Palmitoylation in Osteoblast Figure 5. Overexpression of Osx in p532/2 osteoblasts rendered resistance to the inhibitory effect of 2BP on differentiation. A. 2BP treatment did not significantly down-regulate the mRNA levels of Osx. Primary osteoblasts were cultured in the presence or absence of 2BP for different periods of time and total RNA was isolated and RT-PCR was carried out to determine the mRNA levels of Osx. The value of control (lane 1) was set at 1.00. B. PAT inhibitor did not markedly affect ALP staining in p532/2 osteoblasts. p532/2 osteoblasts were treated with different amounts of 2BP for 7 days and then stained for ALP. C. PAT inhibitor did not markedly affect bone nodule formation in p532/2 osteoblasts. p532/2 osteoblasts were treated with different amounts of 2BP for 21 days and then stained for mineralization. doi:10.1371/journal.pone.0004135.g005 expression of Osx and the induction of Osx by BMP2. These results also support the notion that Osx might be a critical transcription factor that integrates many cues to control osteoblast differentiation. For example, c-Abl, p53, Atm, and p38 MAPK are all involved in osteoblast differentiation and they all regulate the expression of Osx, but not Runx2 [16,19,33,34]. One explanation for the difference between Runx2 and Osx could be that Runx2 induction by BMPs is usually transient and modest, whereas Osx induction can be long lasting and robust (Fig. 4 and 6). BMPs are synthesized and secreted by osteoblasts and are the main driving force of in vitro differentiation [19]. The observation that PAT inhibitor compromised Osx induction by BMP2 suggests that protein palmitoylation might be involved in signaling pathways activated by BMPs. However, PAT inhibitor did not show a marked effect on the canonical BMP-Smad signaling. Instead, it compromised MAPK activation at the basal level and in the presence of BMP2. One possibility is that components of the non-canonical pathway such as Tak1, Tab1/2, Xiap, MAPKKs, are palmitoylated [35]. Another possibility is that BMPRs are palmitoylated and their palmitoylation differentially regulates the Smad pathway and the MAPK pathway. Indeed, we found that BMPRI and II could be palmitoylated. We are currently mapping the palmitoylation sites and studying their role in regulating BMPs triggered Smad and MAPK pathways (Leong et al, unpublished results). Further studies are needed to determine how protein palmitoylation affects MAPK activation through BMP receptors. This is because BMP-Smad signaling and MAPK activation can be complex: i) MAPKs are regulated by many cues including growth factors that are present in the culture medium, and some types of stress; ii) BMPs have been reported to either activate or inhibit MAPKs, depending on the cell type and context [19,36,37]; iii) BMPs are known to require cooperation with other signaling pathways to control the expression of target genes due to the nature of Smad binding elements (only 4 bp) [38]; iv) a crosstalk exists between BMP-Smad signaling and MAPK [38,39]. Thus it is important that caution be exercised in interpreting these results. Nevertheless, this study provides a new dimension in p38 MAPK activation and osteoblast differentiation. Future study will focus on identifying the palmitoylation target and studying the function of the palmitoylation in regulating Osx expression and osteoblast differentiation. In summary, this is the first study to show that protein palmitoylation plays an important role in osteoblast differentiation and function. It seems that the potential palmitoylation substrates might regulate the MAPK pathway, especially p38 MAPK, to regulate Osx expression and osteoblast differentiation. Given the importance of BMPs activated signaling pathways in bone remodeling and cancer development, it is worth the effort to further study how protein palmitoylation affects these signaling events. Materials and Methods Isolation, culture and treatment of calvarial osteoblasts To prepare primary osteoblasts, calvaria were excised from 4–5 newborn pups (p532/2 and wild type on a 129/Sv background), washed in PBS and digested in MEM alpha medium containing PLoS ONE | www.plosone.org 6 January 2009 | Volume 4 | Issue 1 | e4135