Methotrexate Inhibits NF-B Activity Via Long Intergenic (Noncoding) RNA–p21 Induction
ARTHRITIS&RHEUMATOLOGY
Vol.66,No.11,November2014,pp2947–2957
DOI10.1002/art.38805
?2014,American College of Rheumatology
Methotrexate Inhibits NF-?B Activity Via Long Intergenic(Noncoding)RNA–p21Induction Charles F.Spurlock III,1John T.Tossberg,1Brittany K.Matlock,2
Nancy J.Olsen,3and Thomas M.Aune1
Objective.To determine interrelationships be-tween the expression of long intergenic(noncoding) RNA–p21(lincRNA-p21),NF-?B activity,and re-sponses to methotrexate(MTX)in rheumatoid arthritis (RA)by analyzing patient blood samples and cell cul-ture models.
Methods.Expression levels of long noncoding RNA and messenger RNA(mRNA)were determined by quantitative reverse transcription–polymerase chain re-action.Western blotting and flow cytometry were used to quantify levels of intracellular proteins.Intracellular NF-?B activity was determined using an NF-?B lucif-erase reporter plasmid.
Results.Patients with RA expressed reduced basal levels of lincRNA-p21and increased basal levels of phosphorylated p65(RelA),a marker of NF-?B activation.Patients with RA who were not treated with MTX expressed lower levels of lincRNA-p21and higher levels of phosphorylated p65compared with RA patients treated with low-dose MTX.In cell culture using pri-mary cells and transformed cell lines,MTX induced lincRNA-p21through a DNA-dependent protein kinase catalytic subunit(DNA PKcs)–dependent mechanism.Deficiencies in the levels of PRKDC mRNA in patients with RA were also corrected by MTX in vivo.Further-more,MTX reduced NF-?B activity in tumor necrosis factor?–treated cells through a DNA PKcs–dependent mechanism via induction of lincRNA-p21.Finally,we observed that depressed levels of TP53and lincRNA-p21 increased NF-?B activity in cell lines.Decreased levels of lincRNA-p21did not alter NFKB1or RELA tran-scripts;rather,lincRNA-p21physically bound to RELA mRNA.
Conclusion.Our findings support a model whereby depressed levels of lincRNA-p21in RA contrib-ute to increased NF-?B activity.MTX decreases basal levels of NF-?B activity by increasing lincRNA-p21 levels through a DNA PKcs–dependent mechanism.
Rheumatoid arthritis(RA)is the most common autoimmune disease,affecting?1million adults in the US(1).Although the etiology of RA remains unknown, the disease is characterized by bone erosion(both small and large joints)and disability that manifest across decades.Newer biologic therapies,such as those that inhibit the activity of tumor necrosis factor?(TNF?), have enhanced outcomes and decreased disability(2). However,despite these advances,excess mortality in RA continues,and recent data suggest that the mortality gap between patients with RA and the remainder of the population continues to widen(3).
T cells from patients with RA exhibit loss of genomic integrity via reduced expression of the proteins responsible for cell cycle arrest and DNA damage repair (4,5),including the sentinel DNA damage response proteins ATM kinase and DNA-dependent protein ki-nase catalytic subunit(DNA PKcs),the tumor suppres-sor protein p53,the cyclin-dependent kinase inhibitors p21and p27,and stress kinases such as JNKs that also respond to DNA damage(5–7).
Genes that encode these proteins are also meth-
Supported by the NIH(grants R42-AI-53948,R01-AI-044924,and R21-AR-063846),the Rheumatology Research Founda-
tion(Within Our Reach program grant ACR124405),National Center
for Advancing Translational Sciences Award UL1TR000445,and the National Science Foundation Graduate Research Fellowship Program.
The Vanderbilt Medical Center Flow Cytometry Shared Resource is supported by the Vanderbilt Ingram Cancer Center(P30-CA-68485)
and the Vanderbilt Digestive Research Center(DK-058404).
Charles F.Spurlock III,PhD,John T.Tossberg,MS,Thomas
M.Aune,PhD:Vanderbilt University School of Medicine,Nashville, Tennessee;2Brittany K.Matlock,BS:Vanderbilt University Medical Center,Nashville,Tennessee;3Nancy J.Olsen,MD:Penn State Milton
S.Hershey Medical Center,Hershey,Pennsylvania.
Address correspondence to Thomas M.Aune,PhD,MCN
T3113,Vanderbilt University Medical Center,116121st Avenue South,Nashville,TN37232.E-mail:tom.aune@https://www.360docs.net/doc/ed1659764.html,.
Submitted for publication December18,2013;accepted in revised form July24,2014.
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otrexate(MTX)response genes(8,9).MTX is a potent inhibitor of dihydrofolate reductase(DHFR),causing depletion of tetrahydrofolate and cell cycle arrest(10). Adenosine synthesis and activation of adenosine recep-tors may also contribute to the antiinflammatory effects of MTX(11–14).Inhibition of DHFR by MTX also inhibits the reduction of dihydrobiopterin(BH2)to tetrahydrobiopterin(BH4),leading to nitric oxide syn-thase“uncoupling,”which decreases production of nitric oxide and increases production of reactive oxygen spe-cies(ROS).Increased production of ROS activates JNK,which increases the expression of proapoptotic proteins such as p53and enhances cellular apoptotic responses(8,9).
The effects of p53are mediated in part by induction of long noncoding RNAs(lncRNAs),long intergenic(noncoding)RNA–p21(lincRNA-p21),and PANDA(15,16).LncRNAs represent a new class of RNAs,and thousands of lncRNA genes exist in mam-malian genomes(17,18).Genes encoding lncRNAs may contain exons and introns,and exons are spliced to produce mature lncRNAs.LncRNAs do not code for proteins due to the presence of multiple translational stop codons(18).One function of lncRNAs is to activate or repress transcription of protein-coding genes,and thus lncRNAs play key roles in determining cellular transcriptional programs(19).For example,lincRNA-p21and PANDA play critical roles in mediating cellular responses to p53(15,16).
