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HIF1-regulated ATRIP expression is required for hypoxia induced ATR activation
FEBS Letters 587 (2013) 930–935
journal homepage: www.FEBSLetters.org
HIF1-regulated ATRIP expression is required for hypoxia induced ATR activation Gang Ding a,b,c, He-Dai Liu a, Hong-Xiang Liang a, Ru-Feng Ni a, Zhao-Yan Ding a, Guo-Ying Ni a, Hong-Wei Hua a, Wei-Guo Xu d,⇑ a
Department of Oncology, Chongming Branch of Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, PR China Shanghai Translational Research Institute for Cancer Pain, Shanghai, PR China Shanghai Jiaotong University School of Medicine, Shanghai, PR China d Department of Respiratory, Xinhua Hospital of Shanghai Jiaotong University School of Medicine , Shanghai 200092, PR China b c
a r t i c l e
i n f o
Article history: Received 20 October 2012 Revised 1 February 2013 Accepted 8 February 2013 Available online 20 February 2013 Edited by Angel Nebreda Keywords: Hypoxia HIF-1 ATRIP ATR
a b s t r a c t The ATR–ATRIP protein kinase complex plays a crucial role in the cellular response to replication stress and DNA damage. Recent studies found that ATR could be activated in response to hypoxia and be involved in hypoxia-induced genetic instability in cancer cells. However, the underlying mechanisms for ATR activation in response to hypoxic stress are still not fully understood. We reported that ATRIP is a direct target of HIF-1. Silencing the expression of HIF-1a in cancer cells by RNA interference abolished hypoxia-induced ATRIP expression. Silencing the expression of ATRIP by RNA interference abolished hypoxia induced ATR activation and CHK1 phosphorylation in cancer cells. Taken together, these data shed novel insights on the mechanism of hypoxia-induced activation of the ATR pathway. Ó 2013 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
1. Introduction In eukaryotic cells, several checkpoint-control mechanisms help to preserve the integrity of the genome [1]. Members of the phosphoinositide kinase-related family of protein kinases (PIKKs), such as ATM and ATR, are key upstream regulators of checkpoint responses [2,3]. ATM is activated following double-strand breaks while ATR activation is associated with single-strand breaks and stalled replication forks [4,5]. ATR is recruited to single-stranded DNA via ATR-interacting protein (ATRIP). In response to genomic stress, the ATR interacting protein (ATRIP) binds and is phosphorylated by the DNA damage- and checkpoint-activated kinase ATR [6]. ATRIP directly interacts with replication protein A (RPA) that results in the recruitment of phosphorylated ATR/ATRIP to stalled replication forks and sites of DNA damage [7]. ATR/ATRIP coordinates DNA repair and cell cycle progression in conjunction with several key regulatory proteins, including Rad17 and the 9-1-1 complex [8]. ATR and ATRIP are essential for the DNA damage response and replication-induced cell cycle arrest [7,9]. Intratumoral hypoxia is a hallmark of solid cancer. A hypoxic microenvironment initiates multiple cellular responses, including proliferation and angiogenesis, resulting in the development and ⇑ Corresponding author. E-mail address: [email protected] (W.-G. Xu).
progression of cancer [10]. The homeostatic response to hypoxia is predominantly mediated by the transcription factor hypoxiainducible factor (HIF)-1 which is a heterodimer that consists of two basic helix-loop-helix proteins of the PAS family—a hypoxia regulated a-subunit (HIF-1a or HIF-2a) and a constitutive b-subunit (HIF-1b) [11]. Under normoxic conditions, HIF-a subunits are hydroxylated on proline residues by a class of prolyl hydroxylases. Then prolyl-hydroxylated HIF-a is recognized and targeted for ubiquitin-mediated destruction by the von Hippel–Lindau (VHL) tumor suppressor protein, which is a substrate recognition component of an E3 ubiquitin ligase. Under hypoxic conditions, HIF-a remains unmodified by prolyl hydroxylases and thereby escapes recognition by VHL and destruction [12]. Thus escaped HIF-a dimerizes with HIF-1b, translocates to the nucleus and activates genes of the hypoxia response by binding to the core sequence (RCGTG) of the hypoxic responsive element (HRE) with additional recruitment of the co-activators CBP/p300 [13]. Mutations in VHL can be found in familial and sporadic clear cell carcinomas of the kidney, hemangioblastomas of the retina and central nervous system, and pheochromocytomas, underscoring its gatekeeper function in the pathogenesis of these tumors. Tissue-specific gene targeting of VHL in mice has demonstrated that efficient execution of pVHL-mediated HIF proteolysis under normoxia is fundamentally important for survival, proliferation, differentiation and normal physiology of many cell types. As a
0014-5793/$36.00 Ó 2013 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. http://dx.doi.org/10.1016/j.febslet.2013.02.020
G. Ding et al. / FEBS Letters 587 (2013) 930–935
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Fig. 1. Hypoxia and hypoxia reagent induce ATRIP expression. (A) mRNA levels of ATRIP in MCF7, HeLa and HepG2 cells under normoxia, hypoxia or 100 lM DFO (deferoxamine mesylate salt) for 24 h were determined by real-time PCR assay. (B–D) Western blot analysis of ATRIP, HIF-1a and Actin in MCF6 (B), HeLa (C) and HepG2 (D) cells under normoxia (N) or hypoxia (H) condition. (E) HepG2 cells were cultured in hypoxia for the indicated length of time. Lysate of cells were assayed by Western blot with indicated antibodies.
result of hypoxia and genetic mutations, HIF-1a is over-expressed in many human cancers [14]. Emerging evidence suggests that hypoxia can also induce genetic instability in cancer cells [15,16]. Several DNA damage response factors are activated in response to hypoxia and subsequent reoxygenation, including ATM/ATR, Chkl/Chk2 and BRCA1 [17–20]. These findings reveal that hypoxia induces an acute DNA damage response in cancer cells by activating key DNA damage sensors. However, how these sensors are activated by hypoxia is largely unknown. So, it is necessary to understand the mechanisms underlying control of ATR kinase activity in response to hypoxic stress. Here, we show that both the mRNA and protein levels of ATRIP are markedly increased in human cancer cell lines in response to hypoxia. We also found that ATRIP is a direct target gene of HIF1. The increased ATRIP then activates the ATR signaling pathway under hypoxia. Our findings thus help us to understand how ATR is activated by hypoxia, which may have profound biological and therapeutic implications for cancer.
