communication between the PTH-cAMP-CREB and BMP2 signaling pathways. Recently, we have reported that osteoblast- specific deficiency of CREB in mice causes reduction of postnatal bone mass and decreases BMP2 expression in osteoblasts [35,36], suggesting that BMP2 could be a critical transcriptional target of CREB in osteoblasts. This is consistent with the findings of a genome-wide study, based on a chromatin immunoprecipitation (ChIP) assay, in which the cAMP-CREB signaling pathway was identified as a positive transcriptional regulator of BMP2 mRNA expression in PC12 cells [37]. Furthermore, we have identified multiple CREs in the BMP2 promoter by DNA sequence analysis. Based on the collective findings reviewed above, it is reasonable to hypothesize that the transcription factor CREB is an activator of BMP2 expression in osteoblasts, and that CREB mediates the anabolic function of PTH-cAMP signaling in bone, at least in part, by this mechanism. This hypothesis was examined in the current investigation through pharmacological and genetic experiments, and promoter analyses. Results PTH signaling activates CREB in osteoblasts CREB activity is controlled by phosphorylation through multiple signaling pathways, including the PTH signaling pathway [11–14]. Here, we determined the effects of manipulating the signaling activity of the PTH-cAMP-signaling cascade on CREB phosphorylation in osteoblasts. First, we determined the effect of PTH on CREB phosphorylation in osteoblasts. Pluripotent mesenchymal precursor C2C12 cells, which are capable of differentiating into an osteoblast lineage, were treated with PTH, and phosphorylation levels of CREB were detected by Western blot, with non-phosphorylated CREB and b-actin as controls. The results showed that treatment with PTH at 0–500 nM substan- tially, and dose-dependently, increased CREB phosphorylation (Fig. 1A). The minimum dose with a discernable effect was approximately 50 nM (Fig. 1A). The PTH-induced CREB phosphorylation in osteoblasts was also time-dependent between 5 and 60 minutes (Fig. 1B). As a second messenger, cAMP is known to mediate PTH signaling. Thus, we determined the effects of the cAMP enhancer 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cAMP phosphodiesterase, on CREB phosphorylation. Western blot showed that incubation of C2C12 cells with IBMX at doses of 10–300 mM increased intracellular levels of phosphory- lated CREB protein (Fig. 1C), and that this stimulation was induced in a time-dependent manner up to 4 hours (Fig. 1D). As a direct downstream target of cAMP, PKA phosphorylates CREB. Next, we examined the effects of inhibition of PKA activity on CREB phosphorylation using KT5720, a PKA inhibitor. We found that the KT5720-induced inhibition of PKA activity markedly reduced levels of phosphorylated CREB in C2C12 cells, in both dose- and time-dependent manners (Fig. 1E, 1F). The levels in the Western blot of phosphor-CREB treated by IBMX and KT5720 were quantitated. The quantitative results confirmed that these cAMP and PKA drugs dose- and time-dependently regulated CREB phosphorylation in osteoblasts (see the quantita- tive data in Figure S1). Phosphorylated CREB migrates into nuclei and transactivates target genes through CRE binding sites. Consequently, we evaluated the effects of PTH, which induces CREB phosphoryla- tion, on transcriptional activity of CREB. The luciferase assay using a CREB-specific reporter CRE-Luc showed that overexpression of CREB in C2C12 cells dose-dependently increased the activity of the CRE reporter, and that treatment with PTH at 50 nM further elevated the CREB-induced reporter activity (Fig. 1G). CREB Activates BMP2 Expression in Osteoblasts PTH-CREB pathway promotes osteoblast differentiation It has been postulated that the anabolic effect of PTH on bone is mediated through osteoblasts. In this series of experiments, we characterized the role of the PTH-CREB signaling pathway in osteoblast differentiation. First, we determined the effects of PTH on alkaline phosphatase (ALP) activity of C2C12 cells by incubating cells with PTH for 24 hours. Measurement of ALP activity in cell lysates showed that treatment with PTH at 0– 200 nM stimulated ALP activity in a pattern that approached dose-dependence, with a maximum effect (2-fold increase) at PTH concentration 100 nM (Fig. 2A). Real time PCR showed that the mRNA levels of osteoblast maturation genes, Runx2 and type I collagen 1a1 (Col1a1), were significantly enhanced by treatment with PTH at doses of 10–100 nM (Fig. 2B). Then, we determined the effects of overexpression of CREB on osteoblast differentiation. Osteoblast precursor C2C12 and 2T3 cells [38–40] were transfected with CREB expression plasmid or its empty vector for 48 hours. Enzymatic quantitation of ALP activity showed that CREB overexpression significantly increased ALP activity in both C2C12 and 2T3 cells, compared with vehicle controls (Fig. 2C). Interestingly, CREB-stimulation of ALP activity was completely reversed in these cells by adding noggin (500 ng/ ml), a natural BMP antagonist, to the culture medium (Fig. 2C). This finding is critical evidence for our hypothesis that the stimulatory effect of CREB on ALP depends on BMP signaling. mRNA measurement of C2C12 cells also showed that CREB transfection significantly increased expression of osteoblast-specific marker genes, including Runx2, Col1a1 and osteocalcin (OCN) (Fig. 2D). Next, we determined whether overexpression of CREB affects bone matrix formation in osteoblast precursor 2T3 cells, which can form mineralized matrices in culture under osteogenic conditions [33,37–40]. Von Kossa staining showed that mineral- ized bone nodule formation was greatly enhanced in 2T3 cell cultures 14 and 21 days after CREB transfection, compared with control 2T3 cells transfected with empty vector (Fig. 2E). In order to confirm the role of CREB in promoting osteoblast differentiation, we performed a loss-of-function study with primary CREB-deficient calvarial osteoblast cells. These cells were isolated from conditional CREB knockout mice (CREB cKO), that were generated by crossing CREB floxed mice with Col1a1-Cre mice. We have recently reported that these osteoblast-specific CREB knockout mice exhibit low bone mass [35,36]. In the present study, we found that CREB deficiency markedly decreased both basal and PTH-induced ALP activity in calvarial osteoblast cells, compared with floxed control cells (Fig. 2F). We also noticed that, despite at a much lower level than PTH treatment in the control group, PTH appeared still capable of increasing ALP activity in the absence of CREB, compared with vehicle treatment. However, the statistical analysis did not show a significant difference between the treatment groups in the CREB deficient cells. This suggests that PTH stimulation of ALP activity was attenuated by removal of the CREB gene in the calvairal osteoblast cells, (Fig. 2F). Together, these results suggest that the PTH-CREB pathway is an activator of osteoblast differentiation. PTH up-regulates BMP2 expression through CREB in osteoblasts The finding that noggin blocked CREB-induced ALP activity in osteoblasts (Fig. 2C) suggested an involvement of the BMP pathway in CREB function. A previous genome-wide study has found that BMP2, a prototype member of the BMP family, is a transcriptional target of the CREB pathway in other cell systems [37]. Given these facts, and the presence of potential CREB binding elements in the BMP2 promoter (vide infra), we PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e20780