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Tissue transglutaminase-induced down-regulation of matrix metalloproteinase-9
Biochemical and Biophysical Research Communications 376 (2008) 743–747
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Tissue transglutaminase-induced down-regulation of matrix metalloproteinase-9 Jun-Seok Ahn a, Min-kyung Kim a, Jang-hee Hahn a, Jung-Hyeun Park a, Kyeong-Han Park a, Byung-Ryul Cho b, Seung-Bae Park c, Dae-joong Kim a,* a
Department of Anatomy and Cell Biology, Kangwon National University, College of Medicine, Hyoja 2 Dong, Chunchon 200-701, Republic of Korea Department of Internal Medicine, Kangwon National University College of Medicine, Chunchon 200-701, Republic of Korea c Department of Surgery, Kangwon National University College of Medicine, Chunchon 200-701, Republic of Korea b
a r t i c l e
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Article history: Received 4 September 2008 Available online 20 September 2008
Keywords: Tissue transglutaminase MMP-9 Transcription AP-1
a b s t r a c t Tissue transglutaminase (TGase 2) has been reported to have multiple functions in addition to its function as a biological adhesive. To identify its roles, we investigated the effects of TGase 2 on gelatinase activity. The MMP-9 activity of certain cell lines was significantly inhibited with retinoic acid treatment, and this effect was reversed in the presence of a TGase 2 inhibitor. Furthermore, TGase 2 overexpression reduced the MMP-9 protein expression levels and inhibited its activity in both culture media and cell lysate. The decreased mRNA levels of MMP-9 and the results of a promoter assay revealed that TGase 2 may be involved in MMP-9 transcription. Further, data obtained in an immunoprecipitation assay and an electrophoretic mobility shift assay demonstrated that TGase 2 binds to c-Jun and suppresses its binding activity toward AP-1. These results suggest that TGase 2 inhibits MMP-9 via downregulation of MMP-9 transcription activity by blocking the binding of the Jun–fos complex to an AP-1 site. Ó 2008 Elsevier Inc. All rights reserved.
The interactions between the extracellular matrix (ECM) macromolecules and cells are important for supporting such cellular activities as growth and development. Thus, it is natural to say that the state of the macromolecules within the ECM is of critical importance. The process of proteolysis and cross-linking is a major factor leading to changes in the ECM; proteolysis can affect the cellular adherence to the ECM, and also release bioactive fragments, sequestered growth factors, and cytokines [1]. Matrix metalloproteinases (MMPs) are proteinases that participate in ECM degradation. MMPs are a family of zinc-dependent endopeptidases that play important roles in physiological and pathological conditions. Under normal physiological conditions, the activity of MMPs is precisely regulated at the levels of transcription, activation of the precursor zymogens, interaction with specific ECM components, and inhibition by endogenous inhibitors [2,3]. A loss of activity control may result in diseases such as arthritis, cancer, atherosclerosis, nephritis, tissue ulcers, and fibrosis [2,4]. Several studies have connected metalloproteinase, particularly gelatinases (gelatinase A, MMP-2, and gelatinase B, MMP-9), to tumor angiogenesis and growth [5,6], as well as acute coronary syndrome in the heart [7,8].
* Corresponding author. Fax: +82 33 241 8808. E-mail address: [email protected] (D. Kim), 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.09.048
Tissue transglutaminase (TGase 2) is another regulator in the ECM network. TGase 2 belongs to a family of Ca2+-dependent enzymes, and it catalyzes the post-translational modification of proteins. Recently, many new functions of TGase 2 have been reported, and accumulating evidence shows that TGase 2 expression increases with inflammation-based diseases, such as ischemia [9], celiac diseases [10], skin diseases [11], and conjunctivitis [12]. In particular, TGase 2 has been shown to cross-link components of the ECM macromolecules, including fibronectin, vitronectin, laminin, and collagen [13–16]. It is localized mainly in the cytoplasm; however, a substantial amount of the TGase 2 protein is also found in the nucleus, plasma membrane, and in the extracellular matrix [17]. Furthermore, numerous recent reports point to the role of TGase 2 in cell–matrix interactions [18]. It has been reported that the surface TGase 2 amplifies integrin-mediated signaling to RhoA/ Rock via integrin clustering and down-regulation of Src-p190RhoGAP regulatory pathway [19]. These findings suggest that TGase 2 released from the inside of the cell may function as an important ECM regulator. However, the functions of TGase 2 in each cellular compartment are not yet clearly defined. The purpose of this study was to evaluate the role of TGase 2 in the nuclear compartment, by evaluating the possible relationship between TGase 2 and MMP-9. Our data demonstrate that TGase 2 may regulate MMP-9 expression by interrupting the transcription factor binding at the AP-1 site.
