Function of PAPSS2 in Osteoblasts Figure 4. PAPSS2 silencing (P–S) suppresses expression of osteoblast marker genes and crosstalk with Smads pathway. 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) Real-time RT-PCR analysis of Samd2, Samd3, Col I, and OPN mRNA in MC3T3-E1 cells induced with OS media for 7 and 14 days, showing that the expression of Samd2, Smad3, Col I, and OPN was attenuated in P–S cells compare with PLB cells. Conversely, the expression levels of Samd2, Smad3, Col I, and OPN were higher in P–O cells than in control cells (a P,0.05). (B) Immunofluorescence of MC3T3-E1 cells revealed that the expression of type 1 collagen (Col1, green fluorescence) and osteopontin (OPN, red fluorescence) was lower in P–S cells than in PLB cells. The expression levels of Col I and OPN were higher in P–O cells than in control cells at 14 days. The cell nuclei were stained using DAPI (blue) to identify the number of cells. (C) Western blot analysis of p-Smad2/3 protein expression. Cells were prepared as A. A marked difference was observed in the levels of p-Smad2/3 between P–O and the control groups and between P–S and the PLB groups. (D) The protein levels of p-Smad2/3 in G were normalized to GAPDH. The levels of p-Smad2/3 in PAPSS2-overexpressed and -silenced cells induced with OS media for 7 days were 2.0 and 0.4 times that in the control group and at14 days they were 1.7 and 0.3 times that in the control group. P,0.05 was observed between P–O and control group, P–S and control group were observed. doi:10.1371/journal.pone.0043475.g004 and bone remodeling [18,27,28]. Smad3, a critical component of the Smads signaling pathway, plays an important role in the regulation of bone formation [28–34]. Smad3 has been shown to bind to the OPN promoter, on which it acts as a sequence-specific activator [35]. Smad2 mediates the signal of the TGF-b, and thus regulates many cellular processes, such as cell proliferation, apoptosis, and differentiation. This protein is recruited to the TGF-beta receptors through its interaction with the Smad anchor for receptor activation protein. The phosphorylation induces the dissociation of this protein from the Smad anchor for receptor PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e43475