spurt characteristic of in Kashin-beck disease, compromised final height, and the increased ratio of sitting height to standing height are indicative of bone dysplasia [14,16]. These observations suggest that PAPSS2 may participate in the control of important physiological processes in bone and cartilage, such as collagen fibrillogenesis and/or matrix calcification and mineralization. However, the role of PAPSS2 in bone develop- ment and formation and the mechanisms underlying this role remain largely unidentified. In order to better understand the role of PAPSS2 in various biochemical pathways, its molecular biology, biochemistry, structure, and function must be thoroughly studied. In this study, evaluated role of PAPSS2 in osteoblasts by detailing the function of the gene in the formation of a mineralization- competent bone matrix through activated pathway [5,17]. Results Expression of PAPSS2 mRNA in bone and role in osteoblast differentiation We assessed the expression of PAPSS2 mRNA in various organs and tissues from lean control mice using quantitative real-time PCR. We found that the expression of PAPSS2 was very high in the bones, liver, and lungs and moderate in the heart. The mRNA level was extremely low in the spleen, muscle, kidneys, and brain (Fig. 1A). The housekeeping gene murine B2m was expressed at an almost constant level in the organs and tissues examined, validating the present real-time PCR method for measurement of PAPSS2 mRNA. These findings were also consistent with those of previous reports [18]. PAPSS2 was more highly expressed in human osteoblasts than in human osteoclasts (Figs. 1B, C, F). These results were confirmed by Western blot analysis in MC3T3 cell lines (Figs. 1D, E). Our results showed that PAPSS2 was prominently expressed in authentic mouse osteoblasts exposed to OS media at both the mRNA and protein levels. This suggests that PAPSS2 might play important role in osteoblast differentiation in vitro. Silence of PAPSS2 attenuates mineralization and the ALP activity To demonstrate the importance of PAPSS2 function in osteoblast differentiation, we used lentivirus-mediated RNA in- terference (RNAi) technology to silence (knock down) PAPSS2 expression in MC3T3-E1 exposed to OS media [19]. After stable transfection with constructs encoding small interfering RNA (siRNA) targeting PAPSS2, we confirmed the effects of RNAi and performed RT-PCR (Figs. 2A, B), Western blot analysis (Fig. 2C, D), and immunofluorescent staining (Fig. 2H) to assess silencing efficiency. As shown in Fig. 2A, PAPSS2 mRNA transcription and protein expression were silenced in stably transfected PAPSS2-silenced cell lines from 3, 7, and 14 days, as indicated by comparison to control cells transfected with the empty vector. In contrast to wild-type cells, we found that the cells infected with PAPSS2-silencing lentivirus contained undetectable levels of PAPSS2 protein at 7 days after transfection (Fig. 2C). To determine the effects of PAPSS2 RNAi on osteoblast gene expression, we also examined the expression of PAPSS2 using immunofluorescent staining 7 days after infection. As shown in Fig. 2E, PAPSS2 silencing blocked PAPSS2 protein expression. These results were consistent with those of RT-PCR and Western blot analyses. ALP activity and mineralization are important to bone formation, as are matrix proteins. ALP hydrolyzes pyrophosphate and provides inorganic phosphate to promote mineralization in osteoblasts. We determined whether PAPSS2 would affect ALP Function of PAPSS2 in Osteoblasts activity and mineralization in MC3T3-E1 cells. ALP activity was evaluated biochemically. Mineralization was examined using Alizarin red staining, Kossa staining, and a quantitative assay of mineralization based on Alizarin red staining (Figs. 3A, B). As shown in Fig. 3A, calcium deposits stained with von Kossa and Alizarin red S consistently showed that both PAPSS2 silencing and PAPSS2 overexpression significantly decreased ALP activity and cell mineralization. ALP activity was measured at 7 days and was found to be significantly different in the silencing and over- expression groups (P,0.05) (Fig. 3C). These data indicate that PAPSS2 silencing in MC3T3-E1 cells can block ALP activity and mineralization in osteoblastic cells. Effects of overexpression of PAPSS2 on ALP activity and mineralization of osteoblastic cells To gain further insight into the molecular mechanisms of PAPSS2 function in osteoblast differentiation, we overexpressed PAPSS2 in MC3T3-E1 clone 4 induced with OS media [19]. The PBMN-I-GFP (vector-only control) and PBMN-I-GFP-PAPSS2 were transfected into the Phoenix ecotropic packaging cell line. After stable transfection with constructs, we confirmed the effect