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Originally published In Press as doi:10.1074/jbc.M403738200 on July 7, 2004

J. Biol. Chem., Vol. 279, Issue 37, 38577-38589, September 10, 2004
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Class II Major Histocompatibility Complex Transactivator (CIITA) Inhibits Matrix Metalloproteinase-9 Gene Expression*

Susan Nozell{ddagger}, Zhendong Ma, Cynthia Wilson, Reesha Shah, and Etty N. Benveniste§

From the Department of Cell Biology, The University of Alabama at Birmingham, Birmingham, Alabama 35294-0005

Received for publication, April 5, 2004 , and in revised form, July 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Matrix metalloproteinases (MMPs) are a family of structurally related proteins with the collective capability to degrade all components of the extracellular matrix. Although MMP-mediated degradation of the extracellular matrix occurs physiologically, numerous pathological conditions exhibit increased MMP levels and excessive matrix degradation. Previous work from our laboratory has shown that interferon-{gamma} inhibits MMP-9 expression in a manner dependent upon STAT-1{alpha}. Here we extend our previous observations and show that the class II major histocompatibility complex transactivator (CIITA), a transcriptional target of STAT-1{alpha}, is also capable of inhibiting MMP-9 expression. By using stable cell lines that inducibly express CIITA or various mutant forms of CIITA, we show that CIITA requires the ability to bind the CREB-binding protein (CBP) to effectively inhibit MMP-9 expression. Furthermore, we show that CIITA-mediated inhibition of the MMP-9 gene does not rely on the transcriptional capability of CIITA. These findings support a model wherein CIITA inhibits MMP-9 expression by binding to and sequestering CBP, which reduces the levels of CBP at the MMP-9 promoter, inhibits levels of acetylated histone 3 at the MMP-9 promoter, and subsequently inhibits MMP-9 expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The matrix metalloproteinases (MMPs)1 are a family of Zn2+-dependent endopeptidases (1). To date, there are more than 20 members of the MMP family, and they are classified into four groups based on structural similarities, substrate preferences, and sequence homology. The four groups include the collagenases, gelatinases, stromelysins, and the membrane-type MMPs (1). The gelatinases comprise the smallest group within the MMP family and consist of only two members, MMP-2 and MMP-9.

Collectively, the members of the MMP family possess the ability to degrade all components of the ECM. For example, MMP-mediated proteolysis of the ECM can cause an adhesive cell to become nonadhesive, promote the release of matrix-bound growth factors, or activate other MMPs (2, 3). Cooperatively, these actions enable normal physiological processes such as cell migration, invasion, proliferation, angiogenesis, and wound healing (1). However, progressive and/or excessive ECM degradation has been implicated in many disease states including ischemia, multiple sclerosis, and human immunodeficiency virus-associated dementia and is also associated with tumor invasion, metastasis, and angiogenesis (4). In particular, high levels of MMP-9 have been demonstrated in several inflammatory diseases, including inflammation of the pulmonary tract, gastrointestinal tract, joints, blood vessels, and nervous system, in addition to numerous cancers including brain tumors, bladder, breast, endometrial, prostate, ovarian, pancreatic and gastric cancers, and basal cell and squamous cell carcinomas (4).

The activity of MMP-9 is typically regulated in the following three ways: transcriptional activation, enzymatic activation, or inhibition by tissue inhibitors of MMPs (TIMPs) (4). Of these mechanisms, transcriptional activation of MMP-9 appears to be the most complex and decisive manner by which MMP-9 expression is regulated. The MMP-9 gene is regulated by a 670-bp promoter region upstream of its transcriptional start site (5). This region contains several transcription factor binding sites including two AP-1 sites, an NF-{kappa}B site, an ETS site, and an Sp1 site, and these elements are sufficient for the transcriptional activation of the MMP-9 gene by various cytokines, mitogens, oncogene products, and phorbol esters (4). In general, all stimuli require the proximal AP-1 site to induce MMP-9 expression (6). Additionally, PMA and TNF-{alpha} also require the coordinate activation of the NF-{kappa}B and Sp1 sites, whereas v-src requires the Sp1-binding site and ras utilizes the ETS site (4). We have recently determined that in addition to NF-{kappa}B, AP-1, and Sp1 binding directly to the MMP-9 promoter, chromatin remodeling, recruitment of coactivators such as p300, CARM1, and the CREB-binding protein (CBP), and histone modifications, which is the enhanced acetylation of histones 3 and 4 (H3 and H4), are all critical features of MMP-9 gene transcription (7).

Previous work from our laboratory has shown that interferon-{gamma} (IFN-{gamma}) suppresses PMA-induced MMP-9 gene expression (8). Upon binding to its receptor, a heterodimer of the subunits IFN-{gamma}R1 and IFN-{gamma}R2, IFN-{gamma} induces the trans-phosphorylation of the JAK1 and JAK2 kinases, which in turn phosphorylate signal transducer and activator of transcription-1{alpha} (STAT-1{alpha}) (9, 10). Upon phosphorylation, STAT-1{alpha} homodimerizes and translocates to the nucleus where it binds to {gamma}-activated sequences (GAS), which are present in the promoters of IFN-{gamma}-responsive genes (9, 10). Despite the absence of any GAS elements within the MMP-9 promoter, our data demonstrated the critical role of STAT-1{alpha} in the process of IFN-{gamma}-mediated suppression of the MMP-9 gene, since IFN-{gamma} is unable to suppress MMP-9 expression in either STAT-1{alpha} null murine astrocytes or STAT-1{alpha}-deficient human fibrosarcoma lines (8).

The class II MHC transactivator (CIITA) is a non-DNA-binding protein and master regulator of class II MHC expression (11, 12). Except in professional antigen-presenting cells, CIITA is not expressed, and class II MHC expression is absent. However, upon stimulation with IFN-{gamma}, STAT-1{alpha} triggers CIITA expression through GAS elements present within its promoter (13, 14). Because CIITA does not bind DNA, CIITA must exert its effects at the class II MHC promoter by interacting with proteins bound to the conserved elements within class II MHC promoters. Although these proteins are constitutively present, they alone are insufficient to induce class II MHC expression (11). The conserved elements of class II MHC promoters include the W, X (consisting of X1 and X2 regions), and Y boxes and are bound by the NF-Y heterotrimer at the W and Y box, the RFX heterotrimer at the X1 region of the X box, and the CREB protein at the X2 portion of the X box (11). When present, CIITA binds to the NF-Y and RFX heterotrimers and is believed to stabilize the minimal class II MHC enhanceosome, which promotes the recruitment of general transcription factors, RNA polymerase II, and various coactivators including CBP, to stimulate expression of the class II MHC molecules (11).

Recently, reports have shown that CIITA can also inhibit the expression of several genes, including interleukin-4 (IL-4), thyroid-specific genes, collagen {alpha}2(I), thymidine kinase, cyclin D1, Fas ligand (Fas-L), and cathepsin E (15–21). Most interestingly, except for collagen {alpha}2(I), the promoters of these genes lack any of the conserved class II MHC promoter elements and have not been shown to bind CIITA directly or indirectly. In the case of the collagen {alpha}2(I) gene, an X box has been identified overlapping the collagen {alpha}2(I) transcriptional start site (22). When analyzed by gel shifts, the investigators showed that CIITA, via RFX5, could bind to the collagen {alpha}2(I) X box and possibly hinder RNA polymerase II activity at the collagen {alpha}2(I) promoter (22). With respect to the collagen {alpha}2(I) and IL-4 genes, CIITA was shown to inhibit their expression via an alternative mechanism, that is the sequestration of CBP (16, 17). CBP is a histone acetyltransferase that is required for the regulated expression of many genes (23, 24). CIITA, which can bind CBP, is able to inhibit IL-4 and collagen {alpha}2(I) expression by directly competing for CBP (16, 17).

