Protein Palmitoylation Regulates Osteoblast Differentiation through BMP-Induced Osterix Expression Wai Fook Leong, Tielin Zhou, Gek Liang Lim, Baojie Li* Cancer and Developmental Biology Division, The Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore, Singapore Abstract Osteoporosis is one of the most common diseases and can be treated by either anti-resorption drugs, anabolic drugs, or both. To search for anabolic drug targets for osteoporosis therapy, it is crucial to understand the biology of bone forming cells, osteoblasts, in terms of their proliferation, differentiation, and function. Here we found that protein palmitoylation participates in signaling pathways that control osterix expression and osteoblast differentiation. Mouse calvarial osteoblasts express most of the 24 palmitoyl transferases, with some being up-regulated during differentiation. Inhibition of protein palmitoylation, with a substrate-analog inhibitor, diminished osteoblast differentiation and mineralization, but not proliferation or survival. The decrease in differentiation capacity is associated with a reduction in osterix, but not Runx2 or Atf4. Inhibition of palmitoyl transferases had little effect in p532/2 osteoblasts that show accelerated differentiation due to overexpression of osterix, suggesting that osterix, at least partially, mediated the effect of inhibition of palmitoyl transferases on osteoblast differentiation. BMPs are the major driving force of osteoblast differentiation in the differentiation assays. We found that inhibition of palmitoyl transferases also compromised BMP2-induced osteoblast differentiation through down-regulating osterix induction. However, palmitoyl transferases inhibitor did not inhibit Smad1/5/8 activation. Instead, it compromised the activation of p38 MAPK, which are known positive regulators of osterix expression and differentiation. These results indicate that protein palmitoylation plays an important role in BMP-induced MAPK activation, osterix expression, and osteoblast differentiation. Citation: Leong WF, Zhou T, Lim GL, Li B (2009) Protein Palmitoylation Regulates Osteoblast Differentiation through BMP-Induced Osterix Expression. PLoS ONE 4(1): e4135. doi:10.1371/journal.pone.0004135 Editor: Stefan W o¨lfl, Universita¨t Heidelberg, Germany Received June 17, 2008; Accepted December 4, 2008; Published January 6, 2009 Copyright: 2009 Leong 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 work was supported by the Agency of Science, Technology, and Research of the Republic of Singapore. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: libj@imcb.a-star.edu.sg Introduction Bone is a dynamic organ and is constantly remodeled. New bones are formed by osteoblasts to replace the old ones, which are resorbed by osteoclasts. A fine balance between bone formation and bone resorption is needed to maintain an optimal bone mass [1,2]. Indeed, there exist multiple coupling mechanisms between osteoblasts and osteoclasts [3]. For example, osteoblasts can synthesize and secrete cytokines such as M-CSF and RANKL to promote osteoclastogenesis from hematopoietic stem cells of the bone marrow. On the other hand, bone resorption releases TGFb and BMPs that are trapped in the bone matrix, facilitating osteoblast migration, differentiation and function [4]. Disruption of the balance between bone resorption and formation usually leads to osteosclerosis or osteoporosis [2]. Osteoporosis affects one out of every two women and one out of every four men over age 50, and is regarded as a major public health threat. While there are some anti-resorption drugs in clinical use, such as SERMs and bisphosphonates, there is a lack of anabolic drugs. To date, parathyroid hormone (and teriparatide) and strontium ranelate are the only available anabolic drugs in clinical use [5,6]. Increasing efforts are being made to search for more efficient anabolic drugs with lesser adverse effects. Osteoblasts are derived from bone marrow mesenchymal stem cells (MSCs) under the influence of growth factors such as BMPs and Wnts [2,7]. The two transcription factors that are relatively specific to osteoblast, Runx2 and osterix (Osx), play essential roles in osteoblast differentiation from MSC [8–10]. Deletion of either one by gene targeting leads to the loss of mature osteoblasts and lack of calcified bones [11,12]. On the other hand, ectopic expression of Runx2 or Osx enhances osteoblast differentiation and mineralization [12,13]. Moreover, there is evidence to support the notion that the levels of Osx determine the differentiation status of osteoblasts [14–16]. Given the importance of Osx in osteoblast differentiation and function, it is important to study the regulation of Osx expression. Recent studies show that Osx can be induced by Notch, BMPs, and TNF and its expression is further controlled by posttranslational regulation [17–21]. The induction of Osx is believed to mediate the effect of BMPs on osteoblast differentiation. BMPs can transactivate Osx through both the canonical BMP-Smad1/5/8 pathway and the non-canonical BMP-MAPK pathway [17–19]. Protein function is affected by its expression level, localization, interaction with other proteins, and its posttranslational modifica- tions. Recent studies indicate that many proteins can be modified by palmitoylation on cysteine residues by a family of proteins that contain a unique zf-DHHC domain. At least 24 members have been identified in mammalian genome [22]. This modification regulates protein localization, trafficking, and stability [23–25]. To understand the importance of protein palmitoylation in osteoblast biology, we analyzed the expression of the 24 palmitoyl acyltransferases (PATs) by RT-PCR and found some of them PLoS ONE | www.plosone.org 1 January 2009 | Volume 4 | Issue 1 | e4135