Smad3 interacts with JunB and Cbfa1/Runx2 for transforming growth factor-β1-stimulated collagenase-3 expression in human breast cancer cells

Nagarajan Selvamurugan, Sukyee Kwok, Nicola Partridge

Research output: Contribution to journalArticle

Abstract

We have previously shown that transforming growth factor (TGF)-β1, 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-β1 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-β1-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-β1 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-β1, 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-β1 in MDA-MB231 cells. Co-transfection of Smad3, JunB, and Cbfa1/Runx2 constructs along with a constitutively active TGF-β type I receptor construct identified functional interaction of these proteins and transcriptional activation of the collagenase-3 gene by TGF-β1. Taken together, our results suggest that TGF-β1 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-β1-stimulation of collagenase-3 expression in MDA-MB231 cells.

Original languageEnglish (US)
Pages (from-to)27764-27773
Number of pages10
JournalJournal of Biological Chemistry
Volume279
Issue number26
DOIs
StatePublished - Jun 25 2004

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Matrix Metalloproteinase 13
Transforming Growth Factors
Cells
Breast Neoplasms
Transcription Factor AP-1
Genes
Bone Neoplasms
Functional analysis
Growth Factor Receptors
Chromatin Immunoprecipitation
RNA-Directed DNA Polymerase
Reverse Transcriptase Polymerase Chain Reaction
Immunoprecipitation
Genetic Promoter Regions
Transcriptional Activation
Chromatin
Transfection
Real-Time Polymerase Chain Reaction
Bone
Western Blotting

ASJC Scopus subject areas

  • Biochemistry

Cite this

Smad3 interacts with JunB and Cbfa1/Runx2 for transforming growth factor-β1-stimulated collagenase-3 expression in human breast cancer cells. / Selvamurugan, Nagarajan; Kwok, Sukyee; Partridge, Nicola.

In: Journal of Biological Chemistry, Vol. 279, No. 26, 25.06.2004, p. 27764-27773.

Research output: Contribution to journalArticle

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T1 - Smad3 interacts with JunB and Cbfa1/Runx2 for transforming growth factor-β1-stimulated collagenase-3 expression in human breast cancer cells

AU - Selvamurugan, Nagarajan

AU - Kwok, Sukyee

AU - Partridge, Nicola

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N2 - We have previously shown that transforming growth factor (TGF)-β1, 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-β1 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-β1-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-β1 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-β1, 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-β1 in MDA-MB231 cells. Co-transfection of Smad3, JunB, and Cbfa1/Runx2 constructs along with a constitutively active TGF-β type I receptor construct identified functional interaction of these proteins and transcriptional activation of the collagenase-3 gene by TGF-β1. Taken together, our results suggest that TGF-β1 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-β1-stimulation of collagenase-3 expression in MDA-MB231 cells.

AB - We have previously shown that transforming growth factor (TGF)-β1, 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-β1 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-β1-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-β1 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-β1, 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-β1 in MDA-MB231 cells. Co-transfection of Smad3, JunB, and Cbfa1/Runx2 constructs along with a constitutively active TGF-β type I receptor construct identified functional interaction of these proteins and transcriptional activation of the collagenase-3 gene by TGF-β1. Taken together, our results suggest that TGF-β1 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-β1-stimulation of collagenase-3 expression in MDA-MB231 cells.

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