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J.-S. Ahn et al. / Biochemical and Biophysical Research Communications 376 (2008) 743–747
Materials and methods Reagents and antibody. The protein A bead was obtained from Pharmacia, the dual luciferase reporter assay system was from Turner Designs, and the monodansyl cadaverine and RA were purchased from Sigma. The anti-TGase 2 antibody was obtained from Neomarkers; and the anti-MMP-9 antibody was from Oncogene. Zymogram assay. The gelatinolytic activity of the conditioned medium was detected through gelatin zymography. Serum-free media conditioned for 24 h (40 ll) were subjected to SDS–PAGE using 7.5% acrylamide gels containing 0.1% gelatin. The gels were then incubated twice, each for 30 min at room temperature in 2.5% Triton X-100, before being transferred to a reaction buffer (50 mM Tris–Cl, pH 8.0, 10 mM CaCl2) for overnight incubation at 37 °C. The gels were then stained with Coomassie Brilliant Blue R-250 and briefly destained in 10% acetic acid and 40% methanol. Gelatinolytic activity was detected as transparent bands on a blue background. Transfection of TGase 2 DNA construct. Human TGase 2 DNA (4– 5 lg) in pcDNA3-Neo (Invitrogen) was transfected to H9c2 cells by using CytoPure-huv according to manufacturer’s instructions (Qbiogene, Inc., Irvine, CA). DNA mixed with the CytoPure-huv reagent was incubated for 15 min at room temperature. Thereafter, the DNA–Cytopure reagent complexes were added to the cells. After 6 h of incubation, the complete culture medium was added to the cell culture. One day after transfection, G418 (Sigma) was added to the culture medium at 300 lg/ml, and the cultures were kept in this medium for 4 weeks. Clones of cells derived from these transfections that survived the G418 selection were subcultured as single clones. After expansion and subculture, the cell lines derived were assayed for the presence of TGase 2 by Western blotting. RT-PCR. Expression of mRNA for MMP-9 in H9c2 was examined using a reverse transcriptase-polymerase chain reaction (RT-RCR). Total cellular RNA was extracted from H9c2, through a modified version of the method provided with the Tri-reagent kit. Extracted RNA was quantified by spectrophotometry, and the A260/A280 ratio of extracted RNA was routinely in the range 1.7–1.8. Agarose gel electrophoresis of extracted RNA confirmed the integrity of the RNA samples. Total cellular RNA was reverse-transcribed using a first-strand cDNA synthesis kit, according to the manufacturer’s instructions. The resulting cDNA was amplified using the Taq kit, using a previously reported primer for MMP-9. The primer sequences were as follows: Rat MMP-9 Forward primer: 50 -AAG GAT GGT CTA CTG GCA C-30 Reverse primer: 50 -AGA GAT TCT CAC TGG GGC-30 Promoter assay and transfection of small interfering RNA (siRNA). In order to investigate the role of TGase 2 on MMP-9 activation in the TGase 2-overexpressed H9c2 cells, we performed a promoter assay using pNF-jB-TA-Luc (BD Biosciences, San Jose, CA, USA) containing the NF-jB-binding site. A transient transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The luciferase GL2 duplex that was used as a negative control product was also provided by Dharmacon Research. After 48 h of incubation with the complete medium, the transfected cells were used in the experiments. The cells were harvested at 48 h post-transfection and washed once with PBS, and the extracts were prepared using a 1 passive lysis buffer (Promega, Madison, WI, USA). The extracts were normalized for protein content and assayed for luciferase activity by using the dual luciferase reporter assay system (Promega) and a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA, USA). A TGase 2 siRNA duplex was designed to target the coding sequences of the human TGase 2 mRNA and was synthesized by Dharmacon
Research (Lafayette, CO). The target sequence of TGase 2 siRNA is 50 -AA GAGCGAGAUGAUCUGGAAC-30 . Immunoprecipitation. Cells were harvested and then washed twice with 10 ml PBS. Cells from one dish were lysed with 1 ml IP buffer (150 mM NaCl, 5 mM EDTA, 1% NP-40, 50 mM Tris–HCl (pH 7.5), and 0.5 mM DTT) containing the following inhibitors: 10 lg/ml, leupeptin, 0.5 mM phenylmethlysulfonyl fluoride (PMSF), 0.5 mM pepstatin A, and 0.5 mM aprotinin. Cells were incubated on ice for 30 min and then centrifuged for 20 min at 13,000 rpm. The supernatant was transferred to fresh tubes, to which was added 1 lg/ml of anti c-Jun polyclonal antibody. Lysate was rotated for 18 h (4 °C), and then 40 ll of 70% slurry protein A beads (Pharmacia) was added. The slurry was rotated for 3 h (4 °C) and then washed five times with 1 ml PBS containing no inhibitors. Bound molecules were eluted by boiling the beads in 5% SDS and resolved by 7.5% SDS–PAGE, under non-reduction conditions. Analysis of AP-1-c-Jun binding activity. Electrophoretic gel mobility shift assay (EMSA) was performed for the measurement of AP1-c-Jun binding activity using 32P-labelled double stranded AP-1 specific oligonucleotide and nuclear extracts. For supershift and competitive assays, nuclear extracts were pre-incubated with 2 lg of monoclonal mouse antibodies to the TGase 2 subunit or an excess (100-fold) of cold probe at room temperature for 30 min. Results TGase 2 inhibits MMP-9 activity Quiescent U937 cells were treated with phorbol 12-myristate 13-acetate (PMA), because these cells secreted gelatinase at a very