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TGF-β1 accelerates the DNA damage response in epithelial cells via Smad signaling
Biochemical and Biophysical Research Communications xxx (2016) 1e6
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TGF-b1 accelerates the DNA damage response in epithelial cells via Smad signaling Jeeyong Lee 1, Mi-Ra Kim 1, Hyun-Ji Kim, You Sun An, Jae Youn Yi* Division of Basic Radiation Bioscience, Korea Institute of Radiation and Medical Sciences, Seoul, 01812, Republic of Korea
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Article history: Received 23 May 2016 Accepted 25 May 2016 Available online xxx
The evidence suggests that transforming growth factor-beta (TGF-b) regulates the DNA-damage response (DDR) upon irradiation, and we previously reported that TGF-b1 induced DNA ligase IV (Lig4) expression and enhanced the nonhomologous end-joining repair pathway in irradiated cells. In the present study, we investigated the effects of TGF-b1 on the irradiation-induced DDRs of A431 and HaCaT cells. Cells were pretreated with or without TGF-b1 and irradiated. At 30 min post-irradiation, DDRs were detected by immunoblotting of phospho-ATM, phospho-Chk2, and the presence of histone foci (gH2AX). The levels of all three factors were similar right after irradiation regardless of TGF-b1 pretreatment. However, they soon thereafter exhibited downregulation in TGF-b1-pretreated cells, indicating the acceleration of the DDR. Treatment with a TGF-b type I receptor inhibitor (SB431542) or transfections with siRNAs against Smad2/3 or DNA ligase IV (Lig4) reversed this acceleration of the DDR. Furthermore, the frequency of irradiation-induced apoptosis was decreased by TGF-b1 pretreatment in vivo, but this effect was abrogated by SB431542. These results collectively suggest that TGF-b1 could enhance cell survival by accelerating the DDR via Smad signaling and Lig4 expression. © 2016 Elsevier Inc. All rights reserved.
Keywords: TGF-b1 DNA damage response Smads Lig4 Radioprotection
1. Introduction Transforming growth factor-b (TGF-b) is a multifunctional cytokine that regulates the growth, differentiation, migration, adhesion, and apoptosis of various cell types [1]. TGF-b transduces signals through two distinct serine-threonine kinase receptors, termed type I (TbRI) and type II (TbRII). Upon ligand binding, TbRII activates TbRI, which then phosphorylates the receptor-regulated Smads (R-Smads), Smad2 and Smad3, which interact with the common mediator Smad, Smad4, and move to the nucleus [1,2]. These nuclear Smad complexes interact with various transcription factors and transcriptional coactivators to regulate the transcription of target genes [3]. Radiotherapy is one of the best therapeutic choices for cancer treatment [4]. The cellular response to radiation-induced DNA damage is a complex process involving a wide and integrated array of signal transduction pathways [5]. The primary transducer of
* Corresponding author. Division of Basic Radiation Bioscience, Korea Institute of Radiation and Medical Sciences, 75 Nowon-gil, Nowon-gu, Seoul, 01812, Republic of Korea. E-mail address: [email protected] (J.Y. Yi). 1 Both authors contributed equally to the paper.
genotoxic stress caused by g-radiation is the nuclear protein kinase, ataxia telangiectasia mutated (ATM) [6]. ATM, which is activated by the double-strand breaks (DSBs) caused by ionizing radiation, phosphorylates numerous substrates, including p53, NBS1, histone 2AX (H2AX), BRCA1, and Chk2 [7e10]. These DNA damage response (DDR) proteins activate a complex program that controls cell cycle checkpoints, apoptosis, and genomic integrity. Several recent studies have begun to unravel the relationship between TGF-b1 and the irradiation-induced DDR, addressing the ability of TGF-b1 to act as either a radiosensitizer or a radioprotector. As a radiosensitizer, TGF-b1 reportedly mediates radiation-induced apoptosis in different cell types [11,12]. The TGFb1-targeted signaling molecule, Smad3, has been shown to obstruct the repair of damaged DNA [13] and modulate pro-survival ERKMAPK signaling [14]. Moreover, the inhibitory Smad, Smad7, has been shown to play a crucial role upstream of ATM and p53 in protecting the genome from g-irradiation [15]. In the context of TGF-b1 as a radioprotector, studies have shown that TGF-b1 treatment significantly enhances clonogenic survival, reduces DNA strand breaks, and increases p27 expression by inducing G1 arrest [16,17]. TGF-b1 was shown to enhance DNA repair activity through Smad signaling, thereby protecting cells from hyperoxic stress [18]. In addition, inhibition of TbRI was shown to induce radiosensitivity
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Please cite this article in press as: J. Lee, et al., TGF-b1 accelerates the DNA damage response in epithelial cells via Smad signaling, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.136
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[19,20] and attenuate the activation of ATM [21]. Finally, we recently showed that TGF-b1 protects cells against g-irradiation by enhancing nonhomologous end-joining (NHEJ) repair [22,23]. Despite the existing pool of knowledge, however, it remains unclear exactly how TGF-b signaling regulates the DDR network. To address this open question, we herein investigated whether TGF-b1 plays a role in the g-radiation-induced DDR of the epithelial cell lines, HaCaT and A431. Here, we report that TGF-b1 accelerates the DDR, as exhibited by the levels of phospho-ATM, phosphoChk2, and gH2AX. In addition, we report that the TGF-b1accelerated DDR is dependent on Smad signaling and DNA ligase IV (Lig4) expression. 2. Materials and methods 2.1. Cell culture, chemicals, and antibodies The HaCaT (human keratinocyte) and A431 (human epidermoid carcinoma) cell lines (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and 1X Zell shield (Minerva Biolabs). Cells were pretreated with the TbRI inhibitor, SB431542 (0.5 mM; Tocris) for 1 h, incubated with 0.5 ng/ml TGF-b1 (R&D Systems) for 24 h, and irradiated with 8 Gy using a Gamma-cell 3000 (Atomic Energy) or a BIOBEAM 8000 (Gamma-Service Medical GmbH) with a [137Cs] source. The antibodies against pATM, ATM, pChk2, Chk2, p21, phospho-H2AX (gH2AX), Smad2, and Smad3 were purchased from Cell Signaling; those against Lig4 and b-actin were purchased from Santa Cruz; and that against PCNA was purchased from Dako.
