Cited2 Modulates TGF-B-Mediated Upregulation of MMP9

Y-T Chou1, H Wang1, Y Chen2, D Danielpour1 and Y-C Yang1

1Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA and 2Department of Medical and Molecular Genetics, Indiana University School of Medicine and the Walther Cancer Institute, Indianapolis, IN, USA

Cited (CBP/p300-interacting transactivators with gluta- domain) gene family consists of four nuclear proteins mic acid (E)/aspartic acid (D)-rich C-terminal domain) 2, – Cited1 (formerly MSG1) (Shioda et al., 1996, 1997; which is a CBP/p300-binding transcription co-activator Dunwoodie et al., 1998), Cited2 (formerly MRG1/ without typical DNA-binding domains, has been impli- p35srj) (Shioda et al., 1997; Dunwoodie et al., 1998; cated in control of cell growth and malignant transforma- Sun et al., 1998; Bhattacharya et al., 1999; Leung et al., tion in Rat1 cells. In this report, we provide evidence that 1999), Cited3 (Andrews et al., 2000), and Cited4 Cited2 is an important regulator of transforming growth (formerly MRG2) (Braganca et al., 2002). Members of factor (TGF)-b signaling. Overexpression of Cited2 this family function as transcriptional co-activators enhanced TGF-b-mediated transcription of a Smad- without classical DNA-binding domains. Cited2 Binding Element-containing luciferase reporter construct, interacts with the LIM domain of Lhx2 to enhance SBE4-Luc. This may occur through a direct physical Lhx2-dependent transcription of LH/FSH glycoprotein association of Cited2 with Smads 2 and 3, as supported by a-subunit genes (Glenn and Maurer, 1999). Cited2 and co-immunoprecipitation, mammalian two-hybrid and glu- Cited4 interact with TFAP2, thereby activating TFAP2- tathione S-transferase-pull down assays. The transcription mediated transcription (Braganca et al., 2002, 2003). factor p300, which binds to Smad3, was shown to further Transcriptional responses by Cited2 can be enhanced enhance the interaction between Cited2 and Smad3, and through its association with nuclear receptors, such as the transcriptional responses of Smad3 by Cited2 in peroxisome proliferator activated receptor (PPAR), to reporter assays. Cited2 enhances TGF-b-mediated enhance transcription of target genes (Tien et al., 2004). upregulation of matrix metalloproteinase 9 (MMP9) in Cited2 expression is induced by many cytokines and Cited2 inducible mouse embryo fibroblasts. Overexpression biological stimuli, including IL-1a, -2, -4, -6, -9 and -11, of Cited2 enhanced TGF-b-mediated MMP9 promoter granulocyte/macrophage colony-stimulating factor, reporter activity. Moreover, knockdown of Cited2 in interferon-g, platelet-derived growth factor, insulin, MDA-MB-231 cells attenuated TGF-b-mediated upregula- serum, lipopolysaccharide, and hypoxia in diverse cell tion of MMP9 and TGF-b-mediated cell invasion. types (Sun et al., 1998; Bhattacharya et al., 1999). Chromatin immunoprecipitation showed that Cited2 and Cited2 has been proposed to play an important Smad3 were recruited to MMP9 promoter upon TGF-b function in regulating the cellular responses to hypoxia. stimulation. This is the first demonstration that Cited2 This is supported by the observation that hypoxia functions as a Smad3/p300-interacting transcriptional co- induces Cited2 expression. Elevated level of Cited2 then activator in modulating the expression of MMP9, which functions to downregulate hypoxia inducible factor could affect tumor cell invasion mediated by TGF-b. (HIF)-1-driven transcription by competing with HIF-1a Oncogene (2006) 25, 5547–5560. doi:10.1038/sj.onc.1209552; for binding to CBP/p300 (Bhattacharya et al., 1999; Yin published online 17 April 2006 et al., 2002; Freedman et al., 2003). We and others have shown that disruption of the gene encoding Cited2 is Keywords: Cited2; TGF-b; MMP9; Smad3; p300 embryonic lethal and causes defects in the development of heart and neural tube (Bamforth et al., 2001; Barbera et al., 2002; Yin et al., 2002). Transforming growth factor (TGF)-bs are a highly conserved 25 kDa family of multifunctional autocrine, Introduction paracrine, and endocrine regulators that signal through interaction with cell surface receptors (Attisano and The Cited (CBP/p300-interacting transactivators with Wrana, 2000; ten Dijke et al., 2000). Ligand-activated glutamic acid (E)/aspartic acid (D)-rich C-terminal TGF-b receptors associate with and phosphorylate Smads 2 and 3, leading to nuclear translocation of these Correspondence:Dr Y-C Yang, Department of Pharmacology, Case transcription factors, which serve as key intracellular Western Reserve University School of Medicine, 2109 Adelbert Road, mediators of TGF-b signaling (Attisano and Wrana, W353, Cleveland, OH 44106-4965, USA. E-mail:[email protected] 2000; ten Dijke and Hill, 2004). Receptor-activated Received 3 October 2005; revised 6 February 2006; accepted 22 February Smad2 and Smad3 form hetero-complexes with Smad4, 2006; published online 17 April 2006 a common partner in the assembly of transcriptional Cited2 is a transcriptional modulator in TGF-b signaling pathway Y-T Chou et al 5548 complexes. These complexes are translocated into the nucleus and interact with a variety of transcription factors, such as AP-1, Sp1, FAST, ATF-3, TFE-3, nuclear receptors and E2F4/5, leading to activation or suppression of transcription (ten Dijke et al., 2000; Zimmerman and Padgett, 2000; Shi and Massague, 2003). In addition to DNA-binding transcription factors, the co-activator function of CBP/p300 is essential for Smad-mediated transcription (Feng et al., 1998; Janknecht et al., 1998; Nishihara et al., 1998; Topper et al., 1998). The profile of Smad-binding cofactors during development or under various growth conditions determines cellular responses to TGF-b (Massague, 2000). Cited1 interacts with Smad4 and enhances Smad4- mediated transcription in reporter assays (Shioda et al., Figure 1 Cited2 enhances Smad3-mediated transactivation. 1 Â 105 MDA-MB-231 cells were plated and transfected with 1998; Yahata et al., 2000). Cited1 is downregulated by 300 ng of pSBE4-Luc together with 300 ng of pRK5-Flag-Smad3, TGF-b in melanoma cells (Shioda et al., 1998). pcDNA3.1-Cited2, or both. At 24 h after transfection, cells were Recently, gene expression microarray analysis revealed treated with or without 2.5 ng/ml of TGF-b for another 24 h, that Cited2 is regulated by TGF-b in a variety of cell followed by luciferase assay. Results are representative of three lines (Chen et al., 2001; Kang et al., 2003; Luo et al., individual experiments. *Po0.05 comparing the indicated two bars. þ Po0.05 comparing the indicated two bars. 2005). These studies suggest that Cited2 may play a role in modulating the TGF-b signaling network. To understand the function of Cited2 in TGF-b-mediated signaling pathway and target gene expression, we Luc activity (Figure 1). In the presence of TGF-b, performed microarray analysis of gene expression activated endogenous receptor Smads translocate to the modulated by TGF-b in mouse embryo fibroblasts nucleus and bind SBE, thereby activating transcription (MEFs) following inducible expression of Cited2, and of SBE4-Luc reporter. Cited2 alone enhanced TGF-b- found that Cited2 effectively regulates responses of dependent activation of SBE4-Luc reporter, implying TGF-b on matrix metalloproteinase 9 (MMP9) expres- that activated endogenous receptor Smads may mediate sion. By knocking down Cited2 in MDA-MB-231 cells, this response. Consistent with this notion, under TGF-b we further discovered that Cited2 modulates TGF-b- stimulation, Smad3-mediated transcription was further mediated expression of MMP9 and may play an enhanced in the presence of Cited2 (Figure 1). These important role in tumorigenesis. experiments suggest that Cited2 could function as a co- activator to enhance Smad3-mediated transcription.