2. Materials and methods 2.1. Cell culture HeLa, MCF7, HepG2, 293T and IMR90 cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) (Gibco, CA, USA) as well as 100 U/ml penicilin and 100 lg/ml streptomycin. For hypoxia treatment, cells were cultured in a hypoxia chamber flushed with 0.1% O2 and 5% CO2 with balance of 95% nitrogen at 37 °C. Control cells were placed in a 5% CO2 and 95% air incubator (20% O2) at 37 °C. 2.2. SiRNA and transfection Small interfering (si) RNA against HIF-1a and ATRIP were purchased from Dharmacon (USA). As a negative control, a siRNA sequence targeting green fluorescent protein (non-targeting siRNA) was used. All transient transfections were performed using Lipo-
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Fig. 2. HIF-1 mediates hypoxia-induced ATRIP expression. (A and B) HIF-1a plasmid was transfected into HeLa (A) and HepG2 (B) cells for 36 h. Lysate of these cells were assayed by Western blot with indicated antibodies. (C) mRNA levels of HIF-1a in HeLa cells transfected with siRNA targeting HIF-1a or non-targeting siRNA were determined by real-time PCR assay. (D) HeLa cells were transfected with siRNA targeting HIF-1a or non-targeting siRNA for 24 h and cultured under hypoxic condition for additional 24 h. Lysate of these cells were assayed by Western blot with indicated antibodies. (E) mRNA and protein levels of ATRIP in HeLa cells transfected with siRNA targeting HIF-1a or non-targeting siRNA under normoxia (N) or hypoxia (H) condition were determined by real-time PCR assay and Western blot. (F) mRNA levels of ATRIP in RCC4 and RCC4/VHL cells were determined by real-time PCR assay. (G) Western blot analysis of ATRIP, HIF-1a, VHL and Actin in RCC4 and RCC4/VHL cells under normoxia condition.
fectamine 2000 (Invitrogen) according to the manufacturer’s instructions. 2.3. RNA isolation and real-time PCR Total RNAs from HepG2 and IMR90 cells were isolated by TRIzol reagent, and reverse transcriptions were performed by Takara RNA PCR kit following the manufacturer’s instructions. In order to quantify the transcripts of the interest genes, real-time PCR was performed using a SYBR Green Premix Ex Taq (Takara, Japan) on Light Cycler 480 (Roche, Switzerland). ATRIP transcript expression was normalized to b-actin mRNA expression using the DCt method and the linearized DCt (i.e. 2 DCt) was used for comparative purposes. The primer sequences used are available upon request. 2.4. Western blot Cells were harvested and lysed with ice-cold lysis buffer (50 mM Tris–HCl, pH 7.4, 100 mM DTT, 2% w/v SDS, 10% glycerol). After centrifugation at 20 000g for 10 min at 4 °C, proteins in the supernatants were quantified and separated by 10% SDS–PAGE, transferred to NC membrane. After blocking with 5% non-fat milk, membranes were immunoblotted with antibodies as indicated, followed by HRP-linked secondary antibodies. The signals were detected by Millipore SuperSignalÒ HRP Substrate kit according to manufacturer’s instructions. 2.5. Chemicals and antibodies Deferoxamine mesylate salt (DFO) was purchased from Sigma–Aldrich (St. Louis, MO), dissolved in DMSO. The antibodies
for ATRIP (#2737), ATR (#2790), Chk1 (2G1D5) (#2360), Phospho-Chk1 (Ser345) (133D3) (#2348), Histone H2A.X (#2595), Phospho-Histone H2A.X (Ser139) (#2577), VHL (#2738), Claspin (#2800) and HIF-1a (#3716) were purchased from Cell Signaling Technology. The antibodies for TopBP1 (ab2402) and Hus1 (ab96297) were purchased from Abcam. Actin (A2228) antibody was purchased from Sigma.
2.6. Luciferase assays The ATRIP promoter was amplified from a human genomic DNA template and inserted into pGL3 basic vector (Promega). A mutant HIF-1 binding motif was generated using a PCR mutagenesis kit (Toyobo) with a primer (mutation sites underlined): 50 -ctcaaa cgcaacacgAAtgaatgctagggcag-30 and a reverse complement primer. For luciferase reporter assays, HepG2 cells were seeded in 12-well plates and transfected with the indicated plasmids. Cells were harvested 30 h after transfection. Luciferase activities were measured using the Dual Luciferase Reporter Assay System (Promega, USA). 2.7. ChIP assays Chromatin immunoprecipitation (ChIP) assay kits were used (Upstate, USA). In short, HepG2 cells were fixed with 1% formaldehyde to cross-link the proteins and DNA, after which DNA was sheared to fragments of 200–800 bp by sonication in an ultrasound bath on ice. The chromatin was then incubated and precipitated with anti-IgG and anti-HIF-1a antibodies. The immunoprecipitated DNA fragments were detected using real-time PCR analysis.
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Fig. 3. Identification and validation of a functional HRE in ATRIP promoter region. (A) Schematic diagrams of the regulating sequences with the putative HRE (black ovals) of ATRIP. Mut: CG was changed into AA. (B) HeLa cells were transfected with the putative ATRIP HRE-luciferase reporter plasmids in the absence or presence of HIF-1a plasmid. After 48 h of transfection, the luciferase activity was measured. Transfection efficiency was normalized by Renilla luciferase expression, and the results are presented as activation over that for an empty vector. (C) The luciferase activity was measured in HepG2 cells with the methods described in (B). (D) HeLa cells were transfected with the putative ATRIP HRE-luciferase reporter plasmids. 24 h after transfection, cells were culture under indicated conditions for additional 24 h, the luciferase activity was measured. (E) Anti-IgG and anti-HIF-1a antibodies were used in the ChIP assays using HepG2 cells incubated with or without hypoxia.