2.5. TUNEL assay TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assays were performed using an ApopTag kit (Millipore) in accordance with the manufacturer’s instructions. Apoptotic bodies were visualized using a DAB reagent set (KPL). 2.6. Immunohistochemistry Tissue sections were deparaffinized in a xylene-to-ethanol gradient and rinsed with PBS. The slides were placed in a domestic pressure cooker containing 1 mM EDTA buffer (pH 8.0) and boiled for 15 min. Endogenous peroxidase was blocked by incubating the sections for 10 min with 3% (v/v) H2O2 in methanol. The tissues were then rinsed with PBS, blocked with PBS containing 20% (v/v) horse serum, and incubated overnight at 4 C with anti-PCNA. After a 30-min incubation with diluted biotinylated secondary antibody, immunoreactivity was detected using a Vectastain ABC kit (Vector Laboratories). Sections were washed with PBS, antibody binding was visualized using a DAB reagent set, counterstaining was performed with hematoxylin, and the results were examined using a light microscope. 2.7. Statistical analysis All data were analyzed using Microsoft Office Excel (Microsoft Corp.) and are presented as means ± standard deviations (SDs). A pvalue (Student’s t-test) less than 0.05 was considered significant. 3. Results
2.2. Immunoblot analysis
3.1. TGF-b1 pretreatment accelerates the g-radiation-induced DDR
Cells were extracted using 1X SDS sample buffer. Equal amounts of proteins were resolved, and immunoreactive proteins were detected using ECL reagents (Amersham Pharmacia Biotechnology) and X-ray films (AGFA).
To investigate the mechanism of the DDR, we examined the levels of phospho-ATM, phospho-Chk2, and gH2AX after irradiation. ATM, which is a serine/threonine protein kinase required for the rapid response to g-radiation-induced DNA damage, can directly phosphorylate Chk2 and H2AX in response to irradiation [24]. As shown in Fig. 1A, the irradiation of epithelial cells increased the phosphorylations of ATM, Chk2, and gH2AX, regardless of TGFb1 pretreatment. At 30 min after irradiation, however, TGF-b1pretreated cells exhibited lower levels of phospho-ATM, phosphoChk2, and gH2AX. Irradiation did not change the total protein levels of ATM or Chk2. The effect of TGF-b1 was confirmed by the expression of p21, a downstream effector of TGF-b signaling. To further understand the effect of TGF-b1 on irradiated epithelial cells, we investigated whether the TbRI inhibitor, SB431542, could affect g-radiation-induced DDR. SB431542 alone did not affect the g-radiation-induced DDR; however, it did abrogate the TGF-b1-induced reductions in phospho-ATM, phosphoChk2, and gH2AX (Fig. 1B). These results show that TGF-b1 accelerated the radiation-induced DDR, and that this effect was TGF-b receptor-dependent.
2.3. siRNA transfection siRNA transfections were performed using LipofectAMINE 2000 (Invitrogen) according to the manufacturer’s instructions. The cells were transfected with control siRNA, Smad2/3 siRNA (Santa Cruz Biotechnology), or Lig4 siRNA (Bioneer Inc.) at a final concentration of 10 nM. 2.4. In vivo study Female athymic mice (6 weeks old) were inoculated subcutaneously in the right thigh with 2 106 A431 cells/0.2 ml PBS. The tumor volume was calculated using the formula, (length width2)/ 2. When tumors reached a size of 100 mm3, the mice were randomized into the following four groups (n ¼ 6 mice/group): Group 1 was injected with 20% dimethyl sulfoxide (DMSO) in PBS; Group 2 was injected with 20 mg SB431542/mouse; Group 3 was injected with 15 ng TGF-b1/mouse; and Group 4 was injected with SB431542 (20 mg/mouse) 1 h before injection with TGF-b1 (15 ng/ mouse). Half of the animals in each group were locally irradiated with 8 Gy delivered by a Theratron 780 (Atomic Energy of Canada) with a [60Co] source. Twenty-four hours after irradiation, the tumors were removed, fixed with 10% neutral-buffered formalin at RT, and embedded in paraffin. All animal procedures were reviewed and approved by the Institutional Animal Care and Ethics Committee of Korea Institute of Radiation and Medical Sciences.
3.2. The TGF-b1-accelerated DDR is Smad-dependent We speculated that the R-Smad proteins, which are well-known downstream molecules of TGF-b1, could be involved in the ability of TGF-b1 pretreatment to accelerate the g-irradiation-induced DDR. To test this, we examined whether Smad2/3 knockdown interfered with the effect of TGF-b1 on the DDR. Cells were transfected with Smad2/3 siRNAs, pretreated with TGF-b1, and exposed to g-radiation. As shown in Fig. 2 and consistent with the above-described results, immunoblot analyses showed that TGF-b1 pretreatment resolved the DNA damage within 30 min after irradiation. However,
Please cite this article in press as: J. Lee, et al., TGF-b1 accelerates the DNA damage response in epithelial cells via Smad signaling, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.136
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Fig. 1. TGF-b1 pretreatment accelerates the g-radiation-induced DNA damage response (DDR). HaCaT and A431 cells were pretreated with (B) or without (A) an inhibitor of TGFb1 type I receptor (SB431542; 0.5 mM) for 1 h, treated with or without TGF-b1 (0.5 ng/ml) for 24 h, and irradiated with 8 Gy. Cells were harvested at 0 or 30 min after g-irradiation, cell lysates were resolved by SDS-PAGE and immunoblotting was performed with antibodies against phospho-ATM, phospho-Chk2, and gH2AX. Total ATM, Chk2, and b-actin were used as controls, and p21 was used as a positive control for TGF-b1 treatment. Results are representative of three independent experiments.
