CREB Activates BMP2 Expression in Osteoblasts Figure 2. PTH-CREB pathway promotes osteoblast differentiation. (A) ALP activity in C2C12 cells, treated with PTH at 0 to 200 nM for 24 hours, was measured with normalization by cell proteins. * p,0.05 (vs vehicle; mean6SE, n = 8). (B) mRNA levels of Runx2 and Col1a1 in C2C12 cells, treated with PTH for 12 hours, were quantitated by real time PCR, normalized by 18S rRNA. * p,0.01 (vs vehicle; mean6SE, n = 6). (C) ALP activity in C2C12 and 2T3 cells was measured after transfection with CREB expression plasmid and treated with noggin at 500 nm/ml for 48 hours. * p,0.01 (CREB vs vector); # p,0.05 (noggin vs vehicle; mean6SE, n = 8). (D) mRNA levels of Runx2, Col1a1 and OCN in C2C12 cells, transfected with CREB for 24 hours, were measured by real time PCR, normalized by 18S rRNA. * p,0.05 (vs vector; mean6SE, n = 6). (E) 2T3 cells were transfected with vector (top) or CREB plasmid (bottom) and cultured under osteogenic conditions for 14 and 21 days. Von Kossa staining was performed to visualize the mineralized matrix formation. (F) Isolated calvarial osteoblastic cells from newborn conditional CREB knockout mice (CREB cKO) and their CREBfloxed littermate controls were cultured for 24 hours and ALP activity was measured. * p,0.05 (cKO vs control); # p,0.05 (PTH on cKO vs PTH on control); ** p,0.05 (PTH va vehicle on control mice; mean6SE, n = 6); NS: not significant. doi:10.1371/journal.pone.0020780.g002 Furthermore, we examined the effects of PTH on BMP2 transcription in the absence of CREB using the CREB-deficient calvarial cells described above. We found that CREB deficiency significantly reduced BMP2 mRNA levels compared with the control cells (Fig. 3E, black bars) and that the stimulation of BMP2 expression by treatment with PTH (50 nM) for 24 hours was largely abolished by inactivation of CREB in the CREB-deficient cells, compared with the control cells (Fig. 3E, gray bars). These data indicate that PTH signaling acts as an upstream stimulator of BMP2 gene expression in osteoblasts, and that this function is mediated through CREB. CREB activates BMP2 transcription in osteoblasts We have shown that overexpression of CREB enhanced BMP2 promoter activity (Fig. 3D). To fully characterize the activating PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e20780