Besides its role in apoptosis,p53possesses anti-inflammatory properties(20).In contrast,NF-?B is a prosurvival and proinflammatory transcription factor (21).Thus,biologically,p53and NF-?B may be con-sidered antagonistic(22,23).However,it is unknown whether p53directly inhibits NF-?B activity and vice versa and how this may occur mechanistically(23,24). Here,we sought to explore the interrelationship be-tween p53,its target lncRNAs,and NF-?B in the context of RA and MTX therapy.We observed that lincRNA-p21is underexpressed in RA,and that MTX restores lincRNA-p21levels to normal in vivo.Similarly,NF-?B activity is increased in RA,and MTX restores NF-?B activity to normal levels in vivo.Analyses in primary cells and transformed cell lines demonstrated that induction of lincRNA-p21by MTX occurs via DNA PKcs activa-tion,and that lincRNA-p21inhibits NF-?B activity,in part,by sequestering RELA messenger RNA(mRNA). Collectively,our results demonstrate that lincRNA-p21 is a key negative regulator of NF-?B activity.We suggest that reduced expression of lincRNA-p21in RA may contribute to disease-dependent inflammation,and that a portion of the antiinflammatory properties of MTX may result from induction of lincRNA-p21.
PATIENTS AND METHODS
Reagents and cell culture.MTX was obtained from Sigma,and human TNF?was obtained from BD Biosciences. KU-55933and NU-7441were obtained from Tocris.The following primary antibodies were used:anti-actin(sc-8432; Santa Cruz Biotechnology),anti–phospho-ATM(NB110-55475;Novus),allophycocyanin-conjugated anti-human CD3 (BD Biosciences),and phycoerythrin-conjugated anti-human CD14(BD Biosciences)mouse monoclonal antibodies;anti-JNK(sc-471;Santa Cruz Biotechnology),anti-p53(NB200-171;Novus),anti–phospho-p65(ab10684;Abcam),anti-p65 (ab7970;Abcam),and anti–phospho–DNA PKcs(ab18192; Abcam)rabbit polyclonal antibodies.The NF-?B-Luc reporter plasmid with5-?B elements was provided by Dean Ballard (Vanderbilt University).
Human Jurkat T cell and THP-1monocyte lines (ATCC)were cultured in RPMI1640medium(1?g/ml folate) as previously described(9).Peripheral blood mononuclear cells(PBMCs)were isolated using Vacutainer CPT tubes(BD Biosciences).Activated T cells were prepared by stimulating PBMCs for72hours with plate-bound anti-CD3(OKT3; ATCC)and30units/ml interleukin-2.Concentrations of 0.1?M or1.0?M MTX were used,and culture periods ranged from24hours(activated primary T cells)to48hours(Jurkat T cells)of continuous exposure to MTX(8,9).Pharmaco-kinetic analysis had demonstrated that ingestion of a20-mg tablet of MTX produces concentrations of MTX in plasma of ?0.5?M after1hour and?0.1?M after10hours(8,25).
Transient transfections and luciferase measurements. Jurkat T cells were transfected with the NF-?B-Luc reporter plasmid and Silencer Select small interfering RNAs(siRNAs; Ambion)for the specified targets using a Cell Line Nucleo-fector Kit V(Amaxa).Plasmid amounts were equalized across transfections.THP-1cells and activated T cells were trans-fected with Silencer Select siRNAs,using Lipofectamine RNAiMAX reagent(Life Technologies)according to the manufacturer’s protocol.Gene silencing by siRNAs,as veri-fied by quantitative reverse transcription–polymerase chain reaction(qRT-PCR),was?50%.Luminescence was measured on a TD-20/20luminometer(Turner Designs)using Steady-Glo(E2510;Promega).
RNA isolation,complementary DNA(cDNA)synthe-sis,and qRT-PCR.Total RNA was isolated using TRI Reagent (MRC Pharmaceuticals)or PAXgene tubes(Qiagen),purified with RNeasy MinElute Cleanup Kits(Qiagen),and quantified using a https://www.360docs.net/doc/ed1659764.html,plementary DNA was reverse transcribed from total RNA with Super-Script III First-Strand Synthesis Kits(Life Technologies)using oligo(dT)and purified with QIAQuick PCR Purification Kits (Qiagen).Quantitative RT-PCR(ABI-7300;Applied Biosys-tems)was performed in duplicate using custom or inventoried TaqMan gene expression assays in25?l with50ng cDNA. GAPDH was used for normalization.
Flow cytometry.Cells were suspended in phosphate buffered saline plus10%fetal bovine serum with0.1%azide, fixed with BD Cytofix Buffer,permeabilized with BD Phosflow
2948SPURLOCK ET AL
Perm/Wash Buffer(BD Biosciences),and labeled with primary antibodies overnight at4°C followed by incubation with fluo-rescein isothiocyanate–labeled secondary antibodies and cell surface markers,where indicated,for1hour at4°C.Cells were analyzed using a3-laser BD LSR II flow cytometer at the Vanderbilt Flow Cytometry Core facility.Analyses were per-formed with FlowJo software(Tree Star).
Biotin lincRNA-p21mRNA pulldown assay and bio-informatic analysis.The vector expressing partial human lincRNA-p21(pcDNA3-lincRNA-p21)was a gift from Dr.Myriam Gorospe(National Institute on Aging,National Institutes of Health).Biotinylated transcripts were synthe-sized using MAXIscript T7(?strand)or MAXIscript SP6 (?strand)kits(Ambion),as previously described(26).Bio-tinylated transcripts were incubated with THP-1total RNA, heated to55°C,and cooled https://www.360docs.net/doc/ed1659764.html,plexes were isolated using streptavidin-coupled Dynabeads(Invitrogen). RNA present in pulldowns was measured using qRT-PCR. BLAST(https://www.360docs.net/doc/ed1659764.html,)was used to determine regions of complementarity between lincRNA-p21and JUNB and RELA mRNA(26).
Study populations.The patient cohorts used for qRT-PCR measurements are shown in Table1.The patients met the American College of Rheumatology(ACR)/European League Against Rheumatism classification criteria for RA(27),the American–European Consensus Group criteria for Sjo¨gren’s syndrome(SS)(28),and the ACR1982revised criteria for the classification of systemic lupus erythematosus(SLE)(29). Western blotting was performed in9control subjects(mean?SD age48?8years,7women and2men,8white individuals and1African American)with no chronic or acute infection and no family history of autoimmune disease,and8patients with RA(age41?12years,7women and1man,6white individuals and2African Americans).The demographic char-acteristics of individuals in the control and disease cohorts did not differ significantly.The studies were approved by the institutional review boards at Vanderbilt University or Penn State.Written informed consent was obtained at the time of blood sample collection.