whether Lig4 knockout interfered with the effect of TGF-b1 in our experimental system. The results are presented in Fig. 3. Consistent with our previous report, Lig4 expression was increased by TGF-b1 treatment. Furthermore, Lig4 knockdown diminished the effects of TGF-b1 on the DDR: at 30 min after irradiation, the phosphorylations of ATM and Chk2 and the level of gH2AX were reduced in TGF-b1-treated cells, but no such change was observed in cells expressing Lig4 siRNAs. These data suggest that TGF-b1-induced Lig4 expression is necessary for the acceleration of the DDR in TGF-b1-treated epithelial cells subjected to IR.
Fig. 2. The TGF-b1-accelerated DDR is Smad-dependent. HaCaT and A431 cells were transfected with control or Smad2/3 siRNAs. After 24 h, the cells were treated with TGF-b1 (0.5 ng/ml) for 24 h and then irradiated with 8 Gy, as indicated. Thirty minutes after g-irradiation, immunoblot analyses were performed using antibodies against phospho-ATM, phospho-Chk2, gH2AX, Smad2, and Smad3. Total ATM, Chk2, and bactin were used as loading controls, and p21 was used as a positive control for TGF-b1 treatment. The data are representative of three independent experiments.
treatment with Smad2/3 siRNAs blocked this effect of TGF-b1. No change was seen in the activations of ATM and Chk2 or the level of gH2AX with or without TGF-b1 in the presence of Smad2/3 siRNAs. These data indicate that TGF-b1 pretreatment and subsequent RSmad activation could accelerate the g-irradiation-induced DDR. 3.3. The TGF-b1-accelerated DDR requires Lig4 We recently reported that TGF-b1 pretreatment enhanced the expression and nuclear retention of Lig4, and thereafter increased the activity of nonhomologous end-joining (NHEJ) repair pathway [23]. Here, we transfected cells with Lig4 siRNAs and examined
Fig. 3. The TGF-b1-accelerated DDR requires DNA Ligase IV. HaCaT and A431 cells were transfected with control or Lig4 siRNAs. After 24 h, the cells were treated with TGF-b1 (0.5 ng/ml) for 24 h and then irradiated with 8 Gy, as indicated. Thirty minutes after g-irradiation, immunoblot analyses were performed using antibodies against phospho-ATM, phospho-Chk2, gH2AX, and Lig4. Total ATM, Chk2, and b-actin were used as loading controls, and p21 was used as a positive control for TGF-b1 treatment. The data are representative of three independent experiments.
Please cite this article in press as: J. Lee, et al., TGF-b1 accelerates the DNA damage response in epithelial cells via Smad signaling, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.136
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Fig. 4. TGF-b1 pretreatment protects epithelial cells from g-radiation in vivo. Nude mice bearing A431 tumors was treated with or without an inhibitor of TGF-b1 type I receptor (SB431542; 20 mg) for 1 h, treated with or without TGF-b1 (15 ng) for 24 h, and then irradiated with 8 Gy at the tumor-bearing sites. Twenty-four hours after g-irradiation, tumor sections were collected and embedded in paraffin. (A) To detect apoptosis, tumor sections were processed with TUNEL staining. The sections were also counterstained with hematoxylin (blue; left panel). TUNEL-positive cells (brown nuclei) were counted from five randomly selective fields, and the results are plotted as percentages (right panel). Asterisk (*) indicates p < 0.005 (Student’s t-test), and error bars indicate standard deviations from three independent experiments. (B) To detect proliferative cells, tumor sections were analyzed by PCNA immunostaining. The sections were also counterstained with hematoxylin (blue; left panel). PCNA-positive cells (brown nuclei) were counted from five randomly selected fields, and the results are plotted as percentages (right panel). Asterisks (*) indicate p < 0.05 (Student’s t-test); error bars indicate standard deviations from three independent experiments.
