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HER2-dependent MMP-7 expression is mediated by activated STAT3
Cellular Signalling 20 (2008) 1284–1291
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Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g
HER2-dependent MMP-7 expression is mediated by activated STAT3 Guogang Yuan a, Lu Qian a, Ming Shi a, Feng Lu b, Dan Li a, Meiru Hu a, Ming Yu a, Beifen Shen a, Ning Guo a,⁎ a b
Institute of Basic Medical Sciences, Beijing 100850, PR China Department of Cellular and Molecular Immunology, Medical School of Henan University, Kaifeng 475004, PR China
a r t i c l e
i n f o
Article history: Received 24 January 2008 Accepted 25 February 2008 Available online 4 March 2008 Keywords: HER2 STAT3 MMP-7 Heregulin Cancer
a b s t r a c t MMP-7 expression is highly regulated at the level of transcription. An understanding of how the MMP-7 gene is regulated is critical to elucidate the mechanisms of MMP-7 overexpression in the early tumor development. In the present study, increased mRNA and protein expressions of MMP-7 were observed in MCF-7 cells stably overexpressing HER2 (MCF-7/HER2). The promoter activity of MMP-7 gene was upregulated in MCF-7/HER2 cells and significantly enhanced by HRG induction. Examination of the MMP-7 promoter sequence revealed three potential STAT3 binding sites within the proximal region. MMP-7 promoter activity was remarkably induced in MCF-7 cells expressing the constitutively activated STAT3 (MCF-7/STAT3C). RT-PCR and Western blot analysis confirmed the expression upregulation of mRNA and protein of MMP-7 in the MCF-7/STAT3C cells. Binding of STAT3 to MMP-7 promoter was verified by ChIP and the critical STAT3 element within the MMP-7 promoter identified by the mutagenesis of the core STAT3 recognition sequence. Increased STAT3 phosphorylation was observed in either HER2 overexpressing cells or HRG-induced cells. The data indicate that HRG-induced HER2-dependent transcriptional upregulation and protein secretion of MMP-7 are mediated by activated STAT3. The expression of MMP-7 may be attributed to HER2/STAT3 activation. © 2008 Elsevier Inc. All rights reserved.
1. Introduction Matrix metalloproteinases (MMPs) are a family of structurally related proteinases. They are capable of degrading virtually all protein components of extracellular matrix. More than 20 distinct MMPs have been identified. Based on their structures, substrate specificities and cellular localization, MMPs are classified into collagenases, gelatinases, stromelysins and membrane-type MMPs [1–3]. MMP-7, also known as matrilysin, is the smallest known member among this family. Unlike other MMPs, which are more commonly expressed by stroma cells, MMP-7 appears to be expressed predominantly in epithelial cells of glandular tissue and is usually overproduced in carcinoma cells at early stages of tumor development. Structurally, MMP-7 contains only the common catalytic domain and Zn2+ binding region, but lacks the haemopexin-like domain, making it distinguished from other MMP family members. MMP-7 possesses strong extracellular matrix-degradative activity and its substrates include proteoglycans, fibronectin, entactin, laminin, gelatin and elastin [4,5]. The role of MMP-7 in tumorigenesis and tumor progression has been demonstrated in vitro and in the animal model [6,7]. It has been reported that MMP-7 was overexpressed in several human malignant tumors, including squamous cell carcinoma of the head and neck region [8], breast [9], lung [10], prostate [11], esophagus [12], stomach [13], endometrium [14], ovary [15] and colorectum carcinomas [16]. A ⁎ Corresponding author. Institute of Basic Medical Sciences, Taiping Road 27, Beijing 100850, PR China. Fax: +86 10 6821 3039. E-mail address: [email protected] (N. Guo). 0898-6568/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2008.02.017
positive correlation between MMP-7 and metastatic potential in colon cancer has been documented [17]. It has been demonstrated that the ablation in MMP-7 expression results in a dramatic reduction in intestinal cancer growth in animals [18,19]. Overexpression of MMP-7 has been shown to provide apoptosis resistance to chemotherapeutic agents in colon carcinoma cells by cleavage of FasL, while MMP-7 antisense oligonucleotides could increase the susceptibility of these cells to Fas-mediated apoptosis [20]. MMP genes are not constitutively expressed in most cell types, but MMP expression is highly regulated at the level of transcription and induced by complex interactions between cytokines, cell surface receptors and extracellular matrix [21]. An understanding of how the MMP-7 gene is regulated is critical to elucidate the mechanisms of MMP-7 overexpression. Previous studies revealed a number of potential transcription factor binding elements, including an activator protein-1 (AP-1) binding site and three E-twenty-six (ETS) binding sites, within the first 350 base pairs (bp) of the transcription start site in human MMP-7 promoter [22]. AP-1 and ETS transcription factors can be activated by the mitogen activated protein kinase (MAPK) pathway mediated by HER receptors, leading to increased levels of these two family members [23–25]. It has been reported that EGFR and HER2 levels were strongly correlated with the expression of MMP-7. It has also been reported that the expression of several MMPs in tumor cell lines was upregulated by major HER ligands [26,27]. Our preliminary study suggested a link between HER2 overexpression and upregulation of MMP-7. We were interested in addressing the question of whether HER2 could enhance MMP-7 transcription and identifying the downstream molecules of HER2 that
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Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's instructions and selected in the presence of G418 (Sigma) at a concentration of 800 μg/ml. Single neomycinresistant clones were picked and cultivated in the presence of G418 (200 μg/ml).
Table 1 The primers used Amplification
Primers
−1267/+44 bp region of MMP-7 promoter Point mutation at position −255/− 245 bp Point mutation at position −169/−159 bp Point mutation at position −137/−122 bp Full length MMP-7 cDNA
P1: 5′-CTAGCTAGCTTGCTGGCTCACAGTGCTC-3′ P2: 5′-CCCAAGCTTTGCCGTCCAGAGACAATTG-3′ P3: 5′-AGCAGTCATTTGACCTCGGCACACAAATGAGGTTTCT-3′ P4: 5′-CTCATAGGTATCATTCAGGAC-3′ P5: 5′-ATACATTGTGTGCTCCCTGCCACTAACGATGTAATACTT-3′ P6: 5′-TTGTCTTTCAAAGGATT-3′ P7: 5′-GATGTAATACTTCCTCGTCCTAGCCAATGCAAAATAACACATAC-3′ P8: 5′-GTTATTGGCAGGAAGCACAC-3′ P9: 5′-TCTTTGGCCTACCTATAACTGG-3′ P10: 5′-CTAGACTGCTACCATCCGTC-3′ P11: 5′-GGTCGGAGTCAACGGATTTG-3′ P12: 5′-ATGAGCCCCAGCCTTCTCCAT-3′ P13: 5′-TATGGTACCATAATGTCCTGAAT-3′ P2: 5′-CCCAAGCTTTGCCGTCCAGAGACAATTG-3′
GAPDH −304/+ 44 bp region of MMP-7 promoter
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are involved in this process. In the present study, the mechanism of the MMP-7 expression induced by HER2 mediated signaling was investigated. 2. Materials and methods 2.1. Cell culture Human breast cancer cell line MCF-7 was obtained from American Type Tissue Culture Collection (Rockville, MD). The MCF-7/STAT3C and MCF-7/pcDNA3.1 cells were established as described previously [28]. The cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). To establish MCF-7 cells stably overexpressing HER2, the cells were transfected with the plasmid pcDNA3/HER2 with
2.2. Antibodies The mouse monoclonal antibody against c-erbB-2/HER-2/neu (Ab-20) is from NeoMarkers. Rabbit polyclonal antibody against STAT3 is from Santa Cruz. Rabbit polyclonal antibody recognizing MMP-7 is from ProteinTech Group, Inc. Rabbit polyclonal antibodies against phosphorylated HER2 (Tyr1248) and phosphorylated STAT3 (Tyr705) are from Signalway Antibody and Cell Signaling Technology, respectively. Mouse monoclonal antibody against GAPDH is from Kangcheng Co., Shanghai. The horseradish peroxidase-conjugated secondary antibodies are from Beijing Zhongshan Golden Bridge Biotechnology Co. LTD. 2.3. Constructs The promoter region (nucleotide −1267 to +44 relative to transcription initiation site) of human MMP-7 was amplified by polymerase chain reaction (PCR) with MCF-7 genomic DNA as a template using the primer P1 containing an NheI site and P2 containing a HindIII site (Table 1) with Pyrobest DNA polymerase (TaKaRa). The resultant product was digested with NheI/HindIII, gel purified and directionally cloned into the NheI and HindIII sites at immediate upstream of a firefly luciferase gene in pGL3-Basic reporter vector (Promega) designated pLuc1267. The 5′-end deletion of the promoter was created by digesting pLuc1267 with KpnI/HindIII and the resulting 345 bp fragment inserted into KpnI/HindIII-digested pGL3-Basic (pLuc296). The point mutations of the potential STAT3 sites at positions −255/−245 bp, −168/−159 bp and −137/−122 bp were generated with the primers P3–P8 (Table 1) using TaKaRa mutanBEST kit as described by the manufacturer. All constructs were verified by sequence analysis. The plasmid pRc/CMV-STAT3C-Flag (constitutively activated STAT3, STAT3C) and STAT3 dominant negative construct (STAT3-YF) are the generous gifts from Drs. Bromberg and Darnell. The plasmid pcDNA3/HER2 was kindly provided by Dr. Y. Jia, Institute of Transfusion Medicine, Beijing. The plasmid pRL-TK (Dual Luciferase assay, Promega) was used as a control plasmid.