Western blot analysis.Immunoblotting was performed as previously described(9).Whole cell lysates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to PVDF membranes.The membranes were blocked with Odyssey Blocking Buffer(Li-Cor)for1hour, incubated with primary antibodies overnight at4°C,and incubated with fluorescence-labeled IRDye700/800antibodies diluted in Odyssey Blocking Buffer in the dark.Blots were washed and suspended in Tris buffered saline before scanning and band quantification,using an Odyssey Infrared Imaging System(Li-Cor).
Microarrays.The microarrays were described previ-ously(GSE21761and GSE3447)(16,30).
Statistical analysis.Differences between groups were determined using Student’s2-tailed t-tests with Bonferroni adjustment for multiple comparisons.P values less than0.05 were considered significant.Unless indicated otherwise,results are representative of at least3independent experiments. Statistical analyses were performed using GraphPad Prism software.
RESULTS
TP53and lincRNA-p21expression in RA.To initiate our studies,we determined transcript levels of
Table1.Demographic characteristics of the patients and healthy control subjects and clinical characteristics of the RA patients*
RA patients
Controls (n?45)No MTX treatment
(n?18)
MTX treatment
(n?18)
SLE patients
(n?24)
SS patients
(n?12)
Demographic characteristics
Age,mean?SD years38?1146?1148?1542?1347?13 Female sex73781009482 Ethnicity
White5883804558 African American2211153325 Hispanic13651117 Asian70011–Clinical characteristics
Disease duration,mean?SD years–10?99?76?48?2 Active disease?–7268––Disease Activity Score in28joints– 4.9?0.4 4.5?0.6––Early RA?–2217––Treatment–––Hydroxychloroquine–4439––Steroids–3961––TNF inhibitors–4428––
*Except where indicated otherwise,values are the percent.RA?rheumatoid arthritis;MTX?methotrexate;SLE?systemic lupus erythematosus; SS?Sj?gren’s syndrome;TNF?tumor necrosis factor.
?Defined as the presence of at least3of the following:morning stiffness lasting?45minutes,?3swollen joints,?6tender joints,and erythrocyte sedimentation rate?28mm/hour.
?Disease duration?1year.
MTX,NF-?B,AND lincRNA-p21IN RHEUMATOID ARTHRITIS2949
TP53and lincRNA-p21across3autoimmune diseases: RA,SLE,and SS.We observed that the expression of TP53and lincRNA-p21was reduced in patients with RA compared with control subjects(Figure1A).This de-creased expression compared with control subjects was not observed in either the SLE cohort or the SS cohort. Thus,TP53and lincRNA-p21levels were significantly reduced in RA,but this deficiency was not a common property of all inflammatory diseases.Furthermore,we measured the expression of PANDA,an additional lncRNA induced by p53activation,and observed no significant difference in expression between the RA cohort and the control cohort.
The original study in which the discovery and function of lincRNA-p21was described included micro-array analyses identifying genes positively or negatively regulated by lincRNA-p21or TP53based on effects of siRNA-mediated knockdown of lincRNA-p21,TP53,or both(16).Therefore,we reasoned that if lincRNA-p21 or TP53levels contribute to the differentially expressed gene profiles in RA,genes repressed by knockdown of lincRNA-p21or TP53would be underexpressed,and genes induced by knockdown of lincRNA-p21or TP53would be overexpressed,and this is exactly what we observed.In patients with RA,?15%of the genes assayed were either overexpressed or underexpressed (Figure1B)(see also Supplementary Table1,available on the Arthritis&Rheumatology web site at http:// https://www.360docs.net/doc/ed1659764.html,/doi/10.1002/art.38805/abstract). Of these overexpressed or underexpressed gene sets,
?25%of differentially expressed genes corresponded to the differentially expressed gene profiles obtained by microarray analysis of cells treated with lincRNA-p21or
TP53siRNA.These results were consistent with the hypothesis that underexpression of lincRNA-p21and TP53plays a large role in establishing the unique PBMC transcriptome observed in RA.
We next investigated whether reduced TP53ex-pression could significantly alter baseline levels of lincRNA-p21expression.Activated T cells were trans-fected with Silencer Select Scrambled Negative Control siRNA(Ambion)or TP53siRNA,and the expression levels of TP53and lincRNA-p21were measured by qRT-PCR.Transfection of TP53siRNA into primary T cells reduced TP53expression by?50%(Figure1C). However,lincRNA-p21expression levels were not
sig-Figure1.Reduced expression of long intergenic(noncoding)RNA–p21(lincRNA-p21;l-p21)in rheumatoid arthritis(RA).Whole blood samples from control(CTRL)subjects(n?45),patients with RA(n?18),patients with systemic lupus erythematosus(SLE)(n?24),and patients with Sj?gren’s syndrome(SS)(n?12)were collected into PAXgene tubes.A,TP53,lincRNA-p21,and PANDA transcript levels,as measured by quantitative reverse transcription–polymerase chain reaction(qRT-PCR)and normalized against GAPDH levels.???P?0.005versus control,by chi-square test.B,Comparison of differentially expressed genes(DEGs)and genes significantly overexpressed(over;OE)or underexpressed(under; UE)in RA cells treated with lincRNA-p21or TP53small interfering RNA(siRNA).C,Transcript levels of TP53and lincRNA-p21in activated T cells transfected with negative control siRNA or TP53siRNA,as measured by qRT-PCR.D,Correlation between lincRNA-p21and TP53transcripts in control subjects(n?20;left)and patients with RA(n?22;right).Values in A,B(left),and C are the mean?SD.NS?not significant. 2950SPURLOCK ET AL
nificantly altered by transfection with TP53siRNA.We further investigated the relationship between TP53and lincRNA-p21expression to determine whether tran-script levels of TP53correlated with expression levels of lincRNA-p21in vivo.In a cohort of control subjects (n ?20)and patients with RA (n ?22),we observed no significant correlation between TP53and lincRNA-p21transcript levels (Figure 1D).Based on these data,we concluded that basal transcript levels of lincRNA-p21are not necessarily dependent on basal levels of TP53,and that p53-independent mechanisms may contribute to basal levels of lincRNA-p21in PBMCs.Therefore,we performed pathway analyses to interrogate overlapping RA,lincRNA-p21,and TP53gene sets (31).Known NF-?B response genes were highly represented in this analysis (see Supplementary Table 1,available on the Arthritis &Rheumatology web site at http://online https://www.360docs.net/doc/ed1659764.html,/doi/10.1002/art.38805/abstract).
