ORIGINAL ARTICLE Cited2 modulates TGF-b-mediated upregulation of MMP9 Y-T Chou 1 , H Wang 1 , Y Chen 2 , D Danielpour 1 and Y-C Yang 1 1 Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA and 2 Department 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- mic acid ( E)/aspartic acid ( D)-rich C-terminal domain) 2, which is a CBP/p300-binding transcription co-activator without typical DNA-binding domains, has been impli- cated in control of cell growth and malignant transforma- tion in Rat1 cells. In this report, we provide evidence that Cited2 is an important regulator of transforming growth factor (TGF)-b signaling. Overexpression of Cited2 enhanced TGF-b-mediated transcription of a Smad- Binding Element-containing luciferase reporter construct, SBE4-Luc. This may occur through a direct physical association of Cited2 with Smads 2 and 3, as supported by co-immunoprecipitation, mammalian two-hybrid and glu- tathione S-transferase-pull down assays. The transcription factor p300, which binds to Smad3, was shown to further enhance the interaction between Cited2 and Smad3, and the transcriptional responses of Smad3 by Cited2 in reporter assays. Cited2 enhances TGF-b-mediated upregulation of matrix metalloproteinase 9 (MMP9) in Cited2 inducible mouse embryo fibroblasts. Overexpression of Cited2 enhanced TGF-b-mediated MMP9 promoter reporter activity. Moreover, knockdown of Cited2 in MDA-MB-231 cells attenuated TGF-b-mediated upregula- tion of MMP9 and TGF-b-mediated cell invasion. Chromatin immunoprecipitation showed that Cited2 and Smad3 were recruited to MMP9 promoter upon TGF-b stimulation. This is the first demonstration that Cited2 functions as a Smad3/p300-interacting transcriptional co- activator in modulating the expression of MMP9, which could affect tumor cell invasion mediated by TGF-b. Oncogene (2006) 25, 5547–5560. doi:10.1038/sj.onc.1209552; published online 17 April 2006 Keywords: Cited2; TGF-b; MMP9; Smad3; p300 Introduction The Cited ( CBP/p300- interacting transactivators with glutamic acid ( E)/aspartic acid ( D)-rich C-terminal domain) gene family consists of four nuclear proteins – Cited1 (formerly MSG1) (Shioda et al., 1996, 1997; Dunwoodie et al., 1998), Cited2 (formerly MRG1/ p35srj) (Shioda et al., 1997; Dunwoodie et al., 1998; Sun et al., 1998; Bhattacharya et al., 1999; Leung et al., 1999), Cited3 (Andrews et al., 2000), and Cited4 (formerly MRG2) (Braganca et al., 2002). Members of this family function as transcriptional co-activators without classical DNA-binding domains. Cited2 interacts with the LIM domain of Lhx2 to enhance Lhx2-dependent transcription of LH/FSH glycoprotein a-subunit genes (Glenn and Maurer, 1999). Cited2 and Cited4 interact with TFAP2, thereby activating TFAP2- mediated transcription (Braganca et al., 2002, 2003). Transcriptional responses by Cited2 can be enhanced through its association with nuclear receptors, such as peroxisome proliferator activated receptor (PPAR), to enhance transcription of target genes (Tien et al., 2004). Cited2 expression is induced by many cytokines and biological stimuli, including IL-1a, -2, -4, -6, -9 and -11, granulocyte/macrophage colony-stimulating factor, interferon-g, platelet-derived growth factor, insulin, serum, lipopolysaccharide, and hypoxia in diverse cell types (Sun et al., 1998; Bhattacharya et al., 1999). Cited2 has been proposed to play an important function in regulating the cellular responses to hypoxia. This is supported by the observation that hypoxia induces Cited2 expression. Elevated level of Cited2 then functions to downregulate hypoxia inducible factor (HIF)-1-driven transcription by competing with HIF-1a for binding to CBP/p300 (Bhattacharya et al., 1999; Yin et al., 2002; Freedman et al., 2003). We and others have shown that disruption of the gene encoding Cited2 is 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, paracrine, and endocrine regulators that signal through interaction with cell surface receptors (Attisano and Wrana, 2000; ten Dijke et al., 2000). Ligand-activated TGF-b receptors associate with and phosphorylate Smads 2 and 3, leading to nuclear translocation of these transcription factors, which serve as key intracellular mediators of TGF-b signaling (Attisano and Wrana, 2000; ten Dijke and Hill, 2004). Receptor-activated Smad2 and Smad3 form hetero-complexes with Smad4, a common partner in the assembly of transcriptional Received 3 October 2005; revised 6 February 2006; accepted 22 February 2006; published online 17 April 2006 Correspondence: Dr Y-C Yang, Department of Pharmacology, Case Western Reserve University School of Medicine, 2109 Adelbert Road, W353, Cleveland, OH 44106-4965, USA. E-mail: yxy36@cwru.edu Oncogene (2006) 25, 5547–5560 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc