Function of PAPSS2 in Osteoblasts Figure 2. Silencing of PAPSS2(P–S) with lentivirus transfection block PAPSS2 expression and overexpression of PAPSS2 (P–O) with adenovirus system significantly increases the PAPSS2 expression. (A) RT-PCR analysis. MC3T3-E1 clone 4 cells were infected with plenti- shRNA PAPSS2 (P–S) or plenti-scrambled shRNA (PLB) viruses for 48 hours and then induced with OS media for 0, 3, 7, and 14 days. The transcription level of PAPSS2 gradually increased in PLB cells (lanes 1, 2, 3, and 4) and were much lower or undetectable in P–S cells (lanes 4, 5, 6, and 7). (B) Values in A are normalized to GAPDH levels. (a P,0.05 between P–O and PLB for 1, 3, 7, and 14 days). (C) Western blot analysis of PAPSS2 protein expression. The cells were treated as described in A. (D) Quantitative analysis of protein levels from immunoblots, as in C. The protein levels of PAPSS2 were normalized to GAPDH. (a P,0.05 between P–O and PLB for 7 and 14 days). The level of PAPSS2 in the silenced group was 426.6 times (7 d) and 6.6 times (14 d) lower than that of the control. (E) Real-time PCR analysis of PAPSS2 expression in MC3T3-E1 cells infected with pBMN-I-GFP (control) or pBMN-PAPSS2 (P–O) for 24 hours. It was then induced with OS media for 14 days. The signals were significantly increased in P–O group compared with that in control group (a P,0.05 between P–O and the control). (F) Western blot analysis of PAPSS2 protein expression after cells were treated as described in E. (G) Quantitative analysis of protein levels from immunblots, as in F. The protein levels of PAPSS2 were normalized to GAPDH. The level of PAPSS2 in the silenced group was 1.9 times (14 days) higher than that of the control. (a P,0.05 between P–O and the control) (H) Immunofluorescence staining revealed that the expression of PAPSS2 was blocked after infection with P–S and overexpression in P–O cells relative to cells without virus transfection (control) and to cells without P–O after 14 days of induction with OS media. The cell nuclei were stained using DAPI (blue) to identify the number of cells. doi:10.1371/journal.pone.0043475.g002 Inactivating mutations for PAPSS2 are associated with severe inherited developmental skeletal disorders, Kanshin Beck disease, spondyloepimetaphyseal dysplasia in humans, and brachymorph- ism in mice [8,12]. The deficiency of PAPSS2 results in osteochondrodysplasias. Osteoechondrodysplasias are a genetically heterogeneous group of disorders that affect skeletal development, linear growth, and the maintenance of cartilage and bone. A large inbred family of mice with a distinct form of recessively inherited, spondyloepimetaphyseal dysplasia (SEMD) was examined. Their condition was attributed to a PAPSS2 isoform located in the chromosome region of 10q23–24 [21]. Diastrophic dysplasia is an autosomal recessive osteochondrodysplasia that constitutes dis- orders of the skeletal system that can have defect on growth or bone density (the physical characteristics include, short limb stature, cleft palate, generalized dysplasia of joints, hitchhiker thumb etc.). The abnormality is due to deficiency in the intracellular sulfate, which then leads to under sulfated pro- teoglycan [22]. Sequence polymorphisms of PAPSS2 with reference to osteoarthritis were investigated by Ikeda et al. [11,23,24]. OA is a musculoskeletal disorder characterized by degeneration of articular cartilage. The SNP analysis of the PAPSS2 from certain Japanese populations suffering from knee OA showed some correlation with PAPSS2. Differential distribu- tions of the two isoforms of PAPS synthase were manifested only in tissues affected by OA [24]. MC3T3-E1 pre-osteoblastic cells undergo differentiation and formation of a mineralizing matrix. These cell cultures are frequently used to investigate the role of specific genes in osteoblast differentiation. In this study, knocking down PAPSS2 with siRNA or by viral transduction with shRNA showed that PAPSS2 plays an important role in promoting osteoblast differentiation. When PAPSS2 is depleted, multiple markers of osteoblast differentiation were significantly reduced. This included mRNA levels of collagen I and OPN. PAPSS2 siRNA significantly reduced the up-regulation of alkaline phosphatase activity that occurred as a result of osteoblast differentiation. These results are unlikely to be due to global changes because PAPSS2 knockdown did not affect that overall protein or total RNA levels per well (data PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e43475