Fig. 1. The expression of MMP-7 associates with HER2 level. A, HER2 expression in the stably transfected MCF-7 cells analyzed by FACS; B, HER2 expression in a stable transfectant analyzed by Western blot; C, mRNA level of MMP-7 in MCF-7 cells analyzed by RT-PCR; D, MMP-7 promoter activity analyzed by luciferase assays. ⁎ P b 0.05.
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2.4. Semi-quantitative RT-PCR
2.7. Cell lysate preparation and Western blot
Total RNA was isolated from MCF-7 cells treated with or without heregulin-β (HRG) (Sigma) using the TRIzol reagents (Invitrogen) following the manufacturer's instructions and quantified by spectrophotometry. cDNA was synthesized from 5 μg of total RNA using M-MuLV reverse transcriptase and random hexamers (Biolabs, NEW ENGLAND). Then the resulting cDNA was subsequently amplified by using the MMP-7 primers P9 and P10 (Table 1) with Taq DNA polymerase (Tiangen Biotech Co., LTD). The PCR products were analyzed on 1.5% agarose gel. Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene using specific primers P11 and P12 (Table 1) was used as an internal control. The experiment was conducted in triplicate.
The cells were treated with HRG for 48 h under serum-free conditions after overnight starvation. For detection of MMP-7 protein secretion, the cultural media were harvested and concentrated about 20-fold using Amicon Ultra-4 devices (Millipore). For detection of HER2, STAT3, phosphorylated HER2, phosphorylated STAT3 and GAPDH, the cell lysates were prepared. Protein concentrations were determined using the BCA™ protein assay kit (PIERCE) to ensure equal loading. The concentrated media in non-reducing sample buffer and lysates in reducing buffer were fractionated by SDSPAGE, transferred onto nitrocellulose membranes. The membranes were blocked, incubated overnight at 4 °C with the primary antibodies followed by washing and then incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies. After washing, the bands were visualized by SuperSignal® West Femto Maximum Sensitivity Substrate (PIERCE).
2.5. Transient transfection and luciferase assays Plasmid DNA was transiently transfected into the cells. For reporter assay, pRL-TK reporter plasmid containing the Renilla luciferase reporter gene was co-transfected along with the firefly luciferase reporter constructs as an internal control. At 5 h after transfection, the medium was replaced with serum containing medium and then the cells were incubated for an additional 48 h. For HRG stimulation, the cells were allowed to recover for 12 h after transfection and then the medium was changed to the serum-free medium. After overnight culture, HRG was added at various concentrations and cells were incubated for 24 h. For luciferase assays, cells were lysed in lysis buffer (Promega). Both firefly and Renilla luciferase activities were measured with a dual luciferase assay kit (Promega) according to the manufacturer's instructions. All transfections were carried out in triplicate and repeated at least three times. Statistical analysis was performed using a two-way analysis of variance (ANOVA) test. The luciferase activities were shown by normalizing to Renilla luciferase activities. 2.6. Chromatin immunoprecipitation (ChIP) assays Preparation of chromatin-DNA from MCF-7 cells expressing STAT3C and ChIP assays were performed by using the chromatin immunoprecipitation assay kit (Upstate Biotechnology, Waltham, MA) following the protocol supplied by the manufacturer. STAT3/DNA complexes were precipitated either by anti-STAT3 antibody or by rabbit IgG as the negative control. Precipitated DNA was amplified by PCR using primers (P13 and P2, Table 1) specific for a 348 bp region (− 304 to + 44) spanning the potential STAT3-binding sites in the MMP-7 promoter. Final products were resolved on a 1.5% agarose gel.
2.8. Flow cytometry (FACS) MCF-7 cells stably expressing HER2 were reacted with anti-HER2 antibody. Following washing, the cells were incubated with FITC-conjugated secondary antibody. Then, the cells were washed and the level of HER2 protein on the cell surface was analyzed by FACS (FACS Calibur, Becton Dickinson).
3. Results 3.1. Expression of MMP-7 associates with HER2 level Increasing evidences suggest a correlation of HER signaling with MMPs. It has been reported that the level of EGFR on tumor cells affected the production of proteolytic enzymes [29]. Aberrant HER2 activation has also been demonstrated to increase invasive and metastatic capacity by stimulating the expression of several key molecules including MMP-1 and MMP-9 [30,31]. To investigate whether HER2 overexpression associates with the transcription of MMP-7, MCF-7 cells were transfected with the plasmid pcDNA3/HER2. The stable clones overexpressing HER2 were selected by G418 and the increased level of HER2 expression confirmed by FACS and Western blot (Fig. 1A and B). Semiquantitative RT-PCR analysis showed that MMP-7 expression at
Fig. 2. The expression of MMP-7 is in response to HRG stimulation. A, the effects of HRG on MMP-7 transcription analyzed by RT-PCR; B, MMP-7 promoter activity induced by HRG in MCF-7 cells transiently transfected with pcDNA3/HER2; C, MMP-7 promoter activity induced by HRG in MCF-7 cells stably expressing HER2; D, MMP-7 protein expression induced by HRG. ⁎⁎ P b 0.01, HRG induction vs. no induction; ## P b 0.01, pcDNA3/HER2 vs. pcDNA3.1.