MTX-induced restoration of lincRNA-p21ex-pression via activation of DNA PKcs.We next investi-gated whether RA patients treated with MTX and those not treated with MTX exhibited differences in lincRNA-p21transcript levels and observed that the levels were significantly higher in RA patients who were receiving MTX (Figure 2A).Furthermore,no significant differ-ence was observed between control subjects and RA patients treated with MTX.We concluded from this cross-sectional study that MTX restores lincRNA-p21expression in vivo in RA.
We next sought to determine whether MTX directly induced lincRNA-p21expression in cultured cells.Treatment of either activated primary T cells
or
Figure 2.Methotrexate (MTX)increases expression of lincRNA-p21.A,LincRNA-p21expression levels in control subjects (n ?45),patients with RA who did not receive MTX (RA ?MTX)(n ?18),and patients with RA who did receive MTX (RA ?MTX)(n ?18),as determined by qRT-PCR.??P ?0.05;???P ?0.005.B,LincRNA-p21transcript levels in activated (Act.)T cells (left)or Jurkat cells (right)cultured with MTX at the indicated concentrations.??P ?0.05versus 0.01?M MTX (left)or untreated (right).C,Levels of JNK and p53in Jurkat cells or activated T cells cultured with 0.1?M MTX,as determined by flow cytometry.Left,Representative flow diagrams showing background fluorescence (left histogram)and results obtained with untreated cells or MTX-treated cells (middle and right histograms,respectively).Right,Fold increase in mean fluorescence intensity (MFI).Results are representative of 4independent experiments.??P ?0.05versus untreated.D,LincRNA-p21expression in Jurkat cells cultured with MTX and the JNK inhibitor BI-78D3(left)or the adenosine receptor antagonists caffeine (CAFF;C)and theophylline (THEO;T)(right).??P ?0.05versus untreated.Values in A,B,C (right),and D are the mean ?SD.FITC ?fluorescein isothiocyanate (see Figure 1for other definitions).
MTX,NF-?B,AND lincRNA-p21IN RHEUMATOID ARTHRITIS 2951
Jurkat cells with increasing concentrations of MTX led to 10–20-fold increases in lincRNA-p21expression (Fig-ure 2B).The degree of induction of lincRNA-p21by MTX in both cell types was greater than the degree of induction of either JNK or p53by MTX (Figure 2C).A previous study showed that MTX-mediated activation of JNK induced increased expression of proteins contrib-uting to cell cycle arrest and apoptosis (9).In certain cell types,MTX also induces increased synthesis of adeno-sine and activation of adenosine receptors.Thus,we investigated whether either inhibition of JNK using the small molecular weight JNK antagonist BI-78D3or activation of adenosine receptors using the general adenosine receptor antagonists caffeine and theophyl-line was sufficient to prevent MTX-mediated induction of lincRNA-p21transcripts.The results showed that inhibition of JNK by BI-78D3or by the adenosine receptor antagonists caffeine and theophylline did not abrogate MTX-mediated induction of lincRNA-p21(Figure 2D).Taken together,these findings suggested that pathways other than activation of JNK or adenosine receptors are activated by MTX to induce lincRNA-p21in these culture models.
The original study in which the function of lincRNA-p21was described showed that p53induces human lincRNA-p21in response to DNA damage.Deficiencies in DNA damage responses have also been observed in RA;these include reduced expression of p53and 2sentinels of the DNA damage response,ATM and DNA PKcs (5,6).Therefore,we sought to determine whether MTX induced phosphorylation of ATM or DNA PKcs.To do so,we treated Jurkat cells or activated T cells with MTX and performed intracellular flow cytometric analysis with specific antibodies to detect phospho-ATM or phospho–DNA PKcs.We observed that MTX treatment of both activated primary T
cells
Figure 3.Methotrexate (MTX)induces lincRNA-p21via activation of DNA-dependent protein kinase catalytic subunit (DNA PKcs).A,Levels of phospho-ATM and phospho–DNA PKcs in activated (Act.)T cells and Jurkat cells treated with the indicated concentrations of MTX,as determined by flow cytometry.Left,Representative flow diagrams showing background fluorescence (left histogram)and results obtained with untreated cells and MTX-treated cells (middle and right histograms,respectively).Right,Fold increase in mean fluorescence intensity (MFI).??P ?0.05versus untreated.B,PRKDC transcript levels relative to GAPDH transcript levels in activated T cells and Jurkat cells treated with MTX.??P ?0.05versus untreated.C,PRKDC transcript levels in control subjects (n ?45),RA patients who did not receive MTX (RA ?MTX)(n ?18),and RA patients who did receive MTX (RA ?MTX)(n ?18).??P ?0.05;???P ?0.005.D,Transcript levels of TP53(left)and lincRNA-p21(right)in MTX-treated Jurkat cells cocultured for 48hours with varying concentrations of KU-55933(KU)or NU-7441(NU),as determined by qRT-PCR.??P ?0.05versus 0.1?M MTX.E,Fold increase in lincRNA-p21expression relative to GAPDH expression in activated T cells cultured with MTX,NU-7441,or MTX plus NU-7441.??P ?0.05versus untreated;???P ?0.05versus 0.1?M MTX.Values in A (right)and B–E are the mean ?SD.FITC ?fluorescein isothiocyanate (see Figure 1for other definitions).
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and Jurkat cells only modestly increased ATM phos-phorylation (Figure 3A).In contrast,MTX markedly increased DNA PKcs phosphorylation in both cell types.
Given these findings,we performed qRT-PCR,using Jurkat cells and activated T cells,to determine whether MTX increased PRKDC expression.The results showed that MTX increased PRKDC expression ?2-fold in both activated T cells and Jurkat cells (Figure 3B).We also compared DNA PKcs (PRKDC )transcript levels in control subjects,RA patients who were not treated with MTX,and RA patients who were treated with MTX.The level of PRKDC expression in RA patients who did not receive MTX was lower than that in control subjects (Figure 3C),and the level of PRKDC expression in RA patients treated with MTX was equivalent to that in control subjects and significantly higher than that in RA patients who did not receive MTX.We concluded from these studies that pharmacologic doses of MTX in patients with RA increased PRKDC transcript levels to near control values.