Please cite this article in press as: J. Lee, et al., TGF-b1 accelerates the DNA damage response in epithelial cells via Smad signaling, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.136
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3.4. TGF-b1 pretreatment protects epithelial cells from g-radiation in vivo We previously reported that TGF-b1 pretreatment suppressed IR-induced apoptosis in a conventional cell culture system [22,23]. Here, we investigated the effect of TGF-b1 in vivo. We injected nude mice with A431 cells and allowed embedded tumors to form. Tumor-bearing mice were treated with TGF-b1 or left untreated, and then locally irradiated. Twenty-four hours after g-irradiation, tumors were collected and analyzed by TUNEL assay. Our results revealed that the frequency of IR-induced apoptosis was decreased by TGF-b1 pretreatment in vivo, and that this effect was abrogated by the inhibitor, SB431542 (Fig. 4A). These data demonstrate that TGF-b1 pretreatment suppresses IR-induced apoptosis in vivo. Finally, we examined the effect of TGF-b1 on the survival of epithelial cells in a same in vivo system for TUNEL assay. Twentyfour hours after g-irradiation, cells were examined immunohistochemically using an antibody against PCNA (proliferating cell nuclear antigen), which is a marker for proliferating cells [25]. As shown in Fig. 4B, un-irradiated samples did not significantly differ in PCNA expression, regardless of TGF-b1 pretreatment. After girradiation, however, TGF-b1-pretreated samples exhibited increased PCNA expression relative to samples exposed to g-irradiation alone, indicating increased proliferation. Co-treatment with SB431542 completely blocked this effect of TGF-b1. Taken together, these results indicate that TGF-b1 pretreatment protects epithelial cells from IR-induced apoptosis and enhances the survival of irradiated cells. 4. Discussion TGF-b initiates and integrates multiple cellular signals that trigger tissue responses to various stimuli, including g-radiation and other types of damage [26]. Here, we report that the activation of the g-radiation-induced DDR in human epithelial cells could be accelerated by the activation of TGF-b signaling. The role of TGF-b1 in irradiation is still a matter of debate, with some studies showing that it acts as a radiosensitizer while others report it functioning as a radioprotector. It was also not previously clear whether TGF-b signaling regulates the DDR network. Here, we show that TGF-b1 acts as a radioprotector by accelerating DDR; TGF-b1 resolves DNA damage-induced signaling, and increases cell survival. We further report that these protective effects of TGF-b1 are mediated by Smad signaling and downstream Lig4 expression. These effects were shown in HaCaT and A431 cells, which are human epithelial cell lines previously shown their responsiveness to TGF-b1 by translocating Smad4 to the nucleus and entering growth arrest at G1 phase [23]. We already showed that there was no difference of gH2AX level right after g-irradiation with or without TGF-b1 pretreatment in our previous study [23]. However, TGF-b1 pretreatment decreased the gH2AX level within 30 min after g-irradiation that mainly caused by TGF-b1-induced Lig4 expression [23]. Since several previous studies showed that TGF-b could regulate the DDR, we investigated whether TGF-b1 could affect DDR signaling molecules. We found that TGF-b1 pretreatment dramatically reduced the activations of ATM and Chk2 in irradiated epithelial cells within 30 min, concomitant with the observed change in gH2AX expression (Fig. 1A). To examine whether these effects required TGF-b signaling, we used SB431542, which is a known TbRI inhibitor [27,28]. SB431542 efficiently sustained the g-irradiation-induced phospho-ATM, phospho-Chk2, and gH2AX in TGF-b1-pretreated cells (Fig. 1B). Moreover, the TGF-b1-accelerated DDR was also abrogated by transfection of Smad2/3 siRNAs (Fig. 2). These results collectively support the notion that TGF-b signaling could regulate
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the DDR. We previously reported that TGF-b1 enhanced g-irradiated epithelial cell survival, at least in part, by increasing NHEJ activity, primarily through the TGF-b1-induced upregulation of Lig4 expression [23]. Here, we investigated whether Lig4 expression could affect the DDR. We obtained similar results in cells transfected with siRNAs against Lig4 and Smad2/3, in which the TGF-b1induced decreases in phospho-ATM, phospho-Chk2, and gH2AX were abrogated to the levels seen in irradiated cells that were not pretreated (Fig. 3). From these results, we conclude that TGF-b1 enhances the recovery of DNA damage by inducing Lig4. Finally, we assessed cell survival in an in vivo mouse model (Fig. 4). We confirmed that TGF-b1 inhibited g-radiation-induced apoptosis, and that treatment with the inhibitor, SB431542, abrogated this effect. We also found that cell proliferation was increased by TGFb1. These results are not unexpected, given that TGF-b1 induced Lig4 expression, enhanced repair activity, and accelerated DDR signaling. Our results are consistent with the previously published data. For example, Buckley et al. reported that TGF-b1 promoted survival and repair after hyperoxic injury [18], suggesting that TGFb1 protected cells against various forms of DNA damage. Moreover, TGF-b1 inhibition prior to g-irradiation was shown to attenuate the DDR, increase cell death, and delay tumor growth [19,20], and TGFb1 pretreatment was reported to protect Mv1Lu epithelial cells from g-irradiation, dependent on de novo protein synthesis [22]. In conclusion, we herein reveal that TGF-b signaling affects IRinduced DDR signaling in both normal epithelial (HaCaT) and carcinoma (A431) cells. TGF-b1 treatment facilitates the DNA repair process and enhances the speed of the DDR. Our data thus support the notion that inhibition of TGF-b may improve radiation therapy. Conflicts of interest statement Manuscript title: “TGF-b1 Accelerates the DNA Damage Response in Epithelial Cells via Smad Signaling”. All authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. Acknowledgments This work was supported by a grant (NRF-2012M2A2A7012377) from the Nuclear Research & Development Program of the Korea Science and Engineering Foundation (KOSEF), funded by the Ministry of Science, ICT and Future Planning (MSIP). References [1] Y. Shi, J. Massague, Mechanisms of TGF-beta signaling from cell membrane to the nucleus, Cell 113 (2003) 685e700. [2] R. Derynck, Y.E. Zhang, Smad-dependent and Smad-independent pathways in TGF-beta family signalling, Nature 425 (2003) 577e584. [3] C.H. Heldin, K. Miyazono, P. Ten Dijke, TGF-beta signalling from cell membrane to nucleus through SMAD proteins, Nature 390 (1997) 465e471. [4] M. Suzuki, D.A. Boothman, Stress-induced premature senescence (SIPS)einfluence of SIPS on radiotherapy, J. Radiat. Res. 49 (2008) 105e112. [5] Y. Shiloh, ATM and related protein kinases: safeguarding genome integrity, Nat. Rev. Cancer 3 (2003) 155e168. [6] E.U. Kurz, S.P. Lees-Miller, DNA damage-induced activation of ATM and ATMdependent signaling pathways, DNA Repair (Amst) 3 (2004) 889e900. [7] M.B. Kastan, D.S. Lim, S.T. Kim, B. Xu, C. Canman, Multiple signaling pathways involving ATM, Cold Spring Harb. Symp. Quant. Biol. 65 (2000) 521e526.
