February 2010
Volume 51, Issue 2
Free
Biochemistry and Molecular Biology  |   February 2010
Trichostatin A–Induced TGF-β Type II Receptor Expression in Retinoblastoma Cell Lines
Author Affiliations & Notes
  • Yoshiko Kashiwagi
    From the Departments of Ocular Cellular Engineering and
  • Kuniko Horie
    the Research Center for Genomic Medicine and
  • Chikako Kanno
    Ophthalmology and Visual Science, Yamagata University Faculty of Medicine, Yamagata City, Japan;
  • Motoko Inomata
    the Faculty of Medicine, Saitama Medical University, Hidaka City, Japan;
  • Takashi Imamura
    the Department of Biochemistry, JFCR Cancer Institute, Tokyo, Japan; and
  • Mitsuyasu Kato
    the Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba City, Japan.
  • Teiko Yamamoto
    From the Departments of Ocular Cellular Engineering and
  • Hidetoshi Yamashita
    Ophthalmology and Visual Science, Yamagata University Faculty of Medicine, Yamagata City, Japan;
  • Corresponding author: Yoshiko Kashiwagi, Department of Ocular Cellular Engineering, Yamagata University Faculty of Medicine, 2–2-2 Iida-nishi, Yamagata City, Yamagata, Japan; kasiwagi@med.id.yamagata-u.ac.jp
Investigative Ophthalmology & Visual Science February 2010, Vol.51, 679-685. doi:10.1167/iovs.09-4073
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      Yoshiko Kashiwagi, Kuniko Horie, Chikako Kanno, Motoko Inomata, Takashi Imamura, Mitsuyasu Kato, Teiko Yamamoto, Hidetoshi Yamashita; Trichostatin A–Induced TGF-β Type II Receptor Expression in Retinoblastoma Cell Lines. Invest. Ophthalmol. Vis. Sci. 2010;51(2):679-685. doi: 10.1167/iovs.09-4073.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Retinoblastoma, an intraocular malignant tumor of childhood, is caused by a mutation in the retinoblastoma tumor-suppressor gene RB. Retinoblastoma cells are thought to be resistant to transforming growth factor-β (TGF-β) because they do not express the TGF-β type II receptor (TβR-II). In several tumor cell lines, trichostatin A (TSA), a potent inhibitor of histone deacetylase, induces expression of the TβR-II gene. The objective of the present study was to determine the effects of TSA on TβR-II gene expression in retinoblastoma cells.

Methods.: Four retinoblastoma cell lines were transfected with a TβR-II promoter-luciferase reporter construct and analyzed for the effect of TSA on TβR-II mRNA expression, TβR-II promoter activity, transforming growth factor (TGF)-β–related signal transduction, and cell growth using RT-PCR, Western blot analysis, chromatin immunoprecipitation, luciferase activity assay, and cell viability assays.

Results.: TSA treatment induced the expression of TβR-II mRNA, activated the TβR-II promoter, and inhibited cell growth in the examined retinoblastoma cell lines. It did not restore TGF-β–related signaling, however.

Conclusions.: These data show that TSA induces the expression of TβR-II mRNA and activates the TβR-II promoter in retinoblastoma cells. However, TSA treatment alone was insufficient to restore TGF-β signaling in these cell lines. The inhibitory effect of TSA on cell growth may be unrelated to its effect on TβR-II expression.

