The ZFHX3 (ATBF1) transcription factor induces PDGFRB ...cerebellum, we investigated a distinct...

RESEARCH ARTICLE

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INTRODUCTIONAtaxia telangiectasia (A-T) is a neurodegenerative, inherited diseasecaused by mutations in the ATM gene (Savitsky et al., 1995), whichencodes a serine-threonine kinase that plays a central role in thecellular response to DNA double-strand breaks (DSBs) (Khannaand Jackson, 2001; Shiloh, 2003). The name A-T represents theclinical symptoms, which include degeneration of the cerebellumcausing ‘ataxia’ and microcapillary aneurysm giving rise to‘telangiectasia’.

Abnormal elevation of alpha-fetoprotein (AFP) is one of thediagnostic markers of A-T patients. The AFP and albumin genesare present in tandem on human chromosome 4 (Kao et al., 1982;Urano et al., 1984). AFP is normally expressed exclusively inembryonic liver and completely suppressed in normal adult liver,but it is abnormally elevated in the serum of adult patients with A-T. The mechanism of gene suppression of AFP must bedysfunctional in A-T, and this mechanism might be involved inother systemic problems of the disease.

We focus here on the functional relationship between thesymptoms of A-T and the transcription regulatory factor ZFHX3(ATBF1). This factor was discovered as a DNA-binding protein thatbinds the AT (adenine and thymine)-rich element of the AFP gene

to suppress its expression and was named ATBF1 (AT-motifbinding factor 1) (Morinaga et al., 1991); subsequently, the full-length transcript ATBF1-A was found to be expressed in a neuronaldifferentiation-dependent manner (Miura et al., 1995; Jung et al.,2005). More recently, the Human Genome Organisation named thetranscription factor as ZFHX3 (zinc finger homeobox 3). Thebinding of ZFHX3 to the AT-rich element of the AFP genesuppresses expression by interfering with the binding of activators(Morinaga et al., 1991; Yasuda et al., 1994). Interplay between p53and TGF- effectors also plays an important role in the suppressionof the AFP gene (Wilkinson et al., 2008). Furthermore, p53 bindsto and cooperates with ZFHX3 to activate the p21Waf1/Cip1 promoterto trigger cell cycle arrest (Miura et al., 2004).

In addition to being elevated in adults with A-T, AFP is expressedin hepatocellular carcinoma (HCC) cells and in specific gastriccancer cells, called AFP-producing gastric cancer (AFP-GC) cells,in adults, and this has been linked with ZFHX3 deficiency. ZFHX3mRNA expression in HCCs was significantly reduced in cancer cellscompared with the corresponding surrounding liver tissues (Kimet al., 2008). In addition, AFP-GC cells showed reduced or absentZFHX3 expression, and an extremely malignant character with ahigh frequency of metastasis, which might be related to alteredfunction of adhesive molecules (Kataoka et al., 2001; Cho et al.,2007).

The most serious issue in A-T is the early onset ofneurodegeneration in the cerebellum. DNA DSBs play an importantrole in inducing neurodegeneration in A-T. However, paradoxicalclinical cases have been reported with mild symptoms, as definedby clinical examination and a quantitative A-T neurological index.Surprisingly, no ATM was detected in such patients’ cells, andsequence analysis revealed that they were homozygous for atruncating ATM mutation that is expected to lead to the classical,

Disease Models & Mechanisms 3, 752-762 (2010) doi:10.1242/dmm.004689© 2010. Published by The Company of Biologists Ltd

1Department of Molecular Neurobiology, Graduate School of Medical Sciences,Nagoya City University, Nagoya, 467-8601, Japan2Department of Pathology, Niigata Rosai Hospital, Touncho, Joetsu, Niigata, 942-8502, Japan3National Institute for Longevity Sciences, Obu, Aichi, 474-0031, Japan4Department of Radiology, Graduate School of Medical Sciences, Nagoya CityUniversity, Nagoya, 467-8601, Japan5Queensland Institute of Medical Research and University of Queensland Centrefor Clinical Research, Brisbane 4029, Queensland, Australia*Author for correspondence ([email protected])

SUMMARY

Ataxia telangiectasia (A-T) is a neurodegenerative disease caused by mutations in the large serine-threonine kinase ATM. A-T patients suffer fromdegeneration of the cerebellum and show abnormal elevation of serum alpha-fetoprotein. Here, we report a novel signaling pathway that links ATMvia cAMP-responsive-element-binding protein (CREB) to the transcription factor ZFHX3 (also known as ATBF1), which in turn promotes survival ofneurons by inducing expression of platelet-derived growth factor receptor (PDGFRB). Notably, AG1433, an inhibitor of PDGFRB, suppressed theactivation of ATM under oxidative stress, whereas AG1433 did not inhibit the response of ATM to genotoxic stress by X-ray irradiation. Thus, theactivity of a membrane-bound tyrosine kinase is required to trigger the activation of ATM in oxidative stress, independent of the response to genotoxicstress. Kainic acid stimulation induced activation of ATM in the cerebral cortex, hippocampus and deep cerebellar nuclei (DCN), predominately inthe cytoplasm in the absence of induction of -H2AX (a marker of DNA double-strand breaks). The activation of ATM in the cytoplasm might playa role in autophagy in protection of neurons against oxidative stress. It is important to consider DCN of the cerebellum in the etiology of A-T, becausethese neurons are directly innervated by Purkinje cells, which are progressively lost in A-T.

The ZFHX3 (ATBF1) transcription factor induces PDGFRB,which activates ATM in the cytoplasm to protectcerebellar neurons from oxidative stressTae-Sun Kim1, Makoto Kawaguchi2, Mitsuko Suzuki1, Cha-Gyun Jung3, Kiyofumi Asai1, Yuta Shibamoto4, Martin F. Lavin5, Kum Kum Khanna5 and Yutaka Miura1,*

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ATM activation in the cytoplasm RESEARCH ARTICLE

severe neurological presentation. Moreover, the cellular phenotypeof these patients was indistinguishable from that of classical A-T:all the tested parameters of the DNA DSBs response were severelydefective, as in typical A-T. This analysis showed that the severityof the neurological component of A-T was determined not only byATM mutations but also by other influences yet to be found(Alterman et al., 2007). Researchers therefore started to search formechanisms other then DNA DSBs to improve understanding ofA-T neurodegeneration.

