MTA1 Coregulation of Transglutaminase 2 Expression and Function during Inflammatory Response*

Although both metastatic tumor antigen 1 (MTA1), a master chromatin modifier, and transglutaminase 2 (TG2), a multifunctional enzyme, are known to be activated during inflammation, it remains unknown whether these molecules regulate inflammatory response in a coordinated manner. Here we investigated the role of MTA1 in the regulation of TG2 expression in bacterial lipopolysaccharide (LPS)-stimulated mammalian cells. While studying the impact of MTA1 status on global gene expression, we unexpectedly discovered that MTA1 depletion impairs the basal as well as the LPS-induced expression of TG2 in multiple experimental systems. We found that TG2 is a chromatin target of MTA1 and of NF-κB signaling in LPS-stimulated cells. In addition, LPS-mediated stimulation of TG2 expression is accompanied by the enhanced recruitment of MTA1, p65RelA, and RNA polymerase II to the NF-κB consensus sites in the TG2 promoter. Interestingly, both the recruitment of p65 and TG2 expression are effectively blocked by a pharmacological inhibitor of the NF-κB pathway. These findings reveal an obligatory coregulatory role of MTA1 in the regulation of TG2 expression and of the MTA1-TG2 pathway, at least in part, in LPS modulation of the NF-κB signaling in stimulated macrophages.

Inflammation is an adaptive immune response triggered by the body against detrimental stimuli and conditions such as microbial infection and tissue injury (1,2). Inflammation is usually a healing response, but it becomes detrimental if targeted destruction and assisted repair are not properly activated (3). Primarily, macrophages and mast cells recognize the infection and produce a wide variety of inflammatory mediators such as chemokines, cytokines, etc., all contributing to the elicitation of an inflammatory response (1). The inflammatory response is characterized by coordinated regulation of signaling pathways that regulate the expression of both the pro-inflammatory and the anti-inflammatory cytokines including IL-1, IL-6, TNF-␣, receptor activator of NF-B ligand (RANKL), etc. (4). The inability of host to regulate inflammatory response results in sepsis, organ dysfunction, and even death (5). These inflammatory cytokines are under the tight control of master gene transcriptional factor NF-B in promoting the inflammation, and in turn, innate immunity (6). Furthermore, transcriptional control of such NF-B genomic targets is also under a tight control of nucleosome-remodeling coregulators and complexes, leading to either the stimulation or the repression of gene transcription at the molecular level (7)(8)(9)(10).
In recent times, metastatic tumor antigen 1 (MTA1) 3 has been recognized as one of the major coregulators in mammalian cells. MTA1 is a ubiquitously expressed chromatin modifier, having an integral role in nucleosome-remodeling and histone deacetylase (NuRD) complexes (11). MTA1 is widely up-regulated in a wide variety of human tumors and has been shown to play a role in tumorigenesis (11)(12)(13)(14). MTA1 regulates transcription of its targets by modifying the acetylation status of the target chromatin and cofactor accessibility to the target DNA. Recent work from this laboratory has shown that MTA1 plays a key role in inflammatory responses both as a target and as a component of the NF-B signaling by regulating a subset of lipopolysaccharide (LPS)-induced proinflammatory cytokines (8) or by directly regulating the MyD88, a proximal component of NF-B signaling (15). In addition to these functions, MTA1 also plays an essential role in Hepatitis B Virus X Protein stimulation of NF-B signaling and in the expression of NF-B target gene products with functions in inflammation and tumorigenesis (16).
