MTA1 Coregulation of Transglutaminase 2 Expression andFunction during Inflammatory Response*□S

Received for publication, October 30, 2010, and in revised form, December 5, 2010 Published, JBC Papers in Press, December 14, 2010, DOI 10.1074/jbc.M110.199273

Krishna Sumanth Ghanta1, Suresh B. Pakala1, Sirigiri Divijendra Natha Reddy, Da-Qiang Li, Sujit S. Nair,and Rakesh Kumar2

From the Department of Biochemistry and Molecular Biology and Institute of Coregulator Biology, The George WashingtonUniversity Medical Center, Washington, D. C. 20037

Although both metastatic tumor antigen 1 (MTA1), a masterchromatin modifier, and transglutaminase 2 (TG2), a multi-functional enzyme, are known to be activated during inflam-mation, it remains unknown whether these molecules regulateinflammatory response in a coordinated manner. Here we in-vestigated the role of MTA1 in the regulation of TG2 expres-sion in bacterial lipopolysaccharide (LPS)-stimulated mamma-lian cells. While studying the impact of MTA1 status on globalgene expression, we unexpectedly discovered that MTA1 de-pletion impairs the basal as well as the LPS-induced expressionof TG2 in multiple experimental systems. We found that TG2is a chromatin target of MTA1 and of NF-�B signaling in LPS-stimulated cells. In addition, LPS-mediated stimulation ofTG2 expression is accompanied by the enhanced recruitmentof MTA1, p65RelA, and RNA polymerase II to the NF-�B con-sensus sites in the TG2 promoter. Interestingly, both the re-cruitment of p65 and TG2 expression are effectively blockedby a pharmacological inhibitor of the NF-�B pathway. Thesefindings reveal an obligatory coregulatory role of MTA1 in theregulation of TG2 expression and of the MTA1-TG2 pathway,at least in part, in LPS modulation of the NF-�B signaling instimulated macrophages.

Inflammation is an adaptive immune response triggered bythe body against detrimental stimuli and conditions such asmicrobial infection and tissue injury (1, 2). Inflammation isusually a healing response, but it becomes detrimental if tar-geted destruction and assisted repair are not properly acti-vated (3). Primarily, macrophages and mast cells recognizethe infection and produce a wide variety of inflammatory me-diators such as chemokines, cytokines, etc., all contributing tothe elicitation of an inflammatory response (1). The inflam-matory response is characterized by coordinated regulation ofsignaling pathways that regulate the expression of both thepro-inflammatory and the anti-inflammatory cytokines in-cluding IL-1, IL-6, TNF-�, receptor activator of NF-�B ligand(RANKL), etc. (4). The inability of host to regulate inflamma-tory response results in sepsis, organ dysfunction, and even

death (5). These inflammatory cytokines are under the tightcontrol of master gene transcriptional factor NF-�B in pro-moting the inflammation, and in turn, innate immunity (6).Furthermore, transcriptional control of such NF-�B genomictargets is also under a tight control of nucleosome-remodel-ing coregulators and complexes, leading to either the stimula-tion or the repression of gene transcription at the molecularlevel (7–10).In recent times, metastatic tumor antigen 1 (MTA1)3 has

been recognized as one of the major coregulators in mamma-lian cells. MTA1 is a ubiquitously expressed chromatin modi-fier, having an integral role in nucleosome-remodeling andhistone deacetylase (NuRD) complexes (11). MTA1 is widelyup-regulated in a wide variety of human tumors and has beenshown to play a role in tumorigenesis (11–14). MTA1 regu-lates transcription of its targets by modifying the acetylationstatus of the target chromatin and cofactor accessibility to thetarget DNA. Recent work from this laboratory has shown thatMTA1 plays a key role in inflammatory responses both as atarget and as a component of the NF-�B signaling by regulat-ing a subset of lipopolysaccharide (LPS)-induced proinflam-matory cytokines (8) or by directly regulating the MyD88, aproximal component of NF-�B signaling (15). In addition tothese functions, MTA1 also plays an essential role in HepatitisB Virus X Protein stimulation of NF-�B signaling and in theexpression of NF-�B target gene products with functions ininflammation and tumorigenesis (16).In addition, MTA1 is a newly added regulator of inflamma-