In this report, we investigated whether CIITA, a transcriptional target of IFN-{gamma}-mediated STAT-1{alpha} activation, was able to suppress MMP-9 expression. Furthermore, because CIITA is capable of binding to CBP, we investigated whether CIITA could inhibit MMP-9 expression through binding to and sequestering the coactivator CBP. Our results indicate that CIITA can inhibit MMP-9 transcription, and this capacity of CIITA requires the ability of CIITA to bind CBP but not its ability to transactivate gene expression. As such, it suggests that the immune system, specifically CIITA, plays an important role in the regulation of the MMP-9 gene.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—The pcDNA3/F-CIITA and pcDNA3/F-{Delta}59–94 expression vectors were generous gifts of Dr. J. P.-Y. Ting, University of North Carolina, Chapel Hill. The pcDNA3/F-{Delta}CT expression vector was created by BamHI restriction digest of pcDNA3/F-CIITA to remove the last 460 nucleotides of F-CIITA. The digested vector was re-ligated to generate pcDNA3/F-{Delta}CT, which expresses a form of CIITA lacking the C-terminal 153 amino acids. To generate 10-3/F-CIITA or 10-3/F-{Delta}59–94, the open reading frames (ORFs) were removed intact from the aforementioned pcDNA3 expression constructs through digestion with EcoRI and XbaI and cloned into the EcoRI and XbaI sites of the 10-3 vector. To generate 10-3/F-{Delta}CT, the ORF was removed intact from the pcDNA3 expression construct through digestion with EcoRI and BamHI and cloned into the EcoRI and BamHI sites of the 10-3 vector. MMP-9-Luc was the generous gift of Dr. D. Boyd, MD Anderson Cancer Center, Houston, TX. The pCMV-5/mCBP-FLAG expression vector was a generous gift of Dr. G. Rosenfeld, University of California, San Diego. The pcDNA3 and pGEM-T vectors were purchased from Promega (Madison, WI). The pUHD10-3, pUH15-1-(neo), and pTRE2hyg-Luc vectors were purchased from Clontech. The pBABE vector was a generous gift of Dr. X. Chen, University of Alabama at Birmingham.

Cell Lines—The STAT-1{alpha}-deficient U3A cell line (25) (the generous gift of Dr. G. Stark, Cleveland Clinic, Cleveland, OH) was maintained in Dulbecco's modified Eagle's medium supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 10 µg/ml streptomycin, and 10% heat-inactivated fetal bovine serum. The U3A tetracycline-inducible cell line UK was generated by transfecting pUH15-1-(neo) into U3A cells with the FuGENE 6 transfection reagent (Roche Applied Science). One day after transfection, cells were selected in 400 µg/ml G418 for 2 weeks. G418-resistant clones were selected and assayed by transient transfection of pTRE2hyg-Luc carrying seven copies of the 19-bp tet operator (tetO) driving expression of the luciferase gene. Clone UK-10 was chosen for further use in these experiments. The FLAG-tagged (F-) CIITA, F-{Delta}59–94, and F-{Delta}CT ORFs were cloned into the pUHD10-3 vector to generate 10-3/F-CIITA, 10-3/F-{Delta}59–94, and F-{Delta}CT. To generate stable inducible cell lines, 5 µg of 10-3/F-CIITA, 10-3/F-{Delta}59–94, or F-{Delta}CT and 0.5 µg of pBABE, which confers puromycin resistance, were transfected into UK-10 cells with the FuGENE 6 transfection reagent. Two days after transfection, cells were selected in 400 µg/ml G418, 2 µg/ml tetracycline (Tet), and 0.5 µg/ml puromycin for 2 weeks. Clones were cultured in medium without Tet for 18 h and assayed for protein expression by immunoblotting with monoclonal antibodies against the FLAG epitope. Several positive clones from each line were expanded and analyzed in this study.

Reagents—Human rTNF-{alpha} was purchased from R & D Systems (Minneapolis, MN). Anti-FLAG mAb, anti-actin mAb, and Tet were purchased from Sigma. The anti-MMP-9 mAb was purchased from Oncogene (San Diego, CA). The anti-PARP Ab was purchased from Pharmingen (San Diego, CA). The secondary peroxidase-conjugated Abs and ECL reagents were purchased from Amersham Biosciences. The anti-caspase 3 Ab and anti-CBP mAb were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-H3 and anti-H4 polyclonal Abs were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Protein A/G beads were purchased from Pierce.

Luciferase Assay—The MMP-9-Luc reporter plasmid containing 670 bp of the human MMP-9 promoter was used in this study (26). Transient transfection was performed using the FuGENE 6 reagent as described previously (8). Cell extracts were assayed in triplicate for luciferase activity as described previously and were normalized to total protein (8). Protein concentrations were measured using the Bio-Rad protein assay. The luciferase activity from the vector control was arbitrarily set at 1 for calculation of fold induction.

Immunoblot Analysis—Transiently transfected cells were washed twice and collected in phosphate-buffered saline (PBS), resuspended in 2x sample buffer, and boiled for 10 min. Where indicated, stably transfected cell lines were cultured for 18–36 h in serum-free media in the absence or presence of Tet (4 µg/ml or as otherwise indicated) and in the absence or presence of TNF-{alpha} (50 ng/ml). Cells were washed twice with PBS and collected in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton-100, 2 mM EDTA, 25 µg/ml aprotinin, 25 µg/ml leupeptin, 1 mM PMSF-20). Protein concentrations were measured using the Bio-Rad protein assay. Equal concentrations of cell extracts were resuspended in 5x sample buffer, boiled for 10 min, and analyzed by SDS-PAGE and immunoblot analysis. To detect protein expression, immunoblots were probed with anti-FLAG mAb (1:2500) to detect F-CIITA, F-{Delta}59–94, and F-{Delta}CT or anti-actin mAb (1:3000) to detect actin. To analyze MMP-9 protein expression, cultured supernatants were collected, concentrated, and quantitated. Equal amounts of concentrated protein were analyzed by SDS-PAGE and immunoblotted with anti-MMP-9 mAb (1:2000) to detect MMP-9 expression.

Fluorescence-activated Cell Sorting Analysis—Cells were seeded at a density of 3 x 105 cells per well in 6-well plates and grown overnight in serum-free media. At 18 h, supernatants were aspirated and cells trypsinized and collected. Cells were washed twice with PBS and stained for class II MHC expression as described previously using fluorescein isothiocyanate-labeled anti-human HLA-DR, -DP, and -DQ (Pharmingen) for 30 min at 4 °C (27). Stained cells were washed one time with PBS and fixed with 2% paraformaldehyde and analyzed within 1 h by flow cytometry using FACScan (BD Biosciences). Flow cytometric data were quantitated using ModFit software from BD Biosciences.

Fluorescent Microscopy—Cells were seeded on 8-well chamber slides and grown overnight in the absence or presence of Tet (2 µg/ml). At 24 h, the slides were washed with PBS and fixed for 15 min in 10% formalin. The slides were washed twice with PBS and blocked in PBS supplemented with 10% BSA for 15 min. Human class II MHC expression was detected using fluorescein isothiocyanate-labeled anti-human HLA-DR, -DP, and -DQ (Pharmingen). Nuclei were visualized by staining with Hoechst.

Nuclear and Cytoplasmic Fractionation and Immunoblot Analysis— Cells were seeded at 3 x 106 cells per 10-cm plate and grown overnight in serum-containing media. At 18 h, cells were cultured in serum-free media for 4 h and then incubated overnight in serum-free media in the absence or presence of Tet (4 µg/ml) and the absence or presence of TNF-{alpha} (50 ng/ml) for 18 h. Cells were collected, and nuclear and cytoplasmic fractions were purified using the NE-PER kit (Pierce) according to the manufacturer's instructions. Twenty µg of protein from each fraction were analyzed by SDS-PAGE and immunoblot analysis. F-CIITA, F-{Delta}59–94, and F-{Delta}CT were detected as described above. To characterize the purity of nuclear and cytoplasmic fractions, all samples were analyzed for the expression of caspase-3, which is restricted to the cytoplasm (28), and poly-ADP-ribose polymerase (PARP) expression, which is restricted to the nucleus (29). To detect caspase-3 protein, membranes were immunoblotted with anti-caspase-3 Ab (1:2000), and for PARP protein, membranes were immunoblotted with anti-PARP mAb (1:2000).