ZFHX3 was found to be one of several hundred substrates ofATM (Matsuoka et al., 2007). The functional meaning of itsphosphorylation could be similar to the phosphorylation of p53 byATM, because both ZFHX3 and p53 share the same consensussequence as a target of ATM and function as a member oftranscription factors in the nucleus. Phosphorylation stabilizes p53by reducing the interaction with its negative regulator, theoncoprotein MDM2 (Banin et al., 1998; Shieh et al., 1997). Weestimated that ATM might also stabilize and activate ZFHX3. Weare interested in the clinical symptoms of A-T patients, which couldbe partly explained by the abnormal function of ZFHX3. Recently,variants in ZFHX3 have been reported to be associated with geneticsusceptibility to Kawasaki disease (also known as mucocutaneouslymph-node syndrome), which induces high risk of coronaryaneurysmal dilatation (Burgner et al., 2009). The report suggestedthat ZFHX3 played a role in protection against the formation ofaneurysms.

The relationship between ZFHX3 and various symptoms of A-T suggests an important role of ZFH3 in the pathogenesis of A-T, and we therefore sought to understand its transcription and identify its target genes. We describe here a transcriptionalregulatory mechanism that operates ZFHX3 gene expression inresponse to retinoic acid via ATM activation, and report thatZFHX3 regulates expression of adhesion molecules and platelet-derived growth factor receptor (PDGFRB). Furthermore, to clarifythe reason why A-T shows neurodegeneration specifically in thecerebellum, we investigated a distinct expression of PDGFRB inthe cerebellum and analyzed the biological meaning of thisexpression. It has been known that the activation of p53 byoxidative stress involves PDGFRB-mediated ATM kinase activation(Chen, K. et al., 2003). Oxidative stress induces activation of ATMin the cytoplasm (Alexander et al., 2010). Collectively, theseobservations suggested an important role of ATM in the cytoplasmin response to oxidative stress in neurons independent from a rolein repairing DNA DSBs in the nucleus (Dupre et al., 2006). Weinvestigated a newly identified signal pathway that activates ATMin the cytoplasm for the protection of neurons against excitotoxicity.The mechanism should be responsible for the survival of neuronsto protect organelles from oxidative stress in the cytoplasm.

RESULTSThe ZFHX3 promoter is activated by CREB in response to ATMactivationRetinoic acid (RA) strongly affects the expression of Hox homeoticgenes. Hox gene expression by neuronal stem cells in the hindbrainand branchial region of the head in the mouse embryo is particularlysensitive to RA (Marshall et al., 1996). The induction of neuronaldifferentiation by RA has been established using cell lines includinghuman and mouse embryonal carcinoma cells (Andrews et al., 1984;

Andrews, 1984). The regulatory mechanism of gene expression withRA treatment is based on nuclear retinoic acid receptors (RARs orRXRs) that recognize particular sequence motifs within targetgenes, called retinoic acid response elements (RAREs) (Marshallet al., 1996). ZFHX3 is a nonclustered homeotic factor expressedin post-mitotic neurons in the embryonic brain (Jung et al., 2005).Importantly, the ZFHX3 promoter is known to lack RAREs, butZFHX3 expression is induced by RA (Miura et al., 1995). Recently,a new RA-activated pathway, dependent on ATM and cAMP-responsive-element-binding protein (CREB), has been identifiedthat regulates RA-induced neuronal differentiation (Fernandes etal., 2007). Notably, we observed a CREB-binding element (CRE) inthe 5� flanking sequence of the human ZFHX3 promoter andtherefore hypothesized that signaling from RA via ATM and CREBactivation might play an important role in initiating ZFHX3 geneexpression after RA treatment.

We cloned various lengths of the human ZFHX3 promoter (Fig.1; supplementary material Fig. S1) upstream of the luciferase geneas a reporter (Fig. 1B), transiently transfected these constructs intomouse embryonal carcinoma P19 cells, and measured reporteractivity with or without stimulation by increasing amounts of RA.RA activated the neuron-specific 5.6-kb promoter in a dose-dependent manner (Fig. 1B, –5.6), whereas a 5� 1.6-kb deletionreduced the promoter activity (Fig. 1B, –4.0) to the basal levelsobserved with 0.42-kb promoters (Fig. 1B, –0.42), which did notrespond to RA. A 5� addition of a 1.6-kb sequence including a CREconsensus sequence to the 0.42-kb basal promoter sequencemarkedly enhanced the response to RA (Fig. 1B, –5.6). Thisresponse was abolished by pre-incubation with the ATM-specifickinase inhibitor KU-55933 (Fig. 1C). To confirm the involvementof CREB, a key survival factor for differentiating neurons, weoverexpressed dominant-negative CREB (dn-CREB) by using thepCMV-CREB133 vector, which expressed a mutant variant of theCREB protein in which a serine was mutated to alanine at aminoacid 133. This mutation blocks phosphorylation of CREB byprotein kinase A, thus preventing cAMP activation of transcription.We observed a significant reduction in transactivation of the RA-responsive promoter construct after RA treatment in cellsexpressing the dn-CREB (Fig. 1D, –5.6). In addition, mutation ofthe CREB-binding site in the promoter significantly impairedpromoter activation in response to RA with or without expressionof dn-CREB (Fig. 1D, –5.6mut). These results indicate that ATMand CREB pathway activation is the mechanism employed by RAto transactivate the neuron-specific ZFHX3 promoter.

We expected to observe maximum promoter activation with the5.6-kb promoter sequence because it contains multiple consensuselements of activators in addition to CRE (see supplementarymaterial Fig. S1). However, the activity of the 5.6-kb promoter waslower than that of a short CRE-containing fragment (5.6),suggesting unknown negative regulatory elements in the promotersequence.

ZFHX3 activates adhesion molecules and PDGFRBRA stimulation of P19 cells produced aggregated structures calledembryonic bodies on bacterial-grade dishes over 4 days, which weretransferred to cell-culture-grade dishes to generate neuron-like cellsthat adhere to the dishes (Fig. 2B1). We observed suppression ofcell adhesion to culture dishes by treatment with KU-55933 (Fig.