Retinoblastoma, an intraocular malignant tumor of childhood, 1 is caused by a heritable mutation of the retinoblastoma tumor suppressor gene RB.2 Although inactivation of the two RB alleles is the most important event in the oncogenesis of retinoblastoma, 2 other oncogenes or tumor suppressor genes may also contribute to the aggressive nature of this tumor. 
Transforming growth factor-β (TGF-β) is a multifunctional cytokine that acts as a potent growth inhibitor in various cell types. 3 Intracellular signaling by TGF-β begins with the assembly of a heterotetrameric complex of two types of transmembrane TGF-β receptors, type I and type II (TβR-I, TβR-II, respectively), 4 each of which has an intracellular serine/threonine kinase domain. On ligand-induced formation of the heteromeric complex, TβR-I is activated by the constitutively active TβR-II. 5  
The Smad proteins are major signaling molecules that act downstream of serine/threonine kinase receptors. 4 There are three types of Smads: receptor-regulated Smads (Smad2, Smad3, and Smad4), common-partner Smads, and inhibitory Smads (Smad6 and Smad7). 4 Resistance to TGF-β–mediated growth suppression in tumor cells is often associated with the functional loss of TGF-β receptors and their downstream Smad molecules. 6 Restoration of the functional TGF-β signaling pathway can repress the tumorigenic phenotype of colon, breast, and pancreatic cancer cells that were previously insensitive to TGF-β. 711 Therefore, TGF-β and its downstream signaling molecules act as tumor suppressors. 810  
Although normal retinal cells have intact TGF-β signaling pathways, TGF-β does not bind to the cell surface receptors of retinoblastoma cells and, consequently, does not inhibit the growth of these cells. 12 We previously demonstrated that the TGF-β resistance exhibited by the retinoblastoma cell lines Y-79 and WERI-Rb-1 (WERI) is caused by a lack of TβR-II. 13 We also showed that sodium butyrate, a potent inhibitor of histone deacetylase (HDAC), induces TβR-II mRNA expression in Y-79 cells. 
HDAC plays a major role in the regulation of gene transcription, 14 and HDAC inhibitors are a promising new class of anticancer agents. 15,16 One such inhibitor, suberoylanilide hydroxamic acid (SAHA), has recently been approved for clinical use, and others are currently being investigated in clinical trials for leukemia, lymphoma, and breast, prostate, ovarian, and other cancers. 17,18 HDAC inhibitors have been reported to inhibit cell growth and to induce apoptosis in retinoblastoma cell lines. 19,20 Trichostatin A (TSA), a potent inhibitor of HDAC, 14 has been found to induce expression of the TβR-II gene in several tumor cell lines. 2126 In the present study, we found that TSA also induces the mRNA expression of TβR-II in four human retinoblastoma cell lines. 
Methods
Cell Culture
The retinoblastoma cell lines Y-79 and WERI were obtained from American Type Culture Collection (Manassas, VA). NCC-RBC-51 (R51), NCC-RBC-54 (R54), and HaCaT human keratinocyte cell line were provided by Motoko Inomata, Kuniko Horie, and Mitshyasu Kato. Y-79 and WERI cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS; JRA Bioscience, Lenexa, KS), and R51 and R54 cells were cultured in RB2+ medium. 27 HaCaT cells were cultured in Dulbecco's modified Eagle's medium (Gibco) containing 10% FBS. Cells were grown under a humidified 5% CO2 atmosphere at 37°C. 
Treatment with Trichostatin A
TSA was obtained from Sigma-Aldrich (St. Louis, MO), dissolved in ethanol at a concentration of 0.5 mM, and added to the medium at a concentration 0.5 μM. When ethanol was added to the cell growth medium at concentrations up to 0.1%, it had no effect on cell proliferation and gene expression. 
Luciferase Assay for Smad Signaling
Cells were seeded in duplicate into 24-well plates (CellStar; Greiner Bio-One GmbH, Frickenhausen, Germany) at 5 × 105 cells/well in their respective growth medium. To assess Smad signaling, we used a plasmid construct in which a luciferase reporter gene was joined to 12 repeats of the Smad-binding CAGA motif. This plasmid, pGL3–(CAGA)12–luciferase, was transfected into cells in the presence or absence of TβR-I or TβR-II cDNA using transfection agent (FuGENE6; Roche Diagnostics, Basel, Switzerland). The Renilla luciferase reporter plasmid pRL-CMV (Promega, Madison, WI) was cotransfected as an internal control for transfection efficiency. Cells were incubated for 24 hours after transfection and then treated with or without 10 ng/mL TGF-β1 (R&D Systems, Minneapolis, MN) for 24 hours. Luciferase activity in the cells was measured with an assay system (Dual-Luciferase Reporter Assay System; Promega) according to the manufacturer's instructions using a luminometer (LB9507; Berthold Technologies GmbH, Bad Wildbad, Germany). 
Alternatively, to examine the effect of TSA treatment on Smad signaling, the cells were transfected with the pGL3–(CAGA)12–luciferase reporter construct alone, and 24 hours later they were treated with or without 0.5 μM TSA for 36 hours. They were then treated with 10 ng/mL TGF-β1 for 24 hours, and luciferase activity was measured as described. 
Luciferase Assay for TβR-II Promoter Activity
pGL3-TβR-II promoter constructs containing the luciferase reporter gene joined to the TβR-II promoter region spanning nucleotides −295 to +50 were cotransfected with plasmid pRL-CMV (Promega) into retinoblastoma cells, as described. Twenty-four hours after transfection, the cells were incubated with 0.5 μM TSA for another 24 hours before luciferase activity was measured, as described. 
Reverse Transcriptase-Polymerase Chain Reaction Analysis