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transcriptional level was very low in MCF-7 cells. When the cells were cultured in serum-free medium, the signal was barely detected. Incubation with the medium containing 10% FBS could result in slightly increased mRNA expression of MMP-7 in parental cells. However, MMP7 mRNA expression was significantly increased in MCF-7/HER2 cells (Fig. 1C) cultured in the media either with or without serum. To determine whether overexpression of HER2 affects MMP-7 promoter activity, the plasmid containing a luciferase reporter gene driven by MMP-7 promoter (pLuc1267) was constructed and MCF-7/ HER2 cells were transfected with pLuc1267. Parental MCF-7 cells transfected with pLuc1267 was used as a control. The effect of HER2 on the MMP-7 promoter activity was assessed by luciferase assay. Luciferase activities from MCF-7/HER2 cells showed an over twofold increase compared with the control cells (Fig. 1D), suggesting that HER2 overexpression induced MMP-7 promoter activity. 3.2. MMP-7 expression is in response to HRG stimulation HRG can regulate growth, differentiation and survival of breast cancer cells and various other cancer cells. It binds to HER3 and HER4 with low and high affinity, respectively [32]. Although HRG does not bind directly to HER2, it can activate HER2 through the formation of the heterodimer with ligand-bound HER3 and HER4. It has been demonstrated that HRG stimulates phosphorylation of the HER2 and HER3 receptors and activates ERK and PI3K in MCF-7 cells, which express HER3 and HER4 [32]. Multiple signaling pathways involved in
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activation of MMP-9 by HRG in human breast cancer cells have been reported [33]. To determine whether HRG has any effects on MMP-7 transcription, RT-PCR was performed. The data showed that untreated MCF-7/HER2 cells had a basal signal (Fig. 2A). The treatment of HRG (100 ng/ml) resulted in an increased MMP-7 mRNA expression in parental MCF-7 cells. However, MMP-7 expression was significantly upregulated by HRG induction in MCF-7/HER2 cells (Fig. 2A). To clarify whether HRG mediated increase of MMP-7 mRNA level is due to increased transcription from the MMP-7 promoter, MCF-7 cells transiently co-transfected with pcDNA3/HER2 and pLuc1267 were treated with HRG for 24 h and then luciferase activities were measured. Treatment with HRG resulted in significant increase in luciferase activity in both control cells and pcDNA3/HER2 transfected cells compared with their corresponding untreated controls (Fig. 2B). An over three-fold increase of MMP-7 promoter activity by HRG (10 ng/ml) induction was detected in MCF-7 stably expressing HER2, indicating that HRG indeed upregulated MMP-7 promoter activity and overexpression of HER2 mediated enhanced signals (Fig. 2C). To determine whether increased levels of MMP-7 mRNA upregulated by HRG lead to a similar increase in protein production of MMP7, the MMP-7 protein in the serum-free media of the cells was analyzed by Western blot. As shown in Fig. 2D, MMP-7 protein could be detected in MCF-7/HER2 cells but not in parental MCF-7 cells. HRG induction resulted in remarkable up-regulation of MMP-7 expression in parental cells. However, higher level of MMP-7 protein was detected in MCF-7/HER2 cells, suggesting that high level of HER2 associated
Fig. 3. Constitutively activated STAT3 stimulates MMP-7 expression. A, three potential STAT3 binding sites (in bold) found in the MMP-7 promoter; B, the expression of STAT3 in MCF7 cells; C, MMP-7 promoter activity in MCF-7/STAT3C cells; D, MMP-7 mRNA expression in MCF-7/STAT3C cells; E, MMP-7 protein expression in MCF-7/STAT3C cells. ⁎⁎ P b 0.01.
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with MMP-7 expression and HER2 contributed to both basal and HRGinduced expression of MMP-7. The HER2 receptor has been shown to play an essential role in the signaling stimulated by HRG. Above data showed that the expression of MMP-7 was significantly increased either through overexpression of HER2 or HRG induction, suggesting that HER2 mediated signaling pathway was important in MMP-7 expression. Several signal transduction pathways activated by HRG have been reported recently. Previous studies showed that ERK pathway contributed to HRGinduced activation of MMP-9 [34]. MMP-7 and MMP-9 share the similarity in the flanking region of their promoters. We utilized PD98059, which is known to selectively block the activity of MAP kinase kinase (MEK, an activator of ERK kinase) to examine whether ERK signaling pathway could be involved in induction of MMP-7 by HRG. Serum-deprived MCF-7/HER2 cells were pretreated with PD98059 for 1 h and then stimulated with 100 ng/ml HRG. Surprisingly, our data from luciferase assays consistently showed that PD98059 had no striking inhibitory effect on MMP-7 promoter
activity (data not shown). It suggested that other signaling pathways might be involved in HRG-induced MMP-7 transcription. 3.3. Constitutively activated STAT3 stimulates MMP-7 expression Our previous study has demonstrated that the functional cooperation of the STAT3 and AP-1 transcription factors is required for the transcription of MMP-9 gene [35]. In further examining the promoter sequence of MMP-7 gene, three putative STAT3 binding sites with the consensus sequence TTN(4–6)AA, which have not been reported previously, were found in the −137/−122 (TTTTAGTTAATGAAAA), −168/ −159 (TTCCTGCCAA), and −255/−245 (TTTGGCAAAAA) regions of the MMP-7 promoter, respectively (Fig. 3A). A recent study indicates that STAT3 is a direct downstream molecule of HER2 receptor signaling [36]. We speculated that HER2 might stimulate STAT3, which may in turn activate MMP-7. Thus, we utilized the MCF-7 cells stably expressing the constitutively activated STAT3 mutant STAT3C (Fig. 3B) to determine whether STAT3 activity by itself was sufficient to regulate MMP-7
Fig. 4. STAT3 binds to MMP-7 promoter and activation of the MMP-7 promoter depends on the functional STAT3 sites. A, binding of STAT3 to the MMP-7 promoter analyzed by ChIP; B, schematic representation of the mutagenesis of STAT3 binding sites; C, identification of the functional STAT3 binding sites by luciferase assays; D, identification of the functional STAT3 binding elements in HRG induction by luciferase assays; E, the inhibition of HRG-induced MMP-7 promoter activity by the dominant negative STAT3. SBE1, SBE2 and SBE3, putative STAT3 binding elements 1, 2 and 3; mSBE1, mSBE2 and mSBE3, mutated putative STAT3 binding elements 1, 2 and 3; Wt, wild type; Mt, mutant. ⁎ P b 0.05; ⁎⁎ P b 0.01, dominant negative STAT3 vs. the control.
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Fig. 4 (continued ).
promoter activity. The plasmid pLuc1267 was transfected into MCF-7/ STAT3C cells. An approximate five-fold increase of MMP-7 promoterdriven luciferase activity was observed in MCF-7/STAT3C cells compared with the parental cells (Fig. 3C). Furthermore, MMP-7 mRNA expression was strikingly increased in MCF-7/STAT3C cells (Fig. 3D). Western blot analysis showed that MMP-7 secretion was only detected in the cell culture medium from MCF-7/STAT3C cells, but not in that from control cells (Fig. 3E). 3.4. Activation of MMP-7 promoter depends on the functional STAT3 sites A previous study reported that STAT3 did not bind to the MMP-7 promoter [37]. We performed ChIP assays to test if STAT3 could bind directly to the STAT3 binding site in the MMP-7 promoter in vivo. As shown in Fig. 4A that immunoprecipitation with anti-STAT3 antibody followed by PCR yielded a distinct ~350 bp band, demonstrating the
association of STAT3 with the MMP-7 promoter in vivo. In contrast, immunoprecipitation with an irrelevant antibody resulted in the absence of this band, confirming the specificity of the interaction between STAT3 and the MMP-7 promoter. In order to judge which of the potential binding sites for STAT3 may have relevance in transcription activation of MMP-7 gene, we first constructed a truncated MMP-7 promoter fragment (− 296 bp upstream from the transcription initiation site) and cloned it in front of the firefly luciferase gene of pGL3-basic (pLuc296). Three putative STAT3 binding sites are all present in this fragment. Then, we undertook the mutagenesis of STAT3 binding sites based on pLuc296 and the contribution of each of three elements to the STAT3-mediated regulation of MMP-7 was assessed through mutating the coresequence of STAT3. The resultant plasmids were designated as pLuc296mSBE1, pLuc296mSBE2 and pLuc296mSBE3 (Fig. 4B). Luciferase assays showed that pLuc296 did not change the basal MMP-7
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4. Discussion
Fig. 5. HRG induces phosphorylation and activation of HER2 and STAT3.
promoter activity in transfected MCF-7/STAT3C cells compared to pLuc1267 (Fig. 4C). The single mutation of putative STAT3 binding site at −255/−245 region had no effect on the transcriptional signal. An about 33% depression of signal was observed with single mutation at −168/−159 site and 22% at −137/−122 site (Fig. 4C). To verify that HRG stimulates MMP-7 expression through HER2 mediated STAT3 activation, we transfected MCF-7/HER2 cells with pLuc296mSBE1, pLuc296mSBE2 and pLuc296mSBE3, respectively. After transfection for 24 h, the cells were treated with HRG. Consistent with above data, the treatment with HRG showed no remarkable decrease in luciferase activity when the core-sequence at −255/−245 site was mutated (Fig. 4D). The mutagenesis at −168/−159 and −137/ −122 sites resulted in 51% and 28% decrease of luciferase activity in response to HRG, respectively. It indicates that STAT3 binding site at −168/−159 region is the most important for HRG enhancement of MMP-7 expression. We then determined whether the activity of the MMP-7 promoter could be inhibited by a dominant negative STAT3 with the mutation at the tyrosine phosphorylation site, which is critical for STAT3 activation. After transient cotransfections with pLuc1267 and the dominant negative STAT3 into MCF-7/HER2 cells, HRG-induced transactivation of the MMP-7 promoter was measured. As shown in Fig. 4E, that the expression of the dominant negative STAT3 significantly reduced HRG-induced MMP-7 promoter activity, further confirming that binding of STAT3 to its cognate sites in the MMP-7 promoter facilitates strong MMP-7 induction and STAT3 plays an important role in MMP-7 expression.