In addition, MTA1 is a newly added regulator of inflammation, and it is also regulated by a number of genes including transglutaminase 2 (TG2) (17). TG2 is a multifunctional enzyme involved in several cellular functions such as apoptosis (18), signaling (19), signal transduction (20), cytoskeleton rearrangements and extracellular matrix stabilization (17), and wound healing (21). Aberrant activation and functions of TG2 have been linked with a variety of inflammatory diseases that include celiac disease, diabetes, multiple sclerosis, rheumatoid arthritis, and sepsis (5,22). Results from a mouse model system revealed that TG2 is also involved in the NF-B activation, which induces the transcription of proinflammatory cytokines, causing continuous activation of inflammatory process 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) contains supplemental Tables 1-4. 1 Both authors contributed equally to this work. 2 To whom correspondence should be addressed. E-mail: bcmrxk@gwumc. edu. (5). Apart from its role in inflammation, elevated levels of TG2 are associated with many types of cancers (17,(23)(24)(25), a property shared with MTA1. In addition to this, increased expression of TG2 in cancer cells leads to increased drug resistance, metastasis, and poor patient survival (23,24,26), a property also shared with MTA1. Although TG2 expression parallels with MTA1 during inflammation, it remains unclear whether these molecules are trans-regulated by inflammation. Here we report that MTA1 is an obligatory coregulator of TG2 expression and that the MTA1-TG2 pathway plays a mechanistic role, at least in part, in bacterial LPS modulation of the NF-B signaling in stimulated macrophages.

EXPERIMENTAL PROCEDURES
Cell Culture, Antibodies, and Reagents-All cells used in this study were cultured in Dulbecco's modified Eagle's medium/F12 medium supplemented with 10% fetal bovine serum. Raw264.7 and MCF7 cells were obtained from American Type Culture Collection, whereas HC11-pcDNA and HC11-MTA1, MTA1 ϩ/ϩ , and MTA1 Ϫ/Ϫ mouse embryonic fibroblasts (MEFs) have been described in our earlier studies (27). Transglutaminase 2 (catalogue number 3557) and NF-B p65 (catalogue number sc-372) antibodies were purchased from Cell Signaling Technology and Santa Cruz Biotechnology, respectively. Antibodies against MTA1 (catalogue number A300-280A) and RNA polymerase II (pol II) (catalogue number A300-653A) were purchased from Bethyl Laboratories, whereas normal mouse IgG, rabbit IgG, and antibodies against vinculin were from Sigma. Bacterial LPS was purchased from Sigma. Whenever needed, LPS was used at the concentration of 1 g/ml of the medium. MTA1 siRNA (catalogue number, M-004127-01) and NF-B p65 siRNA (catalogue number, sc-29411) were purchased from Dharmacon RNAi Technologies (Lafayette, CO) and Santa Cruz Biotechnology, respectively.
Microarray Expression Profiling and Analysis-The microarray expression profiling and analysis were carried out as described elsewhere (28). The RNA isolated by the TRIzol method was further checked for purity by analyzing on a 2100 Bioanalyzer (Agilent Technologies). From the 2 g of the total purified RNA, rRNA reduction was performed using RiboMinus TM transcriptome isolation kit (Invitrogen). Reduced rRNA was now labeled using the GeneChip WT cDNA synthesis/amplification kit and hybridized on a GeneChip Mouse Exon 1.0 ST array. Scanning of the hybridized arrays was carried out using Affymetrix GeneChip scanner 3000 7G. The obtained microarray data were analyzed using GeneSpring GX10.0.2. Gene ontology (GO) analysis was performed on statistically 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 by using Trizol reagent (Invitrogen), and first-strand cDNA synthesis was carried out with SuperScript II reverse transcriptase (Invitrogen) using 2 g of total RNA and oligo(dT) primer. cDNA from macrophages was synthesized using the FastLane 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 a 7900HT sequence detection system (Applied Biosystems). The levels of mRNA of all the genes were normalized to that of ␤-actin mRNA.
Cloning of Murine TG2 Promoter-Murine TG2 promoter was PCR-amplified from mouse genomic DNA and cloned into pGL3 basic vector using the In-Fusion 2.0 dry-down PCR cloning kit (Clontech). The PCR amplification was carried out using the primers listed in supplemental Table 2.
Isolation of Peritoneal Macrophages-Peritoneal macrophages were isolated as described elsewhere (8). After LPS treatment, peritoneal lavage was done with 10 ml of sterile ice-cold PBS, and the peritoneal lavage fluid was collected. The cells were washed and resuspended in Dulbecco's modified Eagle's medium/F12 medium supplemented with 10% fetal bovine serum, cultured overnight, and then washed to remove nonadherent cells.
siRNA Transfection-siRNA against MTA1 and negative control siRNA were purchased from Dharmacon. Raw or MCF-7 cells were seeded at 40% density in 6-well plates, the day before transfection, and siRNA transfections were performed with Oligofectamine reagent (Invitrogen) according to the manufacturer's instructions. After 48 h of transfection, cells were harvested for Western blot analysis or used for either confocal or reporter assays.