Total RNA was prepared by disruption of cells in reagent (Isogen; Nippon Gene, Toyama, Japan) according to the manufacturer's method. Total RNA (2 μg) was reverse-transcribed with 200 U reverse transcriptase (Promega), 0.5 μg oligo dT(16) primer (Promega), and 20 U RNase inhibitor (Takara-Bio, Shiga, Japan) for 60 minutes. The resultant cDNA was PCR-amplified in 25 μL reaction mixtures containing 1.5 μL cDNA, 200 μM dNTP, 1 μM primers (sequences shown below), and 1 U enzyme blend (FastStart High Fidelity; Roche Diagnostics). PCR was performed using 30 cycles (or 35 cycles for TβR-I and TβR-II) of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and elongation 72°C for 30 seconds, followed by a final elongation step at 72°C for 7 minutes. The forward (Fw) and reverse (Rv) primer sequences for SMAD2 (accession no. NM_005901) were as follows: forward primer (Fw) 5′-AGTATGGACACAGGCTCTCC-3′, reverse primer (Rv) 5′-GTCTGCCTTCGGTATTCTG-3′, respectively, to yield a 554-bp product. The other primer sequences and predicted product lengths were as follows: SMAD3 (NM_005902): Fw 5′-ACTGCGAGCCGGCCTTCTGG-3′, Rv 5′-CACTCTGCGAAGACCTCCCC-3′, to yield a 244-bp product; SMAD4 (NM_005359): Fw 5′-ATCTGAGTCTAATGCTACCAGC-3′, Rv 5′-TTCTTTGATGCTCTGTCTTGG-3′, to yield a 952-bp product; SMAD6 (NM_005585): Fw 5′-CCACTGGATCTGTCCGATTC-3′, Rv 5′-AAGTCGAACACCTTGATGGAG-3′, to yield a 455-bp product; SMAD7 (NM_005904): Fw 5′-TCCTGCTGTGCAAAGTGTTC-3′, Rv 5′-AGTAAGGAGGAGGGGGAGAC-3′, to yield a 166-bp product; TβR-I (NM_004612): Fw 5′-AGATTACCAACTGCCTTATT-3′, Rv 5′-TATCCTTCTGTTCCCTCTCA-3′, to yield a 354-bp product; TβR-II (NM_003242): Fw 5′-GCAGTGGGAGAAGTAAAAGA-3′, Rv 5′-CCAGCCTGCCCCATAAGAGC-3′, to yield a 337-bp product; GAPDH (NM_002046): Fw 5′-CAAAGTTGTCATGGATGACC-3′, Rv 5′-CCATGGAGAAGGCTGGGG-3′, to yield a 195-bp product. The PCR products were electrophoresed in 2% agarose gels and stained with ethidium bromide. 
Western Blot Analysis of Acetylated Histone H4 Levels
Cells were collected and lysed in sample buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate) containing protease inhibitor cocktail (Roche Diagnostics) and 1 mM phenylmethylsulfonyl fluoride (PMSF; Wako Pure Chemical Industries, Osaka, Japan) at 4°C. Protein concentrations in the cell lysates were determined using a protein assay kit (Bio-Rad, Hercules, CA), and 20-μg aliquots were denatured and subjected to electrophoresis in 16% polyacrylamide gel. After electrophoresis, samples were transferred to polyvinylidene difluoride (PVDF) membranes (Immuno-blot PVDF Membrane; Bio-Rad). which were then incubated with a primary antibody against acetylated histone H4 (AcH4) (1:1000 dilution; Millipore, Billerica, MA) or β-actin (20 ng/mL; Sigma–Aldrich) followed by horseradish peroxidase-conjugated secondary antibody (1 μg/mL; GE Healthcare, Little Chalfont, UK) in blocking solution. Immunoreactive bands were detected using a Western blotting detection system (ECL Plus; GE Healthcare). 
Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation (ChIP) assay was carried out as described by Zhao et al. 24 The chromatin-protein complexes were immunoprecipitated with anti–AcH4 antibody (Upstate Biotechnology, Lake Placid, NY). Primers used for the TβR-II promoter (gene accession no. U37070) were 5′-GTAAATACTTGGAGCGAGGAAC-3′ (Fw) and 5′-ACTCACTCAACTTCAACTCAGC-3′ (Rv), yielding a 236-bp product. 24 Primers used for the β-actin promoter (E06566) were 5′-CCAACGCCAAAACTCCC-3′ (Fw) and 5′-AGCCATAAAAGGCAACTTTCG-3′, yielding a 170-bp product. 25 PCR was performed using 33 cycles of denaturation at 95°C for 30 seconds, annealing at 50°C for 30 seconds, and elongation 72°C for 30 seconds, followed by a final elongation step at 72°C for 7 minutes The PCR products were electrophoresed in 2% agarose gels and stained with ethidium bromide. 
Cell Titer Blue Assay
An assay kit (Cell Titer-Blue Viability Assay; Promega) was used to evaluate cell viability. Cells were seeding in duplicate into 96-well plates (CellStar; Greiner Bio-One GmbH) at a density of 5 × 104 cells/well in RPMI 1640 containing 10% FBS and cultured with or without 0.5 μM TSA for 24 or 48 hours. Twenty microliters of reagent solution (Cell Titer-Blue Reagent; Promega) was added to each well, and the cells were incubated for 4 hours at 37°C under a 5% CO2 atmosphere. Cellular fluorescence was measured using a microplate spectrofluorometer (Gemini EM; Molecular Devices, Sunnyvale, CA) with excitation and emission wavelengths of 560 and 590 nm, respectively. 
Smad and Phospho-Smad (pSmad) Western Blot Analysis
Cells were collected and lysed in sample buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 1% Nonidet-P 40, 0.25% sodium deoxycholate) 28 containing phosphatase inhibitor (PhosSTOP; Roche Diagnostics), a protease inhibitor cocktail, and 1 mM PMSF at 4°C. Protein concentrations in the cell lysates were determined using a Bio-Rad protein assay kit, and 50-μg aliquots were subjected to electrophoresis in 10% polyacrylamide gels. After the separated proteins were transferred to PVDF membranes (Bio-Rad), the membranes were reacted with a primary antibody (diluted 1:1000) against SMAD2, SMAD3, or pSMAD3 (all three from Cell Signaling Technology, Danvers, MA), pSMAD2 (a gift from TI), or β-actin (control). The membranes were then incubated with an alkaline phosphatase-conjugated secondary antibody (1 μg/mL; Vector Laboratories, Burlingame, CA) in blocking solution. Immunoreactive bands were detected using nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (Vector Laboratories). 
TβR-II Western Blot Analysis
Western blot analysis of TβR-II expression was carried out as described for analysis of Smad expression, except that the sample buffer did not contain phosphatase inhibitor (PhosSTOP; Roche Diagnostics). Aliquots (100 μg) were used for electrophoresis, and the primary antibody was anti–TβR-II antibody (1:50 dilution; Santa Cruz Biotechnology). Anti–β-actin antibody was used as a control. 
Results
Transient Overexpression-Induced TGF-β Signal Restoration in Retinoblastoma Cell Lines
As we previously reported, overexpression of TβR-II in the WERI retinoblastoma cell line restores TGF-β sensitivity in these cells. 13 To determine whether overexpression of TβR-II in other retinoblastoma cell lines would also restore the TGF-β response, we performed luciferase assays in Y-79, NCC-RBC-51 (R51), and NCC-RBC-54 (R54) cells transfected with a luciferase reporter gene joined to 12 repeats of the Smad-binding CAGA motif, which was identified in TGF-β–responsive Smad-binding sites in the PAI-1 and junB promoters. 29,30 Ectopic expression of TβR-II activated the luciferase activity of the (CAGA)12–luciferase construct in Y-79, WERI, and R51 cells (Fig. 1A), and marked ligand-dependent activation was observed in Y-79 and WERI cells. However, activation of the (CAGA)12–luciferase construct was not observed in R54 cells ectopically expressing either TβR-I or TβR-II (data not shown). However, Smad binding-induced luciferase activity was observed in R54 cells when both TβR-II and SMAD3 were ectopically expressed (Fig. 1B). Thus, ectopic expression of TGF-β receptors in retinoblastoma cell lines induced TGF-β/Smad–mediated signaling (Fig. 1). 
Figure 1.
 
