CREB Activates BMP2 Expression in Osteoblasts Figure 3. PTH up-regulates BMP2 expression through CREB. (A, B) BMP2 mRNA levels in C2C12 cells, treated with PTH at 0 to 200 nM for 12 hours (A); or PTH at 50 nM for 0 to 12 hours (B), were measured by real time PCR with GAPDH normalization. * p,0.01 (vs vehicle; mean6SE, n = 6). (C) BMP2 promoter reporter activity in C2C12 cells, transfected with 22712/+165-Luc and treated with PTH at 0 to 200 nM for 24 hours, was measured with b-gal normalization. * p,0.01 (vs vehicle; mean6SE, n = 8). (D) BMP2 promoter reporter activity in C2C12 cells, co-transfected with CREB plasmid or vector and reporter 22712/+165-Luc, and treated with IBMX at 50 mM or KT5720 at 5 mM for 24 hours, was measured. * p,0.01 (CREB vs vector); # p,0.01 (CREB+IBMX vs CREB+vehicle); ** p,0.05 (CREB+KT5720 vs CREB+vehicle; mean6SE, n = 8). (E) BMP2 mRNA levels in calvrial cells, isolated from CREB cKO and control mice, and treated with PTH at 50 nM for 24 hours, were measured by real time PCR. * p,0.01 (cKO vs control); # p,0.01 (PTH on cKO vs PTH on control); ** p,0.05 (PTH vs vehicle on control mice; mean6SE, n = 6); NS: not significant. doi:10.1371/journal.pone.0020780.g003 function of CREB on the BMP2 gene, we performed multiple gain- and loss-of-function experiments to determine the effects of manipulating CREB levels on BMP2 gene expression in osteoblasts. We found that overexpression of CREB in osteoblastic cell lines, including C2C12, 2T3 and UMR106 cells, substantially increased BMP2 mRNA levels in these cells, compared with vector controls (Fig. 4A). Next, we evaluated the effects of CREB transfection on BMP2 promoter activity. The luciferase assay with 22712/+165-Luc reporter showed that CREB markedly increased transcriptional activity of the BMP2 promoter in these osteoblastic cells, with a maximal stimulation of approximately 15-fold in C2C12 cells (Fig. 4B). In contrast, small interfering RNA (siRNA) knockdown of endogenous CREB in these osteoblast cell lines had significant inhibitory effects on BMP2 PLoS ONE | www.plosone.org 5 June 2011 | Volume 6 | Issue 6 | e20780