2.8. Statistical analysis Data were summarized as mean ± S.E.M. Statistical significance was analyzed by one-way ANOVA followed by Turkey’s test using SPSS 11.0 software (Chicago, IL, US). A value of P < 0.05 was considered significant. Statistical significance is displayed as ⁄P < 0.05, ⁄⁄ P < 0.01 or ⁄⁄⁄P < 0.001. 3. Results 3.1. Hypoxia and hypoxia reagent induce ATRIP expression To test whether ATRIP expression is regulated by hypoxia, MCF7, HeLa and HepG2 cells were incubated under normoxia (21% O2), hypoxia (1% O2) or medium containing 100 lM DFO (deferoxamine mesylate salt) for 24 h. Real-time-PCR data revealed an increased ATRIP mRNA level in the cells under hypoxic or DFO treatment conditions (Fig. 1A). An increased ATRIP protein level accompanying increased HIF-1a protein levels was also observed in the cells under hypoxic condition (Fig. 1B–D). Indeed, hypoxia induced the expression on ATRIP in HepG2 cells in a time dependent manner (Fig. 1E). Moreover, ATRIP was the only DNA-damage related protein that induced during hypoxia (Fig. S1). Taken together, these results demonstrate that ATRIP can be induced in cells by hypoxia. 3.2. HIF-1 mediates hypoxia-induced ATRIP expression HIF-1 is a global transcriptional regulator of the hypoxic response. To test whether HIF-1 also mediates hypoxia-regulated ATRIP expression, HIF-1a-expressing plasmid was transiently
transfected into HeLa and HepG2 cells. Notably, ectopic expression of HIF-1a could significantly up-regulate ATRIP expression in both cell lines (Fig. 2A and B). To further investigate the role of HIF-1 in the regulation of the expression of ATRIP, HeLa cells were transfected with siRNA targeting HIF-1a or non-targeting siRNA to silence endogenous HIF-1a expression. The inhibition rate was evaluated by both real-time PCR and Western blot (Fig. 2C and D). As expected, ATRIP mRNA and protein levels were markedly decreased when HIF-1a was silenced under normoxic and hypoxic conditions (Fig. 2E). Similar results were also observed in MCF7 and HepG2 cells (Data not shown). To determine if ATRIP expression was affected by pVHL status, ATRIP mRNA and protein levels in normoxia were examined in RCC4 renal cancer cells with mutant pVHL, and in RCC4/VHL cells, an isogenic stable revertant of RCC4 expressing wild-type pVHL [21]. Levels of ATRIP mRNA and proteins were reduced in cells with wild-type versus mutant pVHL (Fig. 2F and G). Taken together, these data suggested that regulation of ATRIP by hypoxia is HIF-1 dependent. 3.3. Identification and validation of a functional HRE in ATRIP promoter region We next determined whether HIF-1a could be a transcriptional regulator of the ATRIP gene. There is one potential HRE at 0.4 kb region upstream to the transcription start site of ATRIP (Chr3:48,487,864-48,487,868, around 0.4 Kb upstream the TSS) (Fig. 3A). HeLa cells were transfected with a reporter vector encoding luciferase under control of the ATRIP promoter (WT-Luc). Concurrent expression of HIF-1a with the ATRIP reporter construct increased ATRIP promoter activity (Fig. 3B), which was abrogated by mutation of this HIF-1a DNA-binding site (CG-AA) in the ATRIP
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Fig. 4. Hypoxia-induced ATR activation was mediated by ATRIP. (A) mRNA and protein levels of ATRIP in HepG2 cells transfected with siRNA targeting ATRIP or non-targeting siRNA were determined by real-time PCR assay and Western blot. (B) HepG2 cells transfected with siRNA targeting ATRIP or non-targeting siRNA were under normoxia (N) or hypoxia (H) condition. Lysate of these cells were assayed by Western blot with indicated antibodies.
promoter (Mut-Luc). The stimulatory effect of HIF-1a on ATRIP promoter activity was also shown in HepG2 cells (Fig. 3C). Moreover, hypoxia alone could significantly stimulate the activity of WT-Luc but not Mut-Luc (Fig. 3D). These observations indicated that HIF-1a could bind to the promoter of ATRIP. To further demonstrate that HIF-1 binds to the HRE in the ATRIP promoter, we performed ChIP assay using antibodies against HIF-1a (IgG as a negative control) in normoxic- and hypoxic-cultured HepG2 cells. As depicted in Fig. 3E, HIF-1a but not IgG specifically bound to the HRE sequence in the ATRIP promoter. In summary, these results show that ATRIP is directly induced by HIF-1 under hypoxia. 3.4. Hypoxia-induced ATR activation is mediated by ATRIP Several studies have demonstrated that ATM and ATR are activated in response to hypoxia [17,20]. ATR is recruited to singlestranded DNA via ATRIP [6]. Both ATR and ATRIP are essential for the DNA damage response and replication-induced cell cycle arrest [7]. On the basis of this, we hypothesized that hypoxia promotes ATR activation through induction of ATRIP. To test this possibility, HepG2 cells were transfected with siRNA targeting ATRIP or nontargeting siRNA to silence endogenous ATRIP expression. The inhibition rate was evaluated by both real-time PCR and Western blot (Fig. 4A and B). In agreement with the previous study, ATR signaling pathway was activated in HepG2 cells incubated under hypoxia, as evidenced by increased phosphorylation of ATR targets including CHK1 and H2AX (Fig. 4B). However, these hypoxia-induced effects were almost completely abolished when ATRIP was silenced in HepG2 cells (Fig. 4B). Taken together, these data suggested that ATRIP was required for hypoxia-induced ATR activation. 4. Discussion In the present study, we showed that ATRIP was induced by hypoxia in human cancer cells. We provided evidence that ATRIP is a direct target gene of HIF-1. Hypoxia and ectopically expressed HIF1a protein significantly increased ATRIP expression at both mRNA and protein levels in cancer cell lines. Then, silencing of HIF-1a by specific siRNA antagonized hypoxia-induced ATRIP expression. Using luciferase reporter and ChIP assay, we found that HIF-1a directly bound to the HRE located at 386 to 381 of ATRIP gene,