We then investigated whether ATM-or DNA
PKcs–specific inhibitors could reverse induction of TP53or lincRNA-p21by MTX in Jurkat cells.To accomplish this,we used KU-55933,which at low concentrations (10–20n M )inhibits phosphorylation of ATM but at high concentrations (5–10?M )also inhibits phosphorylation of DNA PKcs (32–34).Supplementation of MTX-treated cultures with KU-55933at low concentrations did not significantly alter induction of TP53or lincRNA-p21by MTX (Figure 3D).In contrast,treatment with MTX and high concentrations of KU-55933significantly reduced induction of TP53and lincRNA-p21transcripts by MTX,suggesting that induction of TP53and lincRNA-p21by MTX resulted from activation of DNA PKcs rather than ATM.To directly test this hypothesis,we used the specific DNA PKcs inhibitor NU-7441(35,36)and observed that treatment of Jurkat cells with NU-7441directly inhibited MTX-mediated induction of TP53and lincRNA-p21transcripts.Additionally,acti-vated primary T cells treated with MTX and NU-7441exhibited significantly lower lincRNA-p21levels com-pared with those treated with MTX alone (Figure 3E).Taken together,these results indicated that MTX in-duced expression of lincRNA-p21,at least in part,via activation of DNA
PKcs.
Figure 4.Methotrexate (MTX)reduces NF-?B activity via activation of DNA-dependent protein kinase catalytic subunit (DNA PKcs)and induction of lincRNA-p21.A,NF -?B activity in Jurkat cells transfected with a scrambled negative control siRNA,TP53siRNA,or lincRNA-p21siRNA,as measured using an NF-?B luciferase reporter construct.The cultures were left untreated or were treated with MTX for 48hours,and tumor necrosis factor ?(TNF ?)was added during the last 24hours of culture.B and C,NF-?B activity in Jurkat cells (B )or activated T cells (C )transfected with an NF-?B luciferase reporter construct.The cultures were treated as in A,in the presence or absence of KU-55933(KU)or NU-7441(NU).Results in A–C are expressed as relative light units.D,Levels of phospho-p65in whole cell lysates prepared using peripheral blood mononuclear cells from healthy control (HC)subjects (n ?9),RA patients who did not receive MTX (RA ?MTX)(n ?4),and RA patients who did receive MTX (RA ?MTX)(n ?4),as determined by Western blotting.E,Levels of phospho-p65in control subjects (n ?20),RA patients who did not receive MTX (n ?6),and RA patients who did receive MTX (n ?6),as determined by intracellular flow cytometry with gating on CD3?or CD14?cells.Values in A–C,D (right),and E (right)are the mean ?SD.??P ?0.05.FITC ?fluorescein isothiocyanate (see Figure 1for other definitions).
MTX,NF-?B,AND lincRNA-p21IN RHEUMATOID ARTHRITIS 2953
MTX-induced inhibition of NF-?B activity via lincRNA-p21activation.Because p53deficiencies exac-erbate inflammatory disease in murine models,because RA is characterized by p53deficiency,and because NF-?B is a critical proinflammatory and prosurvival transcrip-tion factor,we postulated that TP53and/or lincRNA-p21may directly interfere with NF-?B activity.To investigate this hypothesis,we used a luciferase reporter construct to measure the activity of NF-?B.We initiated our studies in cell lines cotransfected with the NF-?B luciferase reporter and Silencer Select siRNAs for TP53and lincRNA-p21.The cultures were treated with MTX for 48hours,with TNF ?added during the last 24hours of culture to stimulate NF-?B activity.The cultures treated with MTX plus TNF ?exhibited reduced activa-tion of NF-?B compared with the cultures treated with TNF ?alone (Figure 4A).The addition of TP53siRNA or lincRNA-p21siRNA significantly abrogated MTX-mediated inhibition of NF-?B activity in Jurkat cells.
Because we observed that MTX induced lincRNA-p21via activation of DNA PKcs,we questioned whether inhibition of DNA PKcs using KU-55933or NU-7441blocked MTX-induced inhibition of NF-?B activity.Low concentrations of KU-55933,which inhibited only ATM phosphorylation,failed to prevent MTX-mediated inhibition of NF-?B activity.In contrast,high concen-trations of KU-55933,which inhibited both ATM and DNA PKcs activity,or the addition of the DNA PKcs–specific inhibitor NU-7441effectively blocked inhibition of NF-?B activity by MTX (Figure 4B).We further examined these effects in activated primary T cells.Activated T cells transfected with the NF-?B luciferase reporter were treated with MTX and TNF ?for 24hours in the presence or absence of KU-55933or NU-7441.We observed that treatment with high concentrations of KU-55933or NU-7441also reduced inhibition of NF-?B activity by MTX in activated T cells (Figure 4C).
One of the most abundant NF-?B family mem-bers is the p50/p65complex.Phosphorylation of p65(RelA)in response to exogenous stimuli such as TNF ?increases NF-?B transcriptional activity (37).Therefore,we performed Western blotting,using this biomarker (phospho-p65)to measure basal levels of NF-?B activity in control subjects and patients with RA.Whole cell lysates were prepared from the PBMCs of control subjects and patients with RA.Surprisingly,we
detected
Figure 5.Association of RELA and lincRNA-p21transcripts.A,NF-?B activity in THP-1cells (left)and Jurkat cells (right)transfected with an NF-?B luciferase reporter construct,in the presence of negative control siRNA,lincRNA-p21siRNA,or TP53siRNA.Tumor necrosis factor ?(TNF ?)was added 24hours after transfection,and luminescence was quantified 48hours after transfection.??P ?0.05.B,Transcript levels of NFKB1and RELA in THP-1cells transfected with a lincRNA-p21siRNA,as determined by qRT-PCR 48hours after transfection.??P ?0.05versus control.C,Sites of high complementarity between RELA or JUNB mRNA and lincRNA-p21.D,Relative enrichment of RELA mRNA and JUNB mRNA purified with a biotinylated lincRNA-p21probe,as determined by qRT-PCR.??P ?0.05versus 18S and NFKB1.E,Total p65protein levels in whole cell lysates prepared from peripheral blood mononuclear cells isolated from RA patients who did not receive methotrexate (MTX;RA ?MTX)(n ?4)and RA patients who did receive MTX (RA ?MTX)(n ?4),as determined by Western blotting.??P ?0.05.Values in A,B,D,and E (right)are the mean ?SD.See Figure 1for other definitions.