Total RNA Isolation and RNase Protection Assay (RPA)—Total RNA was isolated using TRIzol reagent (Invitrogen) as described previously (8). The probes for human MMP-9 and GAPDH were generated as described previously (8). A pGEM-T vector containing the CIITA ORF was linearized with AvaII and used to generate a radiolabeled antisense RNA probe of 498 nucleotides corresponding to nucleotides 2908–3545 with T7 RNA polymerase. A pGEM-T vector containing the {Delta}CT ORF was linearized by EcoRI and used to generate a radiolabeled antisense RNA probe of 450 nucleotides corresponding to nucleotides 988–1361 with Sp6 RNA polymerase. 10–20 µg of total RNA was hybridized with MMP-9 (50 x 103 cpm), GAPDH (25 x 103 cpm), CIITA (50 x 103 cpm), or {Delta}CT (50 x 103 cpm) riboprobes at 42 °C overnight. The hybridized mixtures were then treated with RNase A/T1 (1:200) at 37 °C for 30 min, precipitated, and analyzed by 5% denaturing (8 M urea) PAGE. The gels were dried and exposed to PhosphorImager cassettes. Quantification of protected RNA fragments was performed using the PhosphorImager (Amersham Biosciences). Values for MMP-9, CIITA, {Delta}59–94, and {Delta}CT mRNA expression were normalized to GAPDH mRNA levels for each experimental condition.

Immunoprecipitation Assay—Lysates of treated cells were prepared as described previously (8). Two hundred micrograms of total protein were incubated with 2 µg of human {alpha}-CBP mAb overnight at 4 °C. Protein A/G-agarose beads were added for 2 h at 4 °C. The immunoprecipitates were washed three times with lysis buffer, eluted from the agarose beads by boiling in 2x SDS sample buffer, and analyzed by SDS-PAGE and immunoblotting for F-CIITA, F-{Delta}59–94, F-{Delta}CT, as described previously, or CBP protein expression using anti-CBP mAb (1:1000).

Chromatin Immunoprecipitation (ChIP) Assay—ChIP assays were performed as described previously (7, 30). Nuclei from cross-linked cells were resuspended in TE buffer and sonicated. The soluble chromatin was adjusted into RIPA buffer (0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, and 140 mM NaCl) and precleared. Immunoprecipitation was performed with 2–5 µg of appropriate antibodies, and the immune complexes were absorbed with protein A beads (Upstate Biotechnology, Inc.) or protein A/G beads (Pierce) blocked with BSA and salmon sperm DNA. Immunoprecipitated DNA was subjected to semiquantitative PCR. The PCR products were resolved in 1.5% agarose gels in 1x TAE buffer, and the gels were stained with ethidium bromide. Densitometry was used to quantify the PCR results, and all results were normalized by respective input values.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
TNF-{alpha} Induces MMP-9 Promoter Activity—The minimal 670-bp MMP-9 promoter contains a number of transcription factor binding sites, including an NF-{kappa}B site, an Sp1 site, two AP-1 sites, and an ETS site (4). Previous studies (31) have shown that TNF-{alpha}, through NF-{kappa}B, is a potent inducer of MMP-9 expression. We used the luciferase reporter assay to measure MMP-9 promoter activity in response to TNF-{alpha}. U3A cells, which are STAT-1{alpha}-deficient (25), were transiently transfected with a vector encoding the luciferase gene under the regulation of the 670-bp region of the MMP-9 promoter (MMP-9-Luc) in the absence or presence of TNF-{alpha}. As shown in Fig. 1A, TNF-{alpha} treatment stimulated MMP-9 promoter activity by 3.5-fold. These data confirm that TNF-{alpha} is able to induce MMP-9 promoter activity in U3A cells.



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  		FIG. 1.
MMP-9 promoter activity is inhibited by CIITA. A, U3A cells were transiently transfected with 200 ng of MMP-9-Luc; cells were incubated in serum-free media in the absence (–) or presence (+) of 50 ng/ml TNF-{alpha} for 18 h. Luciferase activity was determined in triplicate, as described under "Experimental Procedures." Results of at least three experiments are shown as fold induction (mean ± S.E.). B, U3A cells were transiently transfected with 200 ng of MMP-9-Luc and the indicated amounts of F-CIITA (0–800 ng). Empty pcDNA3 vector was used to normalize the amount of DNA transfected per experiment. At 18 h post-transfection, cells were incubated in serum-free media in the absence or presence of 50 ng/ml TNF-{alpha} for 18 h. To calculate % inhibition, the fold induction from the TNF-{alpha}-stimulated versus -unstimulated pcDNA3 control sample was arbitrarily set at 100%. The effect of CIITA is expressed as the percentage of TNF-{alpha}-induced MMP-9 expression. C, U3A cells were transiently transfected with 50 ng of HLA-DR{alpha}-Luc and the indicated amounts of F-CIITA (0–800 ng). Empty pcDNA3 expression vector was used to normalize the amount of DNA transfected per experiment. At 18 h post-transfection, luciferase activity was determined in triplicate, as described under "Experimental Procedures." Results of at least three experiments are shown as fold induction (mean ± S.E.). D, CIITA is a 1,130-amino acid protein organized into four domains: an acidic domain (AD), proline/serine/threonine-rich domain (PST), GTP binding domain (GBD), andleucine-rich region (LRR). The domain responsible for binding CBP (CBP-BD) is located between amino acids 59 and 94 (marked with an asterisk). {Delta}59–94 is a CIITA mutant that lacks the amino acids between 59 and 94. {Delta}CT is a CIITA mutant that lacks the C-terminal 153 amino acids. E, U3A cells were transiently transfected with 200 ng of MMP-9-Luc and the indicated amounts of F-{Delta}59–94 or F-{Delta}CT (0–800 ng) and analyzed as described in B. F, U3A cells were transiently transfected with 50 ng of HLA-DR{alpha}-Luc and the indicated amounts of F-{Delta}59–94 or F-{Delta}CT (0–800 ng) and analyzed as described in C. G, U3A cells were transiently transfected with 1 µg of empty pcDNA3 vector or 1 µg of the pcDNA3 vector encoding F-CIITA, F-{Delta}59–94, or F-{Delta}CT. Total cell lysates were collected and analyzed by immunoblotting as described under "Experimental Procedures." Results of at least three experiments are shown.

 
CIITA Inhibits MMP-9 Promoter Activity—We have recently shown that CBP binding to the MMP-9 promoter is an important aspect in the induction of MMP-9 gene expression (7). Moreover, previous studies have shown that CIITA, through its ability to bind CBP, is able to inhibit the expression of some genes (16, 17). Therefore, we wanted to determine whether CIITA had any effect on MMP-9 promoter activity. U3A cells were transiently transfected with the MMP-9-Luc reporter construct and increasing amounts of an expression vector encoding FLAG-tagged human CIITA (F-CIITA). As shown in Fig. 1B, F-CIITA inhibited TNF-{alpha}-induced MMP-9 promoter activity in a dose-dependent manner, such that 800 ng of F-CIITA was most effective and inhibited MMP-9 promoter activity by >80%. This effect was specific, since we did not see inhibition of the MMP-9 promoter when cells were transfected with increasing amounts of the empty vector (data not shown and Fig. 7, A and B). As a positive control for CIITA activity, reporter assays were performed using a vector encoding the luciferase gene under the regulation of the class II MHC promoter from the HLA-DR{alpha} gene (HLA-DR{alpha}-Luc). F-CIITA is active and able to activate the class II MHC promoter in a dose-dependent manner (Fig. 1C).