which is essential for HIF-1-induced ATRIP expression. Without ATRIP, hypoxia induced ATR activation was dramatically decreased, indicating that ATRIP may be necessary for hypoxia induced ATR activation. Thus, ATR signaling was, at least, partially activated by induced ATRIP under hypoxia stress in cancer cells. During the review process of our study, one interesting paper described a positive regulation of ATR over HIF-1 [22]. Consistent with our observations, ATR protein level was unchanged during hypoxia. Our data support this work by indicating that ATR-activated HIF-1 further amplifies ATR signaling through ATRIP induction to form a positive feedback loop in the process of hypoxia. ATRIP could also be induced in non-transformed hypoxic IMR90 cells (Fig. S2). Both ATR and ATRIP are essential for the DNA damage response and replication-induced cell cycle arrest. Non-transformed hypoxic cells can undergo cell cycle arrest at the G1/S interface without any alteration in their long-term viability [23], suggesting the HIF-1-ATR-ATRIP axis may play a role in hypoxiainduced cell cycle arrest in non-transformed cells. Future work may focus on the role of the HIF-1-ATR-ATRIP axis in the regulation of hypoxic non-transformed cells. HIF-1 is a heterodimer that consists of two basic helix-loop-helix proteins of the PAS family—a hypoxia regulated a-subunit (HIF1a or HIF-2a) and a constitutive b-subunit (HIF-1b). Both HIF-1a and HIF-2a have been reported to be over-expressed in a series of tumors [24,25]. Therefore, it would be interesting to investigate the expression of ATRIP in human cancers. The relevance between HIF-1a/HIF-2a and ATRIP should also be investigated as well in the future. ATRIP is required for the cellular response to DNA damage and replication stress. ATRIP is phosphorylated by ATR and colocalizes with ATR in cells treated with DNA damaging agents [26]. Previous studies have also shown that the single-stranded DNA binding protein RPA (replication protein A) is necessary for the in vitro binding of ATRIP to DNA and that loss of RPA prevents the formation of ATR and ATRIP foci in cells treated with ionizing radiation [27]. However, whether RPA is required for hypoxia-induced ATR activation and whether RPA is induced or activated by hypoxia remains unknown. It would be interesting to understand the role of RPA in hypoxia-induced ATR activation. In summary, these discoveries shed new insights on the mechanism of ATRIP expression regulation and hypoxia induced activation of the ATR signaling pathway. Ultimately, these data may provide the rationale for the development of ATRIP inhibitors to
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prevent hypoxia-induced ATR activation and genetic instability in cancer cells. Conflict of Interest None of the authors has any potential conflicts of interest. Acknowledgements This work was supported by Grant from the Key Project of Shanghai Science and Technology Commission (NO10411956700). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.febslet.2013.02.020. References [1] Medema, R.H. and Macurek, L. (2012) Checkpoint control and cancer. Oncogene 31, 2601–2613. [2] Bensimon, A., Aebersold, R. and Shiloh, Y. (2011) Beyond ATM: the protein kinase landscape of the DNA damage response. FEBS Lett. 585, 1625–1639. [3] Smith, J., Tho, L.M., Xu, N. and Gillespie, D.A. (2010) The ATM-Chk2 and ATRChk1 pathways in DNA damage signaling and cancer. Adv. Cancer Res. 108, 73–112. [4] Flynn, R.L. and Zou, L. (2011) ATR: a master conductor of cellular responses to DNA replication stress. Trends Biochem. Sci. 36, 133–140. [5] Lavin, M.F., Kozlov, S., Gueven, N., Peng, C., Birrell, G., et al. (2005) ATM and cellular response to DNA damage. Adv. Exp. Med. Biol. 570, 457–476. [6] Cortez, D., Guntuku, S., Qin, J. and Elledge, S.J. (2001) ATR and ATRIP: partners in checkpoint signaling. Science 294, 1713–1716. [7] Zou, L. and Elledge, S.J. (2003) Sensing DNA damage through ATRIP recognition of RPA–ssDNA complexes. Science 300, 1542–1548. [8] Yang, X.H. and Zou, L. (2006) Recruitment of ATR–ATRIP, Rad17, and 9-1-1 complexes to DNA damage. Methods Enzymol. 409, 118–131. [9] Motoyama, N. and Naka, K. (2004) DNA damage tumor suppressor genes and genomic instability. Curr. Opin. Genet. Dev. 14, 11–16.