Reporter Assays-TG2 promoter assay was performed according to the manufacturer's instructions (Promega), and the results were normalized against the ␤-galactosidase activity, an internal control. Some assays were performed in the presence of control siRNA or MTA1 siRNA as described previously (8).
Confocal Analysis-After transfecting the MCF-7 cells with MTA1 siRNA, MTA1 and TG2 expression was determined by indirect immunofluorescence. The cells were grown on sterile glass coverslips, fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, and blocked in 10% normal goat serum in PBS. Cells were incubated with MTA1 and TG2 antibodies, washed three times in PBS, and then incubated with secondary antibodies conjugated (TG2,goat anti-rabbit; MTA1, antimouse) with Alexa Fluor 488 (for TG2) and Alexa Fluor 555 (MTA1) from Molecular Probes (Eugene, OR). The DAPI (Molecular Probes) was used as a nuclear stain. Microscopic analysis was performed using an Olympus FV300 laser-scanning confocal microscope (Olympus America Inc., Melville, NY) using sequential laser excitation to minimize fluorescence emission bleed-through.
Chromatin Immunoprecipitation (ChIP) and Western Blot Analysis-ChIP analysis using p65, MTA1, and RNA pol II antibodies and Western blotting were carried out following the methods described previously (8). The primers used are listed in supplemental Table 3.
Electrophoretic Mobility Shift Assay-For electrophoretic mobility shift assay (EMSA), nuclear extracts were prepared using a Nonidet P-40 lysis method (16). EMSA for NF-B DNA binding was performed using the annealed and [␥-32 P]ATP end-labeled oligonucleotides in a 20-l reaction mixture for 15 min at 20°C. Samples were run on a nondenaturing 5% polyacrylamide gel and imaged by au-toradiography. Specific competitions were performed by adding a 100 M excess of competitor to the incubation mixture, and supershift EMSAs were performed by adding 1.5 l of either the NF-B p65 (Santa Cruz Biotechnology 286-H) or the TG2 (catalogue number 3557) or MTA1 antibodies. Oligonucleotides used were listed in supplemental Table 4.
Statistical Analysis and Reproducibility-The results are given as the means Ϯ S.E. Statistical analysis of the data was performed by using Student's t test.

RESULTS AND DISCUSSION
MTA1 Regulates TG2 Expression-From an ongoing separate microarray analysis, we unexpectedly identified decreased expression of TG2 mRNA in MTA1 Ϫ/Ϫ MEFs as compared with the wild-type MEFs (Fig. 1A). Further, we found that depletion of endogenous MTA1 also reduces the levels of TG2 mRNA and protein (Fig. 1, B and C). In addition, MTA1 overexpression in MEFs and murine mammary HC11 cells resulted in a stimulated expression of the TG2 mRNA and protein (Fig. 1, C and D), suggesting that MTA1 affects TG2 expression. As MTA1 is positively regulated by the expression of TG2, we next tested this hypothesis by analyzing the levels of MTA1 and TG2 in a widely studied experimental model of breast cancer progression involving isogenic MCF-10A cells (nonmalignant), MCF-10AT cells (weakly tumorigenic cells), MCF-10CA1D cells (undifferentiated metastatic cells), and MCF-10DCIS cells (highly proliferative, aggressive, and invasive cells) (29). The levels of both MTA1 and  TG2 were progressively up-regulated from noninvasive MCF10A to highly invasive MCF-10DCIS cells (Fig. 1E). In addition, MTA1 silencing in the MCF-7 cells also down-regulated the levels of TG2 mRNA (Fig. 1F) and protein (Fig. 1H). Effective knockdown of MTA1 protein in MCF-7 cells by MTA1 siRNA was shown in Fig. 1G. Taken together, these finding suggest that MTA1 regulates the expression of TG2.