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  		FIG. 7.
Overexpression of CBP restores TNF-{alpha}-induced MMP-9 expression and overcomes the inhibitory effects of F-CIITA and F-{Delta}CT. U3A cells were transfected with 200 ng of MMP-9-Luc, 200 ng of F-CIITA (A), or 400 ng of F-{Delta}CT (B) and increasing amounts of a pCMV-5/CBP expression vector (0–800 ng). Total DNA transfected was normalized by using empty pcDNA3 vector. Cells were incubated in the absence (–) or presence (+) of 50 ng/ml TNF-{alpha} for 18 h. Luciferase activity was determined in triplicate, as described under "Experimental Procedures." Results of at least three experiments are shown.

 
The CBP Binding Domain of CIITA Is Important for Inhibition of MMP-9 Promoter Activity—Previous reports have shown that CIITA is able to bind the transcriptional coactivator CBP through a region near its N terminus (CBP binding domain) (Fig. 1D) (32). To determine whether CIITA requires CBP binding in order to inhibit MMP-9, MMP-9 promoter activity was analyzed in the presence of increasing amounts of the CIITA mutant F-{Delta}59–94. F-{Delta}59–94 lacks amino acids 59–94, which comprise the CBP binding domain, and previous reports (16) have shown that the F-{Delta}59–94 mutant is unable to bind CBP or induce class II MHC expression. As shown in Fig. 1E, increasing amounts of F-{Delta}59–94 were not as efficient as F-CIITA at inhibiting MMP-9 promoter activity. In fact, 800 ng of F-{Delta}59–94 was only 50% effective at inhibiting MMP-9 promoter activity compared with the >80% efficacy of F-CIITA (Fig. 1B). Additionally, the F-{Delta}59–94 mutant is unable to activate class II MHC expression (Fig. 1F).

CIITA Does Not Require Its Ability to Activate Gene Expression in Order to Inhibit MMP-9 Promoter Activity—The F-{Delta}59–94 mutant is unable to bind CBP or induce class II MHC expression. To separate these functions and further describe the role of CBP binding in CIITA-mediated inhibition of MMP-9 promoter activity, we generated another CIITA mutant, F-{Delta}CT, that lacks the C-terminal 153 amino acids (Fig. 1D). In particular, F-{Delta}CT retains the N-terminal CBP binding domain but is unable to induce class II MHC expression, since previous reports (33) have shown the C terminus to be indispensable for this function. MMP-9 promoter activity was analyzed in the presence of increasing amounts of F-{Delta}CT. F-{Delta}CT was effective at inhibiting MMP-9 promoter activity, that is 800 ng of F-{Delta}CT inhibited MMP-9 promoter activity by >80% (Fig. 1E), which is comparable with that seen with F-CIITA (Fig. 1B). However, unlike full-length F-CIITA, F-{Delta}CT lacks the ability to activate the class II MHC promoter (Fig. 1F). From these results, we propose that CIITA is able to inhibit MMP-9 promoter activity in a manner dependent upon its ability to bind CBP but independent of its transcriptional ability.

To rule out the possibility that the differences between the effects of F-CIITA, F-{Delta}59–94, and F-{Delta}CT were due to differences in levels of protein expressed, U3A cells transiently transfected with equal amounts of the expression vectors encoding F-CIITA, F-{Delta}59–94, or F-{Delta}CT were analyzed by immunoblot analysis. F-CIITA, F-{Delta}59–94, and F-{Delta}CT were expressed at comparable levels in U3A cells (Fig. 1G).

Generation of Stable Cell Lines That Inducibly Express F-CIITA, F-{Delta}59–94, and F-{Delta}CT—Because the above data showed that CIITA, {Delta}CT, but to a lesser extent {Delta}59–94, inhibited TNF-{alpha}-induced MMP-9 promoter activity, we wanted to confirm these effects on the endogenous MMP-9 promoter, transcript, and protein. However, except for professional antigen-presenting cells, CIITA is not expressed in most cells unless stimulated with IFN-{gamma} (11). Because our previous studies have shown that IFN-{gamma}, via STAT-1{alpha}, is also able to inhibit MMP-9 expression (8), we chose to bypass the need for IFN-{gamma} stimulation by creating stable cell lines that would inducibly express F-CIITA, F-{Delta}59–94, or F-{Delta}CT. Furthermore, to eliminate any potential effects of STAT-1{alpha}, we generated these lines using the STAT-1{alpha}-deficient U3A cells. We used the Tet-off system to generate these lines and chose representative clones from each line to demonstrate that we can regulate the expression of F-CIITA, F-{Delta}59–94, or F-{Delta}CT through the addition (no expression) or removal (expression induced) of Tet (Fig. 2A). Furthermore, we used immunoblot analyses with an antibody that detects both endogenous CIITA and F-CIITA to compare the levels of transgenic F-CIITA expressed in U3A cells with the levels of endogenous CIITA expressed in response to IFN-{gamma} in parental HT1080 cells. From these studies we determined that in the presence of 400 ng/ml Tet, the levels of F-CIITA in U3A cells were comparable with the levels of IFN-{gamma}-induced CIITA in HT1080 cells (data not shown).



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  		FIG. 2.
U3A cells inducibly expressing F-CIITA, but not F-{Delta}59–94 or F-{Delta}CT, regulate class II MHC expression. A, U3A cells were used to generate Tet-inducible lines as described under "Experimental Procedures." Cells were incubated overnight in the presence of 4 µg/ml Tet to shut-off CIITA protein expression (–) or in the absence of Tet to induce CIITA protein expression (+). Total cell lysates were collected at 18 h and analyzed by immunoblotting using the {alpha}-FLAG mAb and {alpha}-actin mAb. B, U3A cells or U3A cells that inducibly express F-CIITA, F-{Delta}59–94, or F-{Delta}CT were incubated overnight in serum-free media in the absence of Tet. Cells were collected, stained for class II MHC expression, and analyzed by FACS as described under "Experimental Procedures." C, data obtained from FACS analysis were quantitated by using ModFit software (BD Biosciences). D, U3A cells or U3A cells that inducibly express F-CIITA, F-{Delta}59–94, or F-{Delta}CT were incubated overnight in serum-free media in the absence of Tet. Cells were prepared for immunofluorescence as described under "Experimental Procedures." E, U3A cells that inducibly expressF-CIITA, F-{Delta}59–94, or F-{Delta}CT were incubated overnight in the presence or absence of 4 µg/ml of Tet to turn off (–) or induce (+) the CIITA protein, respectively. Cells were untreated (–) or treated (+) with 50 ng/ml TNF-{alpha} for 18 h. Nuclear and cytoplasmic fractions were purified using the NE-PER kit from Pierce. 25 µg of protein from each fraction was analyzed by immunoblotting using anti-caspase 3 or anti-PARP antibodies as described under "Experimental Procedures." Results of at least two experiments are shown.

 
U3A Cell Lines That Express F-CIITA Induce Class II MHC Expression—Previous reports have shown that overexpression of CIITA is sufficient to induce class II MHC expression in the absence of IFN-{gamma} stimulation (11); thus we analyzed the stable cell lines for the expression of class II MHC molecules. U3A cells or U3A cells that inducibly expressed either F-CIITA, F-{Delta}59–94, or F-{Delta}CT were grown in the absence of Tet overnight, stained for cell surface class II MHC molecules, and analyzed by fluorescence-activated cell sorting (FACS) analysis. As shown in Fig. 2, B and C, only cells that expressed F-CIITA were positive for class II MHC expression. Consistent with class II MHC luciferase data (Fig. 1F), U3A cells that expressed F-{Delta}59–94 or F-{Delta}CT failed to express class II MHC molecules. To confirm these data, we analyzed U3A cells or U3A cells expressing F-CIITA, F-{Delta}59–94, or F-{Delta}CT for class II MHC expression using immunofluorescence. As shown in Fig. 2D, neither U3A nor U3A cells expressing F-{Delta}59–94 or F-{Delta}CT expressed class II MHC molecules, whereas U3A cells with F-CIITA expressed class II MHC molecules.