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ATM activation in the cytoplasmRESEARCH ARTICLE

2B2). Because there was no significant alteration of activatedcaspase 3 between cells treated with KU-55933 and control cells(supplementary material Fig. S2), we concluded that the suppressionof cell adhesion did not result from an increased number ofapoptotic cells but from reduction of adhesive molecules bytreatment with KU-55933.

We investigated whether the primary cause of the detachmentof cells was related to the function of ATM or ZFHX3 by performingtwo distinct RNA-silencing experiments, using small interferingRNA (siRNA) targeting (1) Atm RNA and (2) Zfhx3 RNA. AtmsiRNA reduced the total amount of ATM protein (Fig. 2A, lane 2)and suppressed Atm mRNA to 30% of the normal levels (Fig. 2E),but still this treatment kept the sufficient levels of activated ATM(pS-ATM; autophosphorylated on Ser1981) (supplementarymaterial Fig. S3B) and there was no effect on adhesive moleculesby this treatment (Fig. 2B4,E). By contrast, the suppression of kinaseactivity with KU-55933 reduced the level of pS-ATM(supplementary material Fig. S3A). The reduction of pS-ATMinduced the suppression of ZFHX3, which is associated withdecreased levels of adhesive molecules (Fig. 2B2,D). Notably, Atm-siRNA-treated cells stably expressed Zfhx3 and maintained wild-type levels of pS-ATM, and such cells were attached to the culturedish (Fig. 2B4,E). By contrast, suppression of Zfhx3 with KU55933or direct suppression with Zfhx3 siRNA both induced distinctdetachment of cells from culture dishes (Fig. 2B2,B5), which wasassociated with decreased levels of adhesion molecules (Fig. 2D,F).These results led us to conclude that adhesion suppression bytreatment with KU-55933 is essentially the effect of the suppressionof Zfhx3. The expression microarray analysis revealed that genesencoding extracellular matrix (ECM) components and variousenzyme-linked receptors were significantly suppressed by silencingof Zfhx3 in P19-derived neuron-like cells (supplementary materialTable S1). Semiquantitative real-time polymerase chain reaction(RT-PCR) analysis confirmed the significant decrease of expressionof genes for adhesion molecules including procollagen type III 1(Col3a1) and integrin 8 (Itga8), and the platelet-derived growthfactor receptor (Pdgfrb) in ZFHX3-depleted cells (Fig. 2B5,F).

Membrane receptors are required to trigger the activation of ATMin response to oxidative stressAlthough Pdgfrb-mutant mice reach adulthood without apparentanatomical defects, their brains are more vulnerable to damage afterdirect injection of N-methyl-D-aspartate (NMDA) or cryogenicinjury (Ishii et al., 2006), indicating that PDGFRB expression isimportant to protect neurons from glutamatergic excitotoxicity.The association of PDGF receptor and integrin causes receptorclustering, increases PDGF binding and promotes PDGF receptoractivation (Zemskov et al., 2009). ZFHX3 might play a key role ininducing both integrin and PDGFRB expression, which wouldfacilitate the associated signal transduction from these membranereceptors.

Oxidative stress is a causal, or at least an ancillary, factor inseveral adult neurodegenerative disorders, as well as in stroke,trauma and seizures (Coyle and Puttfarcken, 1993). It is consideredto be one of the major causes for deficient survival of Purkinjeneurons from A-T mutant mice (Chen, P. et al., 2003). Oxidativestress induces PDGFRB-mediated ATM kinase activation in varioustypes of cells (Chen, K. et al., 2003). This newly identified PDGFRB-

Fig. 1. ZFHX3 promoter is activated by retinoic acid via signaling fromATM to CREB activation. (A)Schematic diagram shows the genomic structureof the human ZFHX3 gene. Boxes labeled 1-10 indicate exons. Alternativesplicing-1 produces a longer form of ZFHX3 transcript (also called ATBF1-A) andshorter form of ZFHX3 transcript (also called ATBF1-B). Alternative splicing-2was found as a variant in prostatic cancers (Sun et al., 2005). The transcriptioninitiation sites for the two types of mRNAs (ATBF1-B and ATBF1-A) are indicatedby arrows (PB and PA) starting from the exons 0 and 1. The longer form ofZFHX3 (PA) promoter region (5.6 kb) was used for the reporter assay becauseof its neuron-specific activity. (B)The CRE sequence indicated by an open circleis located at 5.5 kb upstream from the transcription initiation site in ATBF1-A-specific exon 1. Promoter fragments for the luciferase reporter assay arenamed as follows: 5.6 is the original promoter sequence from the 5� flankingsequence of the gene (5.6-kb SalI-BamHI), including 177 bp of the noncodingregion of exon 1 sequence; 5.6 has a deletion of an internal fragment (3.8-kbHindIII-StuI) from 5.6; 4.0 has a deletion of 1.6-kb fragment from the 5�terminal sequence; and 0.42 is limited to the proximal 0.42-kb fragment of thepromoter. All values are depicted as mean ± s.e.m. from at least threeindependent experiments and considered significant if *P



mediated signal pathway is likely to be an important issue inunderstanding the mechanism of excitotoxicity. We tested whetherPDGFRB contributed to ATM activation in P19-derived neuron-like cells under conditions of oxidative stress induced by hydrogenperoxide. We observed rapid activation of ATM as assessed by itsphosphorylation on Ser1981 (pS-ATM) within 15 minutes ofexposure of cells to hydrogen peroxide (100 M) (Fig. 3A) and theactivation was increased with increasing dose of hydrogen peroxide(1-500 M) (Fig. 3B). The activation of ATM in response tooxidative stress was strongly suppressed by AG1433, a specificinhibitor of PDGFRB and of VEGFR-2 (vascular endothelial growthfactor receptor 2; also known as FLK-1 and KDR) (Fig. 3C,D). Bycontrast, the activation of ATM in response to X-ray irradiation

was not suppressed by the addition of AG1433 (Fig. 3E,F).Therefore, we concluded that these membrane receptors play animportant role in triggering the activation of ATM under oxidativestress in neuronal cells.