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[10] Bailey, K.M., Wojtkowiak, J.W., Hashim, A.I. and Gillies, R.J. (2012) Targeting the metabolic microenvironment of tumors. Adv. Pharmacol. 65, 63–107. [11] Majmundar, A.J., Wong, W.J. and Simon, M.C. (2010) Hypoxia-inducible factors and the response to hypoxic stress. Mol. Cell 40, 294–309. [12] Bader, H.L. and Hsu, T. (2012) Systemic VHL gene functions and the VHL disease. FEBS Lett. 586, 1562–1569. [13] Fedele, A.O., Whitelaw, M.L. and Peet, D.J. (2002) Regulation of gene expression by the hypoxia-inducible factors. Mol. Interv. 2, 229–243. [14] Rezvani, H.R., Ali, N., Nissen, L.J., Harfouche, G., de Verneuil, H., et al. (2011) HIF-1alpha in epidermis: oxygen sensing, cutaneous angiogenesis, cancer, and non-cancer disorders. J. Invest. Dermatol. 131, 1793–1805. [15] Kumareswaran, R., Ludkovski, O., Meng, A., Sykes, J., Pintilie, M., et al. (2012) Chronic hypoxia compromises repair of DNA double-strand breaks to drive genetic instability. J. Cell. Sci. 125, 189–199. [16] Lee, J.H., Choi, I.J., Song, D.K. and Kim, D.K. (2010) Genetic instability in the human lymphocyte exposed to hypoxia. Cancer Genet. Cytogenet. 196, 83–88. [17] Harding, S.M., Coackley, C. and Bristow, R.G. (2011) ATM-dependent phosphorylation of 53BP1 in response to genomic stress in oxic and hypoxic cells. Radiother. Oncol. 99, 307–312. [18] Bencokova, Z., Kaufmann, M.R., Pires, I.M., Lecane, P.S., Giaccia, A.J., et al. (2009) ATM activation and signaling under hypoxic conditions. Mol. Cell. Biol. 29, 526–537. [19] Gibson, S.L., Bindra, R.S. and Glazer, P.M. (2006) CHK2-dependent phosphorylation of BRCA1 in hypoxia. Radiat. Res. 166, 646–651. [20] Hammond, E.M., Dorie, M.J. and Giaccia, A.J. (2004) Inhibition of ATR leads to increased sensitivity to hypoxia/reoxygenation. Cancer Res. 64, 6556–6562. [21] Arjumand, W. and Sultana, S. (2012) Role of VHL gene mutation in human renal cell carcinoma. Tumour Biol. 33, 9–16. [22] Fallone, F., Britton, S., Nieto, L., Salles, B. and Muller, C. (2012) ATR controls cellular adaptation to hypoxia through positive regulation of hypoxiainducible factor 1 (HIF-1) expression. Oncogene. [23] Gardner, L.B., Li, Q., Park, M.S., Flanagan, W.M., Semenza, G.L., et al. (2001) Hypoxia inhibits G1/S transition through regulation of p27 expression. J. Biol. Chem. 276, 7919–7926. [24] Keith, B., Johnson, R.S. and Simon, M.C. (2012) HIF1alpha and HIF2alpha: sibling rivalry in hypoxic tumour growth and progression. Nat. Rev. Cancer 12, 9–22. [25] Martin, S.K., Diamond, P., Gronthos, S., Peet, D.J. and Zannettino, A.C. (2011) The emerging role of hypoxia, HIF-1 and HIF-2 in multiple myeloma. Leukemia 25, 1533–1542. [26] Itakura, E., Umeda, K., Sekoguchi, E., Takata, H., Ohsumi, M., et al. (2004) ATRdependent phosphorylation of ATRIP in response to genotoxic stress. Biochem. Biophys. Res. Commun. 323, 1197–1202. [27] Namiki, Y. and Zou, L. (2006) ATRIP associates with replication protein Acoated ssDNA through multiple interactions. Proc. Natl. Acad. Sci. USA 103, 580–585.
journal homepage: www.FEBSLetters.org
HIF1-regulated ATRIP expression is required for hypoxia induced ATR activation Gang Ding a,b,c, He-Dai Liu a, Hong-Xiang Liang a, Ru-Feng Ni a, Zhao-Yan Ding a, Guo-Ying Ni a, Hong-Wei Hua a, Wei-Guo Xu d,⇑ a
Department of Oncology, Chongming Branch of Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, PR China Shanghai Translational Research Institute for Cancer Pain, Shanghai, PR China Shanghai Jiaotong University School of Medicine, Shanghai, PR China d Department of Respiratory, Xinhua Hospital of Shanghai Jiaotong University School of Medicine , Shanghai 200092, PR China b c
a r t i c l e
i n f o
Article history: Received 20 October 2012 Revised 1 February 2013 Accepted 8 February 2013 Available online 20 February 2013 Edited by Angel Nebreda Keywords: Hypoxia HIF-1 ATRIP ATR
a b s t r a c t The ATR–ATRIP protein kinase complex plays a crucial role in the cellular response to replication stress and DNA damage. Recent studies found that ATR could be activated in response to hypoxia and be involved in hypoxia-induced genetic instability in cancer cells. However, the underlying mechanisms for ATR activation in response to hypoxic stress are still not fully understood. We reported that ATRIP is a direct target of HIF-1. Silencing the expression of HIF-1a in cancer cells by RNA interference abolished hypoxia-induced ATRIP expression. Silencing the expression of ATRIP by RNA interference abolished hypoxia induced ATR activation and CHK1 phosphorylation in cancer cells. Taken together, these data shed novel insights on the mechanism of hypoxia-induced activation of the ATR pathway. Ó 2013 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
1. Introduction In eukaryotic cells, several checkpoint-control mechanisms help to preserve the integrity of the genome [1]. Members of the phosphoinositide kinase-related family of protein kinases (PIKKs), such as ATM and ATR, are key upstream regulators of checkpoint responses [2,3]. ATM is activated following double-strand breaks while ATR activation is associated with single-strand breaks and stalled replication forks [4,5]. ATR is recruited to single-stranded DNA via ATR-interacting protein (ATRIP). In response to genomic stress, the ATR interacting protein (ATRIP) binds and is phosphorylated by the DNA damage- and checkpoint-activated kinase ATR [6]. ATRIP directly interacts with replication protein A (RPA) that results in the recruitment of phosphorylated ATR/ATRIP to stalled replication forks and sites of DNA damage [7]. ATR/ATRIP coordinates DNA repair and cell cycle progression in conjunction with several key regulatory proteins, including Rad17 and the 9-1-1 complex [8]. ATR and ATRIP are essential for the DNA damage response and replication-induced cell cycle arrest [7,9]. Intratumoral hypoxia is a hallmark of solid cancer. A hypoxic microenvironment initiates multiple cellular responses, including proliferation and angiogenesis, resulting in the development and ⇑ Corresponding author. E-mail address: [email protected] (W.-G. Xu).