tion, and it is also regulated by a number of genes includingtransglutaminase 2 (TG2) (17). TG2 is a multifunctional en-zyme involved in several cellular functions such as apoptosis(18), signaling (19), signal transduction (20), cytoskeleton re-arrangements and extracellular matrix stabilization (17), andwound healing (21). Aberrant activation and functions of TG2have been linked with a variety of inflammatory diseases thatinclude celiac disease, diabetes, multiple sclerosis, rheumatoidarthritis, and sepsis (5, 22). Results from a mouse model sys-tem revealed that TG2 is also involved in the NF-�B activa-tion, which induces the transcription of proinflammatory cy-tokines, causing continuous activation of inflammatoryprocess and contributing to the development of sepsis,whereas depletion of TG2 brings partial resistance to sepsis* This work was supported, in whole or in part, by National Institute of

Health Grants CA98823 and CA98823-S1 (to R. K.).□S The on-line version of this article (available at http://www.jbc.org) con-

tains supplemental Tables 1– 4.1 Both authors contributed equally to this work.2 To whom correspondence should be addressed. E-mail: bcmrxk@gwumc.

edu.

3 The abbreviations used are: MTA1, metastatic tumor antigen 1; TG2, trans-glutaminase 2; pol II, RNA polymerase II; qPCR, quantitative PCR; MEF,mouse embryonic fibroblast.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 9, pp. 7132–7138, March 4, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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(5). Apart from its role in inflammation, elevated levels ofTG2 are associated with many types of cancers (17, 23–25), aproperty shared with MTA1. In addition to this, increasedexpression of TG2 in cancer cells leads to increased drug re-sistance, metastasis, and poor patient survival (23, 24, 26), aproperty also shared with MTA1.Although TG2 expression parallels with MTA1 during in-

flammation, it remains unclear whether these molecules aretrans-regulated by inflammation. Here we report that MTA1is an obligatory coregulator of TG2 expression and that theMTA1-TG2 pathway plays a mechanistic role, at least in part,in bacterial LPS modulation of the NF-�B signaling in stimu-lated macrophages.

EXPERIMENTAL PROCEDURES

Cell Culture, Antibodies, and Reagents—All cells used inthis study were cultured in Dulbecco’s modified Eagle’smedium/F12 medium supplemented with 10% fetal bovineserum. Raw264.7 and MCF7 cells were obtained from Ameri-can Type Culture Collection, whereas HC11-pcDNA andHC11-MTA1, MTA1�/�, and MTA1�/� mouse embryonicfibroblasts (MEFs) have been described in our earlier studies(27). Transglutaminase 2 (catalogue number 3557) and NF-�Bp65 (catalogue number sc-372) antibodies were purchasedfrom Cell Signaling Technology and Santa Cruz Biotechnol-ogy, respectively. Antibodies against MTA1 (catalogue num-ber A300-280A) and RNA polymerase II (pol II) (cataloguenumber A300-653A) were purchased from Bethyl Laborato-ries, whereas normal mouse IgG, rabbit IgG, and antibodiesagainst vinculin were from Sigma. Bacterial LPS was pur-chased from Sigma. Whenever needed, LPS was used at theconcentration of 1 �g/ml of the medium. MTA1 siRNA (cata-logue number, M-004127-01) and NF-�B p65 siRNA (cata-logue number, sc-29411) were purchased from DharmaconRNAi Technologies (Lafayette, CO) and Santa Cruz Biotech-nology, respectively.Microarray Expression Profiling and Analysis—The mi-

