Oncogene (2006) 25, 5547–5560 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE 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 under- stand 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.