Results Cited2 interacts with Smads 2 and 3 We tested whether Cited2 enhances Smad3-mediated Cited2 is a co-activator for Smad3 transcription by interacting with Smads, using a Cellular responses to TGF-b are largely mediated by mammalian two-hybrid system. Gal4-Smad3 fusion Smads, which serve as both transcription factors and protein mediated transcription of Gal4-binding site transcriptional co-regulators. Much of the function of containing reporters, which was further enhanced by Smads is either mediated or modulated through the coexpression of VP16 fused Cited2 (Cited2/VP16) association with a number of other transcription factors (Figure 2a). These data suggest that Cited2 interacts such as junB, ATF-3, and SnoN, whose expression may with Smad3. We constructed Cited2 truncation mutants also be regulated by TGF-b (Jonk et al., 1998; to map the Smad3-interacting region in Cited2. There Stroschein et al., 1999; Kang et al., 2003). In line with are three conserved regions (CR) among different those observations, TGF-b controls the expression of members of the Cited family (Figure 2b). Construct Cited2, as demonstrated in MDA-MB-231 and MCF- aa.1-199 of Cited2/VP16, which includes the CR1 and 10A cells (Chen et al., 2001). Cited1, another member of CR3, but not CR2 domain of Cited2, did not interact Cited family, is a Smad4 co-activator (Shioda et al., with Gal4-Smad3 (Figure 2c). In contrast, construct 1998; Yahata et al., 2000). We thus investigated the role aa.124–269 of Cited2/VP16 containing the CR2, but not Cited2 may play in TGF-b signaling pathway. MDA- CR1 and CR3 domain of Cited2, interacted with Gal4- MB-231 cells were co-transfected with Smad3 and/or Smad3 (Figure 2c), suggesting that the C-terminal Cited2 plus SBE4-Luc, a TGF-b responsive reporter region of Cited2 is essential for the interaction with with four Smad-binding elements (SBE) in the promoter Smad3. Equivalent expression levels of various Cited2 region to which Smad3 binds. Smad3 increased SBE4- constructs were detected in Figure 2c. Luc activity without stimulation of TGF-b (Figure 1), To further conﬁrm the interaction between Cited2 consistent with other reports. Co-transfection of cells and Smad3, glutathione S-transferase (GST) pull-down with Cited2 further enhanced Smad3-mediated tran- assay was performed. Full-length Cited2 and Cited2 scription, although Cited2 alone had no effect on SBE4- mutant without the C-terminal region were in vitro

Oncogene Cited2 is a transcriptional modulator in TGF-b signaling pathway Y-T Chou et al 5549

Figure 2 Cited2 interacts with Smad2 and Smad3. (a)4Â 104 HEK293T cells were co-transfected with 100 ng of MH100 reporter (Gal4 responsive luciferase reporter), pM-Smad3 (Gal4-Smad3), pVP16-HA1 vector containing full-length Cited2 in-frame with VP16- HA1 (Cited2/VP16), and empty pVP16-HA1 vector (100 ng each) in different combination for 48 h, followed by luciferase assay. (b) Schematic representation of Cited2 shows different domains of Cited2 and the deletion constructs used in the experiment. Abbreviation CR represents the conserved region among different members of the Cited family. (c)4Â 104 HEK293T cells were co-transfected with MH100 reporter (100 ng), pM-Smad3 (100 ng), and pVP16-HA1 vector containing different portions of Cited2 (100 ng each) for 48 h, followed by luciferase assay. *Po0.01 comparing to VP16 alone. Cell lystes transfected with different Cited2 deletion VP16-HA fusion construct were subjected to Western blot analysis with antibodies against HA. (d) (Left) Equal volumes of glutathione S-transferase (GST) and GST-Smad3 bound to glutathione sepharose were mixed with in vitro transcribed and translated Myc-tagged wild-type Cited2 (WT) (aa 1–269) and Cited2 mutant (aa1–199). Glutathione S-transferase-pull-down assay was performed as described in Materials and methods. In all, 50% input and pull-down products were separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and detected by Western blot analysis with antibodies specific for Myc. (Right) Equal volumes of GST and GST-Cited2 bound to glutathione sepharose were mixed with in vitro transcribed and translated wild type Myc-tagged Smad3 (WT) (aa 1–425) and Myc-tagged Smad3 mutant without MH2 domain (MH2) (aa 1–225). In all, 50% input and pull-down products were separated by SDS–PAGE and detected by Western blot analysis with antibodies specific for Myc. (e) Top panel shows the binding of Myc-tagged full-length Cited2 to GST-Smad2, GST-Smad3 and GST-Smad4. A total of 50% input and pull-down products were separated by SDS–PAGE and detected by Western blot analysis with antibodies specific for Myc. Bottom panel is Coomassie blue-stained gel showing relative amounts of GST, GST-Smad2, GST-Smad3, and GST-Smad4.