low level. PMA-activated U937 cells in a conditioned medium were treated with various doses (0, 1, 2.5, and 5 lM) of retinoic acid (RA), a well-known activator TGase 2, for 24 h; culture media was then collected for zymography. As shown in Fig. 1A, MMP-9 activity in the soup from the RA-treated cells was dependently reduced in the dose. To evaluate the effect of TGase 2 on MMP-9 activity in the cells that secrete more TGase 2 protein, zymography was performed using human breast carcinoma cells (MCF-7) and rat cardiomyoblast (H9c2), respectively. MMP-9 activity was also reduced in the RA-treated cells (Fig. 1A). To confirm the MMP-9 inhibitory effect of TGase 2, RA-treated U937 cells were treated with the TGase 2 inhibitor monodansyl cadaverine (MDC). Fig. 1B shows that inhibited MMP-9 activity was significantly reversed by the TGase 2 inhibitor. Because RA itself has been reported to be an MMP-9 inhibitor [20], we measured the activity using rat cardiomyoblast (H9c2)-carrying empty vector and a full-length human TGase 2 in pcDNA3 vector (H9c2/TGase 2). The activity of MMP-9 was also significantly reduced in the H9c2/TGase 2 cells (Fig. 1C). Interestingly, MMP-2 activity was also reduced in the H9c2/TGase 2. These results suggest that TGase 2 may be able to act as a regulator in the process of MMP-9 activation. TGase 2 reduces MMP-9 protein in the culture media Gelatinases are expressed and secreted as inactive proenzymes. Expression is regulated by growth factors, cytokines, the cell–matrix, and cell–cell interactions through signal transduction pathways [21]. To investigate whether the reduced activity of MMP-9 was due to the interruption during the process of proenzyme modulation, we performed a Western blot analysis on the culture media. As shown in Fig. 2(A), TGase 2 overexpression appeared to reduce the MMP-9 protein content in the culture media. In addition, TGase 2 overexpression resulted in the reduction of MMP-9 protein level in the whole cell lysate (data not shown), this effect
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9 activity by TGase 2 may be result from the down-regulation during MMP-9 protein expression. Following the immunoblotting, RT-PCR examination was performed to investigate whether the reduction of MMP-9 activity in the H9c2/TGase 2 cells results from the inhibition in MMP-9 mRNA expression. Fig. 2B showed that MMP-9 mRNA was decreased by the TGase 2 protein overexpression (H9c2/TGase 2). This data suggests that TGase 2 may suppress MMP-9 mRNA expression. TGase 2 reduces the transcription of MMP-9 gene through interrupting AP-1 site
Fig. 1. TGase 2 inhibits MMP-9. (A) Retinoic acid (RA)-treated cell lines were incubated in serum-free DMEM in the absence or presence of PMA (20 nM). (B) Gelatinase activity was inhibited in TGase 2-overexpressed cardiomyoblast (H9c2/ TGase 2). (C) MMP-9 activity was recovered by MDC (monodansyl cadaverine; 500 lM, 24 h), TGase 2 selective inhibitor, in U937 cells. Conditioned media from cells were analyzed for MMP-9 expression by gelatin substrate zymography. RA, retinoic acid; PMA, phorbol 12-myristate 13-acetate; MDC, monodansyl cadaverine.
Fig. 2. TGase 2 overexpression decreased MMP-9 protein in the soup, and TGase 2 suppresses transcription of MMP-9 gene. TGase 2-overexpressed H9c2 cells were incubated with or without MDC (2 mM). After 24 h, equal volumes of conditioned media (A) were collected and analyzed for MMP-9 protein content by Western blot analysis. (B) Expression levels of MMP-9 mRNA were analyzed in the H9c2/pcDNA3 and H9c2/TGase 2 by Northern blot analysis. Total mRNA was extracted from samples of conditioned medium, and analyzed for MMP-9 mRNA expression. GAPDH bands were detected as loading controls.
was also reversed by MDC. The inhibitory effect of MDC on the TGase 2 was confirmed by Western blot analysis of the phophoERK 1/2. These results suggest that the inhibitory effect of MMP-
In order to examine transcriptional control as a potential mechanism for the TGase 2-mediated suppression of MMP-9 mRNA levels, a MMP-9 promoter construct was evaluated following transient transfection. In H9c2/TGase 2 cells, MMP-9 promotermediated luciferase activity was decreased approximately 4- or 5-fold, compared to control cells (Fig. 3A). This effect was reversed with TGase 2 siRNA transfection. This result suggests that TGase 2 may suppress the induction of MMP-9 transcription; this inhibition also supports the RT-PCR data described above. To extend this finding further, we examined the inhibitory effect of TGase 2 using a series of AP-1 site deletion mutant MMP-9 promoter constructs. The MMP-9 promoter has several transcription factor-binding motifs, including two AP-1 binding sites [22]. The deletion of a 50 AP-1 site mutant construct amplified the inhibitory effect of TGase 2 more significantly than the wildtype promoter (Fig. 3B). These results prompt us to suggest that the inhibitory effect of TGase 2 may be due to the down-regulation of transcription through the AP-1 site.
Fig. 3. Luciferase assay of MMP-9 in H9c2 cells. TGase 2 overexpression induced about 4- or 5-fold decrease in MMP-9 promoter-mediated luciferase activity in H9c2 cells transfected transiently with the MMP-9 promoter construct. This effect was reversed by TGase 2 siRNA treatment (A). Luciferase activity was generally decreased in the assay using the deletion of a 50 AP-1 site mutant MMP-9 construct (B). *p < 0.05.