The above data indicate that F-{Delta}59–94 and F-{Delta}CT are unable to regulate class II MHC expression. To rule out the possibility that F-{Delta}59–94 and F-{Delta}CT are unable to function correctly perhaps due to mislocalization, we analyzed the subcellular expression patterns of F-CIITA, F-{Delta}59–94, and F-{Delta}CT. U3A cells inducibly expressing F-CIITA, F-{Delta}59–94, or F-{Delta}CT were grown in the absence or presence of Tet and in the absence or presence of TNF-{alpha}. We included TNF-{alpha} in this experiment to determine whether this stimulus, which induces MMP-9 expression, had any effect on F-CIITA, F-{Delta}59–94, or F-{Delta}CT expression or localization. The samples were collected, fractionated to obtain nuclear and cytoplasmic extracts, and analyzed for protein expression by immunoblot analyses. As shown in Fig. 2E, F-CIITA expression is regulated by Tet and is expressed both in the nucleus and cytoplasm (lanes 2 and 6). Most interestingly, the levels of nuclear F-CIITA modestly increased when treated with TNF-{alpha} (Fig. 2E, lane 4), and cytoplasmic levels decreased accordingly (lane 8). F-{Delta}59–94 and F-{Delta}CT were also expressed in the nucleus (Fig. 2E, lane 2), but each displayed diminished expression in the cytoplasm (lane 6). The purity of the nuclear and cytoplasmic fractions was confirmed by the detection of PARP, which is only expressed in the nucleus (Fig. 2E, lanes 1–4) or caspase-3, which is only expressed in the cytoplasm (lanes 5–8).

F-CIITA and F-{Delta}CT, but Not F-{Delta}59–94, Inhibit the Expression of Endogenous MMP-9 Protein—To determine whether CIITA was able to inhibit endogenous MMP-9 protein expression, stable cells inducibly expressing F-CIITA were analyzed by immunoblotting. As shown in Fig. 3A (upper panel), the levels of CIITA are regulated by Tet and are not affected by the addition of TNF-{alpha} (compare lanes 2 and 4). Furthermore, the absence (–) or presence (+) of F-CIITA alone had no effect on MMP-9 expression (Fig. 3A, middle panel, lanes 1 and 2). TNF-{alpha} induced the expression of MMP-9 protein by 2.7-fold (Fig. 3A, middle panel, lane 3). However, in the presence of F-CIITA, TNF-{alpha} failed to induce the expression of MMP-9 protein (Fig. 3A, middle panel, lane 4), indicating that F-CIITA inhibits the expression of MMP-9 protein.



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  		FIG. 3.
F-CIITA requires its CBP binding domain but not its transcriptional capacity to inhibit endogenous MMP-9 protein. U3A cells that inducibly express F-CIITA (A), F-{Delta}59–94 (B), or F-{Delta}CT (C) were incubated for 18 h in serum-free media in the absence or presence of Tet and in the absence (–) or presence (+) of 50 ng/ml TNF-{alpha}. At 36 h, supernatants were harvested and analyzed by immunoblotting using anti-MMP-9 mAb. Total cell lysates from identical experiments were analyzed by immunoblotting using anti-FLAG mAb to detect F-CIITA, F-{Delta}59–94 or F-{Delta}CT, or anti-actin mAb to detect actin. Fold induction was measured by using densitometry and is represented as fold over MMP-9 levels in the absence of F-CIITA, F-{Delta}59–94, F-{Delta}CT, or TNF-{alpha}. U3A cells that inducibly express F-CIITA (D) or F-{Delta}CT (E) were incubated for 18 h in serum-free media in the presence of 4 µg/ml Tet (lanes A and 1) or absence of Tet (lane B) or in the presence of 400 (lane 2), 40 (lane 3), and 4 ng/ml (lane 4), 400 (lane 5), 40 (lane 6), and 4 pg/ml (lane 7), and 400 (lane 8), 40 (lane 9), or 4 fg/ml (lane 10) Tet, and in the absence (lanes A and B) or presence (lanes 1–10) of 50 ng/ml TNF-{alpha}. At 36 h, supernatants and total cell lysates were harvested and analyzed by immunoblotting as described in A. Results of at least three experiments are shown.

 
We next analyzed stable cells that inducibly expressed F-{Delta}59–94 to determine the importance of the CBP binding domain in CIITA-mediated MMP-9 inhibition. As shown in Fig. 3B (upper panel), the levels of F-{Delta}59–94 are regulated by Tet and are not affected by the addition of TNF-{alpha} (compare lanes 2 and 4). Furthermore, F-{Delta}59–94 did not induce MMP-9 protein in the absence of TNF-{alpha} treatment (Fig. 3B, middle panel, lanes 1 and 2). TNF-{alpha} increased the expression of MMP-9 protein (5.8-fold), and the presence of F-{Delta}59–94 had little effect on the ability of TNF-{alpha} to induce expression of the MMP-9 protein (Fig. 3B, middle panel, lanes 3 and 4), indicating that F-{Delta}59–94 is unable to inhibit the expression of endogenous MMP-9 protein. This suggests the CIITA requires the CBP binding domain to inhibit MMP-9 expression.

U3A cells that inducibly express F-{Delta}CT were analyzed next. F-{Delta}CT was able to inhibit TNF-{alpha}-induced MMP-9 protein expression by ~60% (Fig. 3C, middle panel, lanes 3 and 4). These data indicate that CIITA-mediated inhibition of MMP-9 requires the CBP binding domain but not its ability to activate gene expression.

We next determined whether F-CIITA and F-{Delta}CT were able to inhibit MMP-9 protein expression in a dose-dependent manner. To do this, stable cells that inducibly expressed F-CIITA or F-{Delta}CT were grown in the presence or absence of 4 µg/ml Tet (Fig. 3, D and E, lanes A and B) or in the presence of decreasing concentrations of Tet, that is, from 4 µg/ml to 4 fg/ml (Fig. 3, D and E, lanes 1–10). Cells were then left unstimulated (Fig. 3, D and E, lanes A and B) or stimulated with TNF-{alpha} (lanes 1–10) to induce MMP-9 expression. In the presence of 4 µg/ml Tet, F-CIITA and F-{Delta}CT expression is completely undetectable (Fig. 3, D and E, upper panels, lanes A and 1) and maximally expressed in the absence of any Tet (upper panels, lanes B). However, in the presence of 400 ng/ml Tet, F-CIITA and F-{Delta}CT expression is detectable and steadily increases with decreasing amounts of Tet until maximal levels are achieved with 4 ng/ml Tet (Fig. 3, D and E, upper panels, lanes 2–10). In the absence of F-CIITA or F-{Delta}CT expression, TNF-{alpha} induces MMP-9 expression in both lines (Fig. 3, D and E, middle panels, lane 1). However, with the onset of F-CIITA or F-{Delta}CT expression, the levels of MMP-9 are decreased (Fig. 3, D and E, middle panels, lanes 2–10). Moreover, as the levels of F-CIITA or F-{Delta}CT continue to increase, the levels of MMP-9 continue to decrease until each is approximately one-third its maximal expression level (Fig. 3, D and E, middle panels, compare lanes 1 and 10).

To confirm these data, we analyzed Fig. 3, D and E, lanes 1–10, of the immunoblots by using densitometric analyses and normalized the levels to actin expression. The results of these analyses are presented to the right. As can be seen, an inverse relationship exists between F-CIITA or F-{Delta}CT and MMP-9, such that increased levels of F-CIITA and F-{Delta}CT correlate well with decreased levels of MMP-9 protein. These data suggest that both F-CIITA and F-{Delta}CT are able to inhibit MMP-9 expression in a dose-dependent manner.