Oncogene Cited2 is a transcriptional modulator in TGF-b signaling pathway Y-T Chou et al 5550 transcribed and translated (Figure 2d). As shown in Cited2À/À MEF cells were immortalized with retrovirus Figure 2d, wild-type Cited2, but not Cited2 C-terminal expressing SV40 large T antigen (LT), followed by stable deletion mutant (Cited2 aa.1–199), interacted with GST- transfection of a tet-off vector (pBPSTR1) containing Smad3, which further confirms that the C-terminal the Cited2 coding sequence to generate Cited2À/À(LT þ , region of Cited2 is necessary for interaction with Smad3. pBPSTR1-Cited2). Cited2 mRNA and protein were not As the MH2 domain of Smad3 is responsible for expressed in tetracycline-treated Cited2À/À(LT þ , interaction with various transcription factors, we tested pBPSTR1-Cited2), but detected in the absence of whether Cited2 interacts with Smad3 mutant (Smad3 tetracycline (Figure 4a and b). We performed high- aa.1–225), which contains the MH1 domain and the density oligodeoxynucleotide microarray analysis of linker region but not the MH2 domain of Smad3. In RNAs from Cited2À/À(LT þ , pBPSTR1-Cited2) in the vitro transcribed/translated full-length Smad3, but not presence or absence of tetracycline for 24 h and TGF-b Smad3 mutant lacking the MH2 domain, interacted stimulation for another 4 h. Matrix metalloproteinase 9 with GST-Cited2 (Figure 2d), supporting that MH2 was one of the genes highly stimulated by TGF-b in domain of Smad3 interacts with Cited2. We tested Cited2À/À(LT þ , pBPSTR1-Cited2) in the absence of whether other Smads interact with Cited2. As shown in tetracycline. By real-time analysis, expression of Cited2 Figure 2e, Cited2 interacted with GST-Smad2 and GST- significantly enhanced TGF-b-mediated expression of Smad3, but not GST-Smad4 fusion protein. These MMP9 (Figure 4c). The protein expression of MMP9 in experiments support our hypothesis that Cited2 inter- conditioned medium was confirmed by Western blot acts with Smads 2 and 3. analysis (Figure 4d). We also performed gelatin zymographic assays to measure changes in MMP9 (gelatinase B, 95–105 kDa) activity following TGF-b stimulation Cited2 promotes Smad3/p300-mediated transcription in the presence or absence of Cited2 (Figure 4e). The Transcriptional co-activator p300 interacts with Smad3 basal expression of MMP9 was similar in Cited2 on and Cited2 through the C-terminus and the CH1 and off cells; however, under TGF-b stimulation, domain of p300, respectively (Nishihara et al., 1998, Cited2 strongly enhanced TGF-b-mediated expression 1999; Bhattacharya et al., 1999). We thus speculated of MMP9 as measured in the gelatin-containing gel that p300 may modulate the interaction between Cited2 (Figure 4e). These data support the conclusion that and Smad3. As p300 interacts with only the phosphory- Cited2 modulates TGF-b-mediated MMP9 expression lated Smad3 (Shen et al., 1998), we co-transfected in Cited2 inducible MEFs. constitutively activated form of TGF-b receptor 1 Chen et al. (2001) showed that Cited2 is one of TGF-b (ALK5*) with Smad3, Cited2, and/or p300 in HEK293T responsive genes in MDA-MB-231 breast cancer cells. cells. Co-transfection with p300 increased the level of We found that TGF-b downregulated Cited2 4 h Cited2 co-immunoprecipitated with Flag-tagged Smad3 following TGF-b treatment in MDA-MB-231 cells, (Figure 3a), indicating that p300 enhances the interac- which express high levels of this transcriptional co- tion between Cited2 and Smad3. It has been shown that activator (Figure 4f). As Cited2 regulates TGF-b- p300 is important for Smad-mediated transcription mediated MMP9 expression in MEFs, we asked whether (Feng et al., 1998; Janknecht et al., 1998; Nishihara Cited2 also modulates MMP9 expression in MDA-MB- et al., 1998; Topper et al., 1998). Transfection of 231 cells. Knockdown of Cited2 using siRNA specific HEK293T cells with p300 alone modestly enhanced for Cited2 in MDA-MB-231 cells resulted in decreased Smad3-mediated transcription of SBE4-Luc (Figure 3b). expression of Cited2 (Figure 4g, lane 3), but the However, co-transfection of both Cited2 and p300 expression level was not affected by si-Control significantly promoted Smad3-mediated transcription (Figure 4g, lane 5). By treatment of MDA-MB-231 cells of SBE4-Luc (Figure 3b). with TGF-b for 24 h, we observed that TGF-b-mediated We further asked whether p300 or Cited2 enhances MMP9 mRNA expression was significantly attenuated Gal4-Smad3-mediated transcription by tethering Smad3 among cells in which Cited2 was knocked down to Gal4 DNA-binding domain. Interestingly, co-trans- (Figure 4h and i). Zymographic assays showed that fection of cells with both p300 and Cited2 significantly MMP9 protein was induced by TGF-b in MDA-MB- enhanced Gal4-Smad3-mediated transcription even in 231 cells, and the stimulated expression was reduced in the absence of TGF-b (Figure 3c). In contrast, co- cells transfected with si-Cited2 (Figure 4j). These transfection of cells with p300 and a Cited2 mutant data support our model that Cited2 modulates TGF- encoding aa.1–199 did not enhance Smad3-mediated b-mediated MMP9 expression in both MEFs and transcription (Figure 3c). These data support the MDA-MB-231 cells. conclusion that Cited2 enhances Smad3/p300-mediated transcription. Smad pathway is involved in upregulation of MMP9 by TGF-b in MDA-MB-231 cells Cited2 enhances TGF-b-mediated expression of MMP9 Smad proteins are key mediators in TGF-b signaling, In order to understand the function of Cited2 in TGF-b and Smad2/3 is involved in upregulation of MMP13 by signaling, we adopted a novel approach by manipulating TGF-b in MDA-MB-231 cells (Selvamurugan et al., tetracycline-controlled expression of Cited2 in Cited2- 2004a, b). We tested whether Smad pathway plays null MEFs, followed by microarray analysis to search a role in TGF-b-mediated upregulation of MMP9 in for TGF-b responsive genes regulated by Cited2. MDA-MB-231 cells. To block Smad pathway in

Oncogene Cited2 is a transcriptional modulator in TGF-b signaling pathway Y-T Chou et al 5551

Figure 3 Cited2 enhances Smad3/p300-mediated transcription through p300/Cited2/Smad3 interaction. (a)3Â 106 HEK293T cells were transfected with pRK5-Flag-Smad3, ALK5*, pcDNA3.1-Cited2, and HA-tagged p300 expression vector (5 mg each) in different combination. After 48 h, cell lysates were immunoprecipitated (IP) for Flag-Smad3 and associated Cited2 was determined by Western blotting (WB). The input represents 10% of the lysate. ALK5* represents a constitutive active TGF-b type I receptor (T204D). (b) 4 Â 104 HEK293T cells were co-transfected with 300 ng of pSBE4-Luc together with pRK5-Flag-Smad3 (300 ng), ALK5* (300 ng), pcDNA3.1-Cited2 (300 ng), and HA-tagged p300 expression vectors (300 ng) in different combination. At 48 h after transfection, cells were lysed and subjected to luciferase assay. *Po0.01 comparing the two indicated bars. **Po0.01 comparing the two indicated bars. (c)4Â 104 HEK293T cells were co-transfected with 100 ng of MH100 reporter (Gal4 responsive luciferase reporter), 100 ng of pM- Smad3 (Gal4-Smad3), HA-p300 (100 ng), and pcDNA 3.1-Cited2 or Cited2 aa1–199 expression vectors (100 ng each) in different combination for 48 h, followed by luciferase assay. The empty vector was co-transfected to maintain an equal amount of DNA in each experiment. Results are representative of three different experiments.

MDA-MB-231 cells, we overexpressed Smad7, an or deletions, which were then used to study regulation inhibitory Smad. Overexpression of Smad7 attenuated of transcriptional control of MMP9 by TGF-b in MDA- phosphorylation of Smad2 (Figure 5a) and upregulation MB-231 cells. Deletions upstream of À151 bp in of MMP9 mRNA (Figure 5b and c) by TGF-b. the MMP9 promoter region maintained TGF-b- Zymographic assay further demonstrated that protein mediated response (Figure 6a). The proximal (À73/ expression of MMP9 induced by TGF-b was reduced in À79) AP-1 site (TGAGTCA) is well conserved among cells with overexpression of Smad7 (Figure 5d). These human, rat, and mouse MMP9 promoters, and has data support the conclusion that the Smad pathway is high sequence homology to the TGF-b responsive involved in TGF-b-mediated upregulation of MMP9, elements, TGACTCA and TGAGTTCA, in human a target gene for Cited2, in MDA-MB-231 cells. MMP13 and rat PAI-1 promoters, respectively (Uria et al., 1998; Guo et al., 2005). Given that both proximal Cited2 enhances TGF-b-mediated MMP9 promoter AP-1 and NF-kB cis-elements are essential for mitogen- reporter activity induced MMP9 upregulation (Ma et al., 2004), proximal To localize TGF-b responsive region in the MMP9 AP-1 and NFkB mutants of MMP9 promoter were promoter, we designed a series of MMP9 promoter– further tested for TGF-b-mediated response. Matrix luciferase reporter constructs, with point mutations metalloproteinase 9 promoter with a speciﬁc mutation in

