Function of PAPSS2 in Osteoblasts Figure 1. PAPSS2 is prominently expressed in osteoblasts derived from MC3T3 cells stimulated by ascorbic acid and b-glycerol- phosphate (OS media). (A) Tissue distribution of PAPSS2 mRNA expression in lean control mice. Total RNA was extracted from the indicated tissues, reverse transcribed, and assessed using quantitative real-time PCR. PAPSS2 mRNA expression was calculated relative to the expression of B2m mRNA, and values were expressed relative to the kidney level. (a P,0.05 between calvaria/bone and the other tissues) (B) RT-PCR analysis of PAPSS2 mRNA in MC3T3, for 0, 3, 7, and 14 days, showing that PAPSS2 is expressed in OB. (C) Normalized mRNA level of B. (a p,0.05 compared with 0 days) (D) Western blot analysis of PAPSS2 protein expression in OB derived from MC3T3 cells induced with OS media for 0, 3, 7, and 14 days. Equivalent amounts (20 mg) of total protein were loaded in each lane. (E) Quantification of protein levels from immunoblots, as in D. The protein levels of PAPSS2 were normalized to GAPDH. The levels of PAPSS2 were gradually increased in timeline. (a P,0.05 compared with 0 day) (F) Real-time PCR analysis of PAPSS2 expression pattern during differentiation of osteoclast and osteoblasts. RAW264.7 (Raw) cells were induced with 10 ng/ml of RANKL and 20 ng/ml M-CSF for 0, 24, and 96 hours for osteoclastogenesis; and MC3T3-E1 clone 4 cells were induced with OS media for 0, 7, and 21 days, respectively. (a P,0.05 between raw and MC3T3 for 0, 7, 21 days). The results showed that PAPSS2 is specifically and highly expressed in both pre- osteoblasts and osteoblast. doi:10.1371/journal.pone.0043475.g001 suggest that PAPSS2 can facilitate osteoblast differentiation by regulating the expression of Col1 and OPN. Modulation of the Smad pathway in the transfected osteoblastic cells The Smad family proteins are critical components of the TGF signaling pathways. Smad signaling is critical to the regulation of osteoblast differentiation. Smad signal expression is indicative of the function of the PAPSS2 pathway. To determine the mechanism by which PAPSS2 enhances ALP activity and mineralization, we determined whether PAPSS2 silencing and overexpression would affect the protein or mRNA levels of p- Smad2/3 in MC3T3-E1 cells using Western blot analysis and real- time RT-PCR, respectively. Real-time RT-PCR analysis of Smad2 and Smad3 showed that PAPSS2 silencing significantly reduced the mRNA levels of Smad2 and Smad3 in osteoblasts derived from MC3T3-E1 cells (Fig. 4A). Western blot analysis of p-Smad2/3 protein expression in empty vectors and in transfected MC3T3-E1 cells subjected to PAPSS2 silencing and overexpres- sion showed that protein levels of Smad2/3 in these cells differed from that of the control groups (Fig. 4C). The levels of p-Smad2/3 in the overexpression group and in the silencing group, which were stimulated with OS media for 7 d, were 2.0 and 0.4 times that of control cells. For cells stimulated for 14 d, they were 1.7 and 0.3 times of that of control cells (Figs. 4C, D). Transfected cells subjected to PAPSS2 silencing showed lower levels of Smad 3 than cells transfected with empty vector under real-time RT-PCR analysis. These data indicate that PAPSS2 might stimulate the expression of bone matrix proteins such as Smad 3 in osteoblastic cells. We demonstrated that the involvement of PAPSS2 in the Smad signaling pathway plays a role in the fast effect of osteoblast differentiation. Discussion During skeletogenesis, bone is formed in two different ways, intramembranous ossification and endochondral ossification. In intramembranous ossification, osteogenesis occurs within the condensed mesenchymal cells. This type of ossification produces the flat bones and the additional bone. In endochondral ossification, mesenchymal cells first condense to form a cartilage model. Then bone slowly forms to replace this cartilage. This type of ossification produces most of the bones of the body, including the axial and appendicular skeletons. PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e43475