spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

Right arrow Help viewing high resolution images
Right arrow Return to article
(Downloading may take up to 30 seconds.
If the slide opens in your browser, select File -> Save As to save it.)
Click on image to view larger version.



Fig. 1. Specificity of the BMP response element (BRE) to Smad1/5-mediated BMP signaling in vitro. (A) The BRE sequence consists of two repeats of 2xSBE (bold), 1xCGCC (underlined), 2xCAGC (bold, italic) and 1xGGCGCC (double underlined). 2C2P2C is a similar sequence that lacks 2xSBE + 1xCGCC binding sites and does not respond to BMPs in vitro (see Korchynskyi and ten Dijke, 2002). (B) In C2C12 cells, the BRE-luc reporter is specifically upregulated by BMP2, 4, 6 and 7 (100 ng/ml). 10 ng/ml of TGFß, EGF or FGF do not activate reporter transcription. (C) BRE-luc reporter activity is induced by overexpression of either Smad1 or Smad5 and to a lower extent, by Smad8 in C2C12 cells. BRE-luc reporter activity is further increased upon addition of BMP6 to control cells or cells overexpressing Smad1, 5 or 8. (D) Overexpression of Smad1+Smad4 or Smad5+Smad4 is sufficient to induce BRE-luc activity in C2C12 cells. Smad4 alone does not influence inducibility by BMP6 (100 ng/ml). (E) Binding of Smad4 to the BRE sequence is critical for the reporter response to ligand-dependent and ligand-independent Smad1 or Smad5 overexpression. In the presence of the Smad4-D4 DNA-binding mutant (harboring the K81R and R88K mutations), neither Smad1 nor Smad5 are able to drive BRE-luc expression in MDA-MB468 cells. Expression of wild-type Smad4 is sufficient to restore reporter inducibility either in response to BMP6 or to co-expressed Smad1 or Smad5. (F) In HepG2 cells, constitutively active (ca) type I receptors caALK1, caALK2, caALK3 and caALK6 specifically induce BRE-luc reporter activity, while caALK4 or caALK5, do not. (G) Specific modulation of the BRE-luc reporter in response to activation or inhibition of BMP signaling in ES cells. 10 ng/ml of BMP4 and caALK3 induce while Smad7 blocks basal and BMP induced reporter activity in transient assays. As expected, the 2C2P2C-luc reporter showed no response to modulation. **P≤0.005; *P≤0.05; #P≤0.1.





Right arrow Return to article