2954SPURLOCK ET AL
elevated basal levels of phospho-p65in RA patients who did not receive MTX but not in control subjects(Figure 4D).Increased levels of phospho-p65were not detected in the cohort of RA patients treated with MTX.
We further examined increased phospho-p65ex-pression in patients with RA,using phospho-flow cytom-etry with gating on CD3?T cells and CD14?mono-cytes.Phospho-p65expression in T cells from RA patients who did not receive MTX was significantly higher than that in T cells from control subjects(Figure 4E).However,this increase in phospho-p65activity was not observed in T cells from RA patients treated with MTX.Although phospho-p65expression was not in-creased in RA monocytes compared with control mono-cytes,phospho-p65levels were reduced in RA patients receiving MTX compared with control subjects(Figure 4E).Thus,we concluded that long-term NF-?B activa-tion occurs in RA and is corrected by MTX therapy in vivo.
Our results demonstrated that induction of lincRNA-p21and/or TP53transcripts by MTX contrib-uted to MTX-mediated inhibition of NF-?B activation by TNF?.However,they did not determine whether basal levels of lincRNA-p21and/or TP53transcripts contributed to basal or stimulus(TNF?)–dependent NF-?B activity.We therefore used siRNA to specifically reduce basal levels of lincRNA-p21or TP53transcripts in2different cell lines:THP-1cells(monocytes)and Jurkat cells.In THP-1cells,siRNA-mediated specific reduction of lincRNA-p21or TP53transcripts resulted in increased basal and TNF?-induced NF-?B activity (Figure5A).In Jurkat cells,siRNA-mediated reduction of lincRNA-p21levels,but not TP53levels,resulted in increased basal and TNF?-induced NF-?B activity.This difference is attributable,in part,to decreased efficiency of siRNA-mediated knockdown of TP53transcripts in Jurkat cells.These studies demonstrated that lincRNA-p21is a direct negative regulator of both basal and stimulus-induced NF-?B activity.
Previous studies demonstrated that lincRNA-p21 induced apoptosis in response to DNA damage by inhibiting expression of genes encoding prosurvival pro-teins.Furthermore,lincRNA-p21binds to mRNAs,such as JUNB mRNA and CTNNB1mRNA,to inhibit their translation(16,26).Thus,we reasoned that lincRNA-p21may inhibit NF-?B activity by altering transcript levels of genes that encode proteins critically involved in NF-?B signaling pathways,or that lincRNA-p21may actually bind to these mRNAs and lower the rates of translation.We performed two studies to help discrim-inate between these possibilities,using lincRNA-p21–specific siRNA to knock down levels of lincRNA-p21.
We observed that loss of lincRNA-p21did not produce a corresponding gain in NFKB1or RELA transcript levels,suggesting that lincRNA-p21did not interfere with cellular NF-?B activity by lowering NFKB1or RELA transcript levels(Figure5B).To test the alternate hypothesis,that lincRNA-p21may associ-ate with RELA mRNA and/or NFKB1mRNA and inhibit their translation,we used a bioinformatics ap-proach to identify regions of complementarity between lincRNA-p21and JUNB(as control)and RELA,the gene whose protein product encodes for p65.We iden-tified8regions of high complementarity between lincRNA-p21and JUNB,mirroring published findings (26),and6sites of high complementarity between lincRNA-p21and RELA(Figure5C).
The interactions between endogenous levels of lincRNA-p21and JUNB and RELA were quantified using antisense biotinylated lincRNA-p21,as described previously(26).Similar to the known ability of lincRNA-p21to associate with JUNB,we observed that lincRNA-p21had a significantly higher interaction with RELA transcripts than with18S ribosomal RNA or NFKB1 transcripts(Figure5D).We performed Western blot analysis to examine total p65(RelA)protein levels in patients with RA who were receiving MTX and those who were not receiving MTX.Patients with RA who were treated with MTX exhibited significantly lower total p65levels compared with the patients who did not receive MTX(Figure5E).These observations suggest that one manner in which lincRNA-p21may function is through binding to mRNA that encodes proteins critical for NF-?B transcriptional activity.
DISCUSSION
In RA patients who are not receiving MTX therapy,the expression of phospho-p65(a necessary component of NF-?B transcriptional activity)is in-creased,and the expression of lincRNA-p21is de-creased.Treatment with MTX corrects both defects in vivo.Our results support a model whereby MTX induces lincRNA-p21expression via a DNA PKcs–dependent pathway and also support the notion that lincRNA-p21 directly inhibits both basal and TNF?-stimulated NF-?B activity.Inhibition of NF-?B activity results,at least in part,from the ability of lincRNA-p21to sequester RELA mRNA.Thus,reduced expression of lincRNA-p21and p53contributes to increased activity of the proinflammatory and prosurvival transcription factor NF-?B.We propose that reduced expression of these proapoptotic and antiinflammatory RNAs and proteins
MTX,NF-?B,AND lincRNA-p21IN RHEUMATOID ARTHRITIS2955
in RA contributes to the chronic inflammation observed in patients with this disease.
MTX is generally considered an anchor therapy in patients with RA.Its antiinflammatory effects are incompletely understood.Two known pathways acti-vated by MTX are stimulation of adenosine release and activation of adenosine receptors,which have antiin-flammatory properties,and activation of JNK enzymes to induce proapoptotic proteins,leading to heightened sensitivity to apoptosis(9).Activation of DNA PKcs by MTX appears to be independent of these two pathways. Phosphorylation(activation)of DNA PKcs and ATM in response to DNA damage is reasonably well understood. However,phosphorylation of ATM is not observed in response to MTX,suggesting that induction of DNA damage by MTX was not responsible for phosphoryla-tion of DNA PKcs in our culture systems.DNA PKcs activation also plays a critical role in cellular responses to replicative stress(38).Thus,it is possible that MTX may induce replicative stress via inhibition of DHFR or other mechanisms that activate DNA PKcs independent of https://www.360docs.net/doc/ed1659764.html,ing selective inhibitors of DNA PKcs and ATM,we clearly show that induction of lincRNA-p21 and TP53by MTX is dependent on DNA PKcs and not ATM,and that MTX-mediated inhibition of NF-?B activity in our culture systems requires DNA PKcs.Our interpretation is that MTX activates DNA PKcs,leading to induction of TP53and lincRNA-p21.In turn,in-creased activity of p53and lincRNA-p21inhibits basal and stimulus-induced NF-?B activity.