croarray expression profiling and analysis were carried out asdescribed elsewhere (28). The RNA isolated by the TRIzolmethod was further checked for purity by analyzing on a 2100Bioanalyzer (Agilent Technologies). From the 2 �g of the to-tal purified RNA, rRNA reduction was performed usingRiboMinusTM transcriptome isolation kit (Invitrogen). Re-duced rRNA was now labeled using the GeneChip WT cDNAsynthesis/amplification kit and hybridized on a GeneChipMouse Exon 1.0 ST array. Scanning of the hybridized arrayswas carried out using Affymetrix GeneChip scanner 3000 7G.The obtainedmicroarray data were analyzed using GeneSpringGX10.0.2. Gene ontology (GO) analysis was performed on statis-tically significant samples using GeneSpring GX10.0.2.Quantitative Real Time PCR (qPCR) and RT-PCR Analysis—

For qPCR and RT-PCR analysis, the total RNA was isolated byusing Trizol reagent (Invitrogen), and first-strand cDNA syn-thesis was carried out with SuperScript II reverse transcrip-tase (Invitrogen) using 2 �g of total RNA and oligo(dT)primer. cDNA from macrophages was synthesized using theFastLane cell cDNA synthesis kit (Qiagen). qPCR and RT-PCR were performed using gene-specific primers listed in

supplemental Table 1. qPCR analysis was carried out using a7900HT sequence detection system (Applied Biosystems).The levels of mRNA of all the genes were normalized to thatof �-actin mRNA.Cloning of Murine TG2 Promoter—Murine TG2 promoter

was PCR-amplified from mouse genomic DNA and clonedinto pGL3 basic vector using the In-Fusion 2.0 dry-down PCRcloning kit (Clontech). The PCR amplification was carried outusing the primers listed in supplemental Table 2.Isolation of Peritoneal Macrophages—Peritoneal macro-

phages were isolated as described elsewhere (8). After LPStreatment, peritoneal lavage was done with 10 ml of sterileice-cold PBS, and the peritoneal lavage fluid was collected.The cells were washed and resuspended in Dulbecco’s modi-fied Eagle’s medium/F12 medium supplemented with 10%fetal bovine serum, cultured overnight, and then washed toremove nonadherent cells.siRNA Transfection—siRNA against MTA1 and negative

control siRNA were purchased from Dharmacon. Raw orMCF-7 cells were seeded at 40% density in 6-well plates, theday before transfection, and siRNA transfections were per-formed with Oligofectamine reagent (Invitrogen) according tothe manufacturer’s instructions. After 48 h of transfection,cells were harvested for Western blot analysis or used for ei-ther confocal or reporter assays.Reporter Assays—TG2 promoter assay was performed ac-

cording to the manufacturer’s instructions (Promega), and theresults were normalized against the �-galactosidase activity,an internal control. Some assays were performed in the pres-ence of control siRNA or MTA1 siRNA as described previ-ously (8).Confocal Analysis—After transfecting the MCF-7 cells with

MTA1 siRNA, MTA1 and TG2 expression was determined byindirect immunofluorescence. The cells were grown on sterileglass coverslips, fixed in 4% paraformaldehyde, permeabilizedin 0.1% Triton X-100, and blocked in 10% normal goat serumin PBS. Cells were incubated with MTA1 and TG2 antibodies,washed three times in PBS, and then incubated with second-ary antibodies conjugated (TG2,goat anti-rabbit; MTA1, anti-mouse) with Alexa Fluor 488 (for TG2) and Alexa Fluor 555(MTA1) fromMolecular Probes (Eugene, OR). The DAPI(Molecular Probes) was used as a nuclear stain. Microscopicanalysis was performed using an Olympus FV300 laser-scan-ning confocal microscope (Olympus America Inc., Melville,NY) using sequential laser excitation to minimize fluores-cence emission bleed-through.Chromatin Immunoprecipitation (ChIP) and Western Blot