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Please cite this article in press as: J. Lee, et al., TGF-b1 accelerates the DNA damage response in epithelial cells via Smad signaling, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.136
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TGF-b1 accelerates the DNA damage response in epithelial cells via Smad signaling Jeeyong Lee 1, Mi-Ra Kim 1, Hyun-Ji Kim, You Sun An, Jae Youn Yi* Division of Basic Radiation Bioscience, Korea Institute of Radiation and Medical Sciences, Seoul, 01812, Republic of Korea
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a b s t r a c t
Article history: Received 23 May 2016 Accepted 25 May 2016 Available online xxx
The evidence suggests that transforming growth factor-beta (TGF-b) regulates the DNA-damage response (DDR) upon irradiation, and we previously reported that TGF-b1 induced DNA ligase IV (Lig4) expression and enhanced the nonhomologous end-joining repair pathway in irradiated cells. In the present study, we investigated the effects of TGF-b1 on the irradiation-induced DDRs of A431 and HaCaT cells. Cells were pretreated with or without TGF-b1 and irradiated. At 30 min post-irradiation, DDRs were detected by immunoblotting of phospho-ATM, phospho-Chk2, and the presence of histone foci (gH2AX). The levels of all three factors were similar right after irradiation regardless of TGF-b1 pretreatment. However, they soon thereafter exhibited downregulation in TGF-b1-pretreated cells, indicating the acceleration of the DDR. Treatment with a TGF-b type I receptor inhibitor (SB431542) or transfections with siRNAs against Smad2/3 or DNA ligase IV (Lig4) reversed this acceleration of the DDR. Furthermore, the frequency of irradiation-induced apoptosis was decreased by TGF-b1 pretreatment in vivo, but this effect was abrogated by SB431542. These results collectively suggest that TGF-b1 could enhance cell survival by accelerating the DDR via Smad signaling and Lig4 expression. © 2016 Elsevier Inc. All rights reserved.
Keywords: TGF-b1 DNA damage response Smads Lig4 Radioprotection
1. Introduction Transforming growth factor-b (TGF-b) is a multifunctional cytokine that regulates the growth, differentiation, migration, adhesion, and apoptosis of various cell types [1]. TGF-b transduces signals through two distinct serine-threonine kinase receptors, termed type I (TbRI) and type II (TbRII). Upon ligand binding, TbRII activates TbRI, which then phosphorylates the receptor-regulated Smads (R-Smads), Smad2 and Smad3, which interact with the common mediator Smad, Smad4, and move to the nucleus [1,2]. These nuclear Smad complexes interact with various transcription factors and transcriptional coactivators to regulate the transcription of target genes [3]. Radiotherapy is one of the best therapeutic choices for cancer treatment [4]. The cellular response to radiation-induced DNA damage is a complex process involving a wide and integrated array of signal transduction pathways [5]. The primary transducer of
* Corresponding author. Division of Basic Radiation Bioscience, Korea Institute of Radiation and Medical Sciences, 75 Nowon-gil, Nowon-gu, Seoul, 01812, Republic of Korea. E-mail address: [email protected] (J.Y. Yi). 1 Both authors contributed equally to the paper.
genotoxic stress caused by g-radiation is the nuclear protein kinase, ataxia telangiectasia mutated (ATM) [6]. ATM, which is activated by the double-strand breaks (DSBs) caused by ionizing radiation, phosphorylates numerous substrates, including p53, NBS1, histone 2AX (H2AX), BRCA1, and Chk2 [7e10]. These DNA damage response (DDR) proteins activate a complex program that controls cell cycle checkpoints, apoptosis, and genomic integrity. Several recent studies have begun to unravel the relationship between TGF-b1 and the irradiation-induced DDR, addressing the ability of TGF-b1 to act as either a radiosensitizer or a radioprotector. As a radiosensitizer, TGF-b1 reportedly mediates radiation-induced apoptosis in different cell types [11,12]. The TGFb1-targeted signaling molecule, Smad3, has been shown to obstruct the repair of damaged DNA [13] and modulate pro-survival ERKMAPK signaling [14]. Moreover, the inhibitory Smad, Smad7, has been shown to play a crucial role upstream of ATM and p53 in protecting the genome from g-irradiation [15]. In the context of TGF-b1 as a radioprotector, studies have shown that TGF-b1 treatment significantly enhances clonogenic survival, reduces DNA strand breaks, and increases p27 expression by inducing G1 arrest [16,17]. TGF-b1 was shown to enhance DNA repair activity through Smad signaling, thereby protecting cells from hyperoxic stress [18]. In addition, inhibition of TbRI was shown to induce radiosensitivity
http://dx.doi.org/10.1016/j.bbrc.2016.05.136 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: J. Lee, et al., TGF-b1 accelerates the DNA damage response in epithelial cells via Smad signaling, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.136
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[19,20] and attenuate the activation of ATM [21]. Finally, we recently showed that TGF-b1 protects cells against g-irradiation by enhancing nonhomologous end-joining (NHEJ) repair [22,23]. Despite the existing pool of knowledge, however, it remains unclear exactly how TGF-b signaling regulates the DDR network. To address this open question, we herein investigated whether TGF-b1 plays a role in the g-radiation-induced DDR of the epithelial cell lines, HaCaT and A431. Here, we report that TGF-b1 accelerates the DDR, as exhibited by the levels of phospho-ATM, phosphoChk2, and gH2AX. In addition, we report that the TGF-b1accelerated DDR is dependent on Smad signaling and DNA ligase IV (Lig4) expression. 2. Materials and methods 2.1. Cell culture, chemicals, and antibodies The HaCaT (human keratinocyte) and A431 (human epidermoid carcinoma) cell lines (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and 1X Zell shield (Minerva Biolabs). Cells were pretreated with the TbRI inhibitor, SB431542 (0.5 mM; Tocris) for 1 h, incubated with 0.5 ng/ml TGF-b1 (R&D Systems) for 24 h, and irradiated with 8 Gy using a Gamma-cell 3000 (Atomic Energy) or a BIOBEAM 8000 (Gamma-Service Medical GmbH) with a [137Cs] source. The antibodies against pATM, ATM, pChk2, Chk2, p21, phospho-H2AX (gH2AX), Smad2, and Smad3 were purchased from Cell Signaling; those against Lig4 and b-actin were purchased from Santa Cruz; and that against PCNA was purchased from Dako.
