activation and its association with other Smad family members. The association with Smad2/3 is important to the translocation of this protein into the cell nucleus, where it binds to target promoters and forms a transcription repressor complex with other cofactors. This protein can also be phosphorylated by activin type 1 receptor kinase, and it mediates the signal from the activin. Alternatively spliced transcript variants encoding the same protein have been observed [36]. Like other Smads, Smad2 and Smad3 play roles in the transmission of extracellular signals from ligands of the TGF-b superfamily of growth factors into the cell nucleus [37]. Binding of a subgroup of TGF-b superfamily ligands to extracellular receptors triggers the phosphorylation of Smad2 at a serine-serine-methionine-serine motif at its extreme C-terminus [33,38,39]. Smad2/3 is a key transcriptional factor in osteoblast differentiation, as indicated by its relationship with PAPSS2. Our results suggest that, Smad signal pathways in osteoblast are at least partially mediated by PAPSS2. This study demonstrated the regulatory mechanism of PAPSS2 acts during osteoblast differentiation and mineralization, which is one of principal issues remaining to be elucidated in the control of bone formation for better understanding of pathogenesis of osteoporosis and for the development of therapeutic procedures. In conclusion, these results indicate that PAPSS2 plays an essential role in Smads signaling pathways during osteoblast differentiation. Ongoing research in this laboratory will determine the mechanism by which PAPSS2 affects collagen assembly during bone formation and function, which transcription factors or growth factors regulate PAPSS2 gene expression, synthesis, accumulation, and protein degradation, and the manner in which PAPSS2 regulates TGF pathways. Materials and Methods Cells and cell culture This study was approved by the Animal Experiment Committee of Xian Jiaotong University. MC3T3-E1 cell line, which is an osteoblast line derived from murine calvaria that differentiates in culture, and RAW264.7, which is a mouse monocytic cell line, and ecotropic Phoenix packaging cells were obtained from American Type Culture Collection(ATCC, Bethesda, MD, U.S.) [40]. The cells were seeded at a density of 16104 cells/cm2 and maintained in growth medium consisting of a-modified Eagle’s medium plus 10% fetal bovine serum (FBS, Gibco), 100 U/ml penicillin, and 100 mg/ml streptomycin in a humidified atmosphere containing 5% CO2 at 37uC. They were passaged every 5–7 days and not used beyond passage 20. For osteoblast differentiation, the MC3T3-E1 clone 4 cells were induced with osteogenic medium (OS media), specifically, the growth medium containing 50 mg/ml ascorbic acid and 10 mM b-glycerol-phosphate. For osteoclast differentiation, RAW264.7 cells were induced with 10 ng/ml RANKL and 20 ng/ml M-CSF. Reverse Transcription and Real-Time PCR Analysis Total RNA from mouse tissues (lung, spleen, kidney, muscle, liver, heart, brain, calvaria, bone) and from the OS media-induced MC3T3-E1 clone 4 cells, or RANKL/M-CSF induced RAW264.7 cells was isolated using Trizol reagent (Invitrogen). A two-step reverse transcription real-time PCR (Invitrogen) was used for the amplification of mRNA expression. The sequences of primers are as follows: for PAPSS2 (forward primer, 59- TGTAAAACGACGGCCAGT-39; reverse primer, 59-CAG- GAAACAGCTATGACC-39), the a1 chain of collagen type I (Col1, forward primer, 59-CACCCTCAAGAGCCTGAGTC-39; reverse primer, 59-GCTTCTTTTCCTTGGGGTTC-39), osteo- Function of PAPSS2 in Osteoblasts pontin (OPN, forward primer, 59-TCACCATTCGGAT- GAGTCTG-39, reverse primer, 59-ACTTGTGGCTCT- GATGTTCC-39), Smad2 (forward primer, 59- GAGGAGCAGCTCGCCAA-39; reverse primer, 59- CTGTCAAGGTCCGGCCAGCG-39), Smad3 (forward primer, 59-TG GTCCTCTGGGCATCTCAGGC-39; reverse primer, 59- GGTGAACCTGCTGTTGCCCTCA-39, GAPDH (forward primer, 59-ACCACAGTCCATGCCATCAC-39; reverse primer, 59-TCCACCACCCTGTTGCTGTA-39). PCR-amplified prod- ucts were separated by 1.2% agarose gel electrophoresis. To estimate the degree of accuracy of each transcript, relevant bands were quantified using the NIH Image J software, and the resulting products were normalized against GAPDH levels. For real-time PCR, specific primers for PAPSS2 were synthesized as shown above. Real-time PCR was performed on an ABI PRISM 7500 sequence detection system with SYBR GREEN PCR Master Mix (Applied Biosystems) according to the manufacturer’s instructions. The PCR conditions were as follows: 94uC for 1 min followed by 35 cycles of 95uC for 30 s and 58uC for 40 s. All of the reactions were run in triplicate and normalized to the housekeeping gene GAPDH. The evaluation of relative differences of PCR results was calculated using the comparative cycle threshold method. Papss2 shRNA lentivirus packaging, tittering and cell infection To determine the role of PAPSS2 gene in osteoblast differen- tiation and osteoblastic signaling pathways, we used lentivirus vector mediated PAPSS2 shRNA (Open Biosystems) for packaging PAPSS2 recombinant lentivirus. It was used in accordance with the manufacturer’s instructions. Briefly, the 293FT producer cell line was co-transfected with the expression constructs (pLenti- scrambled shRNA, pLenti-P2a, or pLenti-P2b) and packaging mixture. The viral supernatant was harvested after 48–72 hours and titers were determined by infecting HeLa or 293T cells with serial dilutions of concentrated lentivirus. After 48 hours of incubation, the cells were analyzed by RT-PCR, Western blotting, and immunostaining to determine silent efficiency of the PAPSS2 gene. For osteoblast differentiation, the MC3T3 cells were infected with viral supernatant plus 6 m/ml polybrene (Sigma) for 24 hours were induced with OS media for the indicated times based on different experiments. Construction of overexpression vectors, and production of recombinant retrovirus Retroviral vector pBMP-PAPSS2 was constructed by inserting a full-length 3.64 kb PAPSS2 cDNA (access no. NM_011864) into the EcoRI and Not I site of pBMN-I-GFP (Addgene), and packaging was performed as the protocol from Dr. Garry Nolan Laboratory, Stanford University. Briefly, retrovirus vectors pBMN-I-GFP (control vector) and pBMN-PAPSS2 were sepa- rately transfected into the Phoenix-eco tropic packaging cells by CaCl2 precipitation method. Following the transfection, the cells were placed in a 32uC humidified incubator for 48 hours (32uC aids in stabilizing the virus). The media containing infectious virus was harvested and filtered through a 0.45 mm filter for titration assay and infecting MC3T3-E1 clone 4 cells. GFP and PAPSS2 protein expression were confirmed by observation of GFP+ cells, immunostaining, and Western blot analysis. The retroviruses carrying pBMP-PAPSS2 and pBMN-I-GFP were then used to infect 70–80% subconfluent MC3T3-E1 clone 4 cells in the presence of 6 mg/ml Polybrene. PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e43475