Analysis—ChIP analysis using p65, MTA1, and RNA pol IIantibodies and Western blotting were carried out followingthe methods described previously (8). The primers used arelisted in supplemental Table 3.Electrophoretic Mobility Shift Assay—For electrophoretic

mobility shift assay (EMSA), nuclear extracts were pre-pared using a Nonidet P-40 lysis method (16). EMSA forNF-�B DNA binding was performed using the annealedand [�-32P]ATP end-labeled oligonucleotides in a 20-�lreaction mixture for 15 min at 20 °C. Samples were run ona nondenaturing 5% polyacrylamide gel and imaged by au-

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toradiography. Specific competitions were performed byadding a 100 M excess of competitor to the incubation mix-ture, and supershift EMSAs were performed by adding 1.5�l of either the NF-�B p65 (Santa Cruz Biotechnology286-H) or the TG2 (catalogue number 3557) or MTA1 an-tibodies. Oligonucleotides used were listed in supplementalTable 4.

Statistical Analysis and Reproducibility—The results aregiven as the means � S.E. Statistical analysis of the data wasperformed by using Student’s t test.

RESULTS AND DISCUSSION

MTA1 Regulates TG2 Expression—From an ongoing sepa-rate microarray analysis, we unexpectedly identified de-creased expression of TG2mRNA in MTA1�/� MEFs ascompared with the wild-type MEFs (Fig. 1A). Further, wefound that depletion of endogenous MTA1 also reduces thelevels of TG2mRNA and protein (Fig. 1, B and C). In addi-tion, MTA1 overexpression in MEFs and murine mammaryHC11 cells resulted in a stimulated expression of the TG2mRNA and protein (Fig. 1, C and D), suggesting that MTA1affects TG2 expression. As MTA1 is positively regulated bythe expression of TG2, we next tested this hypothesis by ana-lyzing the levels of MTA1 and TG2 in a widely studied experi-mental model of breast cancer progression involving isogenicMCF-10A cells (nonmalignant), MCF-10AT cells (weakly tu-morigenic cells), MCF-10CA1D cells (undifferentiated meta-static cells), and MCF-10DCIS cells (highly proliferative, ag-gressive, and invasive cells) (29). The levels of both MTA1 and

FIGURE 1. MTA1 regulation of TG2 expression. A, TG2 mRNA expression inwild-type and MTA1�/� MEFs identified by using microarray gene expres-sion profiling. B, qPCR analysis of TG2 mRNA expression in wild-type andMTA1�/� MEFs. Results represent a -fold change decrease in TG2 mRNAexpression in MTA1�/� MEFs relative to the wild type. Each value repre-sents the means � S.E. of three independent experiments. C, Western blotanalysis of TG2 protein expression from the cell lysates isolated from wild-type, MTA1�/�, and MTA1�/� MEFs overexpressing MTA1. D, RT-PCR analy-sis of TG2 expression in HC11 cells stably expressing pcDNA and MTA1. RT-PCR analysis was carried out with the cDNA synthesized from the 2 �g oftotal RNA isolated from the HC11/pcDNA and HC11/MTA1 stable cells usingthe gene-specific primers. E, qPCR analysis of TG2 and MTA1 mRNA expres-sion in MCF10A, MCAF10AT1, MCF10CA1D, and MCF10DCIS cells. qPCR wascarried out for the TG2, MTA1, and �-actin mRNAs from the RNA isolatedfrom the above mentioned cell lines, and results were presented in terms of-fold change after normalizing with �-actin mRNA levels. 2 �g of total RNAfrom each cell line was used for cDNA synthesis. Each value represents themeans � S.E. of three independent experiments. F, qPCR analysis of TG2expression in MCF-7 cells after selective knockdown of MTA1 using MTA1siRNA. Results presented were the means � S.E. of three independent ex-periments. The upper panel represents the Western blot analysis showingthe effective knockdown of MTA1 by using MTA1-specific siRNA. G, Westernblot analysis for MTA1 knockdown using MTA1 siRNA in the MCF-7 cellsused in panel H. H, confocal analysis representing the TG2 protein expres-sion in MCF-7 cells after selective knockdown of MTA1 using MTA1 siRNA.