TGF-β signal transduction in retinoblastoma cell lines is restored by transient overexpression of TGF-β receptors and Smad3. (A) Transcriptional activation of the Smad response sequence (CAGA) in three retinoblastoma cell lines cotransfected with cDNAs encoding TGF-β receptor (TβR)-I and/or TβR-II and a (CAGA)12–luciferase reporter gene. Transcriptional activation was determined using luciferase activity assays. (B) Transcriptional activation of the Smad response sequence in R54 cells cotransfected with the (CAGA)12–luciferase reporter gene and a cDNA encoding TβR-II or SMAD2, 3, or 4. For the cDNAs, − and + indicate no transfection and transfection, respectively, and for TGF-β1, − and + indicate no treatment for 24 hours or treatment with TGF-β1 (10 ng/mL) for 24 hours, respectively. Error bars represent the SD; n = 3. *P < 0.05, Mann–Whitney U test.
Figure 1.
 
TGF-β signal transduction in retinoblastoma cell lines is restored by transient overexpression of TGF-β receptors and Smad3. (A) Transcriptional activation of the Smad response sequence (CAGA) in three retinoblastoma cell lines cotransfected with cDNAs encoding TGF-β receptor (TβR)-I and/or TβR-II and a (CAGA)12–luciferase reporter gene. Transcriptional activation was determined using luciferase activity assays. (B) Transcriptional activation of the Smad response sequence in R54 cells cotransfected with the (CAGA)12–luciferase reporter gene and a cDNA encoding TβR-II or SMAD2, 3, or 4. For the cDNAs, − and + indicate no transfection and transfection, respectively, and for TGF-β1, − and + indicate no treatment for 24 hours or treatment with TGF-β1 (10 ng/mL) for 24 hours, respectively. Error bars represent the SD; n = 3. *P < 0.05, Mann–Whitney U test.
Effects of TSA on the mRNA Expression in Retinoblastoma Cell Lines
We next investigated the expression of TGF-β receptors and Smad proteins at the mRNA level using RT-PCR analysis. HaCaT cells, which are responsive to TGF-β, were therefore used as a positive-response model for TGF-β and were also used as positive controls for TGF-β signaling-related gene expression in the RT-PCR analysis. Although expression of the mRNAs encoding TβR-I and SMAD2, 3, 4, 6, and 7 was observed in all four of the retinoblastoma cell lines, expression of TβR-II mRNA was not observed in any of the retinoblastoma cell lines (Fig. 2A). To investigate whether TSA would induce TβR-II mRNA expression in these cell lines, we treated them with 0.5 μM TSA and analyzed TβR-II mRNA expression. RT-PCR analysis revealed that TSA treatment for 24 hours induced the expression of TβR-II mRNA in all the cell lines but had no effect on the expression of TβR-I mRNA (Fig. 2B). 
Figure 2.
 
TSA induces TβR-II mRNA expression and activates the TβR-II promoter in four retinoblastoma cell lines. (A) RT-PCR analysis of TβR-I, TβR-II, and Smad mRNA levels in four retinoblastoma cell lines. HaCaT cells are positive controls. (B) RT-PCR analysis of TβR-I and TβR-II mRNA levels in retinoblastoma cells treated with (+) or without (−) TSA for 24 hours. (C) Luciferase activity in retinoblastoma cells transfected with a TβR-II promoter–luciferase reporter gene (filled bar) or the empty pGL3 vector (negative control; open bar) and treated with TSA for 24 hours. The data are normalized for transfection efficiency using a cotransfected Renilla luciferase reporter gene. Error bars represent the SD; n = 3. *P < 0.05, Mann–Whitney U test. (D) RT-PCR analysis of Smad mRNA levels in cells treated with (+) or without (−) TSA for 24 hours.
Figure 2.
 