Oncogene Cited2 is a transcriptional modulator in TGF-b signaling pathway Y-T Chou et al 5552

Figure 4 Cited2 modulates transforming growth factor (TGF)-b-mediated upregulation of matrix metalloproteinase 9 (MMP9). (a) Cited2 inducible mouse embryo fibroblasts (MEFs), Cited2À/À(LT þ , pBPSTR1-Cited2), were generated by retroviral infection of Cited2À/À MEFs with pBPSTR1-Cited2. Tetracycline-mediated downregulation of Cited2 was monitored by Western blot analysis using anti-Cited2 antibody. (b) Cited2À/À(LT þ , pBPSTR1-Cited2) MEFs from (a) were cultured with or without tetracycline for 24 h, followed by adding 2.5 ng/ml of TGF-b or reconstitution buffer to the media for another 4 h. Total RNA was isolated and subjected to Northern analysis with specific probes for Cited2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (c) Total RNA from (b) was subjected to real-time PCR with specific primers for mouse MMP9 and normalized with mouse b-actin. *Po0.01 comparing ÀTet (Cited2 on) to þ Tet (Cited2 off) in the presence of TGF-b.(d) Cited2À/À(LT þ , pBPSTR1-Cited2) MEFs were treated with tetracycline for 24 h, followed by stimulation with 2.5 ng/ml of TGF-b for another 24 h in absence of serum. The serum-free conditioned media were collected and condensed with centricon 3, followed by Western blot analysis with specific antibodies against MMP9. The relative intensity of specific bands was quantified with densitometry. (e) Cited2À/À(LT þ , pBPSTR1- Cited2) MEFs were treated with tetracycline for 24 h, followed by stimulation with 2.5 ng/ml of TGF-b for another 24 h in the absence or presence of serum. The conditioned media were collected, followed by gelatin zymographic assay. (f) MCF-10A, MDA-MB-231, and MCF-7 cells were treated with or without 2.5 ng/ml of TGF-b for 4 h. Total RNA was isolated, followed by Northern analysis with specific probes for Cited2 and GAPDH. (g) MDA-MB-231 cells were transfected with si-Cited2 (lane 3, 4) or si-Control (lane 5, 6; negative control) for 60 h, followed by TGF-b stimulation for another 4 h (lane 2, 4, 6). Total RNA was extracted by Trizol, and Northern analysis was performed. (h) MDA-MB-231 cells were transfected with si-Cited2 or si-Control for 60 h, followed by TGF-b stimulation for another 24 h. Total RNA was extracted, followed by quantitative real time PCR with specific primers for human MMP9 and normalized with human GAPDH. (i) The RT– PCR products of RNA samples from (h) were loaded onto a 12% polyacrylamide gel to demonstrate the relative mRNA levels of MMP9, Cited2, and GAPDH. (j) Serum-free conditioned media from (h) were collected and subjected to gelatin zymographic assay. Results are representative of four individual experiments.

Oncogene Cited2 is a transcriptional modulator in TGF-b signaling pathway Y-T Chou et al 5553

Figure 4 (Continued) the proximal AP-1 site not only decreased the basal how Cited2 regulates the expression of endogenous level expression but also abolished TGF-b-mediated genes upon TGF-b stimulation, we performed real-time upregulation of MMP9 transcription (Figure 6a). polymerase chain reaction (PCR) to monitor the Co-transfection of Cited2 was able to enhance TGF-b- expression of MMP9 and Cited2 mRNA (Figure 7a). mediated expression of wild-type but not MMP9 During the first 4 h of TGF-b stimulation, MMP9 promoter mutated at the AP-1 site (Figure 6b). mRNA expression was not significantly enhanced, Smad7 and dominant negative Smad4 have been shown whereas Cited2 was downregulated by TGF-b. Interest- to inhibit Smad pathway by interfering with phos- ingly, as Cited2 mRNA expression started to resume at phorylation and translocation of endogenous Smads, 12–24 h after stimulation, MMP9 expression was respectively (Hayashi et al., 1997; Zhang et al., strongly enhanced (Figure 7a). As it has been reported 1997). Coexpression of Smad7 or dominant negative before that Smad3 is recruited to the MMP13 promoter Smad4 attenuated Cited2-mediated expression of upon TGF-b stimulation (Selvamurugan et al., 2004a, MMP9 promoter reporter, suggesting that endogenous b), we tested whether Smads and Cited2 are recruited to Smads are required for functional interaction the promoter region of MMP9. Chromatin immuno- with Cited2 to enhance MMP9 expression (Figure 6b). precipitation with the MMP9 promoter was performed These data further confirm that Cited2 functions as a using antibodies against Cited2 and Smads. At 4 h after co-activator to enhance TGF-b-mediated upregulation TGF-b stimulation, occupancy of Smad2/3 on MMP9 of MMP9. promoter was increased (Figure 7b). Interestingly, 24 h after stimulation, occupancy of Cited2 was sharply Transforming growth factor-b regulates MMP9 increased on the MMP9 promoter (Figure 7b), during expression by recruiting Cited2 and Smad3 to which an increased Smad2/3 on the promoter was also the MMP9 promoter observed. These data, therefore, would support our Our findings suggest that Cited2 enhances TGF-b- hypothesis that Cited2 functions as a transcriptional mediated expression of MMP9. To further understand modulator of Smad2/3 by simultaneously binding to the

Oncogene Cited2 is a transcriptional modulator in TGF-b signaling pathway Y-T Chou et al 5554

Figure 5 Smad pathway is involved in transforming growth factor (TGF)-b-mediated upregulation of matrix metalloproteinase 9 (MMP9). (a) MDA-MB-231 cells were transfected with Flag-tagged Smad7 expression vector by retroviral infection as described in Materials and methods. MDA-MB-231 cells transfected with pBABE-Smad7 or pBABE vector alone were treated with or without 2.5 ng/ml of TGF-b for 24 h. Total cell lysates from pools of cells transfected with pBABE-Smad7 or pBABE were then harvested and subjected to Western blot analysis with speciﬁc antibodies against phospho-Smad2 (p-Smad2), Flag, and b-actin. (b) Total RNA from MDA-MB-231 cells transfected with pBABE-Smad7 or pBABE was isolated and subjected to quantitative real-time PCR analysis with speciﬁc primers for MMP9 and normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (c) The RT–PCR products of RNA samples from (b) were loaded onto a 12% polyacrylamide gel to demonstrate the relative mRNA levels of MMP9 and GAPDH. (d) Serum-free conditioned media from (b) were collected and subjected to gelatin zymographic assay. Different pools of cells transfected with pBABE-Smad7 were used and showed the similar results. Results are representative of three different experiments.