FIGURE 2. Induction of TG2 expression by LPS is MTA1-mediated. A, RT-PCR analysis of TG2 and MTA1 expression in the cDNA synthesized from 2�g of the total RNA isolated from Raw cells after treating with LPS (1 �g/mlof the medium) for 2 and 4 h. B, RT-PCR analysis of TG2 and MTA1 expres-sion in LPS-treated Raw cells (1 �g/ml of the medium) with or withoutMTA1 knockdown. After 24 h of MTA1 siRNA treatment, Raw cells werestimulated with LPS (1 �g/ml of the medium) for 4 h, and RNA was isolatedfor RT-PCR analysis. C, qPCR analysis of TG2 expression in macrophages iso-lated from MTA1�/� and MTA1�/� mice. Results represent the decreased-fold change in the TG2 mRNA expression after normalizing with �-actinmRNA levels in macrophages isolated from MTA1�/� mice after treatingwith LPS for 2 h. Results presented were the means � S.E. of three inde-pendent experiments. D, Western blot analysis for TG2 expression from thelysates of wild-type and MTA1�/� MEFs treated with LPS (1 �g/ml medium)at time intervals of 2, 4, and 8 h.

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TG2 were progressively up-regulated from noninvasiveMCF10A to highly invasive MCF-10DCIS cells (Fig. 1E). Inaddition, MTA1 silencing in the MCF-7 cells also down-regu-lated the levels of TG2mRNA (Fig. 1F) and protein (Fig. 1H).Effective knockdown of MTA1 protein in MCF-7 cells byMTA1 siRNA was shown in Fig. 1G. Taken together, thesefinding suggest that MTA1 regulates the expression of TG2.Mechanistic Role of MTA1 in LPS Induction of TG2

Expression—Aberrant TG2 expression and functions havebeen reported in inflammatory diseases and cancer (5, 22).Having previously demonstrated that MTA1, as a componentof NF-�B signaling, is involved in the regulation of inflamma-tory responses (8, 15) and in TG2 expression (this study), wenext wished to investigate the role of MTA1 during LPS-me-diated expression of TG2. As LPS induces the expression ofMTA1 in Raw264.7 macrophage (Raw) cells (8, 15), we as-sessed the levels of TG2 in LPS-stimulated Raw cells. We

found that both MTA1 expression and TG2 expression wereco-induced by LPS (Fig. 2A). Further, an experimental reduc-tion in the endogenous MTA1 in the Raw cells by specificsiRNA compromised the ability of LPS to induce the expres-sion of TG2mRNA (Fig. 2B). As results of siRNA-mediatedknockdown studies are drawn by the extent of target knock-down, we next validated these findings in genetically MTA1-depleted MEFs and cultured peritoneal macrophages fromwild-type and MTA1�/� mice (8) and investigated the effectof LPS on TG2 expression. We found that MTA1 deficiencysubstantially compromised the ability of LPS to induce TG2mRNA in MTA1�/� macrophages (Fig. 2C) and TG2 proteinin MTA1�/� MEFs (Fig. 2D). These findings suggest thatMTA1 is a required cellular coregulator for LPS induction ofTG2.MTA1 Stimulates TG2 Transcription—To understand the

basis of MTA1 regulation of TG2, we cloned the TG2 pro-moter into a pGL3-basic-luciferase reporter system. We ob-served that LPS is a potent inducer of TG2 transcription andthat reducing the levels of the endogenous MTA1 by MTA1siRNA (Fig. 3A) and in MTA1�/� MEFs (Fig. 3B), compro-mised TG2 transcription as well as the ability of LPS to induceTG2 promoter activity. In addition, we observed an increasedTG2 promoter activity upon MTA1 overexpression (Fig. 3C).These observations suggest that MTA1 regulates TG2 expres-sion at the transcriptional level.To gain a deeper insight into the molecular mechanism