F-CIITA and F-{Delta}CT Inhibit the Expression of Endogenous MMP-9 mRNA—Thus far, our data have shown that F-CIITA and F-{Delta}CT are able to inhibit MMP-9 promoter activity and endogenous MMP-9 protein expression. To determine whether F-CIITA or F-{Delta}CT are able to inhibit endogenous MMP-9 mRNA expression, we analyzed samples using RPA. Consistent with our previous data, we show that the absence or presence of F-CIITA or F-{Delta}CT alone had no effect on MMP-9 mRNA expression (Fig. 4, A and B, middle panels, compare lanes 1 and 2). Moreover, as shown in Fig. 4, A and B, TNF-{alpha}-induced MMP-9 expression was unaffected in the absence of F-CIITA or F-{Delta}CT (middle panels, lane 3). However, TNF-{alpha} was unable to induce MMP-9 mRNA expression in cells that expressed F-CIITA or F-{Delta}CT (Fig. 4, A and B, lane 4). Expression of F-{Delta}59–94 had no effect on TNF-{alpha}-induced MMP-9 mRNA expression (data not shown).



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  		FIG. 4.
F-CIITA requires its CBP binding domain but not its ability to induce gene expression to inhibit endogenous MMP-9 RNA. U3A cells that inducibly express F-CIITA (A) or F-{Delta}CT (B) were incubated for 18 h in serum-free media in the absence or presence of 4 µg/ml Tet and in the absence (–) or presence (+) of 50 ng/ml TNF-{alpha}. Total RNA was isolated and analyzed by RPA for CIITA, {Delta}CT, MMP-9, and GAPDH expression. Quantification was performed by densitometry, and fold change is represented as fold over MMP-9 mRNA levels in the absence of F-CIITA, F-{Delta}CT, or TNF-{alpha}. Results of at least three experiments are shown.

 
To determine whether the effects of F-CIITA and F-{Delta}CT were because of defective TNF-{alpha} signaling or deregulated MMP expression, we used RPA to analyze the expression of other TNF-{alpha}-inducible genes such as IL-8 and RANTES, or the expression of other MMPs such as MMP-1, -2, -7, -8, -12, and -13. The expression of these genes was not altered by coexpression of F-CIITA or F-{Delta}CT (data not shown), suggesting that the effects of F-CIITA and F-{Delta}CT are specific to MMP-9.

F-CIITA and F-{Delta}CT, but Not F-{Delta}59–94, Bind to CBP—Our data suggest that CIITA is able to inhibit specifically MMP-9 expression in a manner dependent on CBP binding but independent of its ability to activate gene expression. To determine whether F-CIITA, F-{Delta}59–94, or F-{Delta}CT were able to bind CBP in vivo, we analyzed samples using coimmunoprecipitation and immunoblotting. Cells that inducibly expressed F-CIITA, F-{Delta}59–94, or F-{Delta}CT were grown in the absence or presence of Tet and the absence or presence of TNF-{alpha}. Cell lysates were prepared, and equal amounts of protein were immunoprecipitated with an antibody specific for CBP and analyzed by immunoblotting for CIITA expression. As shown in Fig. 5A, F-CIITA (lane 2) and F-{Delta}CT (lane 10), but not F-{Delta}59–94 (lane 6), were detectable, indicating that they had coimmunoprecipitated with CBP and hence were bound to CBP. When immunoprecipitation was performed with an isotype control antibody, no F-CIITA, F-{Delta}59–94, or F-{Delta}CT was detected (data not shown). Furthermore, treatment with TNF-{alpha} did not affect the binding interaction (Fig. 5A, upper panels, compare lanes 2 and 4 and lanes 10 and 12). We also analyzed all samples for expression of their respective proteins or CBP. As shown in the Fig. 5A, lower panel, F-CIITA, F-{Delta}59–94, and F-{Delta}CT were expressed under the appropriate conditions (lanes 2, 4, 6, 8, 10, and 12). Additionally, U3A cells express the CBP protein, and treatment with Tet or TNF-{alpha} did not affect the levels of CBP (Fig. 5B). Therefore, we conclude that F-CIITA and F-{Delta}CT, but not F-{Delta}59–94, are able to bind to CBP.



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  		FIG. 5.
F-CIITA and F-{Delta}CT, but not F-{Delta}59–94, bind CBP. U3A cells that inducibly express F-CIITA, F-{Delta}59–94, or F-{Delta}CT were incubated in serum-free media in the presence of Tet to inhibit expression (–), in the absence of Tet to induce expression (+), and in the absence (–) or presence (+) of 50 ng/ml TNF-{alpha}. 200 µg of total protein were immunoprecipitated (IP) with monoclonal antibodies to human CBP and analyzed by immunoblotting (W) using the anti-FLAG mAb (A, upper panel). 20 µg of total protein was analyzed by immunoblotting using the anti-FLAG mAb (A, lower panel). U3A cells were incubated in serum-free media in the absence or presence of Tet and in the absence (–) or presence (+) of 50 ng/ml TNF-{alpha}. 20 µg of total protein was analyzed by immunoblotting using the anti-CBP mAb (B).

 
F-CIITA and F-{Delta}CT Diminish the Presence of CBP and Reduce the Levels of Acetylated Histone 3 at the MMP-9 Promoter—Thus far, our data have shown that F-CIITA and F-{Delta}CT, but not F-{Delta}59–94, are capable of binding to CBP and inhibiting MMP-9 expression. To correlate CBP binding with the inhibition of MMP-9, we analyzed the in vivo effects on the MMP-9 promoter using ChIP assays. We have shown recently (7) that initiation of MMP-9 gene transcription is associated with recruitment of CBP to the MMP-9 promoter and acetylation of H3 and H4 histones (Ac-H3 and Ac-H4, respectively). As shown in Fig. 6A, in the absence of TNF-{alpha} stimulation, F-CIITA had no effect on CBP recruitment or the levels of Ac-H3 and Ac-H4 at the MMP-9 promoter (Fig. 6A, lanes 1 and 2). When stimulated with TNF-{alpha}, the levels of CBP increased 5.4- (Fig. 6A, lane 3) and 5.1-fold (lane 5) and correlated with increased amounts of Ac-H3 at the MMP-9 promoter (3.2-fold, lane 3, and 3.1-fold, lane 5). To analyze the effects of CIITA at the MMP-9 promoter, TNF-{alpha}-stimulated cells were grown in 400 ng/ml Tet to induce minimal F-CIITA expression (see Fig. 3D). Under these conditions, we saw a decrease in the levels of CBP at the MMP-9 promoter to only 1.9-fold over unstimulated and Ac-H3 at the MMP-9 promoter to only 1.1-fold over unstimulated (lane 4). Similarly, when cells were treated with TNF-{alpha} under conditions when F-{Delta}CT was minimally expressed, we saw a decrease in the levels of CBP at the MMP-9 promoter to only 1.4-fold over unstimulated and Ac-H3 at the MMP-9 promoter to only 1.1-fold over unstimulated (Fig. 6A, lane 6). Most interestingly, the levels of Ac-H4 were only modestly increased by TNF-{alpha} stimulation (Fig. 6A, lanes 3 and 5) and were not greatly affected by expression of either F-CIITA or F-{Delta}CT (lanes 4 and 6). As a control, we analyzed equivalent amounts of DNA collected prior to immunoprecipitation. As shown in Fig. 6A, bottom panel, there were no significant variations between the samples. Quantification of the data are shown in Fig. 6B.



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  		FIG. 6.
F-CIITA and F-{Delta}CT reduce the levels of CBP and Ac-H3 at the MMP-9 promoter. A, U3A cells that inducibly express F-CIITA or F-{Delta}CT were incubated in serum-free media overnight in the presence of 4 µg/ml Tet (+) to turn off protein expression or 400 ng/ml Tet (–) to induce F-CIITA or F-{Delta}CT expression. Cells were untreated (–) or treated (+) with 50 ng/ml TNF-{alpha} for 2 h and analyzed by ChIP as described under "Experimental Procedures." Prior to immunoprecipitation (IP), equal amounts of sonicated DNA were analyzed by PCR. Quantification was performed by densitometry, and fold difference is represented as fold over levels in the absence of F-CIITA, F-{Delta}CT, or TNF-{alpha} (A). The mean ± S.D. of the experiment shown in A, and one other is shown in B. C, U3A cells that inducibly express F-CIITA were incubated in serum-free media overnight in the presence of 4 µg/ml Tet (+) to turn off protein expression, or in the absence of Tet (–) to induce maximal F-CIITA expression. Cells were untreated (–) or treated (+) with 50 ng/ml TNF-{alpha} for 2 h and analyzed by ChIP as described in A.

