cardiomyocytes, suggesting that different cell types may have specific sets of palmitoylated proteins [28]. Taken together, these results indicate that osteoblasts express many PATs and some proteins are palmitoylated. Inhibition of PATs impeded osteoblast differentiation and mineralization, but not proliferation or survival To study whether protein palmitoylation plays a role in osteoblast function, we intended to inhibit protein palmitoylation with PATs inhibitors and/or to silence PATs with short hairpin RNA. However, too many PATs are expressed in osteoblasts and antibodies against these PATs are mostly unavailable, it was not feasible, at this stage, to silence individual PATs. Instead, we used 2-bromohexadecanoic acid (2-bromopalmitate, 2BP) to block PAT activity. 2BP is a substrate analog inhibitor that should have an effect on most if not all PATs [29]. We found that 100 mM of 2BP was sufficient to repress protein palmitoylation in osteoblast by 80% (Fig. 1D). In the following experiments, we used 2BP at the concentration of 10 to100 mM in osteoblasts to inhibit PAT activities and check the consequences in two aspects: differentiation and mineralization. We first looked at one of the early markers of osteoblast differentiation, alkaline phosphatase (ALP). It was found that 2BP had a dosage dependent inhibitory effect on ALP expression. Fig. 2A shows the ALP staining of osteoblasts cultured in the differentiation medium, while Fig. 2B shows the quantitative ALP activities that were normalized to the total protein levels. It is obvious that inhibition of protein palmitoylation diminished ALP expression. We then tested the expression of a few other markers by RT-PCR. It was found that 2BP mainly down-regulated the mRNA levels of osteocalcin, but not collagen type 1a or osteopontin (Fig. 2C). More significantly, we found that 2BP severely inhibited bone nodule formation, a later marker of osteoblast differentiation and an indicator of osteoblast bone forming activity (Fig. 2D). These results indicate that protein palmitoylation is required for osteoblast differentiation into mature osteocytes. However, treatment of osteoblast with the same amounts of palmitic acid, the substrate of PATs (.150 mM palmitic acid showed cytotoxicity in primary osteoblasts), showed no significant effect on osteoblast differentiation (Fig. 2E). The reason why higher concentrations of palmitic acid did not enhance osteoblast differentiation could be that the serum provides sufficient amount of palmitic acid for protein palmitoylation. To test whether 2BP has an effect on cell proliferation/survival in the in vitro differentiation assays, which may indirectly influence osteoblast differentiation, we first counted the live cells in osteoblast cultures in the presence or absence of 2BP. No significant difference was observed (Fig. 3A). Moreover, 2BP showed no significant effect on the total protein levels of the osteoblast cultures (Fig. 3B). More importantly, removal of 2BP from cell cultures led to a recovery of osteoblast differentiation. Two sets of osteoblast cultures were treated with increasing concentrations of 2BP for three days. One set was continually cultured in the presence of 2BP for 4 more days, while the other set had 2BP washed off and then cultured for 4 more days in the differentiation medium. It was found that the expression of ALP in osteoblasts recovered rather well and this was confirmed by the quantitative ALP assays (Fig. 3C and data not shown). These results indicate that 2BP, at the concentrations used, has little effect on cell proliferation and/or survival, at least for a short term. Instead, it mainly affects osteoblast differentiation. Down-regulation of Osx by the PAT inhibitor Osteoblast differentiation is controlled by transcription factors such as Runx2, Osx, and Atf4. Elevation in any of these Palmitoylation in Osteoblast transcription factors promotes osteoblast differentiation while deletion of any of them leads to defects in osteoblast maturation and bone calcification [11,12,30]. We used RT-PCR to monitor the mRNA levels of these transcription factors. Both Osx and Runx2, but not Atf4, showed an up-regulation during osteoblast differentiation, with the increase in Osx mRNA levels being much more pronounced (Fig. 4). 2BP treatment did not significantly affect the mRNA levels of Runx2 or Atf4, suggesting that Runx2 and Atf4 are unlikely to mediate the inhibitory effect of 2BP on osteoblast differentiation. In contrast, 2BP treatment diminished Osx mRNA levels. These results suggest that down-regulation of Osx might mediate the effect of 2BP on osteoblast differentiation. It is known that osteoblasts express and secrete cytokines that promote osteoclastogenesis [3]. We also tested whether inhibition of protein palmitoylation had any effect on the synthesis of these cytokines. RT-PCR assays show that both OPG and RankL were down-regulated during differentiation while the levels of M-CSF were not altered (Fig. 4). 2BP treatment only slightly decreased the mRNA levels of RankL (Fig. 4). Since during osteoblast differenti- ation, RankL down-regulation is associated with Osx up-regulation, we believe that the further decrease of RankL induced by 2BP is probably not due to the changes in Osx. These results suggest that inhibition of protein palmitoylation may not greatly affect the ability of osteoblast to promote osteoclastogenesis. p532/2 OBs were resistant to PAT inhibitor in differentiation To further confirm that Osx mediates the effect of 2BP on osteoblast differentiation, we used p532/2 osteoblasts that have been shown to have an increased expression of Osx and enhanced differentiation [16,31,32]. Moreover, elevated expression of Osx, which was directly repressed by p53, was demonstrated to mediate the effect of p53 on differentiation. We found that the elevated levels of Osx was not further induced during osteoblast differentiation in p532/2 osteoblast, and that 2BP showed little effect on the mRNA levels of Osx either (Fig. 5A). Consistent with this result, ALP staining was not markedly inhibited by 2BP (Fig. 5B). Moreover, 2BP showed only a slight effect on bone nodule formation in p532/2 osteoblast cultures (compared to the Fig. 2D) (Fig. 5C). These results indicate that p532/2 osteoblasts are refractory to the negative effect of 2BP, suggesting that Osx mediated the effect of 2BP on osteoblast differentiation. PAT inhibitor compromised BMP2-induced osteoblast differentiation In the in vitro differentiation assays, BMPs from the serum or secreted by osteoblasts themselves are the driving force of differentiation. It has been shown that the addition of noggin and chordin to differentiation cultures diminished osteoblast differenti- ation, which is accompanied by down-regulation of Osx [19]. We then tested whether 2BP has an effect on BMP2 induced differentiation. It was found that 50 ng/ml of BMP2 was able to dramatically up-regulate ALP expression, which was almost abolished by 2BP (Fig. 6A). More interestingly, BMP2 was able to markedly up-regulate the mRNA levels of Osx, which was impeded by 2BP as well (Fig. 6B), supporting the notion that 2BP inhibits osteoblast differentiation by repressing Osx expression. On the other hand, BMP2 had minimal effect on the mRNA levels of Runx2. PAT inhibitor negatively regulated the activation of p38 MAPK but not Smad1/5/8 How does inhibition of protein palmitoylation regulate Osx expression? Several studies indicate that Osx is under the control PLoS ONE | www.plosone.org 3 January 2009 | Volume 4 | Issue 1 | e4135