underlying the noticed MTA1 regulation of TG2 expression,we carried out a detailed ChIP analysis in Raw cells treatedwith or without LPS and mapped the recruitment of MTA1onto three regions of TG2 promoter at �320 to �491, �630to �849, and �1878 to �2139 (Fig. 4, A and B). However, weonly observed enhanced recruitment of MTA1 in response toLPS stimulation onto the �630 to �849 region of TG2 pro-moter (Fig. 4B), indicating a role for this region in LPS regula-tion of TG2. In addition, we noticed the recruitment ofMTA1-pol II coactivator complex only to this region (�630to �849) under basal as well as LPS stimulation. Together,these results suggest the involvement of MTA1 in regulating

FIGURE 3. MTA1 regulates TG2 transcription. A, TG2 promoter-Luc activityin LPS- (1 �g/ml medium for 4 h) stimulated Raw cells with or without MTA1knockdown. After MTA1 knockdown using MTA1 siRNA, Raw cells weretransfected with murine TG2-pGL3 luciferase reporter plasmid and pCMV�vector carrying the �-galactosidase gene as an internal control for transfec-tion efficiency. After 36 h of transfection, cells were treated with LPS (1�g/ml of the medium for 4 h), and luciferase activities in triplicate sampleswere measured. Results were presented in terms of -fold change, and thevalues represent the means � S.E. from three independent transfectionexperiments. B, TG2 promoter-Luc activity in LPS- (1 �g/ml of the mediumfor 4 h) stimulated wild-type and MTA1�/� MEFs. The TG2-pGL3 luciferaseassay was carried out as described for panel A. Results were presented interms of -fold change, and the values represent the means � S.E. from threeindependent transfection experiments. C, Raw cells were transfected withthe indicated plasmids, and after 36 h of transfection, cells were treatedwith LPS (1 �g/ml of the medium) for 4 h. Luciferase activity was calculatedas mentioned earlier. The upper panel represents the Western blot analysisfor T7-MTA1 to show the transfection efficiency.

FIGURE 4. TG2 is an MTA1 target gene. A, line diagram showing the binding of a region of MTA1 on murine TG2 promoter. To carry out ChIP-based pro-moter walk, murine TG2 promoter was divided into five regions. The double-headed arrow represents the MTA1 binding region on murine TG2 promoter.B, recruitment of MTA1 to TG2 chromatin in Raw cells treated with or without LPS for 1 h. Raw cells that were treated with 1% formaldehyde to cross-linkthe histones to DNA were lysed by sonication and immunoprecipitated by either anti-MTA1 antibody or IgG antibody. The immunoprecipitates (IP) werecollected by adding beads; beads were washed, DNA was eluted from the beads, and purified DNA was subjected to PCR. C, double ChIP analysis of recruit-ment of MTA1-pol II complex onto the TG2-chromatin (�630 to �849) in Raw cells treated with LPS for 1 h. The first ChIP was carried out with anti-MTA1antibody followed by second ChIP with anti-RNA polymerase II. From the same elutes of ChIP analysis, qPCR analysis was also performed.

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TG2 transcription by targeting a specific region of TG2 chro-matin in LPS-stimulated macrophages.TG2 Is an NF-�B-regulated Gene—As MTA1 modulates

NF-�B signaling (8, 15, 16) as well as TG2 transcription inLPS-stimulated Raw cells (this study) and because LPS is apotent inducer of NF-�B (30), we next focused on the mecha-nism of LPS regulation of TG2 expression. We found thatparthenolide, a pharmacological inhibitor of NF-�B (31), at-tenuated both basal and LPS-stimulated TG2 protein, mRNAexpression, and promoter activity (Fig. 5, A–C) in the Rawcells, suggesting that LPS may regulate TG2 expression viaNF-�B signaling. In this context, transient expression ofp65RelA increased the TG2 promoter activity in the basal aswell as LPS-stimulated conditions in the Raw cells (Fig. 5D),whereas depletion of NF-�B p65 resulted in decreased TG2

transcription (Fig. 5E), suggesting that TG2 is an NF-�B tar-get gene.To understand the molecular details of NF-�B regulation of