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To investigate whether TGase 2 is involved in the binding process between the AP-1 site and c-Jun or c-fos, the immunoprecipitation was performed with H9c2 cell lysate (nuclear fraction) using a mouse monoclonal anti-c-Jun antibody. The result showed that TGase 2 was immunoprecipitated with c-Jun, and the interaction of c-Jun with c-fos was significantly reduced in the H9c2/ TGase 2 cells (Fig. 4A). To further investigate the role of TGase 2 on the AP-1 interaction, we performed EMSA using a radiolabeled double stranded AP-1 specific oligonucleotide and nuclear extracts. White a moderate level of AP-1 binding was observed in H9c2/ pcDNA3 cells, AP-1 activation and binding were decreased markedly in nuclear extracts from H9c2/TGase 2 cells (Fig. 4B). Taken together, these results suggest that TGase 2 may suppress the transcription of MMP-9 by interfering with AP-1 site activation. Discussion The focus of this study was on evaluating the mechanism of MMP-9 inhibition effect by TGase 2. In this study, we demonstrate that TGase 2 downregulates MMP-9 mRNA, protein, and activity levels in various cells. When PMA-activated U937, H9c2, and MCF-7 cells were treated with 1–5 lM of retinoic acid (RA) for 24 h, significant decrease in the MMP-9 activity was observed. In
Fig. 4. TGase 2 inhibit AP-1 binding and activation. (A) TGase 2 immunoprecipitated with the c-Jun. Immunoprecipitation was performed with anti-c-Jun antibody using nuclear extract both from H9c2/pcDNA3 and H9c2/TGase 2 cells. (B) AP-1 binding was reduced by TGase 2 protein overexpression. EMSA was performed with oligomers containing AP-1 binding site and nuclear extract from H9c2/pcDNA3 and H9c2/TGase 2 cells. For supershift and competitive assays, nuclear extracts were pre-incubated with 2 lg of monoclonal mouse antibodies to the TGase 2 subunit or an excess (100-fold) of cold probe at room temperature for 30 min.
addition, TGase 2 protein overexpression resulted in reduction of MMP-9 protein level as well as gelatinase activity both in the culture media and cell lysate. These results show that the downregulation of MMP-9 activity by TGase 2 may result from the reduction of MMP-9 protein expression. Our Northern blot analysis showed MMP-9 mRNA level was reduced in the H9c2/TGase 2 cell. Depending on the multi-localization and a wide range of TGase 2 substrates, TGase 2 present also in the nucleus [23–25] as well as in cytosol. In TGase 2-overexpressed cells, we have also observed an enhanced TGase 2 activity, and increased nuclear localization of TGase 2 protein (data not shown). In the nucleus, TGase 2 has been reported to be involved in the histone cross-linking [26], and modulate the expression of a variety of transcription factors, including NF-jB and Sp1 [27,28]. Thus, our Northern blot data suggests that TGase 2-induced change in MMP-9 protein expression may be regulated by the transcriptional control. In addition, our data show that TGase 2 inhibited significantly MMP-9 promoter activity (Fig. 3), suggesting also transcriptional regulation. The MMP genes are regulated by the transcription factors AP-1 and NF-jB [22]. Recent study showed that the AP-1 site at 79 bp was functionally dominant compared to the AP-1 site at 533 bp [29] in human squamous carcinoma cells. In our data, amplified inhibitory effect of TGase 2 in the 50 AP-1 site deletion mutant construct suggest that the inhibitory effect of TGase 2 may be due to the downregulation of transcription through the AP-1 site. Our immunoprecipitation data support the implication of TGase 2 on the AP-1 site activation. This data show that TGase 2 can interact with c-Jun, and the Jun–fos complex formation appeared to be reduced in the TGase 2-overexpressed H9c2 cells. Collectively, these results suggest that TGase 2 inhibits MMP-9 gene expression at the transcriptional level through AP-1 site interruption. However, it remains to be elucidated how TGase 2 inhibits MMP-9 transcription. TGase 2 has been reported to regulate expression of a variety of transcription factors such as NF-jB [27] and Sp1 [28]. Thereby, TGase 2 modulates transcription of a multiple genes, indirectly. The role of TGase 2 on the modulation of transcription is supported by our EMSA result showing that AP-1 activation and binding was decreased by overexpressed TGase 2 protein. Han and Park (2000) demonstrated that purified TGase 2 can enhance the binding activity of Sp1 to the target DNA sequence, and they suggest that this effect was due to the ability of TGase 2 to cross-link Sp1. According to our result, however, we suggest another mechanism of TGase 2 on modulating transcription. Our results from EMSA and immunoprecipitation data showed that TGase 2 interact with c-Jun, and inhibit a dimer formation of c-Jun with Fos. Thereby, TGase 2 plays indirectly as a transcription modulator. As we performed this study, it reported that enhanced crosslinking of ECM by tissue transglutaminase and decreased degradation due to reduced active MMP-9 expression may be at least partially responsible for the deposition of fibronectin in the injured glomeruli [30]. However, they did not show why MMP-9 activity was reduced. This report may provide the mechanism leading to the TGase 2-induced alteration of gelatinase activity. In summary, this report shows that TGase 2 can reduce the transcription process by interrupting the binding of the Jun–fos complex to an AP-1 site. These findings point to the new roles of TGase 2 in the nuclear compartment. Acknowledgments This work was supported by Vascular System Research Center in Kangwon National University. We thank Dr. Kwon Soo Hah for a kind gift of TGase 2 siRNA.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Tissue transglutaminase-induced down-regulation of matrix metalloproteinase-9 Jun-Seok Ahn a, Min-kyung Kim a, Jang-hee Hahn a, Jung-Hyeun Park a, Kyeong-Han Park a, Byung-Ryul Cho b, Seung-Bae Park c, Dae-joong Kim a,* a