progression of cancer [10]. The homeostatic response to hypoxia is predominantly mediated by the transcription factor hypoxiainducible factor (HIF)-1 which is a heterodimer that consists of two basic helix-loop-helix proteins of the PAS family—a hypoxia regulated a-subunit (HIF-1a or HIF-2a) and a constitutive b-subunit (HIF-1b) [11]. Under normoxic conditions, HIF-a subunits are hydroxylated on proline residues by a class of prolyl hydroxylases. Then prolyl-hydroxylated HIF-a is recognized and targeted for ubiquitin-mediated destruction by the von Hippel–Lindau (VHL) tumor suppressor protein, which is a substrate recognition component of an E3 ubiquitin ligase. Under hypoxic conditions, HIF-a remains unmodified by prolyl hydroxylases and thereby escapes recognition by VHL and destruction [12]. Thus escaped HIF-a dimerizes with HIF-1b, translocates to the nucleus and activates genes of the hypoxia response by binding to the core sequence (RCGTG) of the hypoxic responsive element (HRE) with additional recruitment of the co-activators CBP/p300 [13]. Mutations in VHL can be found in familial and sporadic clear cell carcinomas of the kidney, hemangioblastomas of the retina and central nervous system, and pheochromocytomas, underscoring its gatekeeper function in the pathogenesis of these tumors. Tissue-specific gene targeting of VHL in mice has demonstrated that efficient execution of pVHL-mediated HIF proteolysis under normoxia is fundamentally important for survival, proliferation, differentiation and normal physiology of many cell types. As a
0014-5793/$36.00 Ó 2013 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. http://dx.doi.org/10.1016/j.febslet.2013.02.020
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Fig. 1. Hypoxia and hypoxia reagent induce ATRIP expression. (A) mRNA levels of ATRIP in MCF7, HeLa and HepG2 cells under normoxia, hypoxia or 100 lM DFO (deferoxamine mesylate salt) for 24 h were determined by real-time PCR assay. (B–D) Western blot analysis of ATRIP, HIF-1a and Actin in MCF6 (B), HeLa (C) and HepG2 (D) cells under normoxia (N) or hypoxia (H) condition. (E) HepG2 cells were cultured in hypoxia for the indicated length of time. Lysate of cells were assayed by Western blot with indicated antibodies.
result of hypoxia and genetic mutations, HIF-1a is over-expressed in many human cancers [14]. Emerging evidence suggests that hypoxia can also induce genetic instability in cancer cells [15,16]. Several DNA damage response factors are activated in response to hypoxia and subsequent reoxygenation, including ATM/ATR, Chkl/Chk2 and BRCA1 [17–20]. These findings reveal that hypoxia induces an acute DNA damage response in cancer cells by activating key DNA damage sensors. However, how these sensors are activated by hypoxia is largely unknown. So, it is necessary to understand the mechanisms underlying control of ATR kinase activity in response to hypoxic stress. Here, we show that both the mRNA and protein levels of ATRIP are markedly increased in human cancer cell lines in response to hypoxia. We also found that ATRIP is a direct target gene of HIF1. The increased ATRIP then activates the ATR signaling pathway under hypoxia. Our findings thus help us to understand how ATR is activated by hypoxia, which may have profound biological and therapeutic implications for cancer.
2. Materials and methods 2.1. Cell culture HeLa, MCF7, HepG2, 293T and IMR90 cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) (Gibco, CA, USA) as well as 100 U/ml penicilin and 100 lg/ml streptomycin. For hypoxia treatment, cells were cultured in a hypoxia chamber flushed with 0.1% O2 and 5% CO2 with balance of 95% nitrogen at 37 °C. Control cells were placed in a 5% CO2 and 95% air incubator (20% O2) at 37 °C. 2.2. SiRNA and transfection Small interfering (si) RNA against HIF-1a and ATRIP were purchased from Dharmacon (USA). As a negative control, a siRNA sequence targeting green fluorescent protein (non-targeting siRNA) was used. All transient transfections were performed using Lipo-
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Fig. 2. HIF-1 mediates hypoxia-induced ATRIP expression. (A and B) HIF-1a plasmid was transfected into HeLa (A) and HepG2 (B) cells for 36 h. Lysate of these cells were assayed by Western blot with indicated antibodies. (C) mRNA levels of HIF-1a in HeLa cells transfected with siRNA targeting HIF-1a or non-targeting siRNA were determined by real-time PCR assay. (D) HeLa cells were transfected with siRNA targeting HIF-1a or non-targeting siRNA for 24 h and cultured under hypoxic condition for additional 24 h. Lysate of these cells were assayed by Western blot with indicated antibodies. (E) mRNA and protein levels of ATRIP in HeLa cells transfected with siRNA targeting HIF-1a or non-targeting siRNA under normoxia (N) or hypoxia (H) condition were determined by real-time PCR assay and Western blot. (F) mRNA levels of ATRIP in RCC4 and RCC4/VHL cells were determined by real-time PCR assay. (G) Western blot analysis of ATRIP, HIF-1a, VHL and Actin in RCC4 and RCC4/VHL cells under normoxia condition.
fectamine 2000 (Invitrogen) according to the manufacturer’s instructions. 2.3. RNA isolation and real-time PCR Total RNAs from HepG2 and IMR90 cells were isolated by TRIzol reagent, and reverse transcriptions were performed by Takara RNA PCR kit following the manufacturer’s instructions. In order to quantify the transcripts of the interest genes, real-time PCR was performed using a SYBR Green Premix Ex Taq (Takara, Japan) on Light Cycler 480 (Roche, Switzerland). ATRIP transcript expression was normalized to b-actin mRNA expression using the DCt method and the linearized DCt (i.e. 2 DCt) was used for comparative purposes. The primer sequences used are available upon request. 2.4. Western blot Cells were harvested and lysed with ice-cold lysis buffer (50 mM Tris–HCl, pH 7.4, 100 mM DTT, 2% w/v SDS, 10% glycerol). After centrifugation at 20 000g for 10 min at 4 °C, proteins in the supernatants were quantified and separated by 10% SDS–PAGE, transferred to NC membrane. After blocking with 5% non-fat milk, membranes were immunoblotted with antibodies as indicated, followed by HRP-linked secondary antibodies. The signals were detected by Millipore SuperSignalÒ HRP Substrate kit according to manufacturer’s instructions. 2.5. Chemicals and antibodies Deferoxamine mesylate salt (DFO) was purchased from Sigma–Aldrich (St. Louis, MO), dissolved in DMSO. The antibodies
for ATRIP (#2737), ATR (#2790), Chk1 (2G1D5) (#2360), Phospho-Chk1 (Ser345) (133D3) (#2348), Histone H2A.X (#2595), Phospho-Histone H2A.X (Ser139) (#2577), VHL (#2738), Claspin (#2800) and HIF-1a (#3716) were purchased from Cell Signaling Technology. The antibodies for TopBP1 (ab2402) and Hus1 (ab96297) were purchased from Abcam. Actin (A2228) antibody was purchased from Sigma.
