Fig. 2.

The Bmp2 3′UTR represses gene expression in immortalized and primary mesenchymal cells. A: The luciferase activities of transfected reporter genes driven by the heterologous, constitutive promoter RPL10 were compared in MC3T3-E1 immortalized preosteoblast cells, human umbilical artery smooth muscle cells (UASMC), or vascular smooth muscle cells (VSMC) from rat aorta. The indicated plasmid regions are to approximate scale. Relative to the promoter only plasmid, plasmids with the full-length human 3′UTR (1,227 nt to polyadenylation (pA) signal 2, 1,323 nt total) inserted downstream of LUC or a shorter fragment bearing the UCS (855 nt) were expressed less. The fragment bearing the two polyadenylation signals did not repress in MC3T3-E1 cells (UASMC and VSMC not done). Relative reporter activity is shown ±SEM. MC3T3-E1 cells, n =4, P <0.0004; UASMC, n =4−6, P <0.006; VSMC, n =6, P <0.03. B: Bmp2Luc-3′UTR and Bmp2Luc-SVpA-3′UTR both contain the distal mouse Bmp2 promoter (−1,237−471 relative to the distal transcription start site), the entire mouse 3′UTR (870 or 1,185 nt depending on which pA signal is used), and downstream sequence (2,212 nt total). These plasmids are identical except that the SV40 pA signal was positioned downstream or upstream of the Bmp2 3′UTR as indicated. In MC3T3-E1 cells (UASMC and VSMC not done), Bmp2Luc-SVpA-3′UTR, whose transcript excludes the 3′UTR, was nearly twice as active as Bmp2Luc-3′UTR whose transcript includes the 3′UTR. n =6, P <0.005.