2.5. TUNEL assay TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assays were performed using an ApopTag kit (Millipore) in accordance with the manufacturer’s instructions. Apoptotic bodies were visualized using a DAB reagent set (KPL). 2.6. Immunohistochemistry Tissue sections were deparaffinized in a xylene-to-ethanol gradient and rinsed with PBS. The slides were placed in a domestic pressure cooker containing 1 mM EDTA buffer (pH 8.0) and boiled for 15 min. Endogenous peroxidase was blocked by incubating the sections for 10 min with 3% (v/v) H2O2 in methanol. The tissues were then rinsed with PBS, blocked with PBS containing 20% (v/v) horse serum, and incubated overnight at 4 C with anti-PCNA. After a 30-min incubation with diluted biotinylated secondary antibody, immunoreactivity was detected using a Vectastain ABC kit (Vector Laboratories). Sections were washed with PBS, antibody binding was visualized using a DAB reagent set, counterstaining was performed with hematoxylin, and the results were examined using a light microscope. 2.7. Statistical analysis All data were analyzed using Microsoft Office Excel (Microsoft Corp.) and are presented as means ± standard deviations (SDs). A pvalue (Student’s t-test) less than 0.05 was considered significant. 3. Results
2.2. Immunoblot analysis
3.1. TGF-b1 pretreatment accelerates the g-radiation-induced DDR
Cells were extracted using 1X SDS sample buffer. Equal amounts of proteins were resolved, and immunoreactive proteins were detected using ECL reagents (Amersham Pharmacia Biotechnology) and X-ray films (AGFA).
To investigate the mechanism of the DDR, we examined the levels of phospho-ATM, phospho-Chk2, and gH2AX after irradiation. ATM, which is a serine/threonine protein kinase required for the rapid response to g-radiation-induced DNA damage, can directly phosphorylate Chk2 and H2AX in response to irradiation [24]. As shown in Fig. 1A, the irradiation of epithelial cells increased the phosphorylations of ATM, Chk2, and gH2AX, regardless of TGFb1 pretreatment. At 30 min after irradiation, however, TGF-b1pretreated cells exhibited lower levels of phospho-ATM, phosphoChk2, and gH2AX. Irradiation did not change the total protein levels of ATM or Chk2. The effect of TGF-b1 was confirmed by the expression of p21, a downstream effector of TGF-b signaling. To further understand the effect of TGF-b1 on irradiated epithelial cells, we investigated whether the TbRI inhibitor, SB431542, could affect g-radiation-induced DDR. SB431542 alone did not affect the g-radiation-induced DDR; however, it did abrogate the TGF-b1-induced reductions in phospho-ATM, phosphoChk2, and gH2AX (Fig. 1B). These results show that TGF-b1 accelerated the radiation-induced DDR, and that this effect was TGF-b receptor-dependent.
2.3. siRNA transfection siRNA transfections were performed using LipofectAMINE 2000 (Invitrogen) according to the manufacturer’s instructions. The cells were transfected with control siRNA, Smad2/3 siRNA (Santa Cruz Biotechnology), or Lig4 siRNA (Bioneer Inc.) at a final concentration of 10 nM. 2.4. In vivo study Female athymic mice (6 weeks old) were inoculated subcutaneously in the right thigh with 2 106 A431 cells/0.2 ml PBS. The tumor volume was calculated using the formula, (length width2)/ 2. When tumors reached a size of 100 mm3, the mice were randomized into the following four groups (n ¼ 6 mice/group): Group 1 was injected with 20% dimethyl sulfoxide (DMSO) in PBS; Group 2 was injected with 20 mg SB431542/mouse; Group 3 was injected with 15 ng TGF-b1/mouse; and Group 4 was injected with SB431542 (20 mg/mouse) 1 h before injection with TGF-b1 (15 ng/ mouse). Half of the animals in each group were locally irradiated with 8 Gy delivered by a Theratron 780 (Atomic Energy of Canada) with a [60Co] source. Twenty-four hours after irradiation, the tumors were removed, fixed with 10% neutral-buffered formalin at RT, and embedded in paraffin. All animal procedures were reviewed and approved by the Institutional Animal Care and Ethics Committee of Korea Institute of Radiation and Medical Sciences.
3.2. The TGF-b1-accelerated DDR is Smad-dependent We speculated that the R-Smad proteins, which are well-known downstream molecules of TGF-b1, could be involved in the ability of TGF-b1 pretreatment to accelerate the g-irradiation-induced DDR. To test this, we examined whether Smad2/3 knockdown interfered with the effect of TGF-b1 on the DDR. Cells were transfected with Smad2/3 siRNAs, pretreated with TGF-b1, and exposed to g-radiation. As shown in Fig. 2 and consistent with the above-described results, immunoblot analyses showed that TGF-b1 pretreatment resolved the DNA damage within 30 min after irradiation. However,
Please cite this article in press as: J. Lee, et al., TGF-b1 accelerates the DNA damage response in epithelial cells via Smad signaling, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.136
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Fig. 1. TGF-b1 pretreatment accelerates the g-radiation-induced DNA damage response (DDR). HaCaT and A431 cells were pretreated with (B) or without (A) an inhibitor of TGFb1 type I receptor (SB431542; 0.5 mM) for 1 h, treated with or without TGF-b1 (0.5 ng/ml) for 24 h, and irradiated with 8 Gy. Cells were harvested at 0 or 30 min after g-irradiation, cell lysates were resolved by SDS-PAGE and immunoblotting was performed with antibodies against phospho-ATM, phospho-Chk2, and gH2AX. Total ATM, Chk2, and b-actin were used as controls, and p21 was used as a positive control for TGF-b1 treatment. Results are representative of three independent experiments.