ATM is activated in the cytoplasm of neurons in respond toneuronal excitationActivated ATM is localized to nuclear foci in response to genotoxicstress but displays a diffuse pattern in response to hypotonicstimulation or chloroquine treatment (Bakkenist and Kastan, 2003),or RA stimulation (Fernandes et al., 2007). We observed nuclearfoci of activated ATM in response to oxidative-stress-induced DNADSBs, which colocalized with -H2AX, a marker of DNA DSBs

Fig. 2. A set of procollagen, integrin and Pdgfrb genes areactivated by ZFHX3. P19-derived neuron-like cells were attachedon culture dishes and showed extension of neurites. Westernblotting revealed that ATM was expressed in the control cells (A,lane 1) and that Atm siRNA suppressed ATM (A, lane 2); in addition,Zfhx3 was highly expressed (A, lane 3) and Zfhx3 siRNA suppressedZFHX3 (B, lane 4). Treatment with KU-55933 induced detachmentof cells from the culture dish (B2), but there was no apparentalteration in attachment as a result of treatment with Atm-siRNA(B4). Zfhx3 siRNA suppressed adhesion on the culture dish, withthe majority of cells becoming detached (B5). Semi-quantitativeanalysis by RT-PCR was used to compare the relative expressionlevels of the genes Zfhx3, the platelet-derived growth factorreceptor (Pdgfrb), integrin 8 (Itga8) and procollagen type III 1(Col3a1) during neuronal differentiation of P19 cells at day 5 andday 7 (C). KU-55933 treatment significantly suppressed Zfhx3,Pdgfrb, Intga8 and Col3a1 expression (D). Treatment with AtmsiRNA suppressed Atm, but the expression levels of other factorswere not altered (E). Zfhx3 siRNA significantly suppressed Zfhx3,Pdgfrb, Intga8 and Col3a1 expression (F). The relative expressionlevels of each mRNA were measured three times and estimated byquantitative RT-PCR as expression ratios of KU-55933 to controlDMSO treatment, Atm siRNA to control siRNA, or Zfhx3 siRNA tocontrol siRNA, after normalization by paired measurement ofglyceraldehyde-3-phosphate dehydrogenase expression(increased ratio from control levels indicated by gray bar).Statistical significance was assessed with Student’s t-test:**P



(supplementary material Fig. S4). Recently, activation of ATM hasbeen demonstrated in the cytoplasm in response to oxidative stress,which was shown to be independent of its activation in the nucleusby the same agent (Alexander et al., 2010). We failed initially todistinguish the distinct activation of ATM in cytoplasm from thatin the nucleus because of the degree of oxidative stress induced(supplementary material Fig. S4). Therefore, we designed more-physiological oxidative stress conditions for neurons to investigateactivation of ATM in the cytoplasm independent of that in thenucleus.

Although multiple factors can precipitate oxidative stress inneurons, the neurotransmitter glutamic acid is a major factor thatinduces excitotoxicity of neurons in the brain. Kainic acid (KA) is

a potent glutamate receptor agonist with selectivity towards non-NMDA-type glutamate receptors (Olney et al., 1974). We applieda KA stimulation model to induce metabolic oxidative stress inmouse neurons (Coyle and Puttfarcken, 1993; Zhang et al., 2002).KA stimulation caused two types of seizures in these animals. Onecase showed repeated bending movement of the head. Another caseshowed abnormal continuous muscle contraction (supplementarymaterial Movie 1), and induced distinct activation of ATM in themouse brain at the cerebral cortex, hippocampus and deepcerebellar nuclei (DCN) (supplementary material Fig. S5). We foundactivated ATM predominantly in the cytoplasm (Fig. 4A1;supplementary material Fig. S6) in the absence of -H2AX induction(Fig. 4A2; supplementary material Fig. S6). By contrast, X-rayirradiation induced genotoxic stress and ATM was activated in thenucleus (Fig. 4B1; supplementary material Fig. S6) associated with-H2AX foci (Fig. 4B2; supplementary material Fig. S6). Weobserved distinct vacuolar formation in neurons in DCN bystimulation with KA (supplementary material Fig. S5B3).

Suppression of PDGFRB in DCN of ATM-deficient miceWe examined in more detail the relationship between ZFHX3 andPDGFRB expression in P19 cells before and after neuronaldifferentiation. A prominent increase of ZFHX3 and PDGFRBprotein expression was observed in the terminally differentiatedP19 neuron-like cells at day 7 (Fig. 5A). Screening analysis by insitu hybridization (http://developingmouse.brain-map.org/data/Pdgfrb.html) showed that PDGFRB was highly expressed in DCN.Immunohistochemistry revealed distinct staining of ZFHX3 (Fig.5C4,C5) and PDGFRB (Fig. 5C6,C7) in large neurons of the DCNof adult control mouse brain, but expression of both proteins wasstrongly suppressed in ATM-deficient mice (Fig. 5C11-C14). BrdUstaining was used in an embryonic rat to distinguish proliferating(Fig. 5B2i) and post-mitotic (Fig. 5B2ii) cells. The correlated

Fig. 3. AG1433 suppressed the activation of ATM in response to oxidativestress. P19 cells were used at day 5 during neuronal differentiation. (A)ATMactivation in response to oxidative stress with hydrogen peroxide (100M) forthe indicated time points (0, 5, 15, 30 and 60 minutes). (B)ATM activation atdifferent concentrations of hydrogen peroxide dose (1, 10, 100, 500M); cellswere harvested 30 minutes after treatment. ‘X’ indicates stimulation with 10Gy X-ray irradiation for comparison. (C)Cells were treated with hydrogenperoxide (100M) for 30 minutes to activate ATM (lane 3-7). The activation ofATM was suppressed by adding AG1433, an inhibitor of PDGFRB and VEGFR-2;AG1433 was dissolved in DMSO and added at different concentrations (lane 4-7). A control experiment was performed with 0.1% DMSO using the sameconcentration of solvent as for AG1433 (5M) (lane 3). (D)The experimentswere repeated and measured in triplicate. The ratios of pS-ATM to ATM wereanalyzed statistically. (E)After treatment of cells with 10 Gy X-rays, andharvesting 1 hour later, ATM was activated (lane 3-7), and activation was notsuppressed by adding AG1433. A control experiment was performed with0.1% DMSO (lane 3). (F)The experiments were repeated and measured intriplicate. The ratios of pS-ATM to ATM were analyzed statistically. The resultsof western blotting were quantified and subjected to statistical analysis usingthe Mann-Whitney U-test with Bonferroni Correction. *P



expression of ZFHX3 and PDGFRB in the post-mitotic neuronswas consistent with the results of the microarray analysis using amodel of neuronal differentiation (supplementary material TableS1). Suppression of PDGFRB in DCN of the ATM-deficient mousemight account for the mechanisms of specific neurodegenerationin the cerebellum.