TG2, we conducted a ChIP-based promoter walk withp65RelA antibody in the Raw cells with or without LPS treat-ment. We observed the recruitment of p65RelA onto the�630 to �839 region of the TG2 promoter, and this was fur-ther enhanced in the presence of LPS (Fig. 6A). We also ob-

FIGURE 5. NF-�B regulates TG2 transcription. A, Western blot analysis forTG2 expression in LPS- (1 �g/ml for 4 h) treated Raw cells in the presenceand absence of parthenolide (5 �M). B, qPCR analysis of TG2 mRNA expres-sion in LPS- (1 �g/ml for 4 h) treated Raw cells in the presence and absenceof parthenolide (5 �M). Parthenolide (5 �M) was added to the cells 1 h priorto the LPS treatment. After the treatment, RNA was isolated, and qPCR anal-ysis was carried as described in the legend for Fig. 1. C, TG2 promoter-Lucactivity in Raw cells treated with LPS (1 �g/ml for 4 h) in the presence ofparthenolide (5 �M). D, TG2 promoter-Luc activity in Raw cells treated withLPS after cotransfecting with p65RelA. Raw cells were transfected with theindicated plasmids, and the luciferase assay was carried out as described inthe legend for Fig. 3. The upper panel represents the Western blot analysisfor T7-p65 to show the transfection efficiency. E, TG2 promoter-Luc activityin Raw cells treated with LPS after NF-�B-p65 knockdown using NF-�B-p65siRNA. The experiment was carried out as described in the legend for Fig. 3.

FIGURE 6. TG2 is an NF-�B target gene. A, ChIP analysis of p65RelA fol-lowed by MTA1 recruitment to TG2-chromatin in Raw cells p65RelA, treatedwith LPS for 1 h. ChIP analysis was carried with either p65RelA antibody ordouble ChIP analysis; the first ChIP was with p65RelA antibody, and the sec-ond ChIP was with MTA1 antibody from the lysates of LPS-stimulated Rawcells. B, recruitment of p65 followed by pol II on to TG2 chromatin (�630 to�849). The lower panel represents qPCR-ChIP. IP, immunoprecipitates. C, ef-fect of parthenolide (5 �M) on the recruitment of p65 onto TG2 promoter (�630to �849) in LPS- (1 �g/ml of the medium for 1 h) stimulated Raw cells. Raw cellstreated with parthenolide 1 h prior to LPS treatment were used to carry outChIP analysis as described in the legend for Fig. 4. The lower panel representsthe qPCR-ChIP. D, Western blot analysis for MTA1 and NF-�B-p65 after immu-noprecipitating with MTA1 in Raw cells treated with LPS. E, in vitro interaction ofMTA1 and p65. The GST-p65 fusion protein and GST were used in a GST pull-down assay with in vitro-translated 35S-labeled full-length MTA1 (MTA1 (S35)).F, EMSA analysis of p65 and MTA1 binding to the mouse TG2 promoter usingthe wild-type and mutant oligonucleotides encompassing NF-�B consensussequences in Raw cells treated with LPS for 1 h. G, summary of ChIP analysiscarried out on the mouse TG2 promoter. This figure summarizes the recruit-ment of MTA1, p65RelA, MTA1-pol II complex, and p65-MTA1 complex ontothe TG2 chromatin region (�630 to �849).

