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1 Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Tokyo;
2 Department of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research, Tokyo;
3 Department of Ophthalmology, Hiroshima University School of Medicine, Hiroshima;
4 Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., Gunma;
5 Genome Science Division, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The signal transduction pathways leading to the growth inhibition have been studied well at the molecular level (2) . TGF-ß binds to two different types of serine/threonine kinase receptors, termed type I and type II. Type I receptor is activated by type II receptor upon ligand binding and transduces signals into cytoplasm through phosphorylation of receptor-regulated Smads. ALK-5 is a ubiquitously expressed type I receptor for TGF-ß, transmitting signals via Smad2/3. ALK-1 is also a type I receptor for TGF-ß; it is predominantly expressed in vascular endothelial cells and transmits signals via Smad1/5 (5) . Phosphorylated receptor-regulated Smads interact with Co-Smad (Smad4), translocate to the nucleus, and regulate transcription of target genes in cooperation with transcriptional activators/repressors as well as with coactivators/corepressors (6) .
In most types of cells, TGF-ß arrests cell cycle progression in the G1 phase by down-regulating expression of c-Myc and cdc25A and up-regulating that of CDKIs, p21WAF1/CIP1 and/or p15Ink4B (2) . A Smad-responsive element was identified in the c-myc promoter (7 , 8) , and the activated Smad complex (Smad2/3-Smad4) has been found to suppress transcription of c-myc in cooperation with p107 and E2F4/5 (8) . Down-regulation of c-Myc thus leads to induction of p21WAF1/CIP1 and p15Ink4B, because c-Myc physically interacts with Smad2/3 and suppresses the function of Sp1-Smad complex in the transcriptional activation of p21WAF1/CIP1 and p15Ink4B (9 , 10) . In contrast, pathways leading to the growth stimulation by TGF-ß have not been well elucidated. Thus far, several mechanisms for this growth stimulation have been proposed. TGF-ß-induced up-regulation of growth factors including PDGF and FGF has been reported in fibroblasts, Ito cells, and prostate cancer cells (11, 12, 13, 14, 15, 16, 17, 18) . Down-regulation of CDKIs by TGF-ß has been reported in rat osteoblasts (p57Kip2; Refs. 19 , 20 ) and H-ras-transfected colon carcinoma cells (p21WAF1/CIP1; Refs. 21 ). Recently, Goumans et al. (22) reported that ALK-1 but not ALK-5 acts as a signaling receptor for TGF-ß in the transduction of cell proliferation signals in endothelial cells.
Osteosarcoma is one of the most common nonhematological primary malignant tumors of bone (23) . Bone is an organ that produces and stores large amounts of TGF-ß (24) . Recent findings suggest that TGF-ß is involved in the progression of osteosarcoma; TGF-ß stimulates the growth of several osteosarcoma cell lines in culture (25, 26, 27) , and overexpression of TGF-ß is observed in osteosarcoma tissue by immunohistochemistry, mRNA in situ hybridization, and RT-PCR (28 , 29) . It thus appears important to elucidate how TGF-ß acts on osteosarcoma cells.
In the present study, we examined the signal transduction mechanism for the growth-stimulating effect of TGF-ß on MG63 human osteosarcoma cells. We found that SB-431542, a novel TGF-ß type I kinase inhibitor, as well as Gleevec, inhibited TGF-ß-stimulated proliferation of MG63 cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin antibody (T9026) was from Sigma-Aldrich (St. Louis, MO). Antisera against type I receptors were described previously (31)
. Recombinant adenoviruses carrying LacZ, a constitutively active form of ALK-1 (ALK-1QD), or ALK-5 (ALK-5TD) were described previously (32
, 33)
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Cell Culture.
MG63 human osteosarcoma cells were maintained in Minimum Essential Medium (Life Technologies, Inc., Carlsbad, CA) containing 10% fetal bovine serum, 1% nonessential amino acids, and 1% penicillin/streptomycin. NIH3T3 cells were maintained in DMEM (Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum, 1% nonessential amino acids, and 1% penicillin/streptomycin.
Cell Proliferation Assay.
