Fig. 4

Binding of BMP3 to ActRIIB activates SMAD2, TAK1, and JNK, which is blocked by specific inhibitor SB431542. a Co-localization of BMP3 with ActRIIB, not BMPR2, was observed in HCT116-BMP3 cells. Enlarged images show co-localization (yellow and white arrows) of BMP3 (green) with ActRIIB (red). No co-localization of BMP3 (green) with BMPR2 (red and blue arrows) was observed (no yellow and blue arrows). White arrows indicate co-localization of BMP3 and ActRIIB and blue arrows indicate BMPR2 membrane receptor. White bar in original image is 12.2 μm and in enlarged image is 7.5 μm. b BMP3 was co-expressed with ActRIIB or BMPR2 in HCT116 cells, which were incubated for 48 h before they were harvested. Cell lysates were precipitated with anti-Flag antibody or anti-Myc antibody to detect the interaction of BMP3 with ActRIIB or BMPR2 by western blot. KM12 cells were also transfected with constructs carrying ActRIIB alone, BMPR2 alone, or both to detect which membrane receptor binds to endogenous BMP3. c Overexpression of BMP3 in HCT116 cells up-regulates the phosphorylation levels of SMAD2, TAK1, and JNK, but not SMAD1/5/8 or p38 (left panel). BMP3 knockdown reversed the up-regulation effects of p-SMAD2, not p-TAk1 or p-JNK in KM12 cells (right panel). d Knockdown of ActRIIB, rather than that of BMPR2, significantly reduced p-SMAD2 and p-TAK1 in the presence of BMP3. e Peak levels of p-SMAD2 and p-TAK1 were reached at 1 h and 24 h, respectively, after treatment of HCT116 cells with recombinant human BMP3 at 100 ng/ml. SB431542 invalidated the phosphorylation effects induced by hBMP3 in a concentration dependent manner. The activation of TAK1 was inhibited by SB431542 at 24 h time point