The MMPs are frequently overexpressed in various human cancers. Enhanced expressions of MMPs have been associated with an aggressive malignant phenotype and adverse prognosis in cancer patients. Growth factors, cytokines, oncogenes and tumor promoters regulate MMPs both positively and negatively at the transcriptional level. Similar inductive responsiveness of MMP genes to a variety of cellular stimuli has been demonstrated. It has been reported that EGF could directly stimulate MMP-7 gene expression in colon cancer cell lines and FGF-2 increased MMP-7 transcripts in human umbilical vein endothelial cells [27,37]. However, the mechanisms of the transcription regulation of MMP-7 gene are largely unknown. In the present study, increased mRNA and protein expressions of MMP-7 were observed in MCF-7 cells overexpressing HER2. The promoter activity of MMP-7 gene was markedly upregulated in MCF7/HER2 cells. It has been known that HER2 overexpression is related to stromal MMP-2 and MMP-9 expression and proteolytic activity [31]. The expressions of several MMPs upregulated by HER ligands have also been documented [26,27]. It has been postulated that the dysregulation of growth factor-mediated paracrine loops may result in the aberrant HER2 expression. As a direct ligand for HER2 remains to be isolated, it has been suggested that HER2 co-operates with other HER receptor family members forming heterodimers and enhances the signal that links to the cellular migration/invasion machinery. HRG is produced by both stroma and tumor cells. It was reported that mRNA transcripts of HRG were detected in about 25% of human breast carcinomas [38], a percentage similar to HER2 overexpression. This may reflect the enhanced HER2 phosphorylation upon HRG treatment in most of the HER2-overexpressing cancer cell lines. When we treated the cells with HRG, an increased mRNA and protein expression of MMP-7 was observed in both parental MCF-7 and MCF-7/HER2 cells. The activity of the MMP-7 promoter was dramatically enhanced in MCF-7/HER2 cells by HRG induction. It strongly indicates that HER2 mediated signaling directly stimulates MMP-7 gene expression in MCF-7 cells. Since ERK signaling pathway is one of the most important intracellular pathways activated by HER2, we used PD98059 to test
3.5. HRG induces phosphorylation of HER2 and STAT3 It has been demonstrated that the association between the phosphorylated/active form of STAT3 and HER2 and a requirement for HER2 kinase activity to establish constitutive STAT3 activation [36]. To determine whether HRG-triggered activation of MMP-7 correlates with increased phosphorylation of HER2 and STAT3, MCF-7/HER2 cells were incubated in the absence or presence of HRG. Equivalent cell lysates were then processed for determination of HER2 and STAT3 phosphorylation levels by SDS-PAGE and Western blot. The phosphorylation of HER2 in parental MCF-7 cells was at very low level, but it was remarkably elevated by the treatment with HRG. The phosphorylated STAT3 was not detected in parental cells, even with HRG induction (Fig. 5). However, in overexpressing HER2 cells the phosphorylation of HER2 was obvious. HRG induced further increased level of phosphorylated HER2. The constitutively activated STAT3 was observable in MCF-7/HER2 cells and the higher level of phosphorylation could be induced in the presence of HRG (Fig. 5). Our data indicate that the activation of STAT3 was associated with the level of HER2 and HER2 dependent MMP-7 activation was mediated by STAT3 signaling pathway.
Fig. 6. The mechanism of MMP-7 transcription regulated by HER2/STAT3. HRG produced by stroma cells and tumor cells induces the phosphorylation of HER2, which in turn stimulates activation of STAT3. Activated STAT3 translocates into the nucleus, binds to its binding sequences in the MMP-7 promoter and regulates MMP-7 transcription.
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whether the expression of MMP-7 is regulated by HER2 mediated ERK signaling. However, the induction of MMP-7 promoter activity by HRG was not inhibited by the MEK1/2 inhibitor. Examination of the MMP-7 promoter sequence revealed three potential sites conforming to the TTN(4–6)AA core-sequence crucial for STAT3 binding in the proximal region. Transfection of pLuc1267 into MCF-7/STAT3C cells resulted in a remarkable induction in MMP-7 promoter activity. RT-PCR and Western blot analysis confirmed the expression upregulation of mRNA and protein of MMP-7 in the MCF-7/ STAT3C cells. Binding of STAT3 to MMP-7 promoter was verified by ChIP and the critical STAT3 element within the MMP-7 promoter identified by the mutagenesis of the core STAT3 recognition sequence. These data suggest that HRG stimulated the transcription activation of MMP-7 through STAT3. STATs are latent cytoplasmic transcription factors, but they can be activated by either Janus activated kinases or tyrosine kinase receptors. Activation of STATs has been demonstrated in response to ligands that activate growth factor receptors with tyrosine kinase activity [39]. Constitutive activation of STAT3 is found in many human cancers, including 30 to 60% of primary breast cancer [40,41]. A positive relation between STAT3 and HER2 expressions has also been reported. Our data revealed that STAT3 was phosphorylated when HER2 was overexpressed and the activation of STAT3 was significantly enhanced with the addition of HRG, indicating that HRG-induced HER2-dependent transcriptional upregulation and protein secretion of MMP-7 are mediated by activated STAT3. Our findings may elucidate a mechanism of MMP-7 transcription in MCF-7 cells. HRG produced by stroma cells and tumor cells induces the phosphorylation of HER2, which in turn stimulates activation of STAT3. Activated STAT3 translocates into the nucleus, binds to its binding sequences in the MMP-7 promoter and regulates MMP-7 transcription (Fig. 6). MMP-7 was consistently detected at earlier stages in several cancers. The mechanism by which MMP-7 contributes to early tumor growth is still unknown. One of the hallmarks of tumor cells is apoptosis resistance [42]. Accumulating evidences demonstrate that high levels of HER2 and STAT3 are associated with intrinsic multiple drug resistance [43] and increased metastatic potential. MMP-7 expression has also been considered as an apoptosis resistant phenotype. It has been indicated that the shedding CD95 and CD95 ligand (CD95L) by MMP-7 results in decreased CD95-mediated apoptosis sensitivity of tumor cells [20,42]. Our previous study demonstrates that STAT3 also positively regulates the expression of HER2 in breast cancer cells, implying that HER2 and STAT3 may form a positive feedback loop to participate in breast oncogenesis [28]. The MMP-7 expression may be attributed to HER2 and STAT3 activation in the early tumor development.
