete inhibition of cell migration, in analogy with data previously reported for HGF, a cytokine that induces scattering of different epithelial cells, including HepG2 cells (41). Along these lines, it is interesting to note that TPO elicited migration of HepG2 cells by inducing both chemotaxis and chemokinesis, as observed by experiments where the concentration of cytokine and the gradients across the filters of the Boyden chamber were manipulated. The fact that TPO induced migration also via chemokinesis and the fact that TPO is expressed by hepatoblastoma cells suggests the contribution of an autocrine loop to tumor spread.

An additional finding of the present study is related to the observation that TPO induces the activation of NF-{kappa}B in HepG2 cells. NF-{kappa}B is a transcription factor involved in the response to oxidative stress, inflammation, and cell survival (15). Using different experimental approaches, including electrophoretic mobility shift assays, we identified NF-{kappa}B as a molecular target downstream of c-Mpl in HepG2 cells. Limited data are available on NF-{kappa}B activation in TPO-stimulated cells. Kirito et al. (33) have recently shown that binding of Rel proteins to the Bcl-xL promoter contributes to transcription of this gene in the human leukemic cell line UT-7/TPO. The data reported herein, using a nonhematopoietic cell line, corroborate the hypothesis that NF-{kappa}B can represent an additional molecular target downstream of Mpl activation. Interestingly, expression of matrix metalloproteinases by HepG2 cells has been demonstrated to be related to NF-{kappa}B activation, suggesting a contribution to the degradation of the extracellular matrix responsible for tumor cell invasion (1, 25). Moreover, constitutive activation of NF-{kappa}B has been found at higher frequency in tumor tissue of patients with hepatocellular carcinoma compared with noninvolved tissue, suggesting a possible role of this factor in hepatocarcinogenesis (51). Taken together, these data suggest that TPO-induced NF-{kappa}B activation may contribute to increase the invasive phenotype of hepatoblastoma cells.

Analysis of signal transduction mechanisms activated by TPO in HepG2 cells revealed the modulation of several pathways potentially involved in cell survival, including PI3K/Akt, ERK, and NF-{kappa}B (28, 40). Moreover, TPO is a survival factor for different hematopoietic cell types (53). On the basis of these data, we investigated whether incubation of HepG2 cells with TPO was capable of modifying apoptosis induced by the combination of TNF-{alpha} and cycloheximide. While this protocol was highly effective in inducing HepG2 apoptosis, no significant modifications were observed in cells exposed to TPO (data not shown). The fact that TPO failed to afford protection against HepG2 apoptosis may have different, and not mutually exclusive, explanations. The combination of TNF-{alpha} and protein synthesis inhibition is a robust apoptotic stimulus and is therefore difficult to modulate with soluble mediators, considering the low levels of Mpl expression in HepG2 cells. In addition, exposure of HepG2 cells to TPO is also associated with activation of JNK, a member of the MAPK family that has been associated with the induction of apoptotic cell death in different cell types, including HepG2 cells (4, 50).

In summary, we have demonstrated that human HepG2 hepatoblastoma cells are a novel target of the system comprised by TPO and the receptor c-Mpl, which generates motogenic signals in this cell type. These data may contribute to a better understanding of the biology of liver cancer cells.


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Funding for this study was provided by the University of Florence and by the Italian Liver Foundation. E. Efsen was supported in part by the Tode Travel Grant, the Direktør Madsen's Grant, and Fhv. Direktør Nielsen's Grant (Denmark).


   ACKNOWLEDGMENTS
 
We are indebted to Dr. Luigi Pegoraro for kindly providing anti-mpl antibodies and Dr. Laura Bonsi for providing B1647 cells.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. Marra, Dipartimento di Medicina Interna, Univ. of Florence, Viale Morgagni, 85, I-50134 Florence, Italy (e-mail: f.marra{at}dmi.unifi.it)

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


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