Transcriptional Regulation of BMP2 Expression by the PTH-CREB Signaling Pathway in Osteoblasts Rongrong Zhang1., James R. Edwards2., Seon-Yle Ko3., Shanshan Dong 1 , Hongbin Liu1, Babatunde O. Oyajobi4, Christopher Papasian5, Hong-Wen Deng 1 , Ming Zhao1,6* 1 Department of Biostatistics and Bioinformatics, Tulane University, New Orleans, Louisiana, United States of America, 2 Department of Medicine, Vanderbilt University, Nashville, Tennessee, United States of America, 3 School of Dentistry, Dankook University, Cheonan, Choongnam, Korea, 4 Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America, 5 Department of Basic Medical Sciences, University of Missouri – Kansas City, Kansas City, Missouri, United States of America, 6 Department of Cellular and Molecular Biology, Tulane University, New Orleans, Louisiana, United States of America Abstract Intermittent application of parathyroid hormone (PTH) has well established anabolic effects on bone mass in rodents and humans. Although transcriptional mechanisms responsible for these effects are not fully understood, it is recognized that transcriptional factor cAMP response element binding protein (CREB) mediates PTH signaling in osteoblasts, and that there is a communication between the PTH-CREB pathway and the BMP2 signaling pathway, which is important for osteoblast differentiation and bone formations. These findings, in conjunction with putative cAMP response elements (CREs) in the BMP2 promoter, led us to hypothesize that the PTH-CREB pathway could be a positive regulator of BMP2 transcription in osteoblasts. To test this hypothesis, we first demonstrated that PTH signaling activated CREB by phosphorylation in osteoblasts, and that both PTH and CREB were capable of promoting osteoblastic differentiation of primary mouse osteoblast cells and multiple rodent osteoblast cell lines. Importantly, we found that the PTH-CREB signaling pathway functioned as an effective activator of BMP2 expression, as pharmacologic and genetic modulation of PTH-CREB activity significantly affected BMP2 expression levels in these cells. Lastly, through multiple promoter assays, including promoter reporter deletion, mutation, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA), we identified a specific CRE in the BMP2 promoter which is responsible for CREB transactivation of the BMP2 gene in osteoblasts. Together, these results demonstrate that the anabolic function of PTH signaling in bone is mediated, at least in part, by CREB transactivation of BMP2 expression in osteoblasts. Citation: Zhang R, Edwards JR, Ko S-Y, Dong S, Liu H, et al. (2011) Transcriptional Regulation of BMP2 Expression by the PTH-CREB Signaling Pathway in Osteoblasts. PLoS ONE 6(6): e20780. doi:10.1371/journal.pone.0020780 Editor: Jean-Marc A. Lobaccaro, Clermont Universite´, France Received March 4, 2011; Accepted May 9, 2011; Published June 9, 2011 Copyright: 2011 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mzhao1@tulane.edu . These authors contributed equally to this work. Introduction Parathyroid hormone (PTH) plays an important role in skeletal metabolism. In mice [1–4] and rats [5,6], intermittent adminis- tration of PTH has anabolic effects on bone mass and bone formation. Similarly, when PTH is administered intermittently to humans, it has been proven to increase bone mass and decrease fracture risk in post-menopausal women, elderly men, and women with glucocorticoid-induced osteoporosis [7–10]. Despite its clinical efficacy, the precise molecular mechanisms responsible for the anabolic effects of PTH on bone, particularly at the transcriptional level, need to be further elucidated. Although multiple intracellular signaling mechanisms are involved in mediating PTH function, the major downstream signaling pathway of PTH in bone cells involves 59-cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and cAMP response element binding protein (CREB). The anabolic function of this cAMP-PKA-CREB pathway in bone has been characterized in vivo and in vitro [11–30]. Increasing activity of this pathway promotes osteoblast differentiation [11–26] and stimulates bone formation [27–30]. In osteoblasts, PTH binds to a PTH receptor and induces formation of cAMP, leading to activation of PKA, which in turn phosphorylates and activates CREB, a member of a large family of basic leucine zipper (bZIP) domain DNA-binding proteins [11–14]. Activated CREB, in association with the other co-activators, binds to target genes through a cAMP response element (CRE) and activates their transcription. Therefore, in this pathway, CREB plays a pivotal role by converting the PTH signal to activation of gene expression. In various cell systems, CREB induces the expression of some osteoblast-related genes, such as bone sialoprotein (BSP) and osteocalcin (OCN), by directly binding to these promoters [13–22]. However, the major downstream targets of CREB transactivation, which have a predominant role in initiating osteoblast differentiation and stimulating bone formation, are unknown. Bone morphogenetic protein 2 (BMP2) is an important growth factor that stimulates osteoblast differentiation and bone formation [31–33]. The PTH-cAMP-CREB signaling pathway synergizes the anabolic signaling of BMP2 for osteoblast differentiation and bone formation [23,25,26,28,29,34], indicating that there is a PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e20780