Objectives: 

Tmeff2 is a protein with unclear function. It has been described that Tmeff2 promoter is silenced by hypermethylation in certain primary tumors.No other mechanism regulating Tmeff2 expression has been described in tumor cells. Here we show that TGF-B regulates TMEFF2 expression in glioma cells by the classical pathway mediated by Smads and through c-Myc binding to TMEFF2 promoter.

Materials: 

TMEFF2 expression in clinical frozen samples or in human glioma cell lines was measured by RT-qPCR. Cell cultures were treated with TGFB1 (100 pM) or vehicle. TGFB receptor inhibitor SB431542 was used at 2 mM to pretreat cell cultures when appropriate TMEFF2 promoter activity was determinated by dual luciferase assays in three cloned fragments of the human promoter. Deletions were made by PCR or site-directed mutagenesis. For silencing, U87MG cells were transfected with 200 nM SMART pool RNAs to Smad2, Smad3, and Smad4 or siRNA3 control. For CHIP assay, 200 mg of chromatin and 3 mg per antibody (non-immune IgG, anti c-Myc or anti-AcH4).After reversal of crosslinking a fragment of TMEFF2 promoter was amplified by PCR

Results: 

Low levels of TMEFF2 expression are associated with high levels of Smad activation in human glioma samples. In gliomas low levels of Tmeff2 are associated with poorer prognosis. Treatment with TGFB causes a decrease in the expression levels of Tmeff2, an effect that is blocked by pretreatment with SB431542 inhibitor. Gene silencing showed that these effects are mediated by Smad proteins. By mutagenesis of the cloned promoter sequence we could identify an E-box located at positions -92 to -88. Integrity of this element is essential to maintain the inhibitory effect of TGFB. Finally, using ChIP we found that c-Myc binds to this E-box in the absence but not in the presence of TGFB Furthermore, the inhibitory effect of TGFB is abolished by ectopic expression of c-Myc.

Conclusions: 

Our results show that TMEFF2 is a target gene of TGFB in glioma cells and support a model in which the inhibitory effect is mediated by the displacement of c-Myc from an E-box located at -92 to -88 of TMEFF2 promoter