Figure 5. CREB transactivates BMP2 through CRE in the BMP2 promoter.

(A) BMP2 promoter reporter activity in C2C12 cells, co-transfected with CREB plasmid and a series of truncated reporter constructs for 24 hours, was measured with β-gal normalization. (B) Sequence of −150/+165 of mouse BMP2 promoter. Putative sequences for CRE1, CRE2 and CRE3 are underlined and bolded. Sequences of ChIP primers are underlined only. (C) Sequences of mutant CRE1, CRE2 and CRE3 (mCRE1, mCRE2 and mCRE3). (D) BMP2 promoter reporter activity in C2C12 cells, co-transfected with wild-type −150/+165-Luc (WT) and mutant −150/+165-Luc in which CREs were mutated (mCRE1, mCRE2 and mCRE2), and with CREB plasmid for 24 hours, was measured. * p<0.01 (CREB transfection on mCRE2 vs WT or mCRE1 or mCRE3 reporters; mean±SE, n = 8). (E) Effects of PTH on BMP2 promoter activity. C2C12 cells were co-transfected with wild-type −150/+165-Luc (WT) and mutant −150/+165-Luc in which CRE2 was mutated (mCRE2) and treated with PTH at 50 nM for 24 hours. * p<0.01 (PTH mCRE2 vs PTH on WT reporter; mean±SE, n = 8).F) ChIP assay. Nuclear DNA-protein complexes were extracted from C2C12 cells treated with PTH at 50 nM for 6 hours and precipitated with anti-CREB antibody. PCR was performed to amplify the region of the BMP2 promoter that contained CRE2, using PCR primers indicated in (B). Input: BMP2 promoter DNA. IgG: Goat IgG as a negative control. (G) EMSA assay. Nuclear extracts of C2C12, 2T3 and MC3T3-E1 cells were incubated with a biotin-labeled DNA probe containing the CRE2 binding site sequence in the BMP2 promoter, in the absence or presence of anti-CREB antibody. The shift and super shift bands were analyzed using a 5% polyacrylamide gel.