To explore the effects of ligands, cells were seeded at a density of 8 x 104 cells/well in 6-well plates and starved (0.1% FCS for MG63 cells and 0.5% FCS for NIH3T3 cells) for 24 h before ligand stimulation. Media containing various ligands were exchanged at 48-h intervals. Cells were trypsinized and counted by a Coulter counter on days 2, 4, and 6 after ligand stimulation. The experiments were performed in triplicate. To explore the effects of constitutively active receptors, cells were seeded at a density of 2 x 105 cells/well in 6-well plates. The next day, cells were infected with adenoviruses carrying various cDNAs at a multiplicity of infection of 100. Cells were trypsinized and counted on day 3.
Thymidine Incorporation Assay.
Cells were seeded at a density of 2 x 104 cells/well in 24-well plates and cultured overnight. Then serum concentration in the medium was decreased to 0.1%, and the cells were incubated for another 24 h, followed by stimulation with various ligands. Forty-eight h after stimulation, the cells were labeled with [3H]thymidine for 2 h. Thymidine incorporation into the TCA-insoluble fraction was analyzed as described previously (34)
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Immunoblotting, Affinity Cross-Linking, and Immunoprecipitation.
Immunoblotting was performed as described previously (34)
. Nuclear extract was prepared using NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce Biotechnology, Rockford, IL). For affinity cross-linking, recombinant TGF-ß1 was iodinated using the Chloramine-T method. MG63 cells were affinity-labeled with [125I]-labeled TGF-ß1 using 0.27 mM disuccinimidyl suberate (Pierce Biotechnology), followed by immunoprecipitation using specific antisera against ALK-1 or ALK-5. Immune complexes were analyzed as described previously (34)
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RNA Extraction and RT-PCR Analysis.
Total RNA was extracted from MG63 cells using ISOGEN (Nippon Gene, Toyama, Japan). For RT-PCR analysis, first-strand DNA was synthesized using the Superscript First-Strand Synthesis System (Invitrogen, Carlsbad, CA) with random hexamer primers. Expression of various signaling components was examined by semiquantitative RT-PCR analysis. PCR products were separated by electrophoresis in agarose gels (1%) and visualized with ethidium bromide. The primer sequences, PCR programs, and expected sizes of PCR products were described previously (35)
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Oligonucleotide Microarray Analysis.
Total RNAs were extracted from MG63 or HaCaT cells at 0, 1, and 4 h after stimulation by TGF-ß3 (1 ng/ml). We used the total RNAs to prepare cRNA and conducted oligonucleotide microarray analysis using GeneChip Human Genome U95A (Affymetrix, Santa Clara, CA) according to the manufacturer’s instructions.
Quantitative Real-Time PCR Analysis.
Total RNAs were extracted from MG63 cells after various treatments, and first-strand cDNAs were synthesized using the Superscript First-Strand Synthesis System (Invitrogen) with random hexamer primers. Quantitative real-time RT-PCR analysis was performed using the GeneAmp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA) as described (33)
. The primer sequences are available upon request.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Cell numbers were significantly increased (1.5-fold at day 4) by TGF-ß treatment (1 ng/ml), although this effect was rather late in onset and first appeared at day 4. We also examined the effect of TGF-ß on DNA synthesis by MG63 cells (Fig. 1B)
. TGF-ß caused a dose-dependent increase in [3H]thymidine incorporation. Maximal response was observed above the TGF-ß concentration of 100 pg/ml. The response was maintained even at higher concentrations up to 5 ng/ml. These results indicate that TGF-ß positively regulates proliferation of MG63 cells.
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MG63 cells were affinity-labeled with [125I]-labeled TGF-ß followed by cross-linking and immunoprecipitation by antisera against ALK-1 or ALK-5 (Fig. 2A)
. Cross-linked bands were observed when anti-ALK-1 or anti-ALK-5 was used but not when preimmune serum was used. This result indicated that ALK-1 as well as ALK-5 is expressed on the MG63 cell surface and functional in binding to TGF-ß.