2.6. Luciferase assays The ATRIP promoter was amplified from a human genomic DNA template and inserted into pGL3 basic vector (Promega). A mutant HIF-1 binding motif was generated using a PCR mutagenesis kit (Toyobo) with a primer (mutation sites underlined): 50 -ctcaaa cgcaacacgAAtgaatgctagggcag-30 and a reverse complement primer. For luciferase reporter assays, HepG2 cells were seeded in 12-well plates and transfected with the indicated plasmids. Cells were harvested 30 h after transfection. Luciferase activities were measured using the Dual Luciferase Reporter Assay System (Promega, USA). 2.7. ChIP assays Chromatin immunoprecipitation (ChIP) assay kits were used (Upstate, USA). In short, HepG2 cells were fixed with 1% formaldehyde to cross-link the proteins and DNA, after which DNA was sheared to fragments of 200–800 bp by sonication in an ultrasound bath on ice. The chromatin was then incubated and precipitated with anti-IgG and anti-HIF-1a antibodies. The immunoprecipitated DNA fragments were detected using real-time PCR analysis.
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Fig. 3. Identification and validation of a functional HRE in ATRIP promoter region. (A) Schematic diagrams of the regulating sequences with the putative HRE (black ovals) of ATRIP. Mut: CG was changed into AA. (B) HeLa cells were transfected with the putative ATRIP HRE-luciferase reporter plasmids in the absence or presence of HIF-1a plasmid. After 48 h of transfection, the luciferase activity was measured. Transfection efficiency was normalized by Renilla luciferase expression, and the results are presented as activation over that for an empty vector. (C) The luciferase activity was measured in HepG2 cells with the methods described in (B). (D) HeLa cells were transfected with the putative ATRIP HRE-luciferase reporter plasmids. 24 h after transfection, cells were culture under indicated conditions for additional 24 h, the luciferase activity was measured. (E) Anti-IgG and anti-HIF-1a antibodies were used in the ChIP assays using HepG2 cells incubated with or without hypoxia.
2.8. Statistical analysis Data were summarized as mean ± S.E.M. Statistical significance was analyzed by one-way ANOVA followed by Turkey’s test using SPSS 11.0 software (Chicago, IL, US). A value of P < 0.05 was considered significant. Statistical significance is displayed as ⁄P < 0.05, ⁄⁄ P < 0.01 or ⁄⁄⁄P < 0.001. 3. Results 3.1. Hypoxia and hypoxia reagent induce ATRIP expression To test whether ATRIP expression is regulated by hypoxia, MCF7, HeLa and HepG2 cells were incubated under normoxia (21% O2), hypoxia (1% O2) or medium containing 100 lM DFO (deferoxamine mesylate salt) for 24 h. Real-time-PCR data revealed an increased ATRIP mRNA level in the cells under hypoxic or DFO treatment conditions (Fig. 1A). An increased ATRIP protein level accompanying increased HIF-1a protein levels was also observed in the cells under hypoxic condition (Fig. 1B–D). Indeed, hypoxia induced the expression on ATRIP in HepG2 cells in a time dependent manner (Fig. 1E). Moreover, ATRIP was the only DNA-damage related protein that induced during hypoxia (Fig. S1). Taken together, these results demonstrate that ATRIP can be induced in cells by hypoxia. 3.2. HIF-1 mediates hypoxia-induced ATRIP expression HIF-1 is a global transcriptional regulator of the hypoxic response. To test whether HIF-1 also mediates hypoxia-regulated ATRIP expression, HIF-1a-expressing plasmid was transiently
transfected into HeLa and HepG2 cells. Notably, ectopic expression of HIF-1a could significantly up-regulate ATRIP expression in both cell lines (Fig. 2A and B). To further investigate the role of HIF-1 in the regulation of the expression of ATRIP, HeLa cells were transfected with siRNA targeting HIF-1a or non-targeting siRNA to silence endogenous HIF-1a expression. The inhibition rate was evaluated by both real-time PCR and Western blot (Fig. 2C and D). As expected, ATRIP mRNA and protein levels were markedly decreased when HIF-1a was silenced under normoxic and hypoxic conditions (Fig. 2E). Similar results were also observed in MCF7 and HepG2 cells (Data not shown). To determine if ATRIP expression was affected by pVHL status, ATRIP mRNA and protein levels in normoxia were examined in RCC4 renal cancer cells with mutant pVHL, and in RCC4/VHL cells, an isogenic stable revertant of RCC4 expressing wild-type pVHL [21]. Levels of ATRIP mRNA and proteins were reduced in cells with wild-type versus mutant pVHL (Fig. 2F and G). Taken together, these data suggested that regulation of ATRIP by hypoxia is HIF-1 dependent. 3.3. Identification and validation of a functional HRE in ATRIP promoter region We next determined whether HIF-1a could be a transcriptional regulator of the ATRIP gene. There is one potential HRE at 0.4 kb region upstream to the transcription start site of ATRIP (Chr3:48,487,864-48,487,868, around 0.4 Kb upstream the TSS) (Fig. 3A). HeLa cells were transfected with a reporter vector encoding luciferase under control of the ATRIP promoter (WT-Luc). Concurrent expression of HIF-1a with the ATRIP reporter construct increased ATRIP promoter activity (Fig. 3B), which was abrogated by mutation of this HIF-1a DNA-binding site (CG-AA) in the ATRIP
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Fig. 4. Hypoxia-induced ATR activation was mediated by ATRIP. (A) mRNA and protein levels of ATRIP in HepG2 cells transfected with siRNA targeting ATRIP or non-targeting siRNA were determined by real-time PCR assay and Western blot. (B) HepG2 cells transfected with siRNA targeting ATRIP or non-targeting siRNA were under normoxia (N) or hypoxia (H) condition. Lysate of these cells were assayed by Western blot with indicated antibodies.
