Function of PAPSS2 in Osteoblasts Figure 3. Influence of PAPSS2 on ALP activity and mineralization in MC3T3-E1 cells. MC3T3-E1 cells were infected with pBMN-I-GFP (control) or pBMN-PAPSS2 (P–O) for 24 hours, plenti-shRNA PAPSS2 (P–S) or plenti-scrambled shRNA (PLB) viruses for 48 hours and then induced with OS media for 7 and 14 days. (A) Effects of P–O on mineralization of MC3T3-E1 cells. The calcium deposits in the mineralized matrix were analyzed with the Aliralin red staining and Von Kossa staining. (B) Quantitative analysis of mineralization from Alizarin red staining. Each value is expressed as a ratio of the value in PAPSS2-silenced cells to that in PLB transfected cells. A P,0.05 was observed between P–O and the control, P–S, and the PLB. (C) Effects of P–O on ALP activity in OS media induced by MC3T3-E1 cells. Each bar is expressed as the mean6 SD (unit/min.mg DNA) of four determinations. a P,0.05 in the value between P–O and control group, P–S and the PLB was observed. doi:10.1371/journal.pone.0043475.g003 not shown). OPN has been implicated as an important factor in bone remodeling [25]. The organic part of bone is about 20% of the dry weight, and counts in, other than OPN, collagen type I, osteocalcin, osteonectin, bone sialo protein and alkaline phospha- tase. Collagen type I accounts for 90% of the protein mass. Osteoblastic differentiation is accompanied by increased expres- sion of the prominent osteoblastic marker, OPN. Different cell types may differ in their methods of regulating the OPN gene. OPN expression in bone occurs predominantly in osteoblasts, osteocyctes, and osteoclasts [26]. PAPSS2 knockdown reduced OPN mRNA levels, sharply, indicating that PAPSS2 acts upstream of OPN induction. It is possible that the biochemical mechanism of PAPSS2 affects osteoblast differentiation. This would include direct targeting of the OPN gene, which was confirmed in immunostaining assays. These assays have indicated that PAPSS2 targets the OPN promoter directly. The dynamics of this interaction correlates specifically with induction of differen- tiation. The reduction of OPN mRNA levels implies that direct targeting of downstream osteogenic genes by OPN is impaired alongside depletion of PAPSS2. These results do not rule out the possibility that there are multiple mechanisms by which PAPSS2 directly or indirectly modulates OPN activity and expression of osteogenic genes, but they establish an early, direct, and essential role for PAPSS2 in osteoblast differentiation. The other genes that were modulated by PAPSS2 knockdown, which includes tran- scription factors, enzymes associated with differentiated function, bone morphogenetic proteins, and extracellular matrix proteins, need be understood more deeply. TGF/Smads is produced by osteoblasts and appears to regulate bone metabolism in various ways, including skeletal development PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e43475