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Natural Science Foundation (No. 7051006) and National Basic Research Program of China (973 Program, No. 2006CB504305). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
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Acknowledgements
[38]
We are very grateful to Drs. Bromberg and Darnell for the gifts of pRc/ CMV-STAT3C-Flag and STAT3 dominant negative construct. We thank Dr. Yianjun Jia for providing us pcDNA3/HER2. This work was supported by National Natural Science Foundation of China (No. 30771981), Beijing
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Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g
HER2-dependent MMP-7 expression is mediated by activated STAT3 Guogang Yuan a, Lu Qian a, Ming Shi a, Feng Lu b, Dan Li a, Meiru Hu a, Ming Yu a, Beifen Shen a, Ning Guo a,⁎ a b
Institute of Basic Medical Sciences, Beijing 100850, PR China Department of Cellular and Molecular Immunology, Medical School of Henan University, Kaifeng 475004, PR China
a r t i c l e
i n f o
Article history: Received 24 January 2008 Accepted 25 February 2008 Available online 4 March 2008 Keywords: HER2 STAT3 MMP-7 Heregulin Cancer
a b s t r a c t MMP-7 expression is highly regulated at the level of transcription. An understanding of how the MMP-7 gene is regulated is critical to elucidate the mechanisms of MMP-7 overexpression in the early tumor development. In the present study, increased mRNA and protein expressions of MMP-7 were observed in MCF-7 cells stably overexpressing HER2 (MCF-7/HER2). The promoter activity of MMP-7 gene was upregulated in MCF-7/HER2 cells and significantly enhanced by HRG induction. Examination of the MMP-7 promoter sequence revealed three potential STAT3 binding sites within the proximal region. MMP-7 promoter activity was remarkably induced in MCF-7 cells expressing the constitutively activated STAT3 (MCF-7/STAT3C). RT-PCR and Western blot analysis confirmed the expression upregulation of mRNA and protein of MMP-7 in the MCF-7/STAT3C cells. Binding of STAT3 to MMP-7 promoter was verified by ChIP and the critical STAT3 element within the MMP-7 promoter identified by the mutagenesis of the core STAT3 recognition sequence. Increased STAT3 phosphorylation was observed in either HER2 overexpressing cells or HRG-induced cells. The data indicate that HRG-induced HER2-dependent transcriptional upregulation and protein secretion of MMP-7 are mediated by activated STAT3. The expression of MMP-7 may be attributed to HER2/STAT3 activation. © 2008 Elsevier Inc. All rights reserved.
1. Introduction Matrix metalloproteinases (MMPs) are a family of structurally related proteinases. They are capable of degrading virtually all protein components of extracellular matrix. More than 20 distinct MMPs have been identified. Based on their structures, substrate specificities and cellular localization, MMPs are classified into collagenases, gelatinases, stromelysins and membrane-type MMPs [1–3]. MMP-7, also known as matrilysin, is the smallest known member among this family. Unlike other MMPs, which are more commonly expressed by stroma cells, MMP-7 appears to be expressed predominantly in epithelial cells of glandular tissue and is usually overproduced in carcinoma cells at early stages of tumor development. Structurally, MMP-7 contains only the common catalytic domain and Zn2+ binding region, but lacks the haemopexin-like domain, making it distinguished from other MMP family members. MMP-7 possesses strong extracellular matrix-degradative activity and its substrates include proteoglycans, fibronectin, entactin, laminin, gelatin and elastin [4,5]. The role of MMP-7 in tumorigenesis and tumor progression has been demonstrated in vitro and in the animal model [6,7]. It has been reported that MMP-7 was overexpressed in several human malignant tumors, including squamous cell carcinoma of the head and neck region [8], breast [9], lung [10], prostate [11], esophagus [12], stomach [13], endometrium [14], ovary [15] and colorectum carcinomas [16]. A ⁎ Corresponding author. Institute of Basic Medical Sciences, Taiping Road 27, Beijing 100850, PR China. Fax: +86 10 6821 3039. E-mail address: [email protected] (N. Guo). 0898-6568/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2008.02.017
positive correlation between MMP-7 and metastatic potential in colon cancer has been documented [17]. It has been demonstrated that the ablation in MMP-7 expression results in a dramatic reduction in intestinal cancer growth in animals [18,19]. Overexpression of MMP-7 has been shown to provide apoptosis resistance to chemotherapeutic agents in colon carcinoma cells by cleavage of FasL, while MMP-7 antisense oligonucleotides could increase the susceptibility of these cells to Fas-mediated apoptosis [20]. MMP genes are not constitutively expressed in most cell types, but MMP expression is highly regulated at the level of transcription and induced by complex interactions between cytokines, cell surface receptors and extracellular matrix [21]. An understanding of how the MMP-7 gene is regulated is critical to elucidate the mechanisms of MMP-7 overexpression. Previous studies revealed a number of potential transcription factor binding elements, including an activator protein-1 (AP-1) binding site and three E-twenty-six (ETS) binding sites, within the first 350 base pairs (bp) of the transcription start site in human MMP-7 promoter [22]. AP-1 and ETS transcription factors can be activated by the mitogen activated protein kinase (MAPK) pathway mediated by HER receptors, leading to increased levels of these two family members [23–25]. It has been reported that EGFR and HER2 levels were strongly correlated with the expression of MMP-7. It has also been reported that the expression of several MMPs in tumor cell lines was upregulated by major HER ligands [26,27]. Our preliminary study suggested a link between HER2 overexpression and upregulation of MMP-7. We were interested in addressing the question of whether HER2 could enhance MMP-7 transcription and identifying the downstream molecules of HER2 that
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Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's instructions and selected in the presence of G418 (Sigma) at a concentration of 800 μg/ml. Single neomycinresistant clones were picked and cultivated in the presence of G418 (200 μg/ml).
Table 1 The primers used Amplification
Primers
−1267/+44 bp region of MMP-7 promoter Point mutation at position −255/− 245 bp Point mutation at position −169/−159 bp Point mutation at position −137/−122 bp Full length MMP-7 cDNA
P1: 5′-CTAGCTAGCTTGCTGGCTCACAGTGCTC-3′ P2: 5′-CCCAAGCTTTGCCGTCCAGAGACAATTG-3′ P3: 5′-AGCAGTCATTTGACCTCGGCACACAAATGAGGTTTCT-3′ P4: 5′-CTCATAGGTATCATTCAGGAC-3′ P5: 5′-ATACATTGTGTGCTCCCTGCCACTAACGATGTAATACTT-3′ P6: 5′-TTGTCTTTCAAAGGATT-3′ P7: 5′-GATGTAATACTTCCTCGTCCTAGCCAATGCAAAATAACACATAC-3′ P8: 5′-GTTATTGGCAGGAAGCACAC-3′ P9: 5′-TCTTTGGCCTACCTATAACTGG-3′ P10: 5′-CTAGACTGCTACCATCCGTC-3′ P11: 5′-GGTCGGAGTCAACGGATTTG-3′ P12: 5′-ATGAGCCCCAGCCTTCTCCAT-3′ P13: 5′-TATGGTACCATAATGTCCTGAAT-3′ P2: 5′-CCCAAGCTTTGCCGTCCAGAGACAATTG-3′
GAPDH −304/+ 44 bp region of MMP-7 promoter
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are involved in this process. In the present study, the mechanism of the MMP-7 expression induced by HER2 mediated signaling was investigated. 2. Materials and methods 2.1. Cell culture Human breast cancer cell line MCF-7 was obtained from American Type Tissue Culture Collection (Rockville, MD). The MCF-7/STAT3C and MCF-7/pcDNA3.1 cells were established as described previously [28]. The cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). To establish MCF-7 cells stably overexpressing HER2, the cells were transfected with the plasmid pcDNA3/HER2 with
2.2. Antibodies The mouse monoclonal antibody against c-erbB-2/HER-2/neu (Ab-20) is from NeoMarkers. Rabbit polyclonal antibody against STAT3 is from Santa Cruz. Rabbit polyclonal antibody recognizing MMP-7 is from ProteinTech Group, Inc. Rabbit polyclonal antibodies against phosphorylated HER2 (Tyr1248) and phosphorylated STAT3 (Tyr705) are from Signalway Antibody and Cell Signaling Technology, respectively. Mouse monoclonal antibody against GAPDH is from Kangcheng Co., Shanghai. The horseradish peroxidase-conjugated secondary antibodies are from Beijing Zhongshan Golden Bridge Biotechnology Co. LTD. 2.3. Constructs The promoter region (nucleotide −1267 to +44 relative to transcription initiation site) of human MMP-7 was amplified by polymerase chain reaction (PCR) with MCF-7 genomic DNA as a template using the primer P1 containing an NheI site and P2 containing a HindIII site (Table 1) with Pyrobest DNA polymerase (TaKaRa). The resultant product was digested with NheI/HindIII, gel purified and directionally cloned into the NheI and HindIII sites at immediate upstream of a firefly luciferase gene in pGL3-Basic reporter vector (Promega) designated pLuc1267. The 5′-end deletion of the promoter was created by digesting pLuc1267 with KpnI/HindIII and the resulting 345 bp fragment inserted into KpnI/HindIII-digested pGL3-Basic (pLuc296). The point mutations of the potential STAT3 sites at positions −255/−245 bp, −168/−159 bp and −137/−122 bp were generated with the primers P3–P8 (Table 1) using TaKaRa mutanBEST kit as described by the manufacturer. All constructs were verified by sequence analysis. The plasmid pRc/CMV-STAT3C-Flag (constitutively activated STAT3, STAT3C) and STAT3 dominant negative construct (STAT3-YF) are the generous gifts from Drs. Bromberg and Darnell. The plasmid pcDNA3/HER2 was kindly provided by Dr. Y. Jia, Institute of Transfusion Medicine, Beijing. The plasmid pRL-TK (Dual Luciferase assay, Promega) was used as a control plasmid.