DISCUSSIONThe data described here provide further support for a role forcytoplasmic ATM activation in protection against neuronal celldeath. It is well established that a significant amount of ATM proteinis present in the cytoplasm of a variety of cell types includingneurons (Kuljis et al., 1999; Barlow et al., 1996; Boehrs et al., 2007)(Gorodetsky et al., 2007). More recently Li et al. (Li et al., 2009)showed that cytoplasmic ATM modulates synaptic function. Theydemonstrated that ATM forms a complex with the synaptic vesicleproteins VAMP2 and synapsin-1 and is responsible forphosphorylation of these proteins. These data suggest that a non-nuclear role for ATM might be important in protecting againstneurodegeneration. Alexander et al. (Alexander et al., 2010) haveshown that reactive oxygen species rapidly activate ATM in thecytoplasm to repress the kinase mTOR in the mammalian targetof rapamycin complex 1 (mTORC1) and induce autophagy. This

does not involve signaling from DNA DSBs and the authors makethe intriguing suggestion that this activation might involve theoxidation of sulfhydryl groups on ATM to alter its conformation.

In this study, we show that ATM induces ZFHX3 expressionduring RA-induced neuronal differentiation of P19 cells byactivation and binding of CREB to a CRE consensus site locatedin the ZFHX3 promoter. We also show that ZFHX3 regulates targetgenes that encode cell adhesion molecules (procollagen type III 1,integrin 8) as well as PDGFRB. PDGFRB is a key regulator notonly for connective tissue cells in the induction of adhesionmolecules but also for neurons in protection from glutamatergicexcitotoxicity (Ishii et al., 2006). Notably, we showed that PDGFRBand/or VEGFR-2 positively supported ATM activation in responseto oxidative stress in neurons. We identified significant elevationof Pdgfrb and Vegfr-2 mRNA in P19-derived neuron-like cells(supplementary material Fig. S7). Although neurons are highlysusceptible to oxidative stress because of their high rate of oxidativemetabolism and low level of antioxidant enzymes (Brooks et al.,2000), membrane receptors might immediately respond to oxidativestress through the activation of ATM in the cytoplasm to protectneurons.

We revealed here the activation of ATM in the cytoplasm inresponse to excessive excitation of neurons. The excitation of

Fig. 5. PDGFRB is expressed in neurons specifically indeep cerebellar nuclei. (A) Western blot analysisshowed that the expression of PDGFRB correlated withZFHX3 and was at the highest level at day 7 of neuronaldifferentiation of P19 cells. Statistical analysis used theMann-Whitney U-test: *P



neurons induces metabolic oxidative stress that is a causal, or at leastan ancillary, factor of chronic neurodegenerative disorders (Coyleand Puttfarcken, 1993). We showed that PDGFRB and/or VEGFR-2 were required for the activation of ATM in response to oxidativestress. The suppression of PDGFRB in DCN of ATM-deficient miceprovides an explanation for the reduction of response to oxidativestress. We observed a high incidence of vacuolar formationspecifically in DCN (supplementary material Fig. S5B3), wherePDGFRB was highly expressed (Fig. 5C6,C7). Neurons in the DCNare directly innovated by Purkinje cells and most output fibers of thecerebellum originate from these nuclei (supplementary material Fig.S8). Damage of DCN would inevitably induce degeneration ofPurkinje cells by loss of direct synaptic connections. We considerthe functional unit of Purkinje cells and DCN as important to theetiology of A-T. The distinct activation of ATM in the cytoplasm atthe DCN caused by KA stimulation indicated the protective responseto oxidative stress induced by the excessive neuronal excitation inthese neurons. In particular, DCN responded most strongly to showvacuolar formation. This phenomenon might be related to the factthat the activation of ATM in the cytoplasm induces autophagy viasuppression of mTOR (Alexander et al., 2010). We observed intensiveaccumulation of microtuble-asssociated protein light chain 3 (LC3)in DCN in response to KA treatment (supplementary material Fig.S9). Although the autophagic response was believed to promote acuteneuron death because of destruction of organelles through thisprocess (Wang et al., 2008), the study of chronic neurodegenerativedisease revealed that autophagy was not the primary cause ofneuron death but rather was a protective mechanism for the functionof neurons by clearing dysfunctioning organelles. Impaired autophagyis implicated in Parkinson’s disease in the accumulation ofdysfunctional mitochondria, leading to neurons loss (Narendra etal., 2008). We should further study the mechanism of the autophagyin response to oxidative stress in relation to the activation of ATMin these neurons.

Expression microarray analysis revealed that ZFHX3 significantlyactivated most types of procollagen genes except type II, anessential component of the extracellular matrix during woundhealing. Because collagen type II is a specific component ofcartilage (Cheah et al., 1985), it is reasonable that this type ofcollagen is not expressed in a model of neuronal differentiation.PDGFRB protein becomes prominent in vessels in the proliferatingtissue zone in wounds as early as the first day after surgery(Reuterdahl et al., 1993). Because ZFHX3 regulates procollagengenes as well as PDGFRB, it would be expected to play a crucialrole during wound healing. Therefore, malfunction of ZFHX3might be expected to induce circulatory diseases. Recently, variantsin ZFHX3 have been identified that are associated with geneticsusceptibility to Kawasaki disease, with increasing risk ofaneurismal dilatation in the heart (Burgner et al., 2009). Variantsin ZFHX3 are also associated with susceptibility to atrial fibrillationand ischemic stroke (Benjamin et al., 2009; Gudbjartsson et al.,2009). These linkage studies suggest an important role of ZFHX3in the wound healing process in damaged blood vessels. Notably,PDGFRB is highly expressed in the cerebellum (Lein et al., 2007),the area of the brain that undergoes the specific neurodegenerationthat characterizes A-T.