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. We also quantified the expression level of mRNA for PAI-1 (as an ALK-5 target) as well as Id1 (as an ALK-1 target; Ref. 22
; also Fig. 4A
) by quantitative real-time PCR analysis (Fig. 2C)
. In response to TGF-ß, expression of PAI-1 as well as Id1 was induced, suggesting that both ALK-5 and ALK-1 signals were transmitted in MG63 cells.
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, SB-431542 inhibited cell proliferation induced by TGF-ß. In contrast, it did not inhibit PDGF-BB-induced cell proliferation, thus excluding the possibility of a nonspecific effect.
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, although it did not inhibit the phosphorylation of Smad1/5 induced by BMP-6. Furthermore, SB-431542 inhibited TGF-ß-induced Id-1 expression as well as PAI-1 (data not shown). These results indicated that SB-431542 affected both ALK-1- and ALK-5-mediated signaling induced by TGF-ß ligand. The type I receptor responsible for growth stimulation could not be identified with the use of SB-431542.
Constitutively Active ALK-5 but not ALK-1 Induced Proliferation of MG63 Cells.
We next examined effects of constitutively active forms of ALK-1 (ALK-1QD) and ALK-5 (ALK-5TD) on proliferation of MG63 cells. MG63 cells were infected with adenoviruses carrying LacZ, ALK-1QD, or ALK-5TD. Expression of ALK-1QD caused induction of Id-1, whereas expression of ALK-5TD caused induction of PAI-1 (Fig. 4A)
, indicating that both receptors transmitted signals in MG63 cells. Results of cell proliferation assay, however, demonstrated that ALK-5 but not ALK-1 induced cell proliferation in MG63 cells (Fig. 4B)
. We thus concluded that ALK-5 is mainly responsible for growth stimulation by TGF-ß in MG63 cells.
Activin-A but not BMP-6 Stimulated Proliferation of MG63 Cells.
We also examined the effects of other ligands in the TGF-ß superfamily, i.e., activin-A and BMP-6, on proliferation of MG63 cells. In most cell types, activin-A transmits signals via ALK-4 and Smad2/3, whereas BMP-6 transmits Smad1/5 signals. The selectivity of signal transduction in MG63 cells was confirmed on the basis of induction of target genes. Activin-A induced PAI-1, although the intensity of induction was weaker than that by TGF-ß, whereas it did not induce Id1. In contrast, BMP-6 induced Id1, but induced PAI-1 only weakly (Fig. 5A)
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. These findings further confirmed that the Smad2/3 pathway but not the Smad1/5 pathway plays a major role in transmitting proliferation signals in MG63 cells.
DNA Microarray Analysis of TGF-ß-regulated Genes in MG63 Cells.
To elucidate the signaling pathway involved in the cell proliferation induced by TGF-ß, we analyzed TGF-ß-regulated genes using DNA microarray. We used HaCaT cells as a reference, because their growth is inhibited by TGF-ß. Several representative target genes of TGF-ß signaling were induced in MG63 cells (Table 1)
, indicating that the signaling pathway of TGF-ß was not greatly altered in the cells. However, we found four unique features:
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(b) Expression of c-Myc was up-regulated in MG63 cells. In most cells including HaCaT cells, expression of c-Myc is down-regulated after TGF-ß stimulation.
(c) Expression of p21WAF1/CIP1 was up-regulated in both MG63 cells and HaCaT cells, only transiently in MG63 cells but in rather sustained fashion in HaCaT cells.
(d) As expected from the observation that MG63 cells express ALK-1, Id proteins were rapidly induced in MG63 cells. As shown in Fig. 4
, the possibility of involvement of ALK-1 in the growth-stimulating signaling had already been excluded. We thus further examined the first three features.
TGF-ß-induced Growth Factors in MG63 Cells: Their Effects on Proliferation of MG63 Cells.
Induction of PDGF-A, FGF-2, HB-EGF, and PlGF by TGF-ß was confirmed by real-time PCR analysis (Fig. 6
and data not shown). These results are consistent with those of DNA microarray analysis. We also observed that induction of these growth factors was completely inhibited by SB-431542 (data not shown). Among the induced growth factors, however, PlGF seemed unlikely to be involved in the growth stimulation of MG63 cells, because the receptor for PlGF (Flt1) is not expressed in the cells.