MMP9 promoter to enhance TGF-b-mediated MMP9 et al., 2005), we tested whether Cited2 is involved in the expression. invasion of MDA-MB-231 cells. Consistent with the data from others, we found that TGF-b enhanced Cited2 plays a role in TGF-b-mediated invasion of MDA-MB-231 invasion (Figure 8b). Interestingly, MDA-MB-231 cells knockdown of Cited2 through si-Cited2 significantly Cited2 is expressed at a higher level in MDA-MB-231 attenuated TGF-b-mediated invasion of MDA-MB-231 cells, an invasive breast cancer cell line, than in non- cells (Figure 8b). These experiments imply that by invasive lines, such as MCF-10A and MCF-7 cells modulating the expression of MMP9, Cited2 could play (Figure 4f). The MDA-MB-231 cell line is one of the a significant role during tumor invasion. breast cancer cell lines that are resistant to TGF-b- mediated growth arrest (Bandyopadhyay et al., 1999; Chen et al., 2001; Tobin et al., 2002). As Cited2 activates Discussion cell growth (Kranc et al., 2003), we knocked down Cited2 by siRNA and tested whether resistance of Smads are key mediators in the TGF-b signaling MDA-MB-231 cells to TGF-b-mediated growth arrest is pathway. Smad-binding cofactors determine the magni- owing to high expression of Cited2 in MDA-MB-231 tude, duration, and direction (activation or suppression) cells. Knockdown of Cited2 by si-Cited2 did not of Smad-driven transcription during TGF-b stimulation significantly affect the growth rate of MDA-MB-231 (Attisano and Wrana, 2000; ten Dijke and Hill, 2004). under TGF-b stimulation (Figure 8a). As TGF-b Transfection studies support that Cited1 functions as a enhances the invasion of MDA-MB-231 cells in the Smad co-activator (Shioda et al., 1998; Yahata et al., matrigel assay (Ilunga et al., 2004), and Cited2 2000). However, a direct demonstration that Cited1 modulates expression of MMP9 (Figure 4), which has regulates endogenous genes was never performed. In this been suggested to play important roles in the cancer cell study, we have provided evidence supporting that invasion (Itoh et al., 1999; Coussens et al., 2000; Cited2 interacts with Smad3 and functions as a Smad3 Hiratsuka et al., 2002; Santibanez et al., 2002; Javelaud co-activator in TGF-b signaling. Cited2 is recruited to

Oncogene Cited2 is a transcriptional modulator in TGF-b signaling pathway Y-T Chou et al 5555

Figure 7 Cited2 and Smad3 are recruited to the matrix metalloproteinase 9 (MMP9) promoter. (a) MDA-MB-231 cells were stimulated with 2.5 ng/ml of transforming growth factor (TGF)-b for the indicated time points. After TGF-b stimulation, total RNA was subjected to real-time PCR with speciﬁc primers for human MMP9 and normalized with human glyceraldehyde-3- phosphate dehydrogenase. (b) MDA-MB-231 cells were stimulated with 1 ng/ml of TGF-b for indicated time points, followed by chromatin immunoprecipitation with antibodies against Smad2/3, Cited2, or normal IgG. The precipitated DNA was subjected to quantitative real-time PCR with primers that amplify a 196 bp product (À195B þ 1) covering the TGF-b responsive region in the MMP9 promoter and normalized with the input products. Results are representative of two different experiments. *Po0.05 compared to zero time point of TGF-b stimulation. **Po0.05 compared to zero time point of TGF-b stimulation.

Figure 6 Cited2 enhances the expression of matrix metalloproteinase 9 (MMP9) promoter reporter. (a) Schematic representation of mutant constructs in the human MMP9 promoter. The promoter DNA-binding transcription factors such as Lhx2 (Glenn was divided into (a–f) subregions. 5 Â 104 MDA-MB-231 cells were and Maurer, 1999), TFAP2 (Braganca et al., 2002; plated and transfected with 300 ng of various deletion constructs of Braganca et al., 2003), the nuclear receptor PPAR (Tien MMP9 promoter reporters as indicated. At 24 h after transfection, cells were treated with or without 2.5 ng/ml of transforming growth et al., 2004), and HIF-1a (Bhattacharya et al., 1999; factor (TGF)-b for another 24 h, followed by luciferase assay and Freedman et al., 2003; Fox et al., 2004) through normalized to sea pansy luciferase activity of co-transfected pRL- interaction with CBP/p300. The role for CBP/p300 as SV40. (b)5Â 104 MDA-MB-231 cells were plated and co-transfected an essential co-activator in Smad driven gene expression with 300 ng of wild-type MMP9 (black bars) or AP-1 mutant (white bars) promoter reporter and together with pcDNA3.1-Cited2 has been well documented by observations that over- (100 ng), Flag-CMV2-Smad7 (100 ng), and 100 ng of dominant expression of E1A antagonizes the function of CBP/ negative Smad4 expression vector (DN-Smad4) in different combi- p300, which leads to attenuation of Smad-driven nation. At 24 h after transfection, cells were treated with or without transcription (Feng et al., 1998; Nishihara et al., 1998; 2.5 ng/ml of TGF-b for another 24 h, followed by luciferase assay Pouponnot et al., 1998; Topper et al., 1998). However, and normalized to sea pansy luciferase activity of co-transfected pRL-SV40. Results are representative of two independent experi- coexpression of CBP/p300 has modest effects on Smad- ments. *Po0.05 compared to wild-type MMP9 promoter in the mediated transcription based on several promoter/ presence of TGF-b.**Po0.05 compared to wild-type MMP9 reporter assays (Feng et al., 1998; Warner et al., 2004). promoter with Cited2 in the presence of TGF-b. The effects of p300 on Smad-driven transcription may rely on the recruitment of other co-activators to the the endogenous MMP9 promoter along with Smads to Smad/p300 complex (Dennler et al., 2005). We show enhance TGF-b-mediated MMP9 expression. Members that p300 enhances the interaction between Cited2 and of the Cited family modulate transcription mediated by Smad3 (Figure 3a). Coexpression of Cited2 and p300