In summary, we propose here three different pathways to triggerthe activation of ATM (Fig. 6). The first, signaling from DNA DSBs,

induces the activation of ATM at foci in the nucleus. The second,retinoic acid stimulation (Fernandes et al., 2007), activates ATMin a diffuse pattern in the nucleus (Bakkenist and Kastan, 2003).The third, oxidative stress, induces activation of ATM in thecytoplasm (Alexander et al., 2010), which is the pathway in responseto neuronal excitation. We found membrane-bound tyrosinekinases are required to trigger the activation of ATM underoxidative stress, which is independent from the response togenotoxic stress. Once activated, ATM can induce autophagy bysuppressing mTOR to enhance clearance of damaged organelles.Overall, the observations support a role for cytoplasmic ATM inprotecting neurons against oxidative stress and chronic neuronaldegeneration.

METHODSCell cultureP19 mouse embryonal carcinoma cells were maintained in -MEM(Rudnicki and McBurney, 1987). To induce neuronal differentiation,P19 cells were grown as aggregates on bacterial-grade dishes for 4days with 0.5 M all-trans retinoic acid (RA) (Sigma, USA) and10% fetal bovine serum (FBS). Cells were cultured in -MEMcontaining 1% FBS without RA on glass slides coated withcombinations of 0.01% poly-L-lysine (P8920, Sigma) for 3 hoursand 1 g/ml bovine plasma fibronectin (Invitrogen, USA) overnight

Fig. 6. A signaling pathway from ATM to ZFHX3 for the expression ofPDGFRB that is required for the activation of ATM in response tooxidative stress in the cytoplasm. Activation of ATM kinase by retinoic acidstimulation relays to phosphorylation of CREB at Ser133 for the activation oftarget genes. By contrast, X-ray irradiation induces activation of ATM at the fociof DNA DSBs. The genotoxic stress induces extra phosphorylation of CREB atThr100, Ser111, Ser121 to decrease transactivation potential (Shi et al., 2004).In the retinoic-acid-stimulated signal pathway, CREB induces ZFHX3expression by binding to CRE (5�-TGACGTCA-3�) in the ZFHX3 (neuron-specific)promoter located 5.5 kb upstream from the initiation site of ZFHX3transcription. ZFHX3 activates genes for cell adhesion molecules(procollagens, integrins) and PDGFRB for the survival of neurons. Theactivation of ATM in the cytoplasm in response to oxidative stress relayssignals from PDGFRB to LKB1, AMPK, TSC2 and mTOR to maintain homeostasisof organelles by activating autophagy (Alexander et al., 2010).

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at room temperature. Where indicated, the ATM kinase inhibitorKU-55933 [2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one; 2-morpholino-6-(thianthren-1-yl)-4H-pyran-4-one (Calbiochem,USA)] (10 M) was added to the culture medium every day,and AG1433 [2-(3,4-dihydroxyphenyl)-6,7-dimethylquinoxaline(Calbiochem, USA)], a specific inhibitor of PDGFRB and VEGFR-2, at the series of concentrations of 0.05, 0.5, 1.0, 5.0 M was added30 minutes before adding hydrogen peroxide.

MiceATM-wild-type and ATM-deficient mice (provided by P. Leder,Harvard Medical School, Boston, MA) were maintained underambient conditions (23°C, 55% humidity) with controlled light anddark cycles. Genomic DNA was isolated from tail tips and used toamplify a PCR product of the mouse Atm gene by using thefollowing primers: KO-1F, 5�-TGGTCAGTGTAACAGTCAT -TGTGC-3�; KO-1R, 5�-AAGGTTGTAGATAGGTCAGCATTG-3�; KO-2R, 5�-AACGAGATCAGCAGCCTCTGTTCC-3�. PrimerKO-1F is located 220 bp upstream (intron 34) of exon 34, primerKO-1R is located 123 bp into exon 34, and primer KO- 2R is located87 bp into the poly(A) region of PGKneo. These primers generatea PCR product of 342 bp from wild-type animals and a targeted PCRproduct of 406 bp from Atm–/– mice. Kainic acid [2-carboxy-3-car-boxymethyl-4-isopropenyl-pyrrolidine (Enzo Life Science, USA)]was administered [30 mg/kg, intraperitoneal (i.p.)] to male DDYmice. The whole brain was isolated from 3-month-old mice afterperfusion fixation with 4% paraformaldehyde. Pregnant rats (StdWister ST) were administered 5-bromo-2�-deoxy-uridine (BrdU, 50mg/kg) for 3 hours to label E14.5 embryos. The embryos were thendissected out, and their brains were fixed in 4% paraformaldehydeand embedded in paraffin.

X-ray irradiationCells or mice were X-ray-irradiated at a dose rate of 10 Gy usingX-ray machine CAX-210 (Chubu Medical Co. Ltd, Yokkaichi,Japan) operating at 210 kV, 10 mA, for 4 minutes 45 seconds withcopper shield.

Transfection and plasmid constructsTo generate a series of luciferase reporter plasmids, various lengthsof human ZFHX3-promoter regions were cloned intopBluescriptIIKS(+) (Agilent Technologies – Stratagene Products,USA) with SalI and BamHI. The BssHII and BamHI fragmentscontaining ZFHX3 promoter regions were subcloned into the 5�prime position of the luciferase gene of pGV-B basic vector (ToyoInk, Japan) between MluI and BglII sites. The CRE site in 1.0 kbpof the ZFHX3 promoter was mutagenized by PCR-based site-directed mutagenesis using a pair of primers, pGV-F (a forwardprimer with 5� flanking sequence of luciferase gene on pGV-B): 5�-CAATGTATCTTATGGTACTG-3�, mCRE-R (a reverse directionat CRE site to introduce mutations): 5�-CGGAAATGACCA-CAGCAAAG-3�, mCRE-F (a forward direction primer at CRE siteintroduce mutations overlapped with mCRE-F): 5�-CTTTGCTGTGGTCATTTCCG-3�, pGV-R1 (a reverse primerincluding the first ATG codon of luciferase gene on pGV-B): 5�-CTTTATGTTTTTGGCGTCTTCC-3�, pGV-R2 (a reverse primerat 35-bp downstream from the pGV-R1 in luciferase gene on pGV-B): 5�-CCATCCTCTAGAGGATAGAATG-3�, to mutate the CRE

site from 5�-TGACGTCA-3� to 5�-TGTGGTCA-3� (supplementarymaterial Fig. S1). The two sets of PCR product were prepared withpGV-F and mCRE-R as a set, and mCRE-F and pVG-R2 as anotherset. These overlapping primary products were denatured togetherand reamplified with a new set of primers with pGV-F and pGV-R1. Reamplified products were trimmed with MluI and HindIII,and were subcloned into the same cloning site on the pGV-B basicvector. The CREB dominant-negative expression vector pCMV-CREB133 was from Clontech (USA; Cat. No. 631925).