TSA induces TβR-II mRNA expression and activates the TβR-II promoter in four retinoblastoma cell lines. (A) RT-PCR analysis of TβR-I, TβR-II, and Smad mRNA levels in four retinoblastoma cell lines. HaCaT cells are positive controls. (B) RT-PCR analysis of TβR-I and TβR-II mRNA levels in retinoblastoma cells treated with (+) or without (−) TSA for 24 hours. (C) Luciferase activity in retinoblastoma cells transfected with a TβR-II promoter–luciferase reporter gene (filled bar) or the empty pGL3 vector (negative control; open bar) and treated with TSA for 24 hours. The data are normalized for transfection efficiency using a cotransfected Renilla luciferase reporter gene. Error bars represent the SD; n = 3. *P < 0.05, Mann–Whitney U test. (D) RT-PCR analysis of Smad mRNA levels in cells treated with (+) or without (−) TSA for 24 hours.
Effects of TSA on the TβR-II Promoter Activation in Retinoblastoma Cell Lines
To determine whether TSA treatment activates the TβR-II promoter, we transfected retinoblastoma cells with a TβR-II promoter–luciferase reporter gene and analyzed luciferase activity after treatment of the cells with 0.5 μM TSA for 24 hours. Cells expressing the TβR-II promoter constructs exhibited a robust increase in luciferase activity over that observed in cells expressing the control vector lacking the TβR-II promoter sequence (Fig. 2C). 
Our results indicated that TSA activates the transcriptional activity of the proximal promoter of TβR-II. Therefore, we next examined cellular levels of AcH4 in the presence or absence of TSA treatment. Western blot analysis showed that TSA treatment markedly increased the accumulation of AcH4 (Fig. 3A). Furthermore, ChIP analysis indicated a TSA-induced increase in AcH4 recruitment to the TβR-II promoter in all the retinoblastoma cell lines (Fig. 3B). These results suggest that TSA treatment promotes histone acetylation, thereby activating the transcription of TβR-II
Figure 3.
 
TSA induces AcH4 accumulation and inhibits cell growth in retinoblastoma cells. (A) Western blot analysis of AcH4 protein levels in retinoblastoma cells treated with (+) or without (−) TSA for 24 hours. β-Actin was used as a loading control. Each lane contains 20 μg protein. (B) ChIP assay analysis of TβR-II promoter/AcH4 binding in retinoblastoma cells treated with (+) or without (−) TSA for 24 hours. AcH4 immunoprecipitation experiments performed with (+) or without (−) anti–AcH4 antibody are shown. (C) Cell growth with (+; ▴) or without (−; ■; control) TSA treatment. Error bars represent the SD; n = 7. *P < 0.05, Mann–Whitney U test.
Figure 3.
 
TSA induces AcH4 accumulation and inhibits cell growth in retinoblastoma cells. (A) Western blot analysis of AcH4 protein levels in retinoblastoma cells treated with (+) or without (−) TSA for 24 hours. β-Actin was used as a loading control. Each lane contains 20 μg protein. (B) ChIP assay analysis of TβR-II promoter/AcH4 binding in retinoblastoma cells treated with (+) or without (−) TSA for 24 hours. AcH4 immunoprecipitation experiments performed with (+) or without (−) anti–AcH4 antibody are shown. (C) Cell growth with (+; ▴) or without (−; ■; control) TSA treatment. Error bars represent the SD; n = 7. *P < 0.05, Mann–Whitney U test.
Expression and Regulation of SMAD6 and SMAD7 by TSA in Retinoblastoma Cell Lines
To investigate whether TSA affects the expression of Smad proteins in retinoblastoma cell lines, we treated retinoblastoma cells with 0.5 μM TSA for 24 hours and analyzed Smad mRNA expression using RT-PCR. TSA did not alter the level of SMAD2, 3, or 4 mRNA (Fig. 2D), but it decreased the levels of the mRNAs encoding the inhibitory Smad proteins (SMAD6 and SMAD7) in all cell lines (Fig. 2D). 
Effects of TSA on Cell Growth in Retinoblastoma Cell Lines
We expected the TSA treatment to activate TGF-β signaling pathways in the retinoblastoma cell lines. Therefore, we next investigated the effect of TSA on the growth of these cells. In the absence of TSA treatment, cell growth (as indicated by fluorescence in the Cell Titer-Blue Viability Assay; Promega) continued to increase in all four cell lines (Fig. 3C). In contrast, fluorescence slowly decreased in cells treated with TSA, indicating the inhibition of cell growth by TSA. 
Effects of TSA on the TβR-II Protein Expression in Retinoblastoma Cell Lines
To determine whether TSA treatment restores the TGF-β response in retinoblastoma cell lines, we performed luciferase reporter assays in TGF-β–stimulated cells transfected with the (CAGA)12–luciferase reporter construct. However, TSA treatment for 24 hours did not alter luciferase activity in any of the examined retinoblastoma cell lines. 
We next examined the phosphorylation of SMAD2 and SMAD3 in response to TGF-β using Western blot analysis. Phosphorylated SMAD2 and SMAD3 (pSMAD2 and pSMAD3, respectively) are markers for the activation of TGF-β receptors. HaCaT cells were used as a TGF-β positive-response model. Treatment of HaCaT cells with TGF-β1 for 24 hours significantly increased the levels of pSMAD2 and pSMAD3. In contrast, no pSMAD2 or pSMAD3 was detected in any of the four retinoblastoma cell lines after TSA treatment for 36 hours, and the pSMAD2 and pSMAD3 levels in TSA-treated retinoblastoma cells did not increase on subsequent treatment with TGF-β1 for 24 hours. No expression of SMAD3 protein was detected in R54 cells. 
Effects of TSA on the TβR-II Protein Expression in Retinoblastoma Cell Lines
We next used Western blot analysis to investigate whether TSA induces TβR-II protein expression in retinoblastoma cells. No expression of TβR-II protein was observed in any of the examined retinoblastoma cell lines before or after TSA treatment (Fig. 4C). 
Figure 4.
 