whether Lig4 knockout interfered with the effect of TGF-b1 in our experimental system. The results are presented in Fig. 3. Consistent with our previous report, Lig4 expression was increased by TGF-b1 treatment. Furthermore, Lig4 knockdown diminished the effects of TGF-b1 on the DDR: at 30 min after irradiation, the phosphorylations of ATM and Chk2 and the level of gH2AX were reduced in TGF-b1-treated cells, but no such change was observed in cells expressing Lig4 siRNAs. These data suggest that TGF-b1-induced Lig4 expression is necessary for the acceleration of the DDR in TGF-b1-treated epithelial cells subjected to IR.
Fig. 2. The TGF-b1-accelerated DDR is Smad-dependent. HaCaT and A431 cells were transfected with control or Smad2/3 siRNAs. After 24 h, the cells were treated with TGF-b1 (0.5 ng/ml) for 24 h and then irradiated with 8 Gy, as indicated. Thirty minutes after g-irradiation, immunoblot analyses were performed using antibodies against phospho-ATM, phospho-Chk2, gH2AX, Smad2, and Smad3. Total ATM, Chk2, and bactin were used as loading controls, and p21 was used as a positive control for TGF-b1 treatment. The data are representative of three independent experiments.
treatment with Smad2/3 siRNAs blocked this effect of TGF-b1. No change was seen in the activations of ATM and Chk2 or the level of gH2AX with or without TGF-b1 in the presence of Smad2/3 siRNAs. These data indicate that TGF-b1 pretreatment and subsequent RSmad activation could accelerate the g-irradiation-induced DDR. 3.3. The TGF-b1-accelerated DDR requires Lig4 We recently reported that TGF-b1 pretreatment enhanced the expression and nuclear retention of Lig4, and thereafter increased the activity of nonhomologous end-joining (NHEJ) repair pathway [23]. Here, we transfected cells with Lig4 siRNAs and examined
Fig. 3. The TGF-b1-accelerated DDR requires DNA Ligase IV. HaCaT and A431 cells were transfected with control or Lig4 siRNAs. After 24 h, the cells were treated with TGF-b1 (0.5 ng/ml) for 24 h and then irradiated with 8 Gy, as indicated. Thirty minutes after g-irradiation, immunoblot analyses were performed using antibodies against phospho-ATM, phospho-Chk2, gH2AX, and Lig4. Total ATM, Chk2, and b-actin were used as loading controls, and p21 was used as a positive control for TGF-b1 treatment. The data are representative of three independent experiments.
Please cite this article in press as: J. Lee, et al., TGF-b1 accelerates the DNA damage response in epithelial cells via Smad signaling, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.136
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Fig. 4. TGF-b1 pretreatment protects epithelial cells from g-radiation in vivo. Nude mice bearing A431 tumors was treated with or without an inhibitor of TGF-b1 type I receptor (SB431542; 20 mg) for 1 h, treated with or without TGF-b1 (15 ng) for 24 h, and then irradiated with 8 Gy at the tumor-bearing sites. Twenty-four hours after g-irradiation, tumor sections were collected and embedded in paraffin. (A) To detect apoptosis, tumor sections were processed with TUNEL staining. The sections were also counterstained with hematoxylin (blue; left panel). TUNEL-positive cells (brown nuclei) were counted from five randomly selective fields, and the results are plotted as percentages (right panel). Asterisk (*) indicates p < 0.005 (Student’s t-test), and error bars indicate standard deviations from three independent experiments. (B) To detect proliferative cells, tumor sections were analyzed by PCNA immunostaining. The sections were also counterstained with hematoxylin (blue; left panel). PCNA-positive cells (brown nuclei) were counted from five randomly selected fields, and the results are plotted as percentages (right panel). Asterisks (*) indicate p < 0.05 (Student’s t-test); error bars indicate standard deviations from three independent experiments.