ImmunohistochemistrySections of 4 m from 4% paraformaldehyde (PFA)-fixed, paraffin-embedded tissue were used. Tissue sections were deparaffinizedand rehydrated and then both those for ZFHX3 (110°C, pressurecooker) and those for ATM (98°C, microwave oven)immunostaining were heated in citrate buffer (0.01 M, pH 6.0). Theheat retrieval step was not applied for PDFGRB. Staining wascarried out at room temperature. All sections were incubated withmethanol containing 0.3% hydrogen peroxide and 1.0% sodiumazide to block endogenous peroxidase activity, then incubated withmouse monoclonal anti-ATM Ser1981 phosphorylation site (pS-ATM) monoclonal antibody (at 1:1200 dilution: 7C10D8; Rockland,USA) followed by incubation with horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (Envision labeled polymer,Dako Cytomation, Denmark), rabbit polyclonal anti-ZFHX3antibody (at 1:50 dilution: D1-120; MBL, Nagoya, Japan) followedby HRP-conjugated anti-rabbit IgG antibody (Envision labeledpolymer, Dako Cytomation, Denmark) or goat polyclonal anti-mouse PDGFR antibody (at 1:100 dilutions: R&D Systems, USA)followed by HRP-conjugated anti-goat IgG antibody [MAX-PO (G),Nichirei, Japan]. Primary antibodies and secondary antibodies wereincubated at room for 1 hour. Immunoreactive products were thenvisualized after adding diaminobenzidine as a chromogen. BrdUlabeling and detection was performed using a kit from Roche asper the manufacturer’s recommendations. Tissue sections werecounterstained with hematoxylin.

ImmunocytochemistryCells were fixed in 4% PFA in PBS at room temperature for 20minutes, then washed with 0.25% Triton-X in PBS, and blockedwith 10% normal goat serum. Cells were then incubated for 1 hourat room temperature with primary antibodies against rabbitpolyclonal anti-ZFHX3 antibody (at 1:100 dilution: D1-120; MBL,Japan), mouse monoclonal anti -tubulin isotype III (at 1:500dilution: 3D10; Sigma, USA), monoclonal anti-ATM Ser1981phosphorylation site (pS-ATM) (at 1:100 dilution: 10H11.E12;Rockland, USA), rabbit polyclonal anti-H2AX (at 1:100 dilution:BETHYL, USA). After three washes with 0.25% Triton-X in PBS,cells were visualized by secondary antibodies – Alexa-Fluor-488-conjugated goat anti-mouse for mouse monoclonal antibodies andAlexa-Fluor-546-conjugated goat anti-rabbit (at 1:1000 dilution;Molecular Probes, Invitrogen, USA) for rabbit polyclonalantibodies for 1 hour in the dark and, after two washes with PBS,nuclei were stained with 2.0 g/ml DAPI (at 1:500 dilution of 1 mg/ml solution: 4�,6-diamino-2-phenylindole; Wako, Japan) for5 minutes.

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Western blottingCells were washed with washing buffer (10 mM 0.1 M phosphatebuffer, 250 mM sucrose, 50 mM NaF), and total cell lysate wasprepared with TNE buffer [20 mM Tris-HCl (pH 7.4), 150 mMNaCl, 2 mM EDTA, 1% Nonident-P40, 50 mM NaF]. The proteinswere incubated on ice for 15 minutes, and then centrifuged at15,000 g for 30 minutes at 4°C. The supernatant was obtainedand stored at –80°C. The total protein content was measuredusing the Bradford Assay (Bio-Rad, Hercules, CA). For proteindetection, each sample was separated on a 5-20% polyacrylamidegradient gel and the proteins were transferred to a polyvinylidenedifluoride (PVDF) membrane (Millipore, Billerica, MA). Themembrane was blocked with 3% BSA in Tris-buffered salinecontaining 0.05% Tween 20 (TBS-T) for 1 hour, washed in TBS-T, and then incubated for 1 hour at room temperature withprimary antibodies for mouse monoclonal anti--tubulin (at1:8000 dilution: B-5-1-2; Sigma, USA), rabbit polyclonal anti-ZFHX3 (at 1:2000 dilution: AT-6; MBL, Japan), mouse monoclonalanti-HA-tag (at 1:500 dilution: 5D8; MBL, Japan), mousemonoclonal anti-ATM (at 1:200 dilution: 5C2; Santa Cruz, USA),mouse monoclonal anti-pS-ATM (at 1:1000 dilution: 10H11.E12;Rockland, USA), goat polyclonal anti-PDGFRB (at 1:1000 dilution:R&D Systems, USA) or rabbit polyclonal anti-caspase 3 (at 1:1000dilution: AAP-113; Assay Designs, USA). After washing in TBS-T, the membrane was incubated with peroxidase-conjugatedanti-mouse IgG [at 1:10,000 dilution: anti-mouse IgG (H+Lchain)-HRP; MBL, Japan] for mouse monoclonal antibodies,anti-rabbit IgG [at 1:5000 dilution: anti-rabbit IgG (H+L chain)-HRP, MBL, Japan] for rabbit polyclonal antibodies or anti-goatIgG [at 1:5000 dilution: anti-goat IgG (H+L chain)-HRP; MBL,Japan] for a goat polyclonal antibody for 1 hour at roomtemperature and washed in TBS-T. Immunoreactive signals werevisualized by Amersham ECL Plus western blotting detectionreagents (GE Healthcare, UK).