Oncogene Cited2 is a transcriptional modulator in TGF-b signaling pathway Y-T Chou et al 5556 degrading extracellular matrix (ECM), and strict regulation of MMP synthesis is critical for the maintenance of proper ECM expression (Sternlicht and Werb, 2001). In disease states such as cancer, there is often high MMP activity at the tumor–stroma interface (Lynch and Matrisian, 2002). Excessive deposition of ECM has been detected in Engelbreth–Holm–Swarm (EHS) tumors and differentiated F9 embryonic carcinoma (F9-PE). Interestingly, Cited1 and Cited2 are highly expressed in EHS tumors and upregulated during the differentiation of F9-PE, suggesting that Cited1 and Cited2 may be involved in the regulation of ECM (Futaki et al., 2003). Matrix metalloproteinase 9 is activated through both proteolytic and non-proteolytic mechanisms. Full-length MMP9 is the major form of MMP9 in cancer tissues (Fridman et al., 2003) and in MDA-MB-231, an invasive breast cancer cell line. Several studies have suggested that without the partici- pation of proteolytic enzymes and removal of the inhibitory peptide, full-length MMP9 can be activated by binding to the substrates to cause conformational changes or oxidative modification of the cysteine side- chain thiol groups, in turn allowing zinc ion to become Figure 8 Cited2 enhances transforming growth factor (TGF)-b- available for the catalytic function (Okamoto et al., mediated invasion of MDA-MB-231 cells. (a) MDA-MB-231 cells 2001; Bannikov et al., 2002; Gu et al., 2002). Transform- were transfected with si-Cited2 or si-Control for 60 h, followed by TGF-b stimulation. At 33 h after TGF-b stimulation, cells were ing growth factor-b is a potent growth inhibitor of pulse labeled with [3H]thymidine to measure the growth rate. (b) normal epithelial cells, but also fosters tumor formation MDA-MB-231 cells were transfected with si-Cited2 or si-Control during later stages of tumorigenesis (Wakefield and for 60 h, followed by TGF-b stimulation for another 24 h. Cells Roberts, 2002). Transforming growth factor-b enhances were detached and resuspended in mimimum essential medium the expression of MMP9, which plays significant roles (MEM) containing 0.1% BSA with antibiotics, with or without TGF-b in the absence of serum. A total of 1 Â 105 cells were added during the dissemination and growth of cancer cells to the upper well of a 24-well matrigel invasion chamber (BD (Itoh et al., 1999; Coussens et al., 2000; Hiratsuka et al., Biosciences) and incubated at 371C for 20 h. The lower chamber 2002). contained MEM with 5% fetal bovine serum. Number of migrated Although Cited2 is a transforming gene, as we cells through matrigel was quantified according to in vitro invasion assay described in Materials and methods. Results are representa- previously showed, and overexpression of Cited2 in tive of five individual experiments. *Po0.5 compared to si-Control Rat1 cells causes tumor formation in nude mice (Sun in the presence of TGF-b. et al., 1998), the mechanism involved in tumor formation was unclear. Our current demonstration that Cited2 modulates TGF-b-mediated upregulation of MMP9 and enhances in vitro tumor cell invasion may in part explain enhances Smad3-mediated transcription (Figure 3b and why overexpression of Cited2 promotes tumor forma- c), which corroborates the notion that Cited proteins tion in nude mice. In addition to MMP9, Cited2 also represent a novel family of transcription modulators modulates MMP13 expression in MDA-MB-231 cells to regulate the activities of various CBP/p300 com- (Supplementary Information). Yokota et al. (2003) plexes. It is interesting that overexpression of p300 showed that, in a human chondrocyte cell line C-28/I2, without Cited2 and in the absence of TGF-b decreased MMP1 and MMP13 are downregulated by TGF-b, Gal4-Smad3-mediated transcription (Figure 3c). With- possibly through Cited2. In addition, they showed that out TGF-b receptor-mediated phosphorylation, wild- Cited2 is upregulated by TGF-b (Yokota et al., 2003), type Smad3 does not interact with p300 (Shen et al., which is different from our study and those of others 1998). Overexpression of p300 may interact with other (Chen et al., 2001; Tardif et al., 2001; Kang et al., 2003; endogenous transcription factor complexes, such as Luo et al., 2005). These discrepancies could be owing to Cited2 and TFAP2, to decrease the endogenous pool the use of different cell lines. Although Cited2 over- of free Cited2, and in turn affect Gal4-Smad3-mediated expression enhances TGF-b-mediated expression of transcription. These data suggest other transcription MMP9 in Cited2 inducible MEFs, MMP9 upregulation factors including Cited2 are involved in Smad3/p300- by TGF-b is not totally abolished in the Cited2 null mediated transcription. background, suggesting that other transcription factors Our microarray analysis with tet-off inducible Cited2 or cofactors may also be involved in MMP9 upregula- MEF and siRNA-mediated knockdown experiments tion by TGF-b. in MDA-MB-231 cells show that Cited2 modulates Smad proteins are the main trans-acting factors TGF-b-mediated expression of MMP9. Matrix metallo- involved in TGF-b-stimulated MMP13 expression in proteinase 9 are matrix metalloproteinases capable of MDA-MB-231 cells (Selvamurugan et al., 2004a, b).

Oncogene Cited2 is a transcriptional modulator in TGF-b signaling pathway Y-T Chou et al 5557 It has been reported that the AP-1 site in the MMP13 signaling. Cited2 functions as a Smad2/3-binding co- promoter region is essential for TGF-b-stimulated activator and is important for Smad3/p300-mediated MMP13 expression (Uria et al., 1998; Selvamurugan transcription. In MEFs and MDA-MB-231 cells, Cited2 et al., 2004b). Here, we demonstrated that mutation of modulates TGF-b-mediated upregulation of MMP9. the proximal AP-1 site in the MMP9 promoter abolishes Knockdown of Cited2 in MDA-MB-231 cells attenuates TGF-b-mediated response. In addition to Smad-binding TGF-b-mediated invasion of MDA-MB-231 cells. elements, Smads are recruited to the promoter through Unraveling the role of Cited2, a novel modulator in AP-1 complexes (Zhang et al., 1998; Yamamura et al., TGF-b signaling, could contribute to the understanding 2000). Although Zhang et al. (1998) have showed that of effects of TGF-b signaling on development and Smad3 directly binds to AP-1 sequences in MMP1 tumorigenesis. promoter, it is also suggested that Smad2/3 may interact with transcription factors, such as junB and Cbfa1/ runx2, rather than directly binding to the DNA for the Materials and methods MMP13 promoter activity (Selvamurugan et al., 2004 b). Whether Smad3 and Cited2 directly bind to the Reagents and antibodies MMP9 promoter or are recruited through other DNA- Recombinant human TGF-b1 was purchased from R&D binding transcription factors requires further investiga- Systems (Minneapolis, MN, USA). Tetracycline and puro- tion. We have observed that more Cited2 are recruited mycin were obtained from Sigma (St Louis, MO, USA). Anti- to the MMP9 promoter in the absence of TGF-b than MMP9 antibodies were ordered from Chemicon, Temeculo, 4 h after stimulation. As Cited2 interacts with transcrip- CA, USA. Anti-Myc (A14), anti-Smad1/2/3 (SC7960), anti- Cited2 (MRG1, JA22) antibodies, and normal IgG1 (SC3877) tion factors such as TFAP2 and Lhx2 other than Smads, were purchased from Santa Cruz Biotechnology (Santa Cruz, we do not rule out the possibility that in the absence of CA, USA). Anti-Flag (M2) and anti-b-actin antibodies were TGF-b, TFAP2 may also recruit Cited2 to the MMP9 obtained from Sigma (St Louis, MO, USA). Anti-MMP13 promoter. (Ab-4) antibodies were purchased from Calbiochem (San TGF-b activates Smads 2 and 3 by rapid (i.e., 15 min) Diego, CA, USA). yet transient phosphorylation (Selvamurugan et al., 2004a, b). It is difficult to reconcile that MMP9 and Cell culture MMP13 expression stimulated by TGF-b does not MDA-MB-231 and MCF-7 cells were obtained from the significantly occur till 24 h following stimulation (Selva- American Type Culture Collection. MCF-10A was provided murugan et al., 2002). Our data here imply that Cited2 by Dr Amy Wilson-Delfosse (Case Western Reserve Uni- plays an essential role in regulating TGF-b-mediated versity). Primary MEF cultures were harvested and maintained MMP9 expression. Cited2 is recruited to the MMP9 as described (Yin et al., 2002). For generation of MEF cell promoter and the kinetics of Cited2 expression corre- lines, primary MEFs were immortalized by retroviral infection with a retroviral vector containing simian virus 40 LT antigen lates with the mRNA levels of MMP9 during TGF-b (Williams et al., 1988), provided by Dr David Williams stimulation. These data suggest that by modulating the (University of Cincinnati). Inducible Cited2 MEFs, Cited2À/À expression of Cited2, TGF-b creates a temporal and (LT þ , pBPSTR1-Cited2), were generated from a SV40 LT- specific control to regulate the expression of late TGF-b immortalized Cited2À/À MEF line infected with the pBPSTR1 responsive genes such as MMP9. Several regulatory vector containing Cited2 cDNA. Mouse embryo fibroblasts mechanisms that create temporal controls in TGF-b and HEK293T cells were maintained in Dulbecco’s modified signaling network have been demonstrated. SnoN, a Eagle’s medium (DMEM) containing 10% heat-inactivated Smad-binding repressor, is rapidly degraded in response fetal bovine serum (FBS) (HyClone, Logon, UT, USA). to TGF-b. At later stages of TGF-b stimulation, TGF-b MDA-MB-231 and MCF-7 cells were maintained in minimum induces SnoN expression, resulting in termination of essential medium supplemented with 1 nM insulin (Sigma) and 10% heat-inactivated FBS. MCF-10A cells were maintained in Smad-mediated transactivation (Stroschein et al., 1999; a 1:1 mixture of DMEM and Ham’s F-12 supplemented with Sun et al., 1999). Smad7, an antagonistic Smad induced 25% horse serum (Invitrogen, Auckland, New Zealand), by TGF-b, interferes with the binding between Smad2/3 10 mg/ml insulin, 0.5 mg/ml hydrocortisone (Sigma), and and TGF-b receptor, to attenuate TGF-b-mediated 20 ng/ml epidermal growth factor (Sigma). responses (Hayashi et al., 1997). We also show that Cited2 is a Smad3/p300 co-activator, but is modulated Plasmids by TGF-b in MDA-MB-231 cells. As Cited2 expression Mouse Cited2 was cloned from mouse genomic DNA, resumes at later time points of TGF-b stimulation, followed by subcloning into pcDNA3.1(À)B, pM, pVP-HA1, Cited2 enhances Smad2/3-mediated upregulation of and PGEX4T1. pM was purchased from CLONTECH, Palo MMP9. Interestingly, Cited1, another member of the Alto, CA, USA and pVP16-HA1 was provided by Dr Richard Cited family and a Smad4-interacting co-activator, is Baer (University of Texas Southwestern Medical Center). pM also downregulated by TGF-b in B16-F1 melanoma was used to generate a fusion protein with the Gal4-DNA- (Shioda et al., 1998). Whether such a temporal control binding domain. MH100, a Gal4 responsive luciferase reporter, was provided by Dr Hung-Ying Kao (Case Western identified in this study reflects a unique property also Reserve University). Cited2 deletion mutants were PCR shared by other Cited family members to modulate amplified and cloned into pVP16-HA1 and pcDNA3.1(À)B. TGF-b responses will require further investigation. Rat Smad3 was subcloned into pM and pcDNA3.1(À)B. Rat In conclusion, the present data provide strong Smad3 mutant lacking MH2 domain was PCR amplified and evidence for the involvement of Cited2 in TGF-b subcloned into pcDNA3.1(À)B. pRK5-Flag-Smad3 is a gift