Department of Anatomy and Cell Biology, Kangwon National University, College of Medicine, Hyoja 2 Dong, Chunchon 200-701, Republic of Korea Department of Internal Medicine, Kangwon National University College of Medicine, Chunchon 200-701, Republic of Korea c Department of Surgery, Kangwon National University College of Medicine, Chunchon 200-701, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 4 September 2008 Available online 20 September 2008
Keywords: Tissue transglutaminase MMP-9 Transcription AP-1
a b s t r a c t Tissue transglutaminase (TGase 2) has been reported to have multiple functions in addition to its function as a biological adhesive. To identify its roles, we investigated the effects of TGase 2 on gelatinase activity. The MMP-9 activity of certain cell lines was significantly inhibited with retinoic acid treatment, and this effect was reversed in the presence of a TGase 2 inhibitor. Furthermore, TGase 2 overexpression reduced the MMP-9 protein expression levels and inhibited its activity in both culture media and cell lysate. The decreased mRNA levels of MMP-9 and the results of a promoter assay revealed that TGase 2 may be involved in MMP-9 transcription. Further, data obtained in an immunoprecipitation assay and an electrophoretic mobility shift assay demonstrated that TGase 2 binds to c-Jun and suppresses its binding activity toward AP-1. These results suggest that TGase 2 inhibits MMP-9 via downregulation of MMP-9 transcription activity by blocking the binding of the Jun–fos complex to an AP-1 site. Ó 2008 Elsevier Inc. All rights reserved.
The interactions between the extracellular matrix (ECM) macromolecules and cells are important for supporting such cellular activities as growth and development. Thus, it is natural to say that the state of the macromolecules within the ECM is of critical importance. The process of proteolysis and cross-linking is a major factor leading to changes in the ECM; proteolysis can affect the cellular adherence to the ECM, and also release bioactive fragments, sequestered growth factors, and cytokines [1]. Matrix metalloproteinases (MMPs) are proteinases that participate in ECM degradation. MMPs are a family of zinc-dependent endopeptidases that play important roles in physiological and pathological conditions. Under normal physiological conditions, the activity of MMPs is precisely regulated at the levels of transcription, activation of the precursor zymogens, interaction with specific ECM components, and inhibition by endogenous inhibitors [2,3]. A loss of activity control may result in diseases such as arthritis, cancer, atherosclerosis, nephritis, tissue ulcers, and fibrosis [2,4]. Several studies have connected metalloproteinase, particularly gelatinases (gelatinase A, MMP-2, and gelatinase B, MMP-9), to tumor angiogenesis and growth [5,6], as well as acute coronary syndrome in the heart [7,8].
* Corresponding author. Fax: +82 33 241 8808. E-mail address: [email protected] (D. Kim), 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.09.048
Tissue transglutaminase (TGase 2) is another regulator in the ECM network. TGase 2 belongs to a family of Ca2+-dependent enzymes, and it catalyzes the post-translational modification of proteins. Recently, many new functions of TGase 2 have been reported, and accumulating evidence shows that TGase 2 expression increases with inflammation-based diseases, such as ischemia [9], celiac diseases [10], skin diseases [11], and conjunctivitis [12]. In particular, TGase 2 has been shown to cross-link components of the ECM macromolecules, including fibronectin, vitronectin, laminin, and collagen [13–16]. It is localized mainly in the cytoplasm; however, a substantial amount of the TGase 2 protein is also found in the nucleus, plasma membrane, and in the extracellular matrix [17]. Furthermore, numerous recent reports point to the role of TGase 2 in cell–matrix interactions [18]. It has been reported that the surface TGase 2 amplifies integrin-mediated signaling to RhoA/ Rock via integrin clustering and down-regulation of Src-p190RhoGAP regulatory pathway [19]. These findings suggest that TGase 2 released from the inside of the cell may function as an important ECM regulator. However, the functions of TGase 2 in each cellular compartment are not yet clearly defined. The purpose of this study was to evaluate the role of TGase 2 in the nuclear compartment, by evaluating the possible relationship between TGase 2 and MMP-9. Our data demonstrate that TGase 2 may regulate MMP-9 expression by interrupting the transcription factor binding at the AP-1 site.