Fig. 1. The expression of MMP-7 associates with HER2 level. A, HER2 expression in the stably transfected MCF-7 cells analyzed by FACS; B, HER2 expression in a stable transfectant analyzed by Western blot; C, mRNA level of MMP-7 in MCF-7 cells analyzed by RT-PCR; D, MMP-7 promoter activity analyzed by luciferase assays. ⁎ P b 0.05.
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2.4. Semi-quantitative RT-PCR
2.7. Cell lysate preparation and Western blot
Total RNA was isolated from MCF-7 cells treated with or without heregulin-β (HRG) (Sigma) using the TRIzol reagents (Invitrogen) following the manufacturer's instructions and quantified by spectrophotometry. cDNA was synthesized from 5 μg of total RNA using M-MuLV reverse transcriptase and random hexamers (Biolabs, NEW ENGLAND). Then the resulting cDNA was subsequently amplified by using the MMP-7 primers P9 and P10 (Table 1) with Taq DNA polymerase (Tiangen Biotech Co., LTD). The PCR products were analyzed on 1.5% agarose gel. Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene using specific primers P11 and P12 (Table 1) was used as an internal control. The experiment was conducted in triplicate.
The cells were treated with HRG for 48 h under serum-free conditions after overnight starvation. For detection of MMP-7 protein secretion, the cultural media were harvested and concentrated about 20-fold using Amicon Ultra-4 devices (Millipore). For detection of HER2, STAT3, phosphorylated HER2, phosphorylated STAT3 and GAPDH, the cell lysates were prepared. Protein concentrations were determined using the BCA™ protein assay kit (PIERCE) to ensure equal loading. The concentrated media in non-reducing sample buffer and lysates in reducing buffer were fractionated by SDSPAGE, transferred onto nitrocellulose membranes. The membranes were blocked, incubated overnight at 4 °C with the primary antibodies followed by washing and then incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies. After washing, the bands were visualized by SuperSignal® West Femto Maximum Sensitivity Substrate (PIERCE).
2.5. Transient transfection and luciferase assays Plasmid DNA was transiently transfected into the cells. For reporter assay, pRL-TK reporter plasmid containing the Renilla luciferase reporter gene was co-transfected along with the firefly luciferase reporter constructs as an internal control. At 5 h after transfection, the medium was replaced with serum containing medium and then the cells were incubated for an additional 48 h. For HRG stimulation, the cells were allowed to recover for 12 h after transfection and then the medium was changed to the serum-free medium. After overnight culture, HRG was added at various concentrations and cells were incubated for 24 h. For luciferase assays, cells were lysed in lysis buffer (Promega). Both firefly and Renilla luciferase activities were measured with a dual luciferase assay kit (Promega) according to the manufacturer's instructions. All transfections were carried out in triplicate and repeated at least three times. Statistical analysis was performed using a two-way analysis of variance (ANOVA) test. The luciferase activities were shown by normalizing to Renilla luciferase activities. 2.6. Chromatin immunoprecipitation (ChIP) assays Preparation of chromatin-DNA from MCF-7 cells expressing STAT3C and ChIP assays were performed by using the chromatin immunoprecipitation assay kit (Upstate Biotechnology, Waltham, MA) following the protocol supplied by the manufacturer. STAT3/DNA complexes were precipitated either by anti-STAT3 antibody or by rabbit IgG as the negative control. Precipitated DNA was amplified by PCR using primers (P13 and P2, Table 1) specific for a 348 bp region (− 304 to + 44) spanning the potential STAT3-binding sites in the MMP-7 promoter. Final products were resolved on a 1.5% agarose gel.
2.8. Flow cytometry (FACS) MCF-7 cells stably expressing HER2 were reacted with anti-HER2 antibody. Following washing, the cells were incubated with FITC-conjugated secondary antibody. Then, the cells were washed and the level of HER2 protein on the cell surface was analyzed by FACS (FACS Calibur, Becton Dickinson).
3. Results 3.1. Expression of MMP-7 associates with HER2 level Increasing evidences suggest a correlation of HER signaling with MMPs. It has been reported that the level of EGFR on tumor cells affected the production of proteolytic enzymes [29]. Aberrant HER2 activation has also been demonstrated to increase invasive and metastatic capacity by stimulating the expression of several key molecules including MMP-1 and MMP-9 [30,31]. To investigate whether HER2 overexpression associates with the transcription of MMP-7, MCF-7 cells were transfected with the plasmid pcDNA3/HER2. The stable clones overexpressing HER2 were selected by G418 and the increased level of HER2 expression confirmed by FACS and Western blot (Fig. 1A and B). Semiquantitative RT-PCR analysis showed that MMP-7 expression at
Fig. 2. The expression of MMP-7 is in response to HRG stimulation. A, the effects of HRG on MMP-7 transcription analyzed by RT-PCR; B, MMP-7 promoter activity induced by HRG in MCF-7 cells transiently transfected with pcDNA3/HER2; C, MMP-7 promoter activity induced by HRG in MCF-7 cells stably expressing HER2; D, MMP-7 protein expression induced by HRG. ⁎⁎ P b 0.01, HRG induction vs. no induction; ## P b 0.01, pcDNA3/HER2 vs. pcDNA3.1.