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, PDGF-AA and FGF-2 were effective in stimulating thymidine incorporation in MG63 cells, whereas HB-EGF was only weakly effective.
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). MG63 cells were cultured in the presence of the inhibitor and treated with TGF-ß, PDGF-AA, FGF-2, or HB-EGF. Gleevec (1 µM) inhibited the growth stimulation induced by TGF-ß as well as that by PDGF-AA but did not inhibit the stimulatory effects of FGF-2 and HB-EGF. A similar result was obtained when AG1296, another inhibitor of the PDGF receptor kinase (38)
, was used (5 µM; data not shown). These results suggested that the effect of TGF-ß was mediated principally through PDGF signaling, although the involvement of FGF signaling cannot be excluded.
We also examined the induction of growth factors by activin-A and BMP-6 (Fig. 6)
. Activin-A, which stimulated growth of MG63 cells, induced PDGF-A but neither FGF-2 nor HB-EGF. BMP-6, which did not stimulate the growth of MG63 cells, induced none of the three growth factors. These results further supported a major role of PDGF signaling in the growth stimulation of MG63 cells by ligands of the TGF-ß superfamily.
Control of Cell Cycle Regulators by TGF-ß in MG63 Cells.
It is also important to elucidate why growth-inhibitory signaling by TGF-ß is ineffective in MG63 cells. As described above, expression of both c-Myc and p21WAF1/CIP1 was up-regulated on DNA microarray analysis. We confirmed the induction of c-Myc and p21WAF1/CIP1 using quantitative real-time PCR analysis and found that it was inhibited by SB-431542 (data not shown). p15 mRNA was not detected in MG63 cells.
We further examined the protein levels of c-Myc and p21WAF1/CIP1 after TGF-ß treatment (Fig. 8A)
. Expression of c-Myc protein was up-regulated from 12 h after TGF-ß treatment, and this up-regulation was maintained even at 24 h after the treatment. Expression of p21WAF1/CIP1 protein was also up-regulated, but maximal expression was observed at 12 h, with decrease to basal level by 24 h. We also confirmed that nuclear p21WAF1/CIP1, which has cell cycle inhibitory activity, was actually up-regulated by cell-fractionation experiment (Fig. 8B)
. These results suggest that, among the growth-inhibitory signals by TGF-ß, the pathway leading to c-Myc down-regulation was abrogated, and that a signal up-regulating c-Myc expression may be transmitted in MG63 cells.
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. These results suggested that TGF-ß-induced growth stimulation is mediated through various mechanisms, which appear to depend on cell types.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Interestingly, ALK-1, a TGF-ß type I receptor preferentially expressed in endothelial cells, was found to be expressed on the MG63 cell surface and to bind TGF-ß. We further confirmed that ALK-1 was functionally active based on the phosphorylation of Smad1/5 as well as gene expression profiles of targets of the Smad1/5 pathway. The up-regulated genes include those for Id proteins (Id1, Id2, and Id3). Id proteins were originally identified as "inhibitor of differentiation," and their positive roles in cell cycle progression have been reported recently (43) . Id proteins negatively regulate CDK inhibitors. In addition, Id2 physically interacts with hypophosphorylated pRb and antagonizes its antiproliferative effect. It thus seemed possible that induction of Id by the ALK-1 pathway is involved in the growth-stimulating effect of TGF-ß in MG63 cells. In the present study, however, we observed that ALK-1 signaling is not sufficient for the growth stimulation of MG63 cells. The function of ALK-5 is sufficient and indispensable for the growth stimulation, although the possibility cannot be excluded that ALK1 cooperates with ALK5 and is involved in the growth stimulation.
Analyses of the TGF-ß-regulated genes in MG63 cells revealed up-regulation of four growth factors; among them, only PDGF-AA and FGF-2 were effective in stimulating proliferation of MG63 cells when exogenously added. Using specific inhibitors of PDGF receptor kinases, Gleevec and AG1296, we found that PDGF-AA mainly contributed to the growth-promoting action of TGF-ß in MG63 cells. This may be because expression of FGF-2 was only transiently up-regulated, whereas that of PDGF-AA was up-regulated in a sustained fashion.