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Materials and methods Reagents and antibody. The protein A bead was obtained from Pharmacia, the dual luciferase reporter assay system was from Turner Designs, and the monodansyl cadaverine and RA were purchased from Sigma. The anti-TGase 2 antibody was obtained from Neomarkers; and the anti-MMP-9 antibody was from Oncogene. Zymogram assay. The gelatinolytic activity of the conditioned medium was detected through gelatin zymography. Serum-free media conditioned for 24 h (40 ll) were subjected to SDS–PAGE using 7.5% acrylamide gels containing 0.1% gelatin. The gels were then incubated twice, each for 30 min at room temperature in 2.5% Triton X-100, before being transferred to a reaction buffer (50 mM Tris–Cl, pH 8.0, 10 mM CaCl2) for overnight incubation at 37 °C. The gels were then stained with Coomassie Brilliant Blue R-250 and briefly destained in 10% acetic acid and 40% methanol. Gelatinolytic activity was detected as transparent bands on a blue background. Transfection of TGase 2 DNA construct. Human TGase 2 DNA (4– 5 lg) in pcDNA3-Neo (Invitrogen) was transfected to H9c2 cells by using CytoPure-huv according to manufacturer’s instructions (Qbiogene, Inc., Irvine, CA). DNA mixed with the CytoPure-huv reagent was incubated for 15 min at room temperature. Thereafter, the DNA–Cytopure reagent complexes were added to the cells. After 6 h of incubation, the complete culture medium was added to the cell culture. One day after transfection, G418 (Sigma) was added to the culture medium at 300 lg/ml, and the cultures were kept in this medium for 4 weeks. Clones of cells derived from these transfections that survived the G418 selection were subcultured as single clones. After expansion and subculture, the cell lines derived were assayed for the presence of TGase 2 by Western blotting. RT-PCR. Expression of mRNA for MMP-9 in H9c2 was examined using a reverse transcriptase-polymerase chain reaction (RT-RCR). Total cellular RNA was extracted from H9c2, through a modified version of the method provided with the Tri-reagent kit. Extracted RNA was quantified by spectrophotometry, and the A260/A280 ratio of extracted RNA was routinely in the range 1.7–1.8. Agarose gel electrophoresis of extracted RNA confirmed the integrity of the RNA samples. Total cellular RNA was reverse-transcribed using a first-strand cDNA synthesis kit, according to the manufacturer’s instructions. The resulting cDNA was amplified using the Taq kit, using a previously reported primer for MMP-9. The primer sequences were as follows: Rat MMP-9 Forward primer: 50 -AAG GAT GGT CTA CTG GCA C-30 Reverse primer: 50 -AGA GAT TCT CAC TGG GGC-30 Promoter assay and transfection of small interfering RNA (siRNA). In order to investigate the role of TGase 2 on MMP-9 activation in the TGase 2-overexpressed H9c2 cells, we performed a promoter assay using pNF-jB-TA-Luc (BD Biosciences, San Jose, CA, USA) containing the NF-jB-binding site. A transient transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The luciferase GL2 duplex that was used as a negative control product was also provided by Dharmacon Research. After 48 h of incubation with the complete medium, the transfected cells were used in the experiments. The cells were harvested at 48 h post-transfection and washed once with PBS, and the extracts were prepared using a 1 passive lysis buffer (Promega, Madison, WI, USA). The extracts were normalized for protein content and assayed for luciferase activity by using the dual luciferase reporter assay system (Promega) and a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA, USA). A TGase 2 siRNA duplex was designed to target the coding sequences of the human TGase 2 mRNA and was synthesized by Dharmacon
Research (Lafayette, CO). The target sequence of TGase 2 siRNA is 50 -AA GAGCGAGAUGAUCUGGAAC-30 . Immunoprecipitation. Cells were harvested and then washed twice with 10 ml PBS. Cells from one dish were lysed with 1 ml IP buffer (150 mM NaCl, 5 mM EDTA, 1% NP-40, 50 mM Tris–HCl (pH 7.5), and 0.5 mM DTT) containing the following inhibitors: 10 lg/ml, leupeptin, 0.5 mM phenylmethlysulfonyl fluoride (PMSF), 0.5 mM pepstatin A, and 0.5 mM aprotinin. Cells were incubated on ice for 30 min and then centrifuged for 20 min at 13,000 rpm. The supernatant was transferred to fresh tubes, to which was added 1 lg/ml of anti c-Jun polyclonal antibody. Lysate was rotated for 18 h (4 °C), and then 40 ll of 70% slurry protein A beads (Pharmacia) was added. The slurry was rotated for 3 h (4 °C) and then washed five times with 1 ml PBS containing no inhibitors. Bound molecules were eluted by boiling the beads in 5% SDS and resolved by 7.5% SDS–PAGE, under non-reduction conditions. Analysis of AP-1-c-Jun binding activity. Electrophoretic gel mobility shift assay (EMSA) was performed for the measurement of AP1-c-Jun binding activity using 32P-labelled double stranded AP-1 specific oligonucleotide and nuclear extracts. For supershift and competitive assays, nuclear extracts were pre-incubated with 2 lg of monoclonal mouse antibodies to the TGase 2 subunit or an excess (100-fold) of cold probe at room temperature for 30 min. Results TGase 2 inhibits MMP-9 activity Quiescent U937 cells were treated with phorbol 12-myristate 13-acetate (PMA), because these cells secreted gelatinase at a very low level. PMA-activated U937 cells in a