CREB Activates BMP2 Expression in Osteoblasts Figure 4. CREB activates BMP2 expression. (A) BMP2 mRNA levels in C2C12, 2T3 and UMR106 cells, transfected with CREB plasmid or vector for 24 hours, were measured by real time PCR with GAPDH106 cells. * p,0.01 (vs vector; mean6SE, n = 6). (B) BMP2 promoter activity in C2C12, 2T3 and UMR106 cells, co-transfected with 22712/+165-Luc and CREB plasmid for 24 hours, was measured by luciferase activity with b-gal normalization. * p,0.01 (vs vector; mean6SE, n = 8). (C) BMP2 mRNA levels in C2C12, 2T3 and UMR106 cells, transfected with CREB siRNA or control siRNA for 36 hours, were measured by real time PCR. * p,0.05 (vs control siRNA; mean6SE, n = 6). (D) BMP2 mRNA levels in calvarial cells of CREB cKO and control mice were measured by real time PCR. * p,0.01 (vs control; mean6SE, n = 6). (E) BMP2 promoter reporter activity in C2C12 cells, co-transfected with 22712/+165-Luc and CREB or ATF4 plasmid for 24 hours, was measured. * p,0.01 (vs vector; mean6SE, n = 8). (F) BMP2 promoter reporter activity in C2C12 cells, co-transfected with 22712/+165-Luc or BMP4-Luc and CREB plasmid for 24 hours, was measured. * p,0.01 (vs vector; mean6SE, n = 8). doi:10.1371/journal.pone.0020780.g004 mRNA transcription, measured by real time PCR, compared with siRNA controls (Fig. 4C). The loss-of-function by siRNA was further confirmed by CREB knockout. mRNA quantitation demonstrated that an osteoblast-specific CREB deletion markedly reduced BMP2 mRNA levels in primary calvarial osteoblastic cells, compared with cells isolated from control mice (Fig. 4D). In addition to CREB and BMP2, other members of the CREB and BMP families also are involved in osteoblast function. The PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e20780