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transcriptional level was very low in MCF-7 cells. When the cells were cultured in serum-free medium, the signal was barely detected. Incubation with the medium containing 10% FBS could result in slightly increased mRNA expression of MMP-7 in parental cells. However, MMP7 mRNA expression was significantly increased in MCF-7/HER2 cells (Fig. 1C) cultured in the media either with or without serum. To determine whether overexpression of HER2 affects MMP-7 promoter activity, the plasmid containing a luciferase reporter gene driven by MMP-7 promoter (pLuc1267) was constructed and MCF-7/ HER2 cells were transfected with pLuc1267. Parental MCF-7 cells transfected with pLuc1267 was used as a control. The effect of HER2 on the MMP-7 promoter activity was assessed by luciferase assay. Luciferase activities from MCF-7/HER2 cells showed an over twofold increase compared with the control cells (Fig. 1D), suggesting that HER2 overexpression induced MMP-7 promoter activity. 3.2. MMP-7 expression is in response to HRG stimulation HRG can regulate growth, differentiation and survival of breast cancer cells and various other cancer cells. It binds to HER3 and HER4 with low and high affinity, respectively [32]. Although HRG does not bind directly to HER2, it can activate HER2 through the formation of the heterodimer with ligand-bound HER3 and HER4. It has been demonstrated that HRG stimulates phosphorylation of the HER2 and HER3 receptors and activates ERK and PI3K in MCF-7 cells, which express HER3 and HER4 [32]. Multiple signaling pathways involved in
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activation of MMP-9 by HRG in human breast cancer cells have been reported [33]. To determine whether HRG has any effects on MMP-7 transcription, RT-PCR was performed. The data showed that untreated MCF-7/HER2 cells had a basal signal (Fig. 2A). The treatment of HRG (100 ng/ml) resulted in an increased MMP-7 mRNA expression in parental MCF-7 cells. However, MMP-7 expression was significantly upregulated by HRG induction in MCF-7/HER2 cells (Fig. 2A). To clarify whether HRG mediated increase of MMP-7 mRNA level is due to increased transcription from the MMP-7 promoter, MCF-7 cells transiently co-transfected with pcDNA3/HER2 and pLuc1267 were treated with HRG for 24 h and then luciferase activities were measured. Treatment with HRG resulted in significant increase in luciferase activity in both control cells and pcDNA3/HER2 transfected cells compared with their corresponding untreated controls (Fig. 2B). An over three-fold increase of MMP-7 promoter activity by HRG (10 ng/ml) induction was detected in MCF-7 stably expressing HER2, indicating that HRG indeed upregulated MMP-7 promoter activity and overexpression of HER2 mediated enhanced signals (Fig. 2C). To determine whether increased levels of MMP-7 mRNA upregulated by HRG lead to a similar increase in protein production of MMP7, the MMP-7 protein in the serum-free media of the cells was analyzed by Western blot. As shown in Fig. 2D, MMP-7 protein could be detected in MCF-7/HER2 cells but not in parental MCF-7 cells. HRG induction resulted in remarkable up-regulation of MMP-7 expression in parental cells. However, higher level of MMP-7 protein was detected in MCF-7/HER2 cells, suggesting that high level of HER2 associated
Fig. 3. Constitutively activated STAT3 stimulates MMP-7 expression. A, three potential STAT3 binding sites (in bold) found in the MMP-7 promoter; B, the expression of STAT3 in MCF7 cells; C, MMP-7 promoter activity in MCF-7/STAT3C cells; D, MMP-7 mRNA expression in MCF-7/STAT3C cells; E, MMP-7 protein expression in MCF-7/STAT3C cells. ⁎⁎ P b 0.01.
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with MMP-7 expression and HER2 contributed to both basal and HRGinduced expression of MMP-7. The HER2 receptor has been shown to play an essential role in the signaling stimulated by HRG. Above data showed that the expression of MMP-7 was significantly increased either through overexpression of HER2 or HRG induction, suggesting that HER2 mediated signaling pathway was important in MMP-7 expression. Several signal transduction pathways activated by HRG have been reported recently. Previous studies showed that ERK pathway contributed to HRGinduced activation of MMP-9 [34]. MMP-7 and MMP-9 share the similarity in the flanking region of their promoters. We utilized PD98059, which is known to selectively block the activity of MAP kinase kinase (MEK, an activator of ERK kinase) to examine whether ERK signaling pathway could be involved in induction of MMP-7 by HRG. Serum-deprived MCF-7/HER2 cells were pretreated with PD98059 for 1 h and then stimulated with 100 ng/ml HRG. Surprisingly, our data from luciferase assays consistently showed that PD98059 had no striking inhibitory effect on MMP-7 promoter
activity (data not shown). It suggested that other signaling pathways might be involved in HRG-induced MMP-7 transcription. 3.3. Constitutively activated STAT3 stimulates MMP-7 expression Our previous study has demonstrated that the functional cooperation of the STAT3 and AP-1 transcription factors is required for the transcription of MMP-9 gene [35]. In further examining the promoter sequence of MMP-7 gene, three putative STAT3 binding sites with the consensus sequence TTN(4–6)AA, which have not been reported previously, were found in the −137/−122 (TTTTAGTTAATGAAAA), −168/ −159 (TTCCTGCCAA), and −255/−245 (TTTGGCAAAAA) regions of the MMP-7 promoter, respectively (Fig. 3A). A recent study indicates that STAT3 is a direct downstream molecule of HER2 receptor signaling [36]. We speculated that HER2 might stimulate STAT3, which may in turn activate MMP-7. Thus, we utilized the MCF-7 cells stably expressing the constitutively activated STAT3 mutant STAT3C (Fig. 3B) to determine whether STAT3 activity by itself was sufficient to regulate MMP-7
Fig. 4. STAT3 binds to MMP-7 promoter and activation of the MMP-7 promoter depends on the functional STAT3 sites. A, binding of STAT3 to the MMP-7 promoter analyzed by ChIP; B, schematic representation of the mutagenesis of STAT3 binding sites; C, identification of the functional STAT3 binding sites by luciferase assays; D, identification of the functional STAT3 binding elements in HRG induction by luciferase assays; E, the inhibition of HRG-induced MMP-7 promoter activity by the dominant negative STAT3. SBE1, SBE2 and SBE3, putative STAT3 binding elements 1, 2 and 3; mSBE1, mSBE2 and mSBE3, mutated putative STAT3 binding elements 1, 2 and 3; Wt, wild type; Mt, mutant. ⁎ P b 0.05; ⁎⁎ P b 0.01, dominant negative STAT3 vs. the control.
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Fig. 4 (continued ).
promoter activity. The plasmid pLuc1267 was transfected into MCF-7/ STAT3C cells. An approximate five-fold increase of MMP-7 promoterdriven luciferase activity was observed in MCF-7/STAT3C cells compared with the parental cells (Fig. 3C). Furthermore, MMP-7 mRNA expression was strikingly increased in MCF-7/STAT3C cells (Fig. 3D). Western blot analysis showed that MMP-7 secretion was only detected in the cell culture medium from MCF-7/STAT3C cells, but not in that from control cells (Fig. 3E). 3.4. Activation of MMP-7 promoter depends on the functional STAT3 sites A previous study reported that STAT3 did not bind to the MMP-7 promoter [37]. We performed ChIP assays to test if STAT3 could bind directly to the STAT3 binding site in the MMP-7 promoter in vivo. As shown in Fig. 4A that immunoprecipitation with anti-STAT3 antibody followed by PCR yielded a distinct ~350 bp band, demonstrating the
association of STAT3 with the MMP-7 promoter in vivo. In contrast, immunoprecipitation with an irrelevant antibody resulted in the absence of this band, confirming the specificity of the interaction between STAT3 and the MMP-7 promoter. In order to judge which of the potential binding sites for STAT3 may have relevance in transcription activation of MMP-7 gene, we first constructed a truncated MMP-7 promoter fragment (− 296 bp upstream from the transcription initiation site) and cloned it in front of the firefly luciferase gene of pGL3-basic (pLuc296). Three putative STAT3 binding sites are all present in this fragment. Then, we undertook the mutagenesis of STAT3 binding sites based on pLuc296 and the contribution of each of three elements to the STAT3-mediated regulation of MMP-7 was assessed through mutating the coresequence of STAT3. The resultant plasmids were designated as pLuc296mSBE1, pLuc296mSBE2 and pLuc296mSBE3 (Fig. 4B). Luciferase assays showed that pLuc296 did not change the basal MMP-7
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4. Discussion
Fig. 5. HRG induces phosphorylation and activation of HER2 and STAT3.
promoter activity in transfected MCF-7/STAT3C cells compared to pLuc1267 (Fig. 4C). The single mutation of putative STAT3 binding site at −255/−245 region had no effect on the transcriptional signal. An about 33% depression of signal was observed with single mutation at −168/−159 site and 22% at −137/−122 site (Fig. 4C). To verify that HRG stimulates MMP-7 expression through HER2 mediated STAT3 activation, we transfected MCF-7/HER2 cells with pLuc296mSBE1, pLuc296mSBE2 and pLuc296mSBE3, respectively. After transfection for 24 h, the cells were treated with HRG. Consistent with above data, the treatment with HRG showed no remarkable decrease in luciferase activity when the core-sequence at −255/−245 site was mutated (Fig. 4D). The mutagenesis at −168/−159 and −137/ −122 sites resulted in 51% and 28% decrease of luciferase activity in response to HRG, respectively. It indicates that STAT3 binding site at −168/−159 region is the most important for HRG enhancement of MMP-7 expression. We then determined whether the activity of the MMP-7 promoter could be inhibited by a dominant negative STAT3 with the mutation at the tyrosine phosphorylation site, which is critical for STAT3 activation. After transient cotransfections with pLuc1267 and the dominant negative STAT3 into MCF-7/HER2 cells, HRG-induced transactivation of the MMP-7 promoter was measured. As shown in Fig. 4E, that the expression of the dominant negative STAT3 significantly reduced HRG-induced MMP-7 promoter activity, further confirming that binding of STAT3 to its cognate sites in the MMP-7 promoter facilitates strong MMP-7 induction and STAT3 plays an important role in MMP-7 expression.