conditioned medium were treated with various doses (0, 1, 2.5, and 5 lM) of retinoic acid (RA), a well-known activator TGase 2, for 24 h; culture media was then collected for zymography. As shown in Fig. 1A, MMP-9 activity in the soup from the RA-treated cells was dependently reduced in the dose. To evaluate the effect of TGase 2 on MMP-9 activity in the cells that secrete more TGase 2 protein, zymography was performed using human breast carcinoma cells (MCF-7) and rat cardiomyoblast (H9c2), respectively. MMP-9 activity was also reduced in the RA-treated cells (Fig. 1A). To confirm the MMP-9 inhibitory effect of TGase 2, RA-treated U937 cells were treated with the TGase 2 inhibitor monodansyl cadaverine (MDC). Fig. 1B shows that inhibited MMP-9 activity was significantly reversed by the TGase 2 inhibitor. Because RA itself has been reported to be an MMP-9 inhibitor [20], we measured the activity using rat cardiomyoblast (H9c2)-carrying empty vector and a full-length human TGase 2 in pcDNA3 vector (H9c2/TGase 2). The activity of MMP-9 was also significantly reduced in the H9c2/TGase 2 cells (Fig. 1C). Interestingly, MMP-2 activity was also reduced in the H9c2/TGase 2. These results suggest that TGase 2 may be able to act as a regulator in the process of MMP-9 activation. TGase 2 reduces MMP-9 protein in the culture media Gelatinases are expressed and secreted as inactive proenzymes. Expression is regulated by growth factors, cytokines, the cell–matrix, and cell–cell interactions through signal transduction pathways [21]. To investigate whether the reduced activity of MMP-9 was due to the interruption during the process of proenzyme modulation, we performed a Western blot analysis on the culture media. As shown in Fig. 2(A), TGase 2 overexpression appeared to reduce the MMP-9 protein content in the culture media. In addition, TGase 2 overexpression resulted in the reduction of MMP-9 protein level in the whole cell lysate (data not shown), this effect
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9 activity by TGase 2 may be result from the down-regulation during MMP-9 protein expression. Following the immunoblotting, RT-PCR examination was performed to investigate whether the reduction of MMP-9 activity in the H9c2/TGase 2 cells results from the inhibition in MMP-9 mRNA expression. Fig. 2B showed that MMP-9 mRNA was decreased by the TGase 2 protein overexpression (H9c2/TGase 2). This data suggests that TGase 2 may suppress MMP-9 mRNA expression. TGase 2 reduces the transcription of MMP-9 gene through interrupting AP-1 site
Fig. 1. TGase 2 inhibits MMP-9. (A) Retinoic acid (RA)-treated cell lines were incubated in serum-free DMEM in the absence or presence of PMA (20 nM). (B) Gelatinase activity was inhibited in TGase 2-overexpressed cardiomyoblast (H9c2/ TGase 2). (C) MMP-9 activity was recovered by MDC (monodansyl cadaverine; 500 lM, 24 h), TGase 2 selective inhibitor, in U937 cells. Conditioned media from cells were analyzed for MMP-9 expression by gelatin substrate zymography. RA, retinoic acid; PMA, phorbol 12-myristate 13-acetate; MDC, monodansyl cadaverine.
Fig. 2. TGase 2 overexpression decreased MMP-9 protein in the soup, and TGase 2 suppresses transcription of MMP-9 gene. TGase 2-overexpressed H9c2 cells were incubated with or without MDC (2 mM). After 24 h, equal volumes of conditioned media (A) were collected and analyzed for MMP-9 protein content by Western blot analysis. (B) Expression levels of MMP-9 mRNA were analyzed in the H9c2/pcDNA3 and H9c2/TGase 2 by Northern blot analysis. Total mRNA was extracted from samples of conditioned medium, and analyzed for MMP-9 mRNA expression. GAPDH bands were detected as loading controls.
was also reversed by MDC. The inhibitory effect of MDC on the TGase 2 was confirmed by Western blot analysis of the phophoERK 1/2. These results suggest that the inhibitory effect of MMP-
In order to examine transcriptional control as a potential mechanism for the TGase 2-mediated suppression of MMP-9 mRNA levels, a MMP-9 promoter construct was evaluated following transient transfection. In H9c2/TGase 2 cells, MMP-9 promotermediated luciferase activity was decreased approximately 4- or 5-fold, compared to control cells (Fig. 3A). This effect was reversed with TGase 2 siRNA transfection. This result suggests that TGase 2 may suppress the induction of MMP-9 transcription; this inhibition also supports the RT-PCR data described above. To extend this finding further, we examined the inhibitory effect of TGase 2 using a series of AP-1 site deletion mutant MMP-9 promoter constructs. The MMP-9 promoter has several transcription factor-binding motifs, including two AP-1 binding sites [22]. The deletion of a 50 AP-1 site mutant construct amplified the inhibitory effect of TGase 2 more significantly than the wildtype promoter (Fig. 3B). These results prompt us to suggest that the inhibitory effect of TGase 2 may be due to the down-regulation of transcription through the AP-1 site.
Fig. 3. Luciferase assay of MMP-9 in H9c2 cells. TGase 2 overexpression induced about 4- or 5-fold decrease in MMP-9 promoter-mediated luciferase activity in H9c2 cells transfected transiently with the MMP-9 promoter construct. This effect was reversed by TGase 2 siRNA treatment (A). Luciferase activity was generally decreased in the assay using the deletion of a 50 AP-1 site mutant MMP-9 construct (B). *p < 0.05.