TSA treatment does not affect TGF-β signal transduction in retinoblastoma cell lines. (A) Transcriptional activity of the Smad response sequence (CAGA) in four retinoblastoma cell lines transfected with a (CAGA)12–luciferase reporter gene, treated with TSA for 36 hours and treated with (+; filled bar) or without (−; open bar) TGF-β1 for 24 hours. The activity of the Smad response sequence was then determined using luciferase activity assays. The data are normalized for transfection efficiency using a cotransfected Renilla luciferase reporter gene. Error bars represent the SD; n = 3. *P < 0.05, Mann–Whitney U test. (B) Western blot analysis of Smad protein phosphorylation in retinoblastoma cells treated with (+) TSA for 36 hours and then with (+) or without (−) TGF-β1 for 24 hours. β-Actin was used as a loading control. Each lane contains 50 μg protein. (C) Western blot analysis of TβR-II protein levels in retinoblastoma cells treated with (+) TSA for 48 hours. β-Actin was used as a loading control. Each lane contains 100 μg protein.
Figure 4.
 
TSA treatment does not affect TGF-β signal transduction in retinoblastoma cell lines. (A) Transcriptional activity of the Smad response sequence (CAGA) in four retinoblastoma cell lines transfected with a (CAGA)12–luciferase reporter gene, treated with TSA for 36 hours and treated with (+; filled bar) or without (−; open bar) TGF-β1 for 24 hours. The activity of the Smad response sequence was then determined using luciferase activity assays. The data are normalized for transfection efficiency using a cotransfected Renilla luciferase reporter gene. Error bars represent the SD; n = 3. *P < 0.05, Mann–Whitney U test. (B) Western blot analysis of Smad protein phosphorylation in retinoblastoma cells treated with (+) TSA for 36 hours and then with (+) or without (−) TGF-β1 for 24 hours. β-Actin was used as a loading control. Each lane contains 50 μg protein. (C) Western blot analysis of TβR-II protein levels in retinoblastoma cells treated with (+) TSA for 48 hours. β-Actin was used as a loading control. Each lane contains 100 μg protein.
Discussion
The resistance of tumor cells to TGF-β–mediated growth suppression is often associated with the functional loss of TGF-β receptors and their downstream Smad proteins. 8 TβR-II has been reported to be epigenetically silenced or downregulated in several different cell types. 2125 We previously reported that resistance to TGF-β in the human retinoblastoma cell lines Y-79 and WERI is caused by a lack of TβR-II. 13 In addition, we reported that overexpression of TβR-II in WERI cells restores TGF-β sensitivity. 13 In the present study, we determined the effects of TSA on TβR-II gene expression in Y-79, WERI, and two other human retinoblastoma cell lines, NCC-RBC-51 (R51) and NCC-RBC-54 (R54). 
Overexpression of TβR-II restored TGF-β sensitivity in Y-79, WERI, and R51 cells (Fig. 1A). In the R54 cell line, however, TβR-II overexpression restored TGF-β sensitivity only when SMAD3 was also overexpressed (Fig. 1B), even though a modest level of SMAD3 protein was detected in R54 cells in the absence of TβR-II overexpression (Fig. 4B). We surmise that retinoblastoma cells have functional losses in several signaling molecules acting downstream of TGF-β. 
We found that TSA induced the mRNA expression of TβR-II and increased its promoter activity in human retinoblastoma cell lines (Figs. 2B, C). We also found that TSA promoted the accumulation of AcH4 protein and its association with the TβR-II promoter sequence (Figs. 3A, B). These results are similar to those previously reported in several tumor cell lines. 2126 In pancreatic ductal adenocarcinoma cells, HDAC1 has been reported to form a complex with Sp1, which binds to the region flanking nucleotide −102 of the TβR-II promoter (Sp1C site) and consequently represses transcriptional activation of TβR-II. 24 Taken together, these findings suggest that histone deacetylation plays a role in repression of the TβR-II gene and that TSA activates TβR-II transcription by inhibiting HDAC in retinoblastoma cells. 