The MMPs are frequently overexpressed in various human cancers. Enhanced expressions of MMPs have been associated with an aggressive malignant phenotype and adverse prognosis in cancer patients. Growth factors, cytokines, oncogenes and tumor promoters regulate MMPs both positively and negatively at the transcriptional level. Similar inductive responsiveness of MMP genes to a variety of cellular stimuli has been demonstrated. It has been reported that EGF could directly stimulate MMP-7 gene expression in colon cancer cell lines and FGF-2 increased MMP-7 transcripts in human umbilical vein endothelial cells [27,37]. However, the mechanisms of the transcription regulation of MMP-7 gene are largely unknown. In the present study, increased mRNA and protein expressions of MMP-7 were observed in MCF-7 cells overexpressing HER2. The promoter activity of MMP-7 gene was markedly upregulated in MCF7/HER2 cells. It has been known that HER2 overexpression is related to stromal MMP-2 and MMP-9 expression and proteolytic activity [31]. The expressions of several MMPs upregulated by HER ligands have also been documented [26,27]. It has been postulated that the dysregulation of growth factor-mediated paracrine loops may result in the aberrant HER2 expression. As a direct ligand for HER2 remains to be isolated, it has been suggested that HER2 co-operates with other HER receptor family members forming heterodimers and enhances the signal that links to the cellular migration/invasion machinery. HRG is produced by both stroma and tumor cells. It was reported that mRNA transcripts of HRG were detected in about 25% of human breast carcinomas [38], a percentage similar to HER2 overexpression. This may reflect the enhanced HER2 phosphorylation upon HRG treatment in most of the HER2-overexpressing cancer cell lines. When we treated the cells with HRG, an increased mRNA and protein expression of MMP-7 was observed in both parental MCF-7 and MCF-7/HER2 cells. The activity of the MMP-7 promoter was dramatically enhanced in MCF-7/HER2 cells by HRG induction. It strongly indicates that HER2 mediated signaling directly stimulates MMP-7 gene expression in MCF-7 cells. Since ERK signaling pathway is one of the most important intracellular pathways activated by HER2, we used PD98059 to test
3.5. HRG induces phosphorylation of HER2 and STAT3 It has been demonstrated that the association between the phosphorylated/active form of STAT3 and HER2 and a requirement for HER2 kinase activity to establish constitutive STAT3 activation [36]. To determine whether HRG-triggered activation of MMP-7 correlates with increased phosphorylation of HER2 and STAT3, MCF-7/HER2 cells were incubated in the absence or presence of HRG. Equivalent cell lysates were then processed for determination of HER2 and STAT3 phosphorylation levels by SDS-PAGE and Western blot. The phosphorylation of HER2 in parental MCF-7 cells was at very low level, but it was remarkably elevated by the treatment with HRG. The phosphorylated STAT3 was not detected in parental cells, even with HRG induction (Fig. 5). However, in overexpressing HER2 cells the phosphorylation of HER2 was obvious. HRG induced further increased level of phosphorylated HER2. The constitutively activated STAT3 was observable in MCF-7/HER2 cells and the higher level of phosphorylation could be induced in the presence of HRG (Fig. 5). Our data indicate that the activation of STAT3 was associated with the level of HER2 and HER2 dependent MMP-7 activation was mediated by STAT3 signaling pathway.
Fig. 6. The mechanism of MMP-7 transcription regulated by HER2/STAT3. HRG produced by stroma cells and tumor cells induces the phosphorylation of HER2, which in turn stimulates activation of STAT3. Activated STAT3 translocates into the nucleus, binds to its binding sequences in the MMP-7 promoter and regulates MMP-7 transcription.
G. Yuan et al. / Cellular Signalling 20 (2008) 1284–1291
whether the expression of MMP-7 is regulated by HER2 mediated ERK signaling. However, the induction of MMP-7 promoter activity by HRG was not inhibited by the MEK1/2 inhibitor. Examination of the MMP-7 promoter sequence revealed three potential sites conforming to the TTN(4–6)AA core-sequence crucial for STAT3 binding in the proximal region. Transfection of pLuc1267 into MCF-7/STAT3C cells resulted in a remarkable induction in MMP-7 promoter activity. RT-PCR and Western blot analysis confirmed the expression upregulation of mRNA and protein of MMP-7 in the MCF-7/ STAT3C cells. Binding of STAT3 to MMP-7 promoter was verified by ChIP and the critical STAT3 element within the MMP-7 promoter identified by the mutagenesis of the core STAT3 recognition sequence. These data suggest that HRG stimulated the transcription activation of MMP-7 through STAT3. STATs are latent cytoplasmic transcription factors, but they can be activated by either Janus activated kinases or tyrosine kinase receptors. Activation of STATs has been demonstrated in response to ligands that activate growth factor receptors with tyrosine kinase activity [39]. Constitutive activation of STAT3 is found in many human cancers, including 30 to 60% of primary breast cancer [40,41]. A positive relation between STAT3 and HER2 expressions has also been reported. Our data revealed that STAT3 was phosphorylated when HER2 was overexpressed and the activation of STAT3 was significantly enhanced with the addition of HRG, indicating that HRG-induced HER2-dependent transcriptional upregulation and protein secretion of MMP-7 are mediated by activated STAT3. Our findings may elucidate a mechanism of MMP-7 transcription in MCF-7 cells. HRG produced by stroma cells and tumor cells induces the phosphorylation of HER2, which in turn stimulates activation of STAT3. Activated STAT3 translocates into the nucleus, binds to its binding sequences in the MMP-7 promoter and regulates MMP-7 transcription (Fig. 6). MMP-7 was consistently detected at earlier stages in several cancers. The mechanism by which MMP-7 contributes to early tumor growth is still unknown. One of the hallmarks of tumor cells is apoptosis resistance [42]. Accumulating evidences demonstrate that high levels of HER2 and STAT3 are associated with intrinsic multiple drug resistance [43] and increased metastatic potential. MMP-7 expression has also been considered as an apoptosis resistant phenotype. It has been indicated that the shedding CD95 and CD95 ligand (CD95L) by MMP-7 results in decreased CD95-mediated apoptosis sensitivity of tumor cells [20,42]. Our previous study demonstrates that STAT3 also positively regulates the expression of HER2 in breast cancer cells, implying that HER2 and STAT3 may form a positive feedback loop to participate in breast oncogenesis [28]. The MMP-7 expression may be attributed to HER2 and STAT3 activation in the early tumor development.
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Natural Science Foundation (No. 7051006) and National Basic Research Program of China (973 Program, No. 2006CB504305). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
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Acknowledgements
[38]
We are very grateful to Drs. Bromberg and Darnell for the gifts of pRc/ CMV-STAT3C-Flag and STAT3 dominant negative construct. We thank Dr. Yianjun Jia for providing us pcDNA3/HER2. This work was supported by National Natural Science Foundation of China (No. 30771981), Beijing
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