CREB Activates BMP2 Expression in Osteoblasts Figure 1. PTH signaling activates CREB phosphorylation in osteoblasts. (A–F) CREB phosphorylation levels in C2C12 cells, treated with PTH at 0 to 500 nM for 1 hour (A); PTH at 50 nM for 5 to 60 min (B); or IBMX at 0 to 300 mM for 1 hour (C); IBMX at 50 mM for 0 to 4 hours (D); or KT5720 at 0 to 10 mM for 1 hour (E); KT5720 at 10 mM for 0 to 2 hours (F), were detected by Western blot with anti-phosphorylated CREB antibody, with normalization by non-phosphorylated CREB and b-actin. (G) C2C12 cells were co-transfected with CRE-Luc reporter and CREB expression vector, and treated with PTH at 50 nM for 36 hours. The reporter luciferase activity was measured with normalization by b-gal activity. # p,0.01 (CREB vs vector; mean6SE, n = 6); * p,0.05 (PTH vs vehicle; mean6SE, n = 6). doi:10.1371/journal.pone.0020780.g001 hypothesized that PTH-CREB signaling may function in bone, at least in part, by up-regulating BMP2 transcription in osteoblasts. To test this hypothesis, we first examined the effects of PTH on BMP2 expression. C2C12 cells were incubated with PTH at 0–200 nM for 12 hours, and real time PCR was performed to quantitate BMP2 mRNA levels. The results showed that PTH treatment increased BMP2 mRNA expression in a dose-dependent manner (Fig. 3A). We also found that stimulation of BMP2 mRNA expression by PTH (50 nM) was time-dependent from 0 to 12 hours (Fig. 3B). In BMP2 promoter assays, C2C12 cells were transfected with a mouse BMP2 promoter reporter, 22712/+165- Luc [38–40], and cultured in the presence or absence of PTH. The promoter reporter luciferase assays showed that treatment with PTH for 24 hours increased BMP2 promoter activity (Fig. 3C), further demonstrating that PTH is an activator of BMP2 transcription in osteoblasts. Next, we determined the effects of drugs, IBMX and KT5720 that regulate PTH signaling activity, on BMP2 promoter activity. C2C12 cells that carry the BMP2 promoter reporter were transfected with CREB expression vector, and then treated with these drugs. We found that CREB transfection substan- tially increased BMP2 promoter activity, and that treatment with the cAMP enhancer IBMX further increased CREB stimulation. In contrast, treatment with the PKA inhibitor KT5720 significantly decreased CREB-induced BMP2 pro- moter activity (Fig. 3D). PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e20780