title: Smad3 interacts with JunB and Cbfa1/Runx2 for transforming growth factor-beta1-stimulated collagenase-3 expression in human breast cancer cells.
abstract: We have previously shown that transforming growth factor (TGF)-beta1, a crucial molecule in metastatic bone cancer, stimulates collagenase-3 expression in the human breast cancer cell line, MDA-MB231. To understand the molecular mechanisms responsible for TGF-beta1 response on collagenase-3 promoter activity, a functional analysis of the promoter region of the collagenase-3 gene was carried out, and we identified the distal runt domain (RD) and proximal RD/activator protein-1 (AP-1) sites as necessary for full TGF-beta1-stimulated collagenase-3 promoter activity. Gel shift, real time reverse transcriptase-PCR, and Western blot analyses showed increased levels of c-Jun, JunB, and Cbfa1/Runx2 upon TGF-beta1 treatment in MDA-MB231 cells. Co-immunoprecipitation in vitro studies identified no physical interaction between JunB and Cbfa1/Runx2, whereas Smad3 interacted with both. Chromatin immunoprecipitation experiments confirmed interaction of Smad3 with JunB and Cbfa1/Runx2. Under basal conditions, Cbfa1/Runx2 bound to both the proximal RD/AP-1 and distal RD sites. In response to TGF-beta1, Cbfa1/Runx2 was seen only at the distal RD site, whereas JunB occupied the proximal RD/AP-1 site. An assemblage of Smad3, JunB, and Cbfa1/Runx2 at the distal RD site of the collagenase-3 promoter occurred in response to TGF-beta1 in MDA-MB231 cells. Co-transfection of Smad3, JunB, and Cbfa1/Runx2 constructs along with a constitutively active TGF-beta type I receptor construct identified functional interaction of these proteins and transcriptional activation of the collagenase-3 gene by TGF-beta1. Taken together, our results suggest that TGF-beta1 stimulated JunB and Cbfa1/Runx2 to bind to their respective DNA consensus sites and that Smad3 is likely to stabilize their interaction to confer functional TGF-beta1-stimulation of collagenase-3 expression in MDA-MB231 cells.
authors.affiliation: Department of Physiology and Biophysics, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA. selvamn2@umdnj.edu
authors.forename: Nagarajan
authors.initials: N
authors.lastname: Selvamurugan
authors.forename: Sukyee
authors.initials: S
authors.lastname: Kwok
authors.forename: Nicola C
authors.initials: NC
authors.lastname: Partridge
mesh_terms: D000818:Animals; D001483:Base Sequence; D001943:Breast Neoplasms / Q000201:enzymology / Q000378:metabolism*; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D017364:Collagenases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D050659:Core Binding Factor Alpha 1 Subunit; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D053509:Matrix Metalloproteinase 13; D009363:Neoplasm Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D017434:Protein Structure, Tertiary; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D051900:Smad3 Protein; D015534:Trans-Activators / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D016755:Proto-Oncogene Proteins c-jun; C497790:SMAD3 protein, human; D051900:Smad3 Protein; C497787:Smad3 protein, rat; C508960:TGFB1 protein, human; C508962:Tgfb1 protein, rat; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D017364:Collagenases; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13; C507083:Mmp13 protein, rat