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served that p65-MTA1 and p65-pol II complexes are also co-recruited to the same region of the TG2 chromatin (Fig. 6, Aand B). Importantly, parthenolide effectively blocked the re-cruitment of p65RelA to this region of the TG2 promoter (Fig.6C). To support these results, we showed that p65 could becoimmunoprecipitated along with MTA1 under both the ba-sal and the LPS-stimulated Raw cells as evident by an in-creased association of p65 in LPS-stimulated cells as com-pared with the level in the control cells (Fig. 6D). Further, theinteraction of MTA1 and p65 was also validated in vitro (Fig.6E) using 35S-labeled MTA1 protein and glutathione S-trans-ferase-NF-�B-p65 fusion protein. These results support thenotion of a direct interaction of MTA1 with NF-�B-p65. Col-lectively, these results suggest that p65-MTA1-pol II may ex-ist in the same complex under physiological condition.Scanning of the MTA1-targeted region of TG2 promoter

(�630 to �839) for available transcriptional factors using theALGGEN-PROMO software revealed the presence of onlyone potential NF-�B site (GGGAATTATC, �758 to �749).To demonstrate the direct binding of p65RelA to the TG2promoter, we next performed an EMSA analysis using oligo-nucleotides encompassing this site (both wild-type and mu-tant NF-�B) using the nuclear extracts from LPS-stimulatedRaw cells (Fig. 6F). We found the appearance of a distinct pro-tein-DNA complex in the LPS-stimulated condition (Fig. 6F,lane 3). The specificity of this protein-DNA complex was ver-ified by supershift analysis using p65RelA or MTA1 antibod-ies. We noticed supershifts with p65 or MTA1 antibodies butnot by the control IgG antibody (Fig. 6F, lanes 4–6). No pro-tein-DNA complex was observed with the oligonucleotideshaving mutant NF-�B sequence (Fig. 6F, lanes 9–14). Theseresults suggest that LPS stimulates TG2 transcription via di-rect recruitment of the p65-MTA1 complex onto the TG2promoter and that TG2 is an NF-�B target. A schematic rep-resentation of recruitment of p65 and MTA1 to the TG2 pro-moter is shown in Fig. 6G. Together, these findings revealedan inherent role of MTA1 in the regulation of TG2 expres-sion, which, at least in part, may constitute one of the mecha-nisms involved in the modulation of LPS-induced NF-�B sig-naling by MTA1 in stimulated macrophages (Fig. 7).In brief, our finding of MTA1 regulation of TG2 expression

introduces a new regulatory player of NF-�B signaling duringinduction of proinflammatory cytokines and innate immunity.Several reports represented a positive correlation between theexpression of NF-�B and TG2 expression (32, 33) and be-tween the expression of NF-�B and MTA1 (8, 16, 34), sug-gesting the existence of a positive regulatory mechanismamong these genes. Increased TG2 activity triggers NF-�Bactivation by inducing the polymerization of I-�B� ratherthan stimulating I-�B� kinase (35). This polymerization ofI-�B� results in a direct activation of NF-�B, resulting in con-stitutive expression of various target genes involved in inflam-mation (5, 35). During sepsis mediated by LPS, NF-�B is acti-vated, leading to the induction of cytokines and inflammatorymediators. The absence of TG2 could be an advantage duringendotoxic shock because this deficiency appears to be associ-ated with an activation of NF-�B that is transient, thus allow-ing the restoration of immunological equilibrium. In this con-

text, studies from the TG2�/� knock-out mice revealed thatTG2 offers a protection against liver injuries caused by CCl4(36, 37). Although many reports have shown an associationbetween the levels of TG2 during inflammatory diseases, itsregulation and its role in inflammatory process remain poorlyunderstood. In this context, our present study demonstratesthe involvement of MTA1 in the modulation of NF-�B signal-ing leading to TG2 transcription and proinflammatory cyto-kines in LPS-stimulated cells. These findings suggest that TG2is a target of MTA1 and that its transcription is positively reg-ulated by TG2 because of a direct binding of the MTA1,p65RelA, and pol II complex to the TG2 promoter (Figs. 4 and6). We propose that MTA1 offers protection against the in-vading pathogens either directly by regulating the strength ofthe NF-�B signaling and/or by regulating its target genes likeTG2.

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Inflammatory ResponseMTA1 Coregulation of Transglutaminase 2 Expression and Function during

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