Oncogene Cited2 is a transcriptional modulator in TGF-b signaling pathway Y-T Chou et al 5558 from Dr Rick Derynck (University of California, San tion of tet-off inducible Cited2 MEF lines, a Cited2À/À MEF Francisco). pSBE4-Luc was a gift from Dr Bert Vogelstein line infected with retrovirus expressing Cited2 was selected (Johns Hopkins University). ALK5*(T204D) and dominant with 2.5 mg/ml of puromycin for 1 week. Individual clones negative Smad4/DPC4(1–514) were provided by Dr Joan were picked and Cited2 protein and mRNA expression levels Massague (Memorial Sloan-Kettering Cancer Center). were monitored 24 h after culturing cells in 2 mg/ml of pBPSTR1 was obtained from Dr Steven Reeves (Harvard tetracycline. For generation of Smad7-overexpression cells, University). Flag-tagged Smad7 with a consensus Kozak MDA-MB-231 cells were infected with retrovirus expressing sequence was PCR amplified from Flag-CMV2 vector and Flag-tagged Smad7, followed by puromycin selection for 1 subcloned into pBABE-puro. A series of deletion mutants of week. In order to avoid clonal variations, pools of transfec- the human MMP9 promoter reporter constructs were made tants expressing Smad7 or vector alone were used and and described previously (Ogawa et al., 2004). Wild-type, subjected to analysis after puromycin selection and Western NF-kB, and AP-1 mutant of MMP9 promoter reporter were blot confirmation. kind gifts from Dr Hiroshi Sato (Cancer Research Institute, Kanazawa University, Kanazawa, Japan) (Sato and Seiki, Western blot analysis 1993). Cells were lysed into radioimmunoprecipitation assay buffer (50 mM Tris-HCL, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM Luciferase assay, DNA transfection, and siRNA sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, For luciferase assays, MDA-MB-231 cells were transfected 20 mg/ml aprotinin, and 20 mg/ml leupeptin). Lysates were using Lipofectamine Plus reagent (Invitrogen) following the fractionated by SDS–PAGE and transferred to poly- manufacturer’s instructions. HEK293T cells were transfected vinylidene difluoride membranes (PVDF-plus; Osmonics Inc., by the calcium phosphate method. Luciferase activity in cell Westborough, MA, USA). Membranes were incubated with lysates was determined by a dual luciferase reporter assay primary antibodies followed by incubation with secondary system (Promega, Madison, WI, USA) and normalized to sea antibodies conjugated to horseradish peroxidase. Reacted pansy luciferase activity of co-transfected pRL-CMV or pRL- secondary antibodies were detected using an enhanced SV40. Statistical significance was determined by Student’s chemiluminescence detection system (Amersham Pharmacia t-test. For siRNA knockdown experiment, MDA-MB-231 Biotech, Piscataway, NJ, USA). cells were transfected with siRNA using Effectene transfection reagent (Qiagen, Hilden, Germany) for 60 h following the Northern blot analysis manufacturer’s instructions. siRNAs for Cited2 and ZNF76 Total RNA was isolated from cells using Trizol reagent (a negative control) were ordered from Dharmacon Research (Invitrogen) following the manufacturer’s instructions. In (Lafayette, CO, USA). The siRNA sequence for Cited2 is total, 10 mg per lane of total RNA was separated by 2.2 M 50-ugacggacuucgugugcaa-30 and for ZNF76 50-ccagcgccaccaa formaldehyde agarose gel electrophoresis, and transferred to a cuauaa-30. The reconstitution of siRNA was performed nylon membrane (Magna; Osmonics Inc.). following the manufacturer’s instructions. SYBER-Green real-time PCR Purification of GST fusions, in vitro transcription/translation, Real time (RT)–PCR primer pairs are:mouse MMP9 and GST pull-down assay (50-tgtctggagattcgacttgaagtc-30 and 50-tgagttccagggcacacca-30); Purification of GST-fusion proteins and GST pull-down assay mouse b-actin (50-accaactgggacgatatggagaaga-30 and 50-cgcac have been described previously (Chipuk et al., 2002). Briefly, gatttccctctcagc-30); human Cited2 (50-accatcaccctgcccacc-30 cDNA from wild type of Cited2 (aa1–269), Cited2 C-terminal and 50-cgtagtgtatgtgctcgccca-30); human MMP9 (50-tgacagcga deletion mutant (aa 1–199), wild type of Smad3 (aa1–425), and caagaagtg-30 and 50-cagtgaagcggtacatagg-30); human MMP13 Smad3 deletion mutant (aa 1–225) were subcloned into (50-ttcctcttcttgagctggactca-30 and 50-tctgcaaactggaggtcttcct-30); pcDNA3.1(À)B and in vitro transcribed and translated using Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) TNT Coupled Reticulocyte Lysate Systems (Promega). Gluta- (50-gaaggtgaaggtcggagtc-30 and 50-gaagatggtgatgggatttc-30). thione S-transferase fusion proteins were immobilized on Total RNA was isolated by the Trizol (Invitrogen) method Glutathion-sepharose beads first. In vitro transcribed and and reverse transcribed using the SuperScriptt First-Strand translated peptides were incubated with 10 mg of GST-fusion Synthesis System for RT–PCR (Invitrogen). Real time PCR protein in phosphate-buffered saline (PBS) supplemented with was performed with iQt SYBER-Green Supermix (Bio-Rad, 0.1% Triton X-100 and rotated overnight at 41C. The beads Hercules, CA, USA) in a MyiQ thermocycler (Bio-Rad) using were washed with the PBS/0.1% Triton X-100 buffer three a SyBr Greent detection protocol as outlined by the times and resuspended in 50 mlof2Â sodium dodecyl sulfate manufacturer. Quantitations were normalized to endogenous (SDS) sample buffer. The pull-down products were heated at GAPDH or b-actin. The relative quantitation value for each 951C for 5 min and applied to SDS–polyacrylamide gel target gene compared to the calibrator for that target is ÀðCtÀCcÞ electrophoresis (SDS–PAGE). expressed as 2 (Ct and Cc are the mean threshold cycle differences after normalizing to GAPDH or b-actin). Retrovirus infection pBPSTR1-Cited2 was generated by inserting Cited2 Zymographic assay cDNA into multiple cloning sites of pBPSTR1, a retroviral Cited2À/À(LT þ , pBPSTR1-Cited2) MEFs treated with tetra- tet-off vector. pBABE-Smad7 was generated by inserting cycline, or MDA-MB-231 cells transfected with siRNA, were Smad7 cDNA into multiple cloning sites of pBABE-puro, a stimulated with or without TGF-b. After 24 h, the conditioned retrovirus vector. Phoenix packaging cells were used to media were collected and mixed with Â 5 loading buffer (4% generate amphotropic retroviruses. Briefly, Phoenix cells were SDS, 20% glycerol, 0.01% bromophenol blue, and 125 mM seeded at a density of 3.5 Â 106 cells per 10-cm dish, Tris-HCl, pH 6.8). The samples were subjected to 7.5% SDS– and transfected with 10 mg of retrovirus vector by the calcium PAGE containing 2 mg/ml gelatin (Sigma). After electrophor- phosphate method the following day. At 48 h after transfec- esis, the gel was washed three times with 2.5% Triton X-100, tion, virus-containing supernatant was collected. For genera- briefly with distilled water, and incubated with reaction buffer

