CREB Activates BMP2 Expression in Osteoblasts performed to amplify the region of the BMP2 promoter that contained CRE2, using PCR primers indicated in (B). Input: BMP2 promoter DNA. IgG: Goat IgG as a negative control. (G) EMSA assay. Nuclear extracts of C2C12, 2T3 and MC3T3-E1 cells were incubated with a biotin-labeled DNA probe containing the CRE2 binding site sequence in the BMP2 promoter, in the absence or presence of anti-CREB antibody. The shift and super shift bands were analyzed using a 5% polyacrylamide gel. doi:10.1371/journal.pone.0020780.g005 in osteoblasts and that this function is regulated by pharmacolog- ical manipulation of the PTH signaling cascade in the upstream of CREB. The results of experiments with the cAMP-enhancer and the PKA-inhibitor suggest that the capacity of CREB to enhance BMP2 transcription in osteoblasts is phosphorylation-dependent. In these studies, however, we found that the PKA-inhibitor (KT5720) failed to completely abolish the capacity of CREB to increase BMP2 transcription in osteoblasts, suggesting that other protein kinases, in addition to PKA (such as protein kinase C), also may be involved in this process. Similar results have been reported with other cell systems [45,46]. In a separate experiment, we found that the cAMP-enhancer IBMX had biphasic effects on the BMP2 promoter, depending on the duration of treatment. In contrast to the stimulatory effect on BMP2 expression observed in this study when cells were exposed to IBMX for only 24 hours, extending exposure up to 48 hours resulted in inhibition of BMP2 promoter activity (data not shown). This latter effect of IBMX on the BMP2 gene may be due to the degradation of transcription factor Gli2, which we previously identified as a stimulator of BMP2 expression in osteoblasts [39,40]. CREB transactivates target genes through a consensus cAMP response element (CRE), by which CREB has been shown to transactivate multiple osteoblast maturation related marker genes, such as BSP and OCN [13–22]. The rationale in this study to focus on the BMP2 gene as a critical transcriptional target of CREB in osteoblasts includes the following: (1) BMP2 is the prototypic member of the BMP family responsible for osteoblast differentiation; (2) cAMP, an upstream activator of CREB, up- regulates BMP2 gene expression in other cells [37], and; (3) multiple putative CRE sites can be found throughout the BMP2 promoter. Therefore, we mapped out the molecular interaction between CREB protein and the BMP2 promoter. Utilizing promoter deletion, mutation, ChIP and EMSA studies, we defined a specific CRE (CRE2) located within the basal promoter loci from 2150 to +165, as a functional binding element responsible for CREB transactivation of the BMP2 gene. We recognize, however, that regulation of the BMP2 gene is complex, and that multiple additional mechanisms, involving Gli proteins [39,40,47], b-catenin/TCF [48] and ERa [49], have been reported to contribute to the regulation of BMP2 transcription. Thus, we do not exclude the possibility that additional untested CRE sequences in the upstream of the BMP2 promoter may also possess potential cis-functions in BMP2 gene transcription in response to other nuclear transcription factors such as those identified above. Interestingly, in this study, we found that ATF4 (a member of the CREB/ATF family) failed to stimulate BMP2 expression. In other cell types, ATF4 and CREB have been shown to share the same DNA binding element (TGACGTCA) in transactivating the promoter activities of target genes [49]. We presume, therefore, that CREB transactivation of the BMP2 gene in osteoblasts may require other co-activators associated within the CREB transcrip- tional machinery that are structurally and functionally distinct from those associated with ATF4 action. However, more comparison studies about the transcriptional mechanisms of these factors on the BMP2 gene are needed to examine this possibility. The experiments in which noggin blocked CREB-induced ALP activity support the concept that the function of CREB in osteoblasts is likely BMP-dependent, because noggin blocks BMP signaling by hindering BMP ligands from binding to BMP receptors [50,51]. The present data showed that CREB, as a powerful stimulator of BMP2 transcription, lacks the capability to induce expression of BMP4, the closest member of BMP2 in the BMP family. However, it is possible that CREB may have a positive regulating function for transcription of other BMP family members. This possibility warrants further investigation. In addition to the capability of increasing BMP2 gene expression in osteoblasts demonstrated in the current study, the PTH-cAMP-CREB pathway also has been shown to enhance BMP2 activity. For example, it has been demonstrated that this pathway is capable of enhancing BMP2-stimulated Smad signaling [34], and synergizing the BMP2-induced anabolic effects on differentiation of osteoblasts [23,25,26] and chondrocytes [23,52] in vitro, and new bone formation in vivo [28,29]. In contrast, BMP2 is also known to have a positive feedback effect on CREB function. In osteoblast precursor C2C12 cells, BMP2 synergizes with PKA- CREB to enhance ALP activity [25]. Therefore, the crosstalk between the PTH-CREB and BMP2 signaling pathways is complex, and could occur at multiple steps in these signaling cascades. In order to further test the role of BMP2 in mediating the function of PTH-CREB signaling in osteoblast differentiation and bone formation, a BMP2 invalidated mouse model is needed. We are now proposing a study using osteoblast-specific BMP2 knockout mice to determine the effects of BMP2 deficiency on the anabolic effects of intermittent PTH on bone mass. The present finding that PTH-CREB up-regulates of BMP2 gene expression in osteoblasts provides new insights into the relation between these important anabolic signaling pathways in bone. In summary, the functional and mechanistic studies in this study demonstrate that PTH signaling is a positive regulator of BMP2 gene expression in osteoblasts, mediated directly by CREB transactivation of BMP2 promoter activity. This transcriptional mechanism contributes to osteoblast differentiation. The collective results of these studies support the hypothesis that the anabolic effects of PTH on bone mass are mediated through the cAMP- PKA-CREB-BMP2 pathways. Materials and Methods DNA constructs and compounds The CREB expression plasmid pRC/RSV-CREB, its empty vector pRC/RSV, and CREB responsive reporter (CRE-Luc) containing multiple CREs were obtained from Dr. Richard Goodman [53]. The ATF4 expression vector was a gift of Dr. Xiangli Yang [42]. Mouse BMP2 promoter luciferase reporter (22712/+165-Luc) was constructed by linking the 59 promoter region (22712/+165) of mouse the BMP2 gene to firefly luciferase in a pGL3 vector [39–40]. The promoter was truncated from the distal end by enzymatic digestion to create a series of deletion constructs, including 22457/+165-Luc, 2199/+165-Luc, 21803/+165-Luc, 2968/+165-Luc, 2838/+165-Luc, 2310/ +165-Luc, and 2150/+165-Luc. Mouse BMP4 promoter reporter (BMP4-Luc) was obtained from Dr. Stephen Harris. The CREB siRNA (CREB ShortCutH siRNA Mix), and negative control siRNA (New England Biolabs, Beverly, MA), IBMX and KT5720 (Calbiochem, San Diego, CA), and PTH and noggin (R&D Systems, Minneapolis, MN), was purchased. PLoS ONE | www.plosone.org 9 June 2011 | Volume 6 | Issue 6 | e20780