closest members in these respective families are activating transcription factor 4 (ATF4) and BMP4. In order to test the specificity of the effects of CREB on BMP2 expression, we examined the effects of ATF4 on BMP2 expression and the effects of CREB on BMP4 expression. The factor ATF4 belongs to the same large bZIP family of transcription factors as CREB, and has proven to be important for osteoblast differentiation [41,42]. We found that, compared with CREB stimulation, ATF4 transfection did not affect BMP2 promoter activity in C2C12 cells (Fig. 4E). In the BMP family, BMP4 is structurally closest to BMP2. Using a BMP4 promoter reporter, BMP4-Luc, we found that, CREB failed to enhance BMP4 promoter activity, in contrast to CREB enhancement of BMP2 promoter activity (Fig. 4F). Collectively, these results strongly support the conclusion that the transcrip- tional factor CREB is a specific and potent enhancer of BMP2 gene expression in osteoblasts. CREB transactivates BMP2 through CRE in the BMP2 promoter CREB is known to transactivate target genes through a specific CRE consensus sequence in their promoters [17–22]. Sequence analysis revealed multiple CREs throughout the mouse BMP2 promoter from 22712 to +165 (data not shown). To identify the functional CRE(s) responsible for CREB transactivation of the BMP2 promoter, we first performed a promoter deletion study. C2C12 cells were transiently co-transfected with CREB expression plasmid or empty vector plus a series of truncated BMP2 promoter reporters representing 22712/+165, 22457/+165, 2199/+165, 21803/+165, 2968/+165, 2838/+165, 2310/+165, and 2150/ +165 of the 59 promoter region of the BMP2 gene. Interestingly, we found that none of these progressively truncated BMP2 promoter reporters lost or reduced their responsiveness to CREB transactivation of luciferase activity (Fig. 5A). These results suggest that the potential CREs may be located within the 2150/+165 region of the BMP2 promoter. Next, we analyzed the sequence of this narrow region in the promoter, and identified three putative CREs (CRE1, CRE2 and CRE3) in this area (Fig. 5B). To determine the role of these putative CREs in CREB transactiva- tion, we performed site-directed mutagenesis. Each of these putative CREs located within the BMP2 promoter-luciferase construct (2150/+165-Luc) was mutated by deleting or replacing core nucleotides as shown in figure 5C. The results of luciferase reporter assays showed that levels of BMP2 transcriptional activity induced by CREB were comparable for mutant CRE1 and CRE3 reporters (mCRE1 and mCRE3) and the wild-type 2150/+165- Luc reporter (Fig. 5D). Mutation of CRE2 (mCRE2), however, resulted in complete abrogation of the response to CREB stimulation (Fig. 5D), indicating that CRE2, in region 2150/ +165 of the BMP2 promoter, is a functional CRE responsible for CREB’s action on the BMP2 gene. Furthermore, we also examined the effects of CRE2 mutation on PTH stimulation of BMP2 promoter activity. As shown in Figure 5E, we found that PTH lost its stimulatory effect on BMP2 promoter activity when the CRE2 is mutated, compared with wild-type CRE2 control. We subsequently performed ChIP and EMSA assays to verify the interaction of CREB with CRE2 in the BMP2 promoter. In the ChIP assay, a 230 bp fragment of the BMP2 promoter containing the specific CRE2 sequence was amplified after the addition of anti- CREB antibody to nuclear DNA-protein complexes extracted from C2C12 cells, compared with non-specific IgG control (Fig. 5F, lane 3 vs lane 2). This suggests that CREB binds directly to CRE2 in the BMP2 promoter. Importantly, treatment of these cells with PTH (50 nM) for 6 hours further increased CREB binding ability to CRE2, evidenced by increased PCR product in the gel (Fig. 5F, lane CREB Activates BMP2 Expression in Osteoblasts 4 vs lane 3). Furthermore, EMSA was performed to obtain direct evidence of physical binding of CREB with CRE2. Nuclear proteins were extracted from C2C12, 2T3 and MC3T3-E1 osteoblast cells and reacted with a biotin-labeled DNA probe containing the CRE2 sequence (213 to +20) of the BMP2 promoter. The addition of the CRE2 probe to these cell extracts induced a shift in mobility of the probe in the gel, (Fig. 5G, lane 2–4 vs lane 1), indicating a direct interaction between the CRE2 probe and nuclear proteins. Further, addition of anti-CREB antibody to the reaction mixture of C2C12 cells produced a super shift in mobility of the band (Fig. 5G, lane 7 vs lane 6), indicating that CREB binds directly to this CRE2 complex. Based on the combined results of luciferase reporter, ChIP and EMSA assays, we propose that the transcriptional factor CREB transactivates the BMP2 gene through a specific CRE2 site in the BMP2 promoter. Discussion These collective results provide evidence that the PTH signaling pathway is an effective activator of BMP2 gene expression in osteoblasts, and this function is mediated by CREB transactivation of the BMP2 promoter. This transcriptional mechanism, at least in part, accounts for the anabolic function of the PTH-CREB pathway in osteoblast differentiation and bone formation. It is well documented that intermittent dosing with PTH has an anabolic effect on bone [1–10], and that the PTH signaling pathway is a physiological regulator of osteoblast function in bone [11–30]. As a major mediator of PTH signaling, the transcrip- tional factor CREB has been found to play a role in osteoblast differentiation by regulating expression of osteoblast-specific genes [13–22], by which CREB may function as an anabolic regulator for bone mass. Several previous in vivo studies have provided evidence for the anabolic function of CREB in bone [30,35,36,43,44]. A recent report showed that small molecules with potent osteogenic activity induce osteoblast differentiation by activating intracellular CREB activity [43]. In studies with Lrp 5 knockout mice that exhibit low bone mass, Yadav et al reported that Lrp5 deficiency increases duodenal production of serotonin, which inhibits osteoblast function by suppressing CREB activity in osteoblasts [44]. Furthermore, they found that Lrp5 and CREB double knockout mice have a significantly lower bone mass than Lrp5 single mutants [44]. Recently, we have reported that osteoblast-specific knockout of CREB in mice produces a skeletal phenotype with low bone mineral density and low bone volume [35,36]. This is consistent with a previously reported similar osteopenic phenotype of the osteoblast-specific ICER (inducible cAMP early repressor) transgenic mice [30]. ICER belongs to the same bZIP family of transcription factors as CREB, and acts as a dominant negative regulator of gene transcription through CRE. Thus, it appears that CREB plays an important role in postnatal bone formation through its effect on osteoblast cells. To explore the mechanisms by which CREB exerts its effects on bone, in the current gain- and loss-of-function studies employing primary osteoblast cells and multiple osteoblast cell lines, we found that manipulation of activity and expression levels of PTH and CREB affects osteoblast function. We also demonstrated that the PTH- CREB signaling pathway is a positive regulator of osteoblast differentiation. These findings further support the hypothesis that promoting osteoblast differentiation is one of cellular mechanisms by which the PTH-CREB signaling pathway exerts its anabolic function in bone. The cAMP-PKA-CREB axis represents a major signaling pathway that mediates PTH signaling [11–30]. In the current study, we demonstrated that PTH induces CREB phosphorylation PLoS ONE | www.plosone.org 7 June 2011 | Volume 6 | Issue 6 | e20780