Oncogene Cited2 is a transcriptional modulator in TGF-b signaling pathway Y-T Chou et al 5559 (50 mM Tris-HCl, pH 7.5, 10 mM CaCl2,1mM ZnCl2, 150 mM volumes of ethanol and 5 ml of 10 mg/ml glycogen. The input NaCl, 1% Triton X-100, and 0.002% sodium azide) for 16 h at lysates were processed as above. DNA was resuspended in 371C. The gel was stained with 0.25% Coomassie Brilliant water and analysed by quantitative real-time PCR. The primer Blue R-250 and destained with 40% methanol, 10% acetic acid sequences of the human MMP9 promoter used are:5 0- as previously described (Heussen and Dowdle, 1980). agagaggaggaggtggtgtaagc-30 and 50-ttggtgagggcagaggtgtc-30.

Microarray measurement [3H]thymidine incorporation assay Cited2À/À(LT þ , pBPSTR1-Cited2) MEFs were incubated with medium containing 2 mg/ml tetracycline or solvent for 24 h, MDA-MB-231 cells were transfected with siRNA. After 60 h, cells were washed with PBS and plated into 12-well plates at a followed by stimulation with 2.5 ng/ml TGF-b or reconstitu- 4 tion buffer for another 4 h. Total RNA was extracted by Trizol density of 2 Â 10 cells per well. The following day, cells were stimulated with or without 2.5 ng/ml of TGF-b for 33 h. Cells and high-density oligodeoxynucleotide microarray analysis 3 was performed in the Gene Expression Analysis Core Facility were labeled with 4 mCi of [ H]thymidine for 3 h and fixed with 1 of Case Western Reserve University. 10% trichloroacetic acid (TCA) for 30 min at 25 C, followed by two washes with 10% TCA. DNA was solubilized by incubation in 500 ml of 0.2 N NaOH for 30 min, and radio- Chromatin immunoprecipitation (ChIP) activity was counted using 400 ml of solubilized DNA in 4 ml 1 Cells were incubated for 10 min at 37 C with medium scintillation fluid. containing 1% formaldehyde. Formaldehyde crosslinking was stopped by adding glycine to a final concentration of 125 mM for 5 min at 371C. Cells were then washed with ice-cold Matrigel invasion assay PBS containing protease inhibitors and 1 mM phenylmethyl- Invasion through BM Matrigel was performed as previously sulfonyl fluoride and resuspended in SDS lysis buffer contain- described with minor modifications (Melchiori et al., 1987). ing protease inhibitors for 5 min on ice. Samples were MDA-MB-231 cells were transfected with siRNA. After 60 h, sonicated to reduce the DNA length to 200–600 bp, cellular cells were stimulated with or without 2.5 ng/ml of TGF-b for debris was removed by centrifugation, and the supernatant another 24 h. Cells were detached and resuspended in was diluted 20-fold in dilution buffer supplemented with mimimum essential medium (MEM) containing 0.1% BSA protease inhibitors. Before ChIP, the samples were precleared with antibiotics, with or without TGF-b in the absence of with protein A-agarose/carrier DNA/tRNA mixture. The serum. A total of 1 Â 105 cells were added to the upper well of a supernatant was recovered and used directly for immunopre- 24-well matrigel invasion chamber (BD Biosciences, Bedford, cipitation experiments by incubation with appropriate anti- MA, USA) and incubated at 371C for 20 h. The lower chamber bodies overnight at 41C. Immune complexes were mixed with contained MEM with 5% FBS. Cells that traversed through protein A-agarose/carrier DNA/tRNA mixture followed by the filter were fixed, stained with 0.5% (w/v) crystal violet and incubation for 1 h at 41C. After immunoprecipitation, beads counted in five random fields per insert. Each assay was were collected and sequentially washed twice with 1 ml each of repeated five times and performed in triplicate. the following buffers:low-salt wash buffer, high-salt wash buffer, LiCl wash buffer, and TE buffer. The immunocom- plexes were eluted twice by adding a 250 ml aliquot of a freshly Acknowledgements prepared solution of 1% SDS/0.1 M NaHCO3. Then, 20 mlof 5 M NaCl and 1 ml of 10 mg/ml RNase were added to the We are grateful to Dr David Donner for critical reading of samples and the crosslinking reaction was reversed by 6 h manuscript; and Drs Edward Stavnezer, Paul MacDonald, and incubation at 651C. The samples were then digested with Monica Montano for helpful discussion and advice. This work proteinase K at 421C for 1 h, and DNA was recovered by was supported by grants from National Institute of Health phenol/chloroform extraction and precipitated with two (RO1 CA78433 to YCY).

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Oncogene