were up-regulated when osteoblasts started to differentiate. More importantly, we found that inhibition of protein palmitoylation with a substrate analog inhibitor impeded osteoblast differentiation and function, which are mediated by altered expression of Osx through the p38 MAPK pathway. This study also suggests that palmitoylation modifies proteins downstream of BMPR and upstream of p38 MAPK and this modification might regulate other cellular events that involve this pathway. Results Expression of palmitoyl transferases during osteoblast differentiation Protein palmitoylation plays an important role in regulating localization, trafficking, and degradation of a protein, and its Table 1. The primer sequences for the 24 mouse PATs and other genes studied here and the sizes of the PCR products. Gene Zdhhc1 Zdhhc2 Zdhhc3 Zdhhc4 Zdhhc5 Zdhhc6 Zdhhc7 Zdhhc8 Zdhhc9 Forward Reverse Size CACCTGCTCTGCTTCCACAT CTCAGCTGATGCCGAGTAGT GGTCTGCCTGATACTCAAGC GTTCCATTCCTCCACAGCAC TACAAGTGTCCCAAG TGCTG GGTCCTTCAGACCACATACT ACCTTCATCGTCTTGCACCT GGAGTGAATGTTCTGGTGGA ACCCTCACCAGTCCGTTATG GGTGTAGGTGCAGAGGTGTG TTCGGATTGGCTGCGTTTGC AGTCACCATCACAGGGACAC GGTGTGGTTCATCCGAGATG TTCTCGCTCTTCAGCCTCTC CGACAATGGGCTGAAAGCTG GTGCAGGTAGGGTGAATGGT 666 CTACCTCTTCATCCTCTCTC TCTTCAGGAATGCTGGTGTC Zdhhc11 AGTACTGCCACCTGTGTGAG TTCGGCGAAAGAGTAGACAC Zdhhc12 GTGCTAAGCTCCCTGCTGCT GCAGCCTTCTCTCCAGCAAC Zdhhc13 GACTGGCTCTCTGGACTTCA TAAGCCAAAGCAGCCACACT Zdhhc14 AGCCTGTGTGATAACTGCGT CATGGTACGGCTATGTGCTA Zdhhc15 CGTTCTCTACTGCCTGTACA AACCTGCTACGTTTCCGACT Zdhhc16 CTCATCCTCCGAACCTACTC TCCAGTTGTCCAAGCAGCCA Zdhhc17 ATGGTTCTTCTGGTTCTGGA TCCTTACACATCCATGGTTG Zdhhc18 TCTCCCTCTCCTTCTTGACG CTCCTACCATGCTGGCGTCT Zdhhc19 ACCTTCTTCAGTCTCGTCTC CTACAGTGTTTAGGACGACG Zdhhc20 TCTACACCACATCAGCTTCA CTGGTTTGCAACAGAAGCTT Zdhhc21 TGCCTGGTTGCCTTAGTGAG ACAGGATCTTCCAACGAGTG Zdhhc22 GTGACCTTCGTACTGCAGCT ATTTGTCCTGCTGCTTCGAG Zdhhc23 TGCTGGCACTCTGGTATTAC CAGCTGGATGAGGAAGATGT 742 Zdhhc24 TTCCTGTGTCTCCTGCTTCA AGTCACAAGACCCACATCAC Zdhhc25 TCACACCTACGGACTATGCT CATGGTGCTCACTCACTTTG b-actin Atf4 Col1a1 M-CSF Ocn Opg Opn Osx RankL Runx2 AGATGTGGACAGCAAGCAG GCGCAAGTTAGGTTTTGTCA TTCCACTCCAGAGCATTCCT CAGGTGGGTCATAAGGTTTG GCAATCGGGATCAGTACGAA CTTTCACGCCTTTGAAGCCA CTGACACAGGCCATGTGGAG GAGAGGGTAGTGGTGGATGT 315 AAGCAGGAGGGCAATAAGGT AGCTGCTGTGACATCCATAC CACCCTGTGTGAAGAGGCCT GCAGGCTCTCCATCAAGGCA TCACCATTCGGATGAGTCTG ACTTGTGGCTCTGATGTTCC TGAGGAAGAAGCCCATTCAC ACTTCTTCTCCCGGGTGTG TACTTTCGAGCGCAGATGGAT GTACGCTTCCCGATGTTTCAT CCGCACGACAACCGCACCAT CGCTCCGGCCCACAAATCTC doi:10.1371/journal.pone.0004135.t001 503 561 527 761 608 612 662 485 606 665 531 848 477 630 657 431 658 589 555 729 434 576 123 280 484 291 310 436 197 483 288 Palmitoylation in Osteoblast interaction with other proteins. To learn whether protein palmitoyla- tion is involved in osteoblast function, we first analyzed the expression of the 24 potential PATs in calvarial osteoblasts at different stages of differentiation by RT-PCR (Table 1 for primer sequences). It was found that 20 out of 24 PATs can be detected (Fig. 1A–C). More interestingly, DHHC1, 2, 6, 7, 15, 23, 25 were up-regulated in differentiating osteoblasts compared to non-differentiated cells (Fig. 1B), suggesting that these PATs might play an active role in this process. On the other hand, DHHC9, 12, 13, 18 were slightly down-regulated compared to non-differentiated osteoblasts, while DHHC4, 5, 8, 14, 16, 17, 19, 20, and 24 showed no alteration at the mRNA levels (Fig. 1A and 1C). DHHC3, 11, 21, 22 were undetectable, suggesting that their expression in osteoblasts could be very low. Note that DHHC10 does not exist. A recent study showed that in most of the human tissues, 17 of these PATs were expressed [26]. Our previous studies showed that both DHHC19 and DHHC6 were ubiquitously expressed in both mouse and human tissues [27]. Osteoblasts have many PAT substrates To confirm that protein palmitoylation does occur in osteo- blasts, we labeled OBs with 3H-palmitic acid for 24 hours. Total proteins were separated onto SDS-PAGE gels, which were dried and exposed to X-ray films. Osteoblasts clearly showed several radio-labeled bands (proteins with covalent palmitoyl modifica- tion). The most prominent ones were at the MW of ,20, 36, 45, 80, 135 kDa (Fig. 1D). This pattern is different from that of Figure 1. Expression of PATs and protein palmitoylation in osteoblasts. A. RT-PCR analysis of the mRNA levels of the 24 known PATs during osteoblast differentiation. Primary osteoblasts were cultured in the differentiation medium for different periods of time. Total RNA was isolated from these cultures and was then used to carry out RT-PCR. These PATs were classified into unaltered expression (A), increased expression (B), and decreased expression (C), in comparison to the day 1 cultures. D. 2BP was able to inhibit protein palmitoylation in primary osteoblasts. Primary osteoblasts were treated with 100 mM of 2BP for 2 hrs and then 3H-palmitic acid was included in the culture medium for 24 hrs. The cells were harvested and the same amounts of total proteins were loaded for control and 2BP treated samples. The SDS-PAGE gel was dried and exposed to x-ray films. The palmitoylated proteins are radio-labeled. Actin was used as a loading control. doi:10.1371/journal.pone.0004135.g001 PLoS ONE | www.plosone.org 2 January 2009 | Volume 4 | Issue 1 | e4135