 
Additional experiments were performed in which cells were grown in the absence of Tet, which results in maximal levels of F-CIITA (Fig. 3D). As shown in Fig. 6C, in the absence of TNF-{alpha}, F-CIITA had no effect on the recruitment of CBP or the levels of Ac-H3 at the MMP-9 promoter (lanes 1 and 2). In the presence of TNF-{alpha}, the levels of CBP increased 5.7-fold and Ac-H3 increased by 3.5-fold as well (Fig. 6C, lane 3). With maximal induction of F-CIITA, CBP recruitment was substantially inhibited, as were Ac-H3 levels (Fig. 6C, lane 4).

In similar experiments using cell lines that inducibly expressed F-{Delta}59–94, we failed to see F-{Delta}59–94 reduce the levels of CBP or Ac-H3 at the MMP-9 promoter (data not shown). Together, these data suggest that CIITA and {Delta}CT are able to inhibit MMP-9 expression by binding to CBP, preventing its requisite use at the MMP-9 promoter, and inhibiting Ac-H3 levels at the MMP-9 promoter.

F-CIITA- and F-{Delta}CT-mediated Inhibition of MMP-9 Promoter Activity Is Reversed by Overexpression of CBP—To confirm our hypothesis that F-CIITA and F-{Delta}CT inhibit MMP-9 expression by binding to and sequestering CBP, we analyzed whether the overexpression of CBP would restore MMP-9 promoter activity. U3A cells were transfected with the MMP-9-Luc reporter construct, F-CIITA or F-{Delta}CT, and increasing amounts of a CBP expression vector. It should be noted that we used 400 ng of F-{Delta}CT compared with 200 ng of F-CIITA so that the inhibition of MMP-9 by both proteins would be comparable (see Fig. 1, B and E). As shown in Fig. 7, A and B, in the absence of CBP, F-CIITA or F-{Delta}CT substantially reduced TNF-{alpha}-induced MMP-9 promoter activity. However, in the presence of increasing amounts of CBP protein, the inhibitory effects of F-CIITA or F-{Delta}CT were diminished. In fact, MMP-9 promoter activity was either fully restored when CBP was expressed in a 4-fold excess over F-CIITA levels (Fig. 7A), or almost fully restored when CBP was expressed in a 2-fold excess over F-{Delta}CT levels (Fig. 7B). When cells were transfected with CBP alone and grown in the absence or presence of TNF-{alpha}, MMP-9 promoter activity was slightly increased when compared with their respective control transfectants (data not shown). Together, these data demonstrate that overexpression of CBP is able to restore MMP-9 expression and overcome the inhibitory effects of F-CIITA or F-{Delta}CT at the MMP-9 promoter.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here we demonstrate that CIITA is capable of inhibiting MMP-9 expression. In our previous work, we showed that IFN-{gamma}, through STAT-1{alpha}, was able to inhibit MMP-9 expression (8). Because the MMP-9 promoter lacks any consensus GAS or IFN-stimulated response elements, this suggested that STAT-1{alpha} was able to inhibit MMP-9 by perhaps inducing unfavorable changes in the chromatin status at the MMP-9 promoter and/or that STAT-1{alpha} was acting indirectly through the induction of a second gene, which in turn was responsible for MMP-9 inhibition. Although we have not yet addressed whether these possibilities are mutually exclusive, we do provide data that show CIITA, a STAT-1{alpha} regulated gene, is capable of inhibiting MMP-9 expression.

CIITA is the master regulator of class II MHC expression. CIITA induces class II MHC expression by interacting with proteins bound to the consensus elements within class II MHC promoters, that is the RFX and NF-Y heterotrimers and the CREB-binding protein, which are bound to the W, X, and Y boxes (11). Recently, studies have shown that CIITA is also capable of inhibiting the expression of some genes including IL-4, Fas-L, collagen {alpha}2(I), cathepsin E, and some thyroid-specific genes (15–21). In particular, CIITA is capable of specifically inhibiting IL-4 and Fas-L expression by binding to CBP, a transcriptional coactivator, and preventing its requisite use by transcription factors that induce IL-4 and Fas-L expression (17, 18). Because CIITA is regulated by STAT-1{alpha} (13), and because CBP has been shown to bind to CIITA as well as NF-{kappa}B and AP-1 (34–37), which regulate MMP-9 expression (4), we wanted to determine whether CIITA was able to inhibit MMP-9 expression by using a similar mechanism.

To measure the effects of CIITA on MMP-9 promoter activity, we used the luciferase reporter assay. In these experiments, we showed that full-length CIITA, which is able to bind CBP and induce class II MHC expression, is able to inhibit TNF-{alpha}-induced MMP-9 promoter activity in a dose-dependent manner. To discriminate between the ability of CIITA to bind CBP and its ability to induce class II MHC expression, we used two CIITA mutants, {Delta}59–94 and {Delta}CT. As shown in previous reports, the {Delta}59–94 CIITA mutant lacks the ability to bind CBP and induce class II MHC expression (16). Indeed, by using luciferase reporter assays, we determined that {Delta}59–94 was unable to induce the class II MHC promoter and appeared to be less efficient than CIITA at inhibiting MMP-9 promoter activity. In contrast, the {Delta}CT mutant, which lacks the ability to activate the class II MHC promoter, but retains the ability to bind CBP, was as effective as CIITA at inhibiting the MMP-9 promoter. As such, these data suggested that CIITA was able to inhibit the MMP-9 promoter and that this function was dependent upon the ability of CIITA to bind CBP but not its transcriptional ability.

To elaborate on these findings, we generated stable cell lines in the U3A cells that would inducibly express F-CIITA, F-{Delta}59–94, or F-{Delta}CT. By using these lines, we analyzed samples by immunoblotting and RPAs, and we determined that F-CIITA and F-{Delta}CT were effective at inhibiting TNF-{alpha}-induced endogenous MMP-9 mRNA and protein expression. However, cell lines that expressed F-{Delta}59–94 were unable to inhibit endogenous MMP-9 expression, underscoring the importance of the ability of CIITA to bind to CBP for the inhibition of MMP-9. Indeed, when analyzed by coimmunoprecipitation and immunoblotting, we determined that F-CIITA and F-{Delta}CT, but not F-{Delta}59–94, were able to bind to CBP.

The in vivo effects of F-CIITA or F-{Delta}CT on the MMP-9 promoter were analyzed by using the ChIP assay. Previously, we have shown (7) that the MMP-9 promoter is organized into discrete nucleosomes that undergo critical and necessary remodeling events during PMA-induced activation of the MMP-9 gene. In this study, we used TNF-{alpha}, which activates the transcription factor NF-{kappa}B and is also a potent inducer of MMP-9 expression (4). Because NF-{kappa}B and AP-1, which are both required for MMP-9 expression (4), are capable of binding to CBP, we hypothesized that their stimuli-induced presence at the MMP-9 promoter would correlate with CBP recruitment and increased levels of Ac-H3 and Ac-H4. Indeed, in the absence of F-CIITA or F-{Delta}CT, we saw a TNF-{alpha}-induced increase in the levels of CBP recruited to the MMP-9 promoter and an increase in the levels of Ac-H3 at the MMP-9 promoter. This is not surprising, since in the absence of transcription, DNA is often condensed into chromatin through electrostatic interactions between the negatively charged DNA and positively charged histones (38–40). However, to initiate transcription from a particular locale, histones are often acetylated in order to reduce their net charge and disrupt DNA-protein interactions (38–40). Therefore, our data are consistent with the premise that TNF-{alpha} stimulation induces histone acetylation at the MMP-9 promoter. However, upon expression of either F-CIITA or F-{Delta}CT, we saw a decrease in the amount of CBP present at the MMP-9 promoter and diminished levels of Ac-H3, suggesting that when F-CIITA or F-{Delta}CT is expressed, the MMP-9 promoter demonstrates less histone acetylation and may be less accessible to the general transcription machinery. This is consistent with reports that associate un-acetylated histones with silenced promoters or inactive transcription (39, 40), as well as our own data that show decreased MMP-9 mRNA and protein under these conditions.