However, TSA treatment for 36 hours did not restore the TGF-β signaling response or induce detectable levels of SMAD2 or SMAD3 phosphorylation in any of the retinoblastoma cell lines (Fig. 4), all which expressed SMAD2, 3, 4, 6, and 7 mRNA (Fig. 2A). Of the receptor-regulated Smad proteins (SMAD2, 3, 4), neither the SMAD2 nor SMAD3 mRNA or protein levels nor the SMAD4 protein level was affected by TSA treatment (Figs. 2D, 4B). However, TSA treatment decreased the expression of SMAD6 and SMAD7, the inhibitory Smads (Fig. 2D). Smad7 has been reported to block Smad2 and Smad3 and to consequently prevent TGF-β signaling. 4 From our Smad expression results, we infer that TSA readily activates TGF-β signaling. However, our results also suggest that TSA does not restore TGF-β signaling regardless of whether the levels of receptor-regulated and inhibitory Smad mRNA are maintained or decreased. SAHA, an HDAC inhibitor, reportedly restores TGF-β sensitivity in breast cancer cells by restoring TβR-I expression. 31 However, few studies have examined the restoration of TGF-β signaling by HDAC inhibitors. We confirmed that the transcriptional activation of TβR-II by TSA resulted in transcription of the correct open reading frame in retinoblastoma cell lines by sequencing the mRNA (data not shown). Despite the transcriptional activation of TβR-II by TSA, however, TSA failed to induce expression of the TβR-II protein, as determined by Western blot analysis (Fig. 4C). These results suggest that TSA does not induce TβR-II protein translation or TGF-β signaling in retinoblastoma cell lines. We do not know why the TβR-II protein was not translated in our cells; retinoblastoma cells might suffer from inhibited TβR-II translation initiation or rapid degeneration of the TβR-II translation product. 
Karasawa et al. 19 and Dalgard et al. 20 have reported that TSA inhibits cell growth and induces apoptosis of retinoblastoma cell lines. We obtained similar results in four retinoblastoma cell lines (Fig. 3C). TSA-mediated inhibition of cell growth in retinoblastoma cell lines might be unrelated to the restoration of TGF-β expression. The major effect of TSA in retinoblastoma cell lines might be the induction of apoptosis. 19,20  
Dalgard et al. 20 advocate clinical testing of HDAC inhibitors in children with retinoblastoma. These compounds may be optimized in the near future for the clinical treatment of retinoblastoma. 
In summary, we have demonstrated that TSA activates the TβR-II promoter and induces TβR-II mRNA expression. However, TSA treatment alone is insufficient to restore TGF-β signaling to retinoblastoma cell lines. Our findings suggest that TSA-mediated inhibition of cell growth is not related to the TSA-mediated expression of TβR-II in retinoblastoma cells. 
Footnotes
 Supported by a grant from SENJU Pharmaceutical Co., Ltd.
Footnotes
 Disclosure: Y. Kashiwagi, None; K. Horie, None; C. Kanno, None; M. Inomata, None; T. Imamura, None; M. Kato, None; T. Yamamoto, None; H. Yam, None
The authors thank Chifumi Kitanaka (Department of Molecular Cancer Science, Yamagata University Faculty of Medicine) and Kaoru Goto and Sachiko Saino-Saito (Department of Anatomy and Cell Biology, Yamagata University Faculty of Medicine) for lead technical experimentation. 
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Figure 1.
 
TGF-β signal transduction in retinoblastoma cell lines is restored by transient overexpression of TGF-β receptors and Smad3. (A) Transcriptional activation of the Smad response sequence (CAGA) in three retinoblastoma cell lines cotransfected with cDNAs encoding TGF-β receptor (TβR)-I and/or TβR-II and a (CAGA)12–luciferase reporter gene. Transcriptional activation was determined using luciferase activity assays. (B) Transcriptional activation of the Smad response sequence in R54 cells cotransfected with the (CAGA)12–luciferase reporter gene and a cDNA encoding TβR-II or SMAD2, 3, or 4. For the cDNAs, − and + indicate no transfection and transfection, respectively, and for TGF-β1, − and + indicate no treatment for 24 hours or treatment with TGF-β1 (10 ng/mL) for 24 hours, respectively. Error bars represent the SD; n = 3. *P < 0.05, Mann–Whitney U test.
Figure 1.
 
TGF-β signal transduction in retinoblastoma cell lines is restored by transient overexpression of TGF-β receptors and Smad3. (A) Transcriptional activation of the Smad response sequence (CAGA) in three retinoblastoma cell lines cotransfected with cDNAs encoding TGF-β receptor (TβR)-I and/or TβR-II and a (CAGA)12–luciferase reporter gene. Transcriptional activation was determined using luciferase activity assays. (B) Transcriptional activation of the Smad response sequence in R54 cells cotransfected with the (CAGA)12–luciferase reporter gene and a cDNA encoding TβR-II or SMAD2, 3, or 4. For the cDNAs, − and + indicate no transfection and transfection, respectively, and for TGF-β1, − and + indicate no treatment for 24 hours or treatment with TGF-β1 (10 ng/mL) for 24 hours, respectively. Error bars represent the SD; n = 3. *P < 0.05, Mann–Whitney U test.
Figure 2.
 