promoter (Mut-Luc). The stimulatory effect of HIF-1a on ATRIP promoter activity was also shown in HepG2 cells (Fig. 3C). Moreover, hypoxia alone could significantly stimulate the activity of WT-Luc but not Mut-Luc (Fig. 3D). These observations indicated that HIF-1a could bind to the promoter of ATRIP. To further demonstrate that HIF-1 binds to the HRE in the ATRIP promoter, we performed ChIP assay using antibodies against HIF-1a (IgG as a negative control) in normoxic- and hypoxic-cultured HepG2 cells. As depicted in Fig. 3E, HIF-1a but not IgG specifically bound to the HRE sequence in the ATRIP promoter. In summary, these results show that ATRIP is directly induced by HIF-1 under hypoxia. 3.4. Hypoxia-induced ATR activation is mediated by ATRIP Several studies have demonstrated that ATM and ATR are activated in response to hypoxia [17,20]. ATR is recruited to singlestranded DNA via ATRIP [6]. Both ATR and ATRIP are essential for the DNA damage response and replication-induced cell cycle arrest [7]. On the basis of this, we hypothesized that hypoxia promotes ATR activation through induction of ATRIP. To test this possibility, HepG2 cells were transfected with siRNA targeting ATRIP or nontargeting siRNA to silence endogenous ATRIP expression. The inhibition rate was evaluated by both real-time PCR and Western blot (Fig. 4A and B). In agreement with the previous study, ATR signaling pathway was activated in HepG2 cells incubated under hypoxia, as evidenced by increased phosphorylation of ATR targets including CHK1 and H2AX (Fig. 4B). However, these hypoxia-induced effects were almost completely abolished when ATRIP was silenced in HepG2 cells (Fig. 4B). Taken together, these data suggested that ATRIP was required for hypoxia-induced ATR activation. 4. Discussion In the present study, we showed that ATRIP was induced by hypoxia in human cancer cells. We provided evidence that ATRIP is a direct target gene of HIF-1. Hypoxia and ectopically expressed HIF1a protein significantly increased ATRIP expression at both mRNA and protein levels in cancer cell lines. Then, silencing of HIF-1a by specific siRNA antagonized hypoxia-induced ATRIP expression. Using luciferase reporter and ChIP assay, we found that HIF-1a directly bound to the HRE located at 386 to 381 of ATRIP gene,
which is essential for HIF-1-induced ATRIP expression. Without ATRIP, hypoxia induced ATR activation was dramatically decreased, indicating that ATRIP may be necessary for hypoxia induced ATR activation. Thus, ATR signaling was, at least, partially activated by induced ATRIP under hypoxia stress in cancer cells. During the review process of our study, one interesting paper described a positive regulation of ATR over HIF-1 [22]. Consistent with our observations, ATR protein level was unchanged during hypoxia. Our data support this work by indicating that ATR-activated HIF-1 further amplifies ATR signaling through ATRIP induction to form a positive feedback loop in the process of hypoxia. ATRIP could also be induced in non-transformed hypoxic IMR90 cells (Fig. S2). Both ATR and ATRIP are essential for the DNA damage response and replication-induced cell cycle arrest. Non-transformed hypoxic cells can undergo cell cycle arrest at the G1/S interface without any alteration in their long-term viability [23], suggesting the HIF-1-ATR-ATRIP axis may play a role in hypoxiainduced cell cycle arrest in non-transformed cells. Future work may focus on the role of the HIF-1-ATR-ATRIP axis in the regulation of hypoxic non-transformed cells. HIF-1 is a heterodimer that consists of two basic helix-loop-helix proteins of the PAS family—a hypoxia regulated a-subunit (HIF1a or HIF-2a) and a constitutive b-subunit (HIF-1b). Both HIF-1a and HIF-2a have been reported to be over-expressed in a series of tumors [24,25]. Therefore, it would be interesting to investigate the expression of ATRIP in human cancers. The relevance between HIF-1a/HIF-2a and ATRIP should also be investigated as well in the future. ATRIP is required for the cellular response to DNA damage and replication stress. ATRIP is phosphorylated by ATR and colocalizes with ATR in cells treated with DNA damaging agents [26]. Previous studies have also shown that the single-stranded DNA binding protein RPA (replication protein A) is necessary for the in vitro binding of ATRIP to DNA and that loss of RPA prevents the formation of ATR and ATRIP foci in cells treated with ionizing radiation [27]. However, whether RPA is required for hypoxia-induced ATR activation and whether RPA is induced or activated by hypoxia remains unknown. It would be interesting to understand the role of RPA in hypoxia-induced ATR activation. In summary, these discoveries shed new insights on the mechanism of ATRIP expression regulation and hypoxia induced activation of the ATR signaling pathway. Ultimately, these data may provide the rationale for the development of ATRIP inhibitors to
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prevent hypoxia-induced ATR activation and genetic instability in cancer cells. Conflict of Interest None of the authors has any potential conflicts of interest. Acknowledgements This work was supported by Grant from the Key Project of Shanghai Science and Technology Commission (NO10411956700). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.febslet.2013.02.020. References [1] Medema, R.H. and Macurek, L. (2012) Checkpoint control and cancer. Oncogene 31, 2601–2613. [2] Bensimon, A., Aebersold, R. and Shiloh, Y. (2011) Beyond ATM: the protein kinase landscape of the DNA damage response. FEBS Lett. 585, 1625–1639. [3] Smith, J., Tho, L.M., Xu, N. and Gillespie, D.A. (2010) The ATM-Chk2 and ATRChk1 pathways in DNA damage signaling and cancer. Adv. Cancer Res. 108, 73–112. [4] Flynn, R.L. and Zou, L. (2011) ATR: a master conductor of cellular responses to DNA replication stress. Trends Biochem. Sci. 36, 133–140. [5] Lavin, M.F., Kozlov, S., Gueven, N., Peng, C., Birrell, G., et al. (2005) ATM and cellular response to DNA damage. Adv. Exp. Med. Biol. 570, 457–476. [6] Cortez, D., Guntuku, S., Qin, J. and Elledge, S.J. (2001) ATR and ATRIP: partners in checkpoint signaling. Science 294, 1713–1716. [7] Zou, L. and Elledge, S.J. (2003) Sensing DNA damage through ATRIP recognition of RPA–ssDNA complexes. Science 300, 1542–1548. [8] Yang, X.H. and Zou, L. (2006) Recruitment of ATR–ATRIP, Rad17, and 9-1-1 complexes to DNA damage. Methods Enzymol. 409, 118–131. [9] Motoyama, N. and Naka, K. (2004) DNA damage tumor suppressor genes and genomic instability. Curr. Opin. Genet. Dev. 14, 11–16.
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