We further examined the expression of cell cycle regulators. Miyazaki et al. (44) reported that TGF-ß stimulates or down-regulates cell growth through down- or up-regulation of p21WAF1/CIP1. p21WAF1/CIP1 is one of the direct targets of Smad proteins (10 , 45) . In MG63 cells, p21WAF1/CIP1 was up-regulated by TGF-ß, but cell proliferation was still stimulated. This result suggested that certain signaling pathways of TGF-ß leading to growth inhibition are still active, but that some mechanism canceling the growth inhibition by p21WAF1/CIP1 may become predominant. In this respect, it should be noted that the induction of p21WAF1/CIP1 by TGF-ß in MG63 cells was only transient. We also observed that c-Myc was up-regulated in a sustained fashion in response to TGF-ß stimulation in MG63 cells. Induction of c-Myc by TGF-ß has already been reported by Leof et al. (11) in AKR-2B cells, which are also growth stimulated by TGF-ß. One of the functions of c-Myc in cell cycle progression is to down-regulate expression of CDKIs including p21WAF1/CIP1. Thus, up-regulation of c-Myc in MG63 cells may cause the transient induction of p21WAF1/CIP1, which may lead, at least in part, to abrogation of growth-inhibitory signaling by TGF-ß.
In the present study, we used SB-431542 to selectively inhibit ALK-5 signaling but observed an inhibitory effect of SB-431542 on ligand-induced ALK-1 signaling in MG63 cells. Inman et al. (36) reported that this inhibitor was not effective on the constitutively active form of ALK-1 in which Gln-201 was mutated to Asp (QD mutant). There appears to be two possible explanations for this observation. One is that SB-431542 has differential effects on ligand-activated ALK-1 kinase and mutationally activated ALK-1 kinase. Alternatively, ALK-1 requires ALK-5 kinase activity for its activation by ligands. The mechanism of inhibition of ALK-1 signaling by SB-431542 remains to be elucidated.
Many tumor cells are not responsive to TGF-ß and sometimes secrete TGF-ß. TGF-ß may induce growth factor secretion from stromal cells, and the secreted growth factors may in turn enhance proliferation of cancer cells. This is thought to be one mechanism by which TGF-ß activity enhances malignancy of cancer (46) . Thus, inhibition of growth factor induction may lead to suppression of tumor growth. Moreover, PDGF was reported to cause high interstitial fluid pressure in tumor tissues. High interstitial fluid pressure acts as a barrier to tumor transvascular transport, decreasing uptake of antitumor reagents by tumors. Recently, Pietras et al. (47) reported that inhibition of PDGF activity by Gleevec enhanced the efficacy of chemotherapeutic treatment by decreasing the tumor interstitial fluid pressure. In the present study, we found that SB-431542, a novel ALK-4/5/7 inhibitor, can block induction of growth factors including PDGF. Thus, synthetic kinase inhibitors of ALK-5 may have additional effects in vivo, mediated through inhibition of PDGF induction, if used together with chemotherapeutic drugs. Development of clinically available ALK-5 kinase inhibitors is thus highly desired.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Requests for reprints: Kohei Miyazono, Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Phone: 81-3-5841-3345; Fax: 81-3-5841-3354; E-mail: miyazono-ind{at}umin.ac.jp
S. Matsuyama and M. Iwadate contributed equally to this work.
6 The abbreviations used are: TGF, transforming growth factor; BMP, bone morphogenetic protein; ALK, activin receptor-like kinase; CDKI, cyclin-dependent kinase inhibitor; PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; RT-PCR, reverse transcription-PCR; HB-EGF, heparin-binding EGF-like growth factor; PAI-1, plasminogen activator inhibitor 1; PlGF, placenta growth factor. 
Received 4/ 4/03. Revised 9/ 3/03. Accepted 9/ 8/03.
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, control samples; 
, SB-431542 (0.3 µM)-treated samples; 
, Gleevec (1 µM)-treated samples.