TSA induces TβR-II mRNA expression and activates the TβR-II promoter in four retinoblastoma cell lines. (A) RT-PCR analysis of TβR-I, TβR-II, and Smad mRNA levels in four retinoblastoma cell lines. HaCaT cells are positive controls. (B) RT-PCR analysis of TβR-I and TβR-II mRNA levels in retinoblastoma cells treated with (+) or without (−) TSA for 24 hours. (C) Luciferase activity in retinoblastoma cells transfected with a TβR-II promoter–luciferase reporter gene (filled bar) or the empty pGL3 vector (negative control; open bar) and treated with TSA for 24 hours. The data are normalized for transfection efficiency using a cotransfected Renilla luciferase reporter gene. Error bars represent the SD; n = 3. *P < 0.05, Mann–Whitney U test. (D) RT-PCR analysis of Smad mRNA levels in cells treated with (+) or without (−) TSA for 24 hours.
Figure 2.
 
TSA induces TβR-II mRNA expression and activates the TβR-II promoter in four retinoblastoma cell lines. (A) RT-PCR analysis of TβR-I, TβR-II, and Smad mRNA levels in four retinoblastoma cell lines. HaCaT cells are positive controls. (B) RT-PCR analysis of TβR-I and TβR-II mRNA levels in retinoblastoma cells treated with (+) or without (−) TSA for 24 hours. (C) Luciferase activity in retinoblastoma cells transfected with a TβR-II promoter–luciferase reporter gene (filled bar) or the empty pGL3 vector (negative control; open bar) and treated with TSA for 24 hours. The data are normalized for transfection efficiency using a cotransfected Renilla luciferase reporter gene. Error bars represent the SD; n = 3. *P < 0.05, Mann–Whitney U test. (D) RT-PCR analysis of Smad mRNA levels in cells treated with (+) or without (−) TSA for 24 hours.
Figure 3.
 
TSA induces AcH4 accumulation and inhibits cell growth in retinoblastoma cells. (A) Western blot analysis of AcH4 protein levels in retinoblastoma cells treated with (+) or without (−) TSA for 24 hours. β-Actin was used as a loading control. Each lane contains 20 μg protein. (B) ChIP assay analysis of TβR-II promoter/AcH4 binding in retinoblastoma cells treated with (+) or without (−) TSA for 24 hours. AcH4 immunoprecipitation experiments performed with (+) or without (−) anti–AcH4 antibody are shown. (C) Cell growth with (+; ▴) or without (−; ■; control) TSA treatment. Error bars represent the SD; n = 7. *P < 0.05, Mann–Whitney U test.
Figure 3.
 
TSA induces AcH4 accumulation and inhibits cell growth in retinoblastoma cells. (A) Western blot analysis of AcH4 protein levels in retinoblastoma cells treated with (+) or without (−) TSA for 24 hours. β-Actin was used as a loading control. Each lane contains 20 μg protein. (B) ChIP assay analysis of TβR-II promoter/AcH4 binding in retinoblastoma cells treated with (+) or without (−) TSA for 24 hours. AcH4 immunoprecipitation experiments performed with (+) or without (−) anti–AcH4 antibody are shown. (C) Cell growth with (+; ▴) or without (−; ■; control) TSA treatment. Error bars represent the SD; n = 7. *P < 0.05, Mann–Whitney U test.
Figure 4.
 
TSA treatment does not affect TGF-β signal transduction in retinoblastoma cell lines. (A) Transcriptional activity of the Smad response sequence (CAGA) in four retinoblastoma cell lines transfected with a (CAGA)12–luciferase reporter gene, treated with TSA for 36 hours and treated with (+; filled bar) or without (−; open bar) TGF-β1 for 24 hours. The activity of the Smad response sequence was then determined using luciferase activity assays. The data are normalized for transfection efficiency using a cotransfected Renilla luciferase reporter gene. Error bars represent the SD; n = 3. *P < 0.05, Mann–Whitney U test. (B) Western blot analysis of Smad protein phosphorylation in retinoblastoma cells treated with (+) TSA for 36 hours and then with (+) or without (−) TGF-β1 for 24 hours. β-Actin was used as a loading control. Each lane contains 50 μg protein. (C) Western blot analysis of TβR-II protein levels in retinoblastoma cells treated with (+) TSA for 48 hours. β-Actin was used as a loading control. Each lane contains 100 μg protein.
Figure 4.
 
TSA treatment does not affect TGF-β signal transduction in retinoblastoma cell lines. (A) Transcriptional activity of the Smad response sequence (CAGA) in four retinoblastoma cell lines transfected with a (CAGA)12–luciferase reporter gene, treated with TSA for 36 hours and treated with (+; filled bar) or without (−; open bar) TGF-β1 for 24 hours. The activity of the Smad response sequence was then determined using luciferase activity assays. The data are normalized for transfection efficiency using a cotransfected Renilla luciferase reporter gene. Error bars represent the SD; n = 3. *P < 0.05, Mann–Whitney U test. (B) Western blot analysis of Smad protein phosphorylation in retinoblastoma cells treated with (+) TSA for 36 hours and then with (+) or without (−) TGF-β1 for 24 hours. β-Actin was used as a loading control. Each lane contains 50 μg protein. (C) Western blot analysis of TβR-II protein levels in retinoblastoma cells treated with (+) TSA for 48 hours. β-Actin was used as a loading control. Each lane contains 100 μg protein.
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