Functional Cloning of the Proto-oncogene Brain Factor-1 (BF-1) As a Smad-binding Antagonist of Transforming Growth Factor-β Signaling

Abstract

Using the plasminogen activator inhibitor (PAI) promoter to drive the expression of a reporter gene (mouse CD2), we devised a system to clone negative regulators of the transforming growth factor-␤ (TGF-␤) signaling pathway. We infected a TGF-␤-responsive cell line (MvLu1) with a retroviral cDNA library, selecting by fluorescence-activated cell sorter single cells displaying low PAI promoter activity in response to TGF-␤. Using this strategy we cloned the proto-oncogene brain factor-1 (BF-1). BF-1 represses the PAI promoter in part by associating with both unphosphorylated Smad3 (in the cytoplasm) and phosphorylated Smad3 (in the nucleus), thus preventing its binding to DNA. BF-1 also associates with Smad1, -2, and -4; the Smad MH2 domain binds to BF-1, and the C-terminal segment of BF-1 is uniquely and solely required for binding to Smads. Further, BF-1 represses another TGF-␤-induced promoter (p15), it upregulates a TGF-␤-repressed promoter (Cyclin A), and it reverses the growth arrest caused by TGF-␤. Our results suggest that BF-1 is a general inhibitor of TGF-␤ signaling and as such may play a key role during brain development. Transforming growth factor-␤ (TGF-␤)1 signals through a series of events that includes transphosphorylation and activation of the TGF-␤ type I receptor (T␤-RI) by the TGF-␤ type II receptor (T␤-RII) and phosphorylation of Smad3 by the T␤-RI, followed by the assembly and nuclear translocation of a Smad3/ Smad4 complex (1, 2-7). This process results in the transcriptional regulation of genes required in development and extracellular matrix production (i.e. up-regulation of extracellular matrix genes) as well as of genes involved in cell cycle regulation (i.e. up-regulation of the cell cycle inhibitor p15) (8 -12).The importance of TGF-␤ in tumorigenesis is underscored by the finding in a variety of cancers of inactivating mutations and a loss of heterozygosity affecting genes involved in the TGF-␤ signaling cascade (13-17). More recently, several laboratories have reported a group of known oncoproteins that repress Smad3-dependent transcription (18, 19 -22). Here we report the use of a novel retroviral functional cloning system to clone the brain factor 1 (BF-1) cDNA as a functional antagonist of TGF-␤ signaling.Recently, Dou et al. (23) also showed that BF-1 expression inhibits TGF-␤ signaling. Although several of their results are similar to ours (BF-1 expression causes resistance to TGF-␤-mediated growth inhibition and reverses TGF-␤-induced transcriptional activation), we differ on one crucial point. Dou et al. could not detect interactions between any Smad proteins and BF-1, and they claim that BF-1 exerts its inhibitory effects by binding with the transcriptional coactivator FAST-2. In contrast, we show that BF-1 inhibits TGF-␤-induced gene activation in part by associating with unphosphorylated Smad3 in the cytoplasm. BF-1 does not affect Smad phosphorylation induced by TGF-␤ binding to its receptor, but it does prevent Smad binding to DNA by associating with phosphorylated Smad...

Overexpression of FOXG1 contributes to TGF-β resistance through inhibition of p21WAF1/CIP1 expression in ovarian cancer

“…Therefore, to exclude the effect of p53 on p21 WAF1/CIP1 expression, we used p21 WAF1/CIP1 promoter (truncated without p53-binding site) luciferase reporter assay to study the effect of FOXG1 on p21 WAF1/CIP1 in HEK293T and A2780cp cell models. Consistent with the findings from FOXG1 on human medulloblastoma and glioblastoma (Rodriguez et al, 2001;Seoane et al, 2004;Adesina et al, 2007b), FOXG1 could reduce TGF-bmediated p21 WAF1/CIP1 expression in a dose-dependent manner. We also showed that the overexpression or RNAi-mediated depletion of FOXG1 could alter the expression of p21 WAF1/CIP1 in ovarian cancer cells.…”

“…These inhibitors are able to block cyclin and CDKs from phosphorylating the retinoblastoma protein (Rb), as well as preventing the progression of the cell cycle (Reynisdottir et al, 1995;Massague et al, 2000). FOXG1 has been shown to inhibit Smad-mediated induction of p21 WAF1/CIP1 expression through association with FoxO -Smad complexes in the nucleus (Rodriguez et al, 2001;Seoane et al, 2004;Adesina et al, 2007b). To investigate the cell growth inhibitory mechanism of FOXG1 on ovarian cancer cells, we evaluated the p21 WAF1/CIP1 expression by quantitative RT -PCR, western blot and immunohistochemical analyses.…”

“…Recent studies have shown that FOXG1 acts as a negative regulator of TGF-b signalling pathway by specifically binding to the Smad MH2 domain and associates with Smad -1, -2, -3 and -4 (Dou et al, 2000;Seoane et al, 2004). This association blocks the binding of Smad proteins to DNA and results in the inhibition of TGF-b signalling (Rodriguez et al, 2001). In addition, FOXG1 has been shown to inhibit expression of the cyclin-dependent kinase (CDK) inhibitor p21…”

Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

“…In the cytoplasm, Foxg1 works as a TGF-β inhibitor by binding to Smad3 (mothers against decapentaplegic homolog 3) (12).…”

The C-terminal region of cellular Qin oligomerizes: correlation with oncogenic transformation and transcriptional repression

“…BF-1 interferes with transforming growth factor b (TGFb) signals by associating with Smad proteins and repressing TGFb-activated promoters. This activity is independent of DNA binding but requires the Cterminal region (Dou et al, 2000;Rodriguez et al, 2001).…”