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To investigate whether TGase 2 is involved in the binding process between the AP-1 site and c-Jun or c-fos, the immunoprecipitation was performed with H9c2 cell lysate (nuclear fraction) using a mouse monoclonal anti-c-Jun antibody. The result showed that TGase 2 was immunoprecipitated with c-Jun, and the interaction of c-Jun with c-fos was significantly reduced in the H9c2/ TGase 2 cells (Fig. 4A). To further investigate the role of TGase 2 on the AP-1 interaction, we performed EMSA using a radiolabeled double stranded AP-1 specific oligonucleotide and nuclear extracts. White a moderate level of AP-1 binding was observed in H9c2/ pcDNA3 cells, AP-1 activation and binding were decreased markedly in nuclear extracts from H9c2/TGase 2 cells (Fig. 4B). Taken together, these results suggest that TGase 2 may suppress the transcription of MMP-9 by interfering with AP-1 site activation. Discussion The focus of this study was on evaluating the mechanism of MMP-9 inhibition effect by TGase 2. In this study, we demonstrate that TGase 2 downregulates MMP-9 mRNA, protein, and activity levels in various cells. When PMA-activated U937, H9c2, and MCF-7 cells were treated with 1–5 lM of retinoic acid (RA) for 24 h, significant decrease in the MMP-9 activity was observed. In
Fig. 4. TGase 2 inhibit AP-1 binding and activation. (A) TGase 2 immunoprecipitated with the c-Jun. Immunoprecipitation was performed with anti-c-Jun antibody using nuclear extract both from H9c2/pcDNA3 and H9c2/TGase 2 cells. (B) AP-1 binding was reduced by TGase 2 protein overexpression. EMSA was performed with oligomers containing AP-1 binding site and nuclear extract from H9c2/pcDNA3 and H9c2/TGase 2 cells. For supershift and competitive assays, nuclear extracts were pre-incubated with 2 lg of monoclonal mouse antibodies to the TGase 2 subunit or an excess (100-fold) of cold probe at room temperature for 30 min.
addition, TGase 2 protein overexpression resulted in reduction of MMP-9 protein level as well as gelatinase activity both in the culture media and cell lysate. These results show that the downregulation of MMP-9 activity by TGase 2 may result from the reduction of MMP-9 protein expression. Our Northern blot analysis showed MMP-9 mRNA level was reduced in the H9c2/TGase 2 cell. Depending on the multi-localization and a wide range of TGase 2 substrates, TGase 2 present also in the nucleus [23–25] as well as in cytosol. In TGase 2-overexpressed cells, we have also observed an enhanced TGase 2 activity, and increased nuclear localization of TGase 2 protein (data not shown). In the nucleus, TGase 2 has been reported to be involved in the histone cross-linking [26], and modulate the expression of a variety of transcription factors, including NF-jB and Sp1 [27,28]. Thus, our Northern blot data suggests that TGase 2-induced change in MMP-9 protein expression may be regulated by the transcriptional control. In addition, our data show that TGase 2 inhibited significantly MMP-9 promoter activity (Fig. 3), suggesting also transcriptional regulation. The MMP genes are regulated by the transcription factors AP-1 and NF-jB [22]. Recent study showed that the AP-1 site at 79 bp was functionally dominant compared to the AP-1 site at 533 bp [29] in human squamous carcinoma cells. In our data, amplified inhibitory effect of TGase 2 in the 50 AP-1 site deletion mutant construct suggest that the inhibitory effect of TGase 2 may be due to the downregulation of transcription through the AP-1 site. Our immunoprecipitation data support the implication of TGase 2 on the AP-1 site activation. This data show that TGase 2 can interact with c-Jun, and the Jun–fos complex formation appeared to be reduced in the TGase 2-overexpressed H9c2 cells. Collectively, these results suggest that TGase 2 inhibits MMP-9 gene expression at the transcriptional level through AP-1 site interruption. However, it remains to be elucidated how TGase 2 inhibits MMP-9 transcription. TGase 2 has been reported to regulate expression of a variety of transcription factors such as NF-jB [27] and Sp1 [28]. Thereby, TGase 2 modulates transcription of a multiple genes, indirectly. The role of TGase 2 on the modulation of transcription is supported by our EMSA result showing that AP-1 activation and binding was decreased by overexpressed TGase 2 protein. Han and Park (2000) demonstrated that purified TGase 2 can enhance the binding activity of Sp1 to the target DNA sequence, and they suggest that this effect was due to the ability of TGase 2 to cross-link Sp1. According to our result, however, we suggest another mechanism of TGase 2 on modulating transcription. Our results from EMSA and immunoprecipitation data showed that TGase 2 interact with c-Jun, and inhibit a dimer formation of c-Jun with Fos. Thereby, TGase 2 plays indirectly as a transcription modulator. As we performed this study, it reported that enhanced crosslinking of ECM by tissue transglutaminase and decreased degradation due to reduced active MMP-9 expression may be at least partially responsible for the deposition of fibronectin in the injured glomeruli [30]. However, they did not show why MMP-9 activity was reduced. This report may provide the mechanism leading to the TGase 2-induced alteration of gelatinase activity. In summary, this report shows that TGase 2 can reduce the transcription process by interrupting the binding of the Jun–fos complex to an AP-1 site. These findings point to the new roles of TGase 2 in the nuclear compartment. Acknowledgments This work was supported by Vascular System Research Center in Kangwon National University. We thank Dr. Kwon Soo Hah for a kind gift of TGase 2 siRNA.
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