LincRNA-p21belongs to the class of lncRNAs induced by p53in response to DNA damage.In blood cells,baseline levels of TP53and lincRNA-p21are not well correlated.In the absence of DNA damage,genes responsive to TP53knockdown or lincRNA-p21knock-down are not identical(16).Both classes of genes are highly overrepresented in the class of genes shown to be overexpressed or underexpressed in RA.Thus,we be-lieve that our results and those of other investigators support the notion that TP53and lincRNA-p21tran-script levels are regulated by both of the common pathways in response to,for example,DNA damage,as well as by independent pathways.Furthermore,gene expression programs regulated by p53and lincRNA-p21 are not identical.Additional experimentation will be required to identify mechanisms that cause reduced expression of TP53and how loss of TP53and lincRNA-p21contributes to RA pathogenesis.
LincRNA-p21affects cellular function by mul-tiple mechanisms.First,lincRNA-p21mediates p53-dependent transcriptional repression of target genes through association with heterogeneous nuclear RNP K(16).Second,lincRNA-p21associates with target mRNAs,such as JUNB mRNA and CTNNB1mRNA,to prevent their translation(26).Third,lincRNA-p21mod-ulates responses to hypoxia by interfering with interac-tions between the transcription factor hypoxia-inducible factor1?and von Hippel-Lindau protein(39).In our analysis,reduced lincRNA-p21levels did not alter tran-script levels of the NF-?B genes NFKB1or RELA. Rather,lincRNA-p21bound RELA mRNA,thus possi-bly interfering with mRNA translation,a result consis-tent with the second mechanism.However,we cannot rule out the possibility that lincRNA-p21may inhibit transcription of other mRNAs that encode proteins required for NF-?B activation,represses translation of these same mRNAs,or interferes with the normal function of the NF-?B activation pathway at the protein level.
The existence of defects in the transcriptional response leading to cell cycle arrest and apoptosis in RA is well established.These same defects are cor-rected,at least in part,by MTX,which is one of the major therapies for RA(8).What has been unclear is how these defects may contribute to the underlying chronic inflammation that is a hallmark of RA.One possibility is that p53and lincRNA-p21are negative regulators of NF-?B activity.As such,a reduction in the levels of p53and lincRNA-p21produces enhanced basal and stimulus-dependent NF-?B activity,and res-toration of the levels of p53and lincRNA-p21by MTX also lowers NF-?B activity,which is a major driver of the proinflammatory state in RA.Future studies will be required to determine whether interplay between these2 pathways can be exploited for therapeutic benefit in RA.
ACKNOWLEDGMENTS
We thank Cheri Stewart and personnel at the Clinical Trials Center at Vanderbilt University Medical Center for assistance collecting patient samples.We also thank Carl McAloose(Penn State Milton S.Hershey Medical Center)for performing flow cytometric analyses and Drs.Howard A. Fuchs and Joseph W.Huston III(Vanderbilt University Medical Center)for access to their clinics and for providing patient samples.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content,and all authors approved the final version to be published.Dr.Aune had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design.Spurlock,Olsen,Aune.
Acquisition of data.Spurlock,Tossberg,Matlock,Olsen.
Analysis and interpretation of data.Spurlock,Olsen,Aune.
2956SPURLOCK ET AL
REFERENCES
1.Helmick CG,Felson DT,Lawrence RC,Gabriel S,Hirsch R,
Kwoh CK,et al.Estimates of the prevalence of arthritis and other rheumatic conditions in the United States:Part I.Arthritis Rheum 2008;58:15–25.
2.Gabriel SE,Crowson CS,Kremers HM,Doran MF,Turesson C,
O’Fallon WM,et al.Survival in rheumatoid arthritis:a population-based analysis of trends over40years.Arthritis Rheum2003;48: 54–8.
3.Gabriel SE.Why do people with rheumatoid arthritis still die
prematurely?Ann Rheum Dis2008;67:30–4.
4.Fujii H,Shao L,Colmegna I,Goronzy JJ,Weyand CM.Telo-
merase insufficiency in rheumatoid arthritis.Proc Natl Acad Sci U S A2009;106:4360–5.
5.Shao L,Fukii H,Ines C,Oishi H,Goronzy JJ,Weyand CM.
Deficiency of the DNA repair enzyme ATM in rheumatoid arthritis.J Exp Med2009;206:1435–49.
6.Shao L,Goronzy JJ,Weyand CM.DNA-dependent protein kinase
catalytic subunit mediates T-cell loss in rheumatoid arthritis.
EMBO Mol Med2010;2:415–27.
7.Shiloh Y.ATM and related protein kinases:safeguarding genome
integrity.Nat Rev Cancer2003;3:155–68.
8.Spurlock CF III,Tossberg JT,Fuchs HA,Olsen NJ,Aune TM.
Methotrexate increases expression of cell cycle checkpoint genes via JNK activation.Arthritis Rheum2012;64:1780–9.
9.Spurlock CF III,Aune ZT,Tossberg JT,Collins PL,Aune JP,
Huston JW,et al.Increased sensitivity to apoptosis induced by methotrexate is mediated by JNK.Arthritis Rheum2011;63: 2606–16.
10.Tian H,Cronstein BN.Understanding the mechanisms of action
of methotrexate:implications for the treatment of rheumatoid arthritis.Bull NYU Hosp Jt Dis2007;65:168–73.
11.Chan SL,Cronstein BN.Methotrexate:how does it really work?
Nat Rev Rheumatol2010;6:175–8.
12.Cronstein BN.Low-dose methotrexate:a mainstay in the treat-
ment of rheumatoid arthritis.Pharmacological Rev2005;57: 163–72.