Mechanistic Role of MTA1 in LPS Induction of TG2 Expression-Aberrant TG2 expression and functions have been reported in inflammatory diseases and cancer (5,22). Having previously demonstrated that MTA1, as a component of NF-B signaling, is involved in the regulation of inflammatory responses (8,15) and in TG2 expression (this study), we next wished to investigate the role of MTA1 during LPS-mediated expression of TG2. As LPS induces the expression of MTA1 in Raw264.7 macrophage (Raw) cells (8,15), we assessed the levels of TG2 in LPS-stimulated Raw cells. We found that both MTA1 expression and TG2 expression were co-induced by LPS ( Fig. 2A). Further, an experimental reduction in the endogenous MTA1 in the Raw cells by specific siRNA compromised the ability of LPS to induce the expression of TG2 mRNA (Fig. 2B). As results of siRNA-mediated knockdown studies are drawn by the extent of target knockdown, we next validated these findings in genetically MTA1depleted MEFs and cultured peritoneal macrophages from wild-type and MTA1 Ϫ/Ϫ mice (8) and investigated the effect of LPS on TG2 expression. We found that MTA1 deficiency substantially compromised the ability of LPS to induce TG2 mRNA in MTA1 Ϫ/Ϫ macrophages (Fig. 2C) and TG2 protein in MTA1 Ϫ/Ϫ MEFs (Fig. 2D). These findings suggest that MTA1 is a required cellular coregulator for LPS induction of TG2.
MTA1 Stimulates TG2 Transcription-To understand the basis of MTA1 regulation of TG2, we cloned the TG2 promoter into a pGL3-basic-luciferase reporter system. We observed that LPS is a potent inducer of TG2 transcription and that reducing the levels of the endogenous MTA1 by MTA1 siRNA (Fig. 3A) and in MTA1 Ϫ/Ϫ MEFs (Fig. 3B), compromised TG2 transcription as well as the ability of LPS to induce TG2 promoter activity. In addition, we observed an increased TG2 promoter activity upon MTA1 overexpression (Fig. 3C). These observations suggest that MTA1 regulates TG2 expression 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 treated with or without LPS and mapped the recruitment of MTA1 onto three regions of TG2 promoter at Ϫ320 to Ϫ491, Ϫ630 to Ϫ849, and Ϫ1878 to Ϫ2139 (Fig. 4, A and B). However, we only observed enhanced recruitment of MTA1 in response to LPS stimulation onto the Ϫ630 to Ϫ849 region of TG2 promoter (Fig. 4B), indicating a role for this region in LPS regulation of TG2. In addition, we noticed the recruitment of MTA1-pol II coactivator complex only to this region (Ϫ630 to Ϫ849) under basal as well as LPS stimulation. Together, these results suggest the involvement of MTA1 in regulating  To carry out ChIP-based promoter 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-link the histones to DNA were lysed by sonication and immunoprecipitated by either anti-MTA1 antibody or IgG antibody. The immunoprecipitates (IP) were collected by adding beads; beads were washed, DNA was eluted from the beads, and purified DNA was subjected to PCR. C, double ChIP analysis of recruitment of MTA1-pol II complex onto the TG2-chromatin (Ϫ630 to Ϫ849) in Raw cells treated with LPS for 1 h. The first ChIP was carried out with anti-MTA1 antibody followed by second ChIP with anti-RNA polymerase II. From the same elutes of ChIP analysis, qPCR analysis was also performed.
TG2 transcription by targeting a specific region of TG2 chromatin 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 in LPS-stimulated Raw cells (this study) and because LPS is a potent inducer of NF-B (30), we next focused on the mechanism of LPS regulation of TG2 expression. We found that parthenolide, a pharmacological inhibitor of NF-B (31), attenuated both basal and LPS-stimulated TG2 protein, mRNA expression, and promoter activity (Fig. 5, A-C) in the Raw cells, suggesting that LPS may regulate TG2 expression via NF-B signaling. In this context, transient expression of p65RelA increased the TG2 promoter activity in the basal as well 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 target gene.