Please cite this article in press as: J. Lee, et al., TGF-b1 accelerates the DNA damage response in epithelial cells via Smad signaling, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.136
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3.4. TGF-b1 pretreatment protects epithelial cells from g-radiation in vivo We previously reported that TGF-b1 pretreatment suppressed IR-induced apoptosis in a conventional cell culture system [22,23]. Here, we investigated the effect of TGF-b1 in vivo. We injected nude mice with A431 cells and allowed embedded tumors to form. Tumor-bearing mice were treated with TGF-b1 or left untreated, and then locally irradiated. Twenty-four hours after g-irradiation, tumors were collected and analyzed by TUNEL assay. Our results revealed that the frequency of IR-induced apoptosis was decreased by TGF-b1 pretreatment in vivo, and that this effect was abrogated by the inhibitor, SB431542 (Fig. 4A). These data demonstrate that TGF-b1 pretreatment suppresses IR-induced apoptosis in vivo. Finally, we examined the effect of TGF-b1 on the survival of epithelial cells in a same in vivo system for TUNEL assay. Twentyfour hours after g-irradiation, cells were examined immunohistochemically using an antibody against PCNA (proliferating cell nuclear antigen), which is a marker for proliferating cells [25]. As shown in Fig. 4B, un-irradiated samples did not significantly differ in PCNA expression, regardless of TGF-b1 pretreatment. After girradiation, however, TGF-b1-pretreated samples exhibited increased PCNA expression relative to samples exposed to g-irradiation alone, indicating increased proliferation. Co-treatment with SB431542 completely blocked this effect of TGF-b1. Taken together, these results indicate that TGF-b1 pretreatment protects epithelial cells from IR-induced apoptosis and enhances the survival of irradiated cells. 4. Discussion TGF-b initiates and integrates multiple cellular signals that trigger tissue responses to various stimuli, including g-radiation and other types of damage [26]. Here, we report that the activation of the g-radiation-induced DDR in human epithelial cells could be accelerated by the activation of TGF-b signaling. The role of TGF-b1 in irradiation is still a matter of debate, with some studies showing that it acts as a radiosensitizer while others report it functioning as a radioprotector. It was also not previously clear whether TGF-b signaling regulates the DDR network. Here, we show that TGF-b1 acts as a radioprotector by accelerating DDR; TGF-b1 resolves DNA damage-induced signaling, and increases cell survival. We further report that these protective effects of TGF-b1 are mediated by Smad signaling and downstream Lig4 expression. These effects were shown in HaCaT and A431 cells, which are human epithelial cell lines previously shown their responsiveness to TGF-b1 by translocating Smad4 to the nucleus and entering growth arrest at G1 phase [23]. We already showed that there was no difference of gH2AX level right after g-irradiation with or without TGF-b1 pretreatment in our previous study [23]. However, TGF-b1 pretreatment decreased the gH2AX level within 30 min after g-irradiation that mainly caused by TGF-b1-induced Lig4 expression [23]. Since several previous studies showed that TGF-b could regulate the DDR, we investigated whether TGF-b1 could affect DDR signaling molecules. We found that TGF-b1 pretreatment dramatically reduced the activations of ATM and Chk2 in irradiated epithelial cells within 30 min, concomitant with the observed change in gH2AX expression (Fig. 1A). To examine whether these effects required TGF-b signaling, we used SB431542, which is a known TbRI inhibitor [27,28]. SB431542 efficiently sustained the g-irradiation-induced phospho-ATM, phospho-Chk2, and gH2AX in TGF-b1-pretreated cells (Fig. 1B). Moreover, the TGF-b1-accelerated DDR was also abrogated by transfection of Smad2/3 siRNAs (Fig. 2). These results collectively support the notion that TGF-b signaling could regulate
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the DDR. We previously reported that TGF-b1 enhanced g-irradiated epithelial cell survival, at least in part, by increasing NHEJ activity, primarily through the TGF-b1-induced upregulation of Lig4 expression [23]. Here, we investigated whether Lig4 expression could affect the DDR. We obtained similar results in cells transfected with siRNAs against Lig4 and Smad2/3, in which the TGF-b1induced decreases in phospho-ATM, phospho-Chk2, and gH2AX were abrogated to the levels seen in irradiated cells that were not pretreated (Fig. 3). From these results, we conclude that TGF-b1 enhances the recovery of DNA damage by inducing Lig4. Finally, we assessed cell survival in an in vivo mouse model (Fig. 4). We confirmed that TGF-b1 inhibited g-radiation-induced apoptosis, and that treatment with the inhibitor, SB431542, abrogated this effect. We also found that cell proliferation was increased by TGFb1. These results are not unexpected, given that TGF-b1 induced Lig4 expression, enhanced repair activity, and accelerated DDR signaling. Our results are consistent with the previously published data. For example, Buckley et al. reported that TGF-b1 promoted survival and repair after hyperoxic injury [18], suggesting that TGFb1 protected cells against various forms of DNA damage. Moreover, TGF-b1 inhibition prior to g-irradiation was shown to attenuate the DDR, increase cell death, and delay tumor growth [19,20], and TGFb1 pretreatment was reported to protect Mv1Lu epithelial cells from g-irradiation, dependent on de novo protein synthesis [22]. In conclusion, we herein reveal that TGF-b signaling affects IRinduced DDR signaling in both normal epithelial (HaCaT) and carcinoma (A431) cells. TGF-b1 treatment facilitates the DNA repair process and enhances the speed of the DDR. Our data thus support the notion that inhibition of TGF-b may improve radiation therapy. Conflicts of interest statement Manuscript title: “TGF-b1 Accelerates the DNA Damage Response in Epithelial Cells via Smad Signaling”. All authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. Acknowledgments This work was supported by a grant (NRF-2012M2A2A7012377) from the Nuclear Research & Development Program of the Korea Science and Engineering Foundation (KOSEF), funded by the Ministry of Science, ICT and Future Planning (MSIP). References [1] Y. Shi, J. Massague, Mechanisms of TGF-beta signaling from cell membrane to the nucleus, Cell 113 (2003) 685e700. [2] R. Derynck, Y.E. Zhang, Smad-dependent and Smad-independent pathways in TGF-beta family signalling, Nature 425 (2003) 577e584. [3] C.H. Heldin, K. Miyazono, P. Ten Dijke, TGF-beta signalling from cell membrane to nucleus through SMAD proteins, Nature 390 (1997) 465e471. [4] M. Suzuki, D.A. Boothman, Stress-induced premature senescence (SIPS)einfluence of SIPS on radiotherapy, J. Radiat. Res. 49 (2008) 105e112. [5] Y. Shiloh, ATM and related protein kinases: safeguarding genome integrity, Nat. Rev. Cancer 3 (2003) 155e168. [6] E.U. Kurz, S.P. Lees-Miller, DNA damage-induced activation of ATM and ATMdependent signaling pathways, DNA Repair (Amst) 3 (2004) 889e900. [7] M.B. Kastan, D.S. Lim, S.T. Kim, B. Xu, C. Canman, Multiple signaling pathways involving ATM, Cold Spring Harb. Symp. Quant. Biol. 65 (2000) 521e526.
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Please cite this article in press as: J. Lee, et al., TGF-b1 accelerates the DNA damage response in epithelial cells via Smad signaling, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.136