13.Cronstein BN,Naime D,Ostad E.The antiinflammatory mecha-
nism of methotrexate.Increased adenosine release at inflamed sites diminishes leukocyte accumulation in an in vivo model of inflammation.J Clin Invest1993;92:2675–82.
14.Montesinos MC,Desai A,Delano D,Chen JF,Fink JS,Jacobson
MA,et al.Adenosine A2A or A3receptors are required for inhibition of inflammation by methotrexate and its analog MX-68.
Arthritis Rheum2003;48:240–7.
15.Hung T,Wang Y,Lin MF,Koegel AK,Kotake Y,Grant GD,et al.
Extensive and coordinated transcription of noncoding RNAs within cell-cycle promoters.Nat Genet2011;43:621–9.
16.Huarte M,Guttman M,Feldser D,Garber M,Koziol MJ,
Kenzelmann-Broz D,et al.A large intergenic noncoding RNA induced by p53mediates global gene repression in the p53 response.Cell2010;142:409–19.
17.Guttman M,Amit I,Garber M,French C,Lin MF,Feldser D,
et al.Chromatin signature reveals over a thousand highly con-served large noncoding RNAs in mammals.Nature2009;458: 223–7.
18.Guttman M,Garber M,Levin JZ,Donaghey J,Robinson J,
Adiconis X,et al.Ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic struc-ture of lincRNAs.Nat Biotechnol2010;28:503–10.
19.Rinn JL,Chang HY.Genome regulation by long noncoding
RNAs.Annu Rev Biochem2012;81:145–66.
20.Komarova EA,Krivokrysenko V,Wang K,Neznanov N,Chernov
MV,Komarov PG,et al.p53is a suppressor of inflammatory response in mice.FASEB J2005;19:1030–2.21.Pahl HL.Activators and target genes of Rel/NF-?B transcription
factors.Oncogene1999;18:6853–66.
22.Cooks T,Pateras IS,Tarcic O,Solomon H,Schetter AJ,Wilder S,
et al.Mutant p53prolongs NF-?B activation and promotes chronic inflammation and inflammation-associated colorectal cancer.
Cancer Cell2013;23:634–46.
23.Ak P,Levine AJ.p53and NF-?B:different strategies for respond-
ing to stress lead to a functional antagonism.FASEB J2010;24: 3643–52.
24.Gudkov AV,Komarova EA.Pathologies associated with the p53
response.Cold Spring Harb Perspect Biol2010;2:a001180.
https://www.360docs.net/doc/ed1659764.html,be B,Edno L,Lafforgue P,Bologna C,Bernard JC,
Acquaviva P,et al.Total and free methotrexate pharmacokinetics, with and without piroxicam,in rheumatoid arthritis patients.Br J Rheumatol1995;34:421–8.
26.Yoon JH,Abdelmohsen K,Srikantan S,Yang X,Martindale JL,
De S,et al.LincRNA-p21suppresses target mRNA translation.
Mol Cell2012;47:648–55.
27.Aletaha D,Neogi T,Silman AJ,Funovits J,Felson DT,Bingham
CO III,et al.2010rheumatoid arthritis classification criteria:an American College of Rheumatology/European League Against Rheumatism collaborative initiative.Arthritis Rheum2010;62: 2569–81.
28.Vitali C,Bombardieri S,Jonsson R,Moutsopoulos HM,Alexan-
der EL,Carsons SE,et al,and the European Study Group on Classification Criteria for Sjo¨gren’s Syndrome.Classification cri-teria for Sjo¨gren’s syndrome:a revised version of the European criteria proposed by the American-European Consensus Group.
Ann Rheum Dis2002;61:554–8.
29.Tan EM,Cohen AS,Fries JF,Masi AT,McShane DJ,Rothfield
NF,et al.The1982revised criteria for the classification of systemic lupus erythematosus.Arthritis Rheum1982;25:1271–7.
30.Maas K,Chan S,Parker J,Slater A,Moore J,Olsen N,et al.
Cutting edge:molecular portrait of human autoimmune disease.
J Immunol2002;169:5–9.
31.Wang L,Zhang B,Wolfinger RD,Chen X.An integrated ap-
proach for the analysis of biological pathways using mixed models.
PLoS Genet2008;4:e1000115.
32.Eaton JS,Lin ZP,Sartorelli AC,Bonawitz ND,Shadel GS.
Ataxia-telangiectasia mutated kinase regulates ribonucleotide re-ductase and mitochondrial homeostasis.J Clin Invest2007;117: 2723–34.
33.Li Y,Yang DQ.The ATM inhibitor KU-55933suppresses cell
proliferation and induces apoptosis by blocking Akt in cancer cells with overactivated Akt.Mol Cancer Ther2010;9:113–25.
34.Gapud EJ,Dorsett Y,Yin B,Callen E,Bredemeyer A,Mahowald
GK,et al.Ataxia telangiectasia mutated(Atm)and DNA-PKcs kinases have overlapping activities during chromosomal signal joint formation.Proc Natl Acad Sci U S A2011;108:2022–7. 35.Zhao Y,Thomas HD,Batey MA,Cowell IG,Richardson CJ,
Griffin RJ,et al.Preclinical evaluation of a potent novel DNA-dependent protein kinase inhibitor NU7441.Cancer Res2006;66: 5354–62.
36.Oksenych V,Kumar V,Liu XY,Guo CG,Schwer B,Zha S,et al.
Functional redundancy between the XLF and DNA-PKcs DNA repair factors in V(D)J recombination and nonhomologous DNA end joining.Proc Natl Acad Sci U S A2013;110:2234–9.
37.Wang D,Westerheide SD,Hanson JL,Baldwin AS Jr.Tumor
necrosis factor?-induced phosphorylation of RelA/p65on Ser529 is controlled by casein kinase II.J Biol Chem2000;275:32592–7.
38.Liu S,Opiyo SO,Manthey K,Glanzer JG,Ashley AK,Amerin C,
et al.Distinct roles for DNA-PK,ATM and ATR in RPA phosphorylation and checkpoint activation in response to replica-tion stress.Nucleic Acids Res2012;40:10780–94.
39.Yang F,Zhang H,Mei Y,Wu M.Reciprocal regulation of HIF-1?
and lincRNA-p21modulates the Warburg effect.Mol Cell2014;
53:88–100.
MTX,NF-?B,AND lincRNA-p21IN RHEUMATOID ARTHRITIS2957