RNA extraction and RT-PCR analysisTotal RNA from cells was isolated using Trizol reagent (Invitrogen,USA), and 1 g of total RNA was transcribed into cDNA using theReady-To-Go You-Prime First-Strand Beads kit (GE Healthcare,UK). Real-time PCR was carried out with qPCR MasterMix Plusfor SYBR Green (Eurogentec, USA) using the company’s manualprocedure: incubation at 95°C for 10 minutes followed by 40 cyclesamplification (15 seconds at 95°C, 1 minute at 60°C, 45 seconds at72°C and 15 seconds at 80°C) for SYBR Green detection. Theprimers used for real-time measurement of PCR were as follows:Gapdh, 5�-TGTGTCCGTCGTGGATCTGA-3� and 5�-CCTGCT -TCACCACCTTCTTGA-3�; Zfhx3, 5�-TTCTTTTCCTCCTC -TCTCCTCATC-3� and 5�-CGGTCCGTCGGACTTTTG-3�;Col3a1, 5�-GCACAGCAGTCCAACGTAGA-3� and 5�-TCTCCA -AATGGGATCTCTGG-3�); Itga8, 5�-AGTGGGAGGACC -TGGAAGTT-3� and 5�-AGTGGGAGGACCTGGAAGTT-3�;Pdfgrb, 5�-AACCCCCTTACAGCTGTCCT-3� and 5�-TAATCCCGTCAGCATCTTCC-3�. The expression of each genewas normalized by the corresponding amount of Gapdh mRNA.The relative amounts of each product were calculated using thecomparative CT (2- CT) method described in User Bulletin #2of the ABI Prism 7500 fast Sequence Detection System (AppliedBiosystems, USA).

RNA interferenceRNA interference (RNAi) was performed using Stealth/siRNA(Invitrogen, USA) duplex oligoribonucleotides against Zfhx3(ATBF1) (5�-UACACUGGUCAGACCACUGUCCUUG-3� and 5�-CAAGGACAGUGGUCUGACCAGUGUA-3�), and three sets ofduplex oligoribonucleotides against Atm (5�-UGAACUUC -CCGAUAAUCCACAAGG-3� and 5�-CCCUUGUGGAUUAU -CAGGAAGUUCA-3�; 5�-UAAACAGAGAGAUACU UUCUC -CUGC-3� and 5�-GCAGGAGAAAGUAUCUCUCUGUUUA-3�;5�-UUAGAAGGCCCACUUCCUCUUUGGC-3� and 5�-GCCAAAGAGGAAGUGGGCCUUCUAA-3�). The transfection

TRANSLATIONAL IMPACT

Clinical issueAtaxia telangiectasia (A-T) is a rare, recessive, neurodegenerative disease, withsymptoms that normally appear in early childhood. Initial indications areusually ataxia (a lack of muscle control leading to loss of balance andcoordination) and telangiectasia (tiny red veins, which are most noticeable onthe whites of the eyes). The most serious clinical problem is loss of Purkinjecells, leading to degeneration of the cerebellum (the body’s motor controlcentre). About 70% of sufferers also have thymic hypoplasia and acompromised immune system. Patients are also highly sensitive to X-irradiation and are susceptible to malignant tumors. Currently, there is no cure,nor any treatment that halts the progression of the disease.

A-T is caused by mutation of the ATM gene, which encodes a ubiquitous370-kD serine-threonine kinase that is essential for normal repair of double-stranded DNA breaks; loss of ATM function results in DNA instability. However,there are atypical cases of A-T in which ATM is completely lacking: in thesecases, DNA repair is defective, but symptoms are mild. These cases suggestthat the severity of the neurological component of A-T might be due to acombination of defects, some of which are unknown.

ResultsATM phosphorylates several hundred substrates, including the transcriptionfactor ZFHX3 (also known as ATBF1). In this study, the authors show thatZFHX3 is indirectly induced by ATM, and that ZFHX3 in turn induces themembrane tyrosine kinase PDGFRB, which is a survival factor in neurons.Neurons are highly susceptible to oxidative stress owing to their high rate ofoxidative metabolism and low levels of antioxidant enzymes, and, in A-Tmutant mice, oxidative stress is one of the major causes of Purkinje cell death.Inhibition of PDGFRB activity results in loss of ATM activity under conditions ofoxidative stress, but not genotoxic stress, suggesting a means whereby ATMcan rapidly autoregulate its activity.

To induce oxidative stress, mice were treated with kainic acid, resulting inpredominantly cytoplasmic activation of ATM in the cerebral cortex,hippocampus and deep cerebellar nuclei (DCN). The authors propose thatactivation of ATM in the cytoplasm might play a role in autophagic protectionof neurons against oxidative stress.

Implications and future directionsThe signaling pathway from ATM to PDGFRB described here might beimportant in determining the response of neurons to oxidative stress, and beone of the hitherto unknown factors contributing to A-T. Therefore, targetingthe action of PDGFRB might yield therapies that protect neurons againstoxidative stress. The use of kainic acid to induce neuronal stress in mousebrains will also mean that Atm-deficient mice can be used to observe the earlyonset of neurodegeneration in the cerebellum; currently, the mice cannot beused as a model, because they die of cancer within 3-4 months, beforemanifestation of neurodegenerative disease. Kainic acid treatment willtherefore be a good experimental tool to speed up screening of effectivetherapeutics.

doi:10.1242/dmm.006353

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was performed with Lipofectamine RNAiMAX four times to P19cells during the neuronal differentiation process on days 0, 1, 3 and5. Stealth RNAi negative control duplex (medium GC duplex) wasalso transfected four times, following the protocol of neuronaldifferentiation of P19 cells (Rudnicki and McBurney, 1987).

StatisticsStatistical analysis of results from luciferase assays and westernblotting was performed using the Mann-Whitney U-test withBonferroni Correction. All values were depicted as mean ± s.e.m.from at least three independent experiments and consideredsignificant if *P



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SUMMARYINTRODUCTIONADDRESS INFO: NO SPACE BETWEEN LINES AND ONLY ONE RULERESULTSThe ZFHX3 promoter is activated by CREB in response toZFHX3 activates adhesion molecules and PDGFRBMembrane receptors are required to trigger the activation of ATMATM is activated in the cytoplasm of neurons in respondSuppression of PDGFRB in DCN of ATM-deficient mice

Fig. 1.Fig. 2.Fig. 3.Fig. 4.DISCUSSIONFig. 5.METHODSCell cultureMiceX-ray irradiationTransfection and plasmid constructsImmunohistochemistryImmunocytochemistryWestern blottingRNA extraction and RT-PCR analysisRNA interferenceStatistics

Fig. 6.TRANSLATIONAL IMPACTSupplementary materialREFERENCES

The ZFHX3 (ATBF1) transcription factor induces PDGFRB ...cerebellum, we investigated a distinct...

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