To understand the molecular details of NF-B regulation of TG2, we conducted a ChIP-based promoter walk with p65RelA antibody in the Raw cells with or without LPS treatment. We observed the recruitment of p65RelA onto the Ϫ630 to Ϫ839 region of the TG2 promoter, and this was further enhanced in the presence of LPS (Fig. 6A). We also ob-  served that p65-MTA1 and p65-pol II complexes are also corecruited to the same region of the TG2 chromatin (Fig. 6, A  and B). Importantly, parthenolide effectively blocked the recruitment of p65RelA to this region of the TG2 promoter (Fig.  6C). To support these results, we showed that p65 could be coimmunoprecipitated along with MTA1 under both the basal and the LPS-stimulated Raw cells as evident by an increased association of p65 in LPS-stimulated cells as compared with the level in the control cells (Fig. 6D). Further, the interaction of MTA1 and p65 was also validated in vitro (Fig.  6E) using 35 S-labeled MTA1 protein and glutathione S-transferase-NF-B-p65 fusion protein. These results support the notion of a direct interaction of MTA1 with NF-B-p65. Collectively, these results suggest that p65-MTA1-pol II may exist in the same complex under physiological condition.
Scanning of the MTA1-targeted region of TG2 promoter (Ϫ630 to Ϫ839) for available transcriptional factors using the ALGGEN-PROMO software revealed the presence of only one potential NF-B site (GGGAATTATC, Ϫ758 to Ϫ749). To demonstrate the direct binding of p65RelA to the TG2 promoter, we next performed an EMSA analysis using oligonucleotides encompassing this site (both wild-type and mutant NF-B) using the nuclear extracts from LPS-stimulated Raw cells (Fig. 6F). We found the appearance of a distinct protein-DNA complex in the LPS-stimulated condition (Fig. 6F,  lane 3). The specificity of this protein-DNA complex was verified by supershift analysis using p65RelA or MTA1 antibodies. We noticed supershifts with p65 or MTA1 antibodies but not by the control IgG antibody (Fig. 6F, lanes 4 -6). No protein-DNA complex was observed with the oligonucleotides having mutant NF-B sequence (Fig. 6F, lanes 9 -14). These results suggest that LPS stimulates TG2 transcription via direct recruitment of the p65-MTA1 complex onto the TG2 promoter and that TG2 is an NF-B target. A schematic representation of recruitment of p65 and MTA1 to the TG2 promoter is shown in Fig. 6G. Together, these findings revealed an inherent role of MTA1 in the regulation of TG2 expression, which, at least in part, may constitute one of the mechanisms involved in the modulation of LPS-induced NF-B signaling 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 during induction of proinflammatory cytokines and innate immunity. Several reports represented a positive correlation between the expression of NF-B and TG2 expression (32,33) and between the expression of NF-B and MTA1 (8,16,34), suggesting the existence of a positive regulatory mechanism among these genes. Increased TG2 activity triggers NF-B activation by inducing the polymerization of I-B␣ rather than stimulating I-B␣ kinase (35). This polymerization of I-B␣ results in a direct activation of NF-B, resulting in constitutive expression of various target genes involved in inflammation (5,35). During sepsis mediated by LPS, NF-B is activated, leading to the induction of cytokines and inflammatory mediators. The absence of TG2 could be an advantage during endotoxic shock because this deficiency appears to be associated with an activation of NF-B that is transient, thus allowing the restoration of immunological equilibrium. In this con-text, studies from the TG2 Ϫ/Ϫ knock-out mice revealed that TG2 offers a protection against liver injuries caused by CCl 4 (36,37). Although many reports have shown an association between the levels of TG2 during inflammatory diseases, its regulation and its role in inflammatory process remain poorly understood. In this context, our present study demonstrates the involvement of MTA1 in the modulation of NF-B signaling leading to TG2 transcription and proinflammatory cytokines in LPS-stimulated cells. These findings suggest that TG2 is a target of MTA1 and that its transcription is positively regulated by TG2 because of a direct binding of the MTA1, p65RelA, and pol II complex to the TG2 promoter (Figs. 4 and 6). We propose that MTA1 offers protection against the invading pathogens either directly by regulating the strength of the NF-B signaling and/or by regulating its target genes like TG2.