Because our data suggest that CIITA is able to inhibit MMP-9 by binding to CBP and sequestering it away from transcription factors that activate the MMP-9 promoter, we wanted to determine whether the overexpression of CBP could reverse the inhibitory effects of CIITA or {Delta}CT and restore MMP-9 expression. We determined that the inhibitory effects of F-CIITA or F-{Delta}CT could be overcome through the addition of exogenous CBP, indicating that CIITA mediates inhibition of MMP-9 through the sequestration of CBP.

How is CIITA able to inhibit specifically the expression of MMP-9, IL-4, Fas-L, and collagen {alpha}2(I) but not the expression of other genes? By using RPA analyses, we showed that CIITA inhibited the expression of TNF-{alpha}-induced MMP-9 but did not affect the expression of TNF-{alpha}-induced IL-8 or RANTES or MMP-1, -2, -7, -11, -12, or -13 gene expression. Moreover, we showed that CIITA required its CBP binding domain but not its transcriptional capabilities to inhibit MMP-9 gene expression. As such, these data suggest that CIITA inhibits MMP-9 expression through sequestration of CBP. However, CBP interacts with numerous transcription factors that regulate the expression of some of the genes unaffected by CIITA expression. Given this, such a simplistic model does not address why interactions between CIITA and CBP fail to inhibit the expression of other genes. Specifically, how do CIITA-CBP interactions inhibit MMP-9 expression without affecting the expression of IL-8, RANTES, or other MMPs? Unfortunately, at present we are unable to the answer this question but can propose several theories. One possibility is that these genes (IL-8, RANTES, other MMPs) may differ with respect to how dependent they are on CBP at their respective promoters. In our previous study, we showed that in the absence of any stimuli, the MMP-9 promoter is occupied by the Sin3A/HDAC-1 and NcoR/HDAC-3 corepressor complexes, which are removed from the MMP-9 promoter within 4 h of stimulation with PMA (7). Concurrent with the exit of these corepressor complexes from the MMP-9 promoter, additional factors such as Brg-1 and Brm, two ATPase subunits of the SWI/SNF chromatin remodeling complex, CBP, JunD, c-Fos, p65, p50, and Sp1 are recruited to the MMP-9 promoter (7). These events coincide with maximal relaxation of chromatin structure and histone acetylation at the MMP-9 promoter and expression of MMP-9 mRNA and demonstrate that activation of the MMP-9 gene is dependent upon the simultaneous release of corepressors and the recruitment of activators and coactivators (7). Therefore, the interactions between CIITA and CBP may have a more pronounced effect on genes that require CBP for their expression compared with those genes whose expression is less influenced by the absence or presence of CBP. In this regard, we have recently determined that MMP-2 gene expression is critically regulated by recruitment of Brg-1 to the MMP-2 promoter, whereas CBP recruitment is modest at best.2

Alternatively, because a single molecule of CBP is unlikely to bind to two transcription factors through the same domain simultaneously, it is possible that CIITA inhibits gene expression by binding to a region of CBP that is utilized by the transcription factors that regulate expression of the other genes. Therefore, with CIITA bound to CBP, CIITA may prevent transcription factors from binding to the same domain of CBP while simultaneously sequestering CBP to CIITA-regulated promoters. Indeed, in previous studies (17, 18), CIITA was shown to inhibit the expression of both IL-4 and Fas-L, two genes that are regulated by the transcription factor NFAT. Moreover, both CIITA and NFAT bind to the CH3 domain of CBP (32, 41–43). Although we have not identified the transcription factor(s) affected by CIITA-CBP interactions, several possibilities exist. The MMP-9 promoter contains binding sites for NF-{kappa}B, Sp1, and AP-1, and all three transcription factors participate in both TNF-{alpha} and PMA-induced MMP-9 expression (4). The NF-{kappa}B family member p65 and the AP-1 family members c-Jun and c-Fos have each been shown to bind CBP (34–37), and our own recent data (7) have shown that activation of the MMP-9 gene requires the coordinated efforts of cell signaling, chromatin remodeling, histone modifications, and recruitment of transcription factors. Therefore, we propose that CIITA inhibits MMP-9 expression by binding to CBP, which reduces the levels of CBP at the MMP-9 promoter and prevents transcription factors and/or transcriptional machinery from accessing the MMP-9 promoter.

What is the significance of CIITA-mediated inhibition of MMP-9? CIITA is the master regulator of class II MHC expression and an important participant in the cell-mediated immune response (11). The absence of CIITA expression causes a severe immunodeficient condition known as Bare Lymphocyte syndrome, wherein class II MHC molecules are absent (11). These findings underscore the contributions of the transcriptional capabilities of CIITA. However, recent data and our own suggest that the contributions of CIITA during the immune response may be more complex and may serve to fine-tune the immune response. For example, CIITA inhibits the expression of IL-4, an inducer and effector of TH2 cells, which are involved in the innate immune response (17, 19, 20). Because CIITA inhibits IL-4 production, CIITA may shift the immune response from a TH2 profile to a TH1 profile (19). Additionally, CIITA inhibits the expression of Fas-L, which may prevent differentiating TH1 cells from undergoing premature Fas-L-induced apoptosis prior to their activation (18). Here, we present data to show that CIITA can affect the expression of MMP-9, a gene that is overexpressed in several disease states (4). In particular, we showed that when expressed within the same cell, CIITA was capable of binding to CBP and preventing its use at the MMP-9 promoter, thus attenuating MMP-9 expression. Perhaps in the absence of CIITA, which occurs in most cells, MMP-9 expression is elevated in certain disease states. However, upon CIITA induction by IFN-{gamma}, CIITA down-regulates MMP-9 expression. Therefore, in diseases with elevated MMP-9 levels, a triggered induction of CIITA may reduce MMP-9 expression.

We have presented here the identification of a new role for CIITA, the master regulator of class II MHC expression, in the inhibition of MMP-9 expression. Although the exact underlying mechanism and biological significance remain to be elucidated, we hope that these studies provide the opportunity for novel therapies involving the use of one's own immune system in treating diseases with excessive ECM degradation.


   FOOTNOTES
 
* This work was supported in part by United States Public Service NCI Grant CA-97247 and NINDS Grant NS-36765 from the National Institutes of Health (to E. N. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported by National Institutes of Health Postdoctoral Fellowship T32 AI-07493. Back

§ To whom correspondence should be addressed: Office of the Chair, Dept. of Cell Biology, University of Alabama at Birmingham, 1530 3rd Ave. S., MCLM 395, Birmingham, AL 35294-0005. Tel.: 205-934-7667; Fax: 205-975-6748; E-mail: tika{at}uab.edu.

1 The abbreviations used are: MMP, matrix metalloproteinase; ECM, extracellular matrix; IFN-{gamma}, interferon-{gamma}; STAT-1{alpha}, signal transducer and activator of transcription-1{alpha}; CBP, CREB-binding protein; H3, histone 3; H4, histone 4; GAS, {gamma}-activated sequence; CIITA, class II MHC transactivator; IL, interleukin; Fas-L, Fas ligand; ORF, open reading frame; Tet, tetracycline; PBS, phosphate-buffered saline; Ab, antibody; MHC, major histocompatibility complex; PARP, poly(ADP-ribose) polymerase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Luc, luciferase; FACS, fluorescence-activated cell sorting; ChIP, chromatin immunoprecipitation; Ac, acetylated; mAb, monoclonal antibody; RANTES, regulated on activation normal T cell expressed and secreted; PMA, phorbol 12-myristate 13-acetate; TNF, tumor necrosis factor; RPA, RNase protection assay. Back

2 S. Nozell, Z. Ma, C. Wilson, R. Shah, and E. N. Benveniste, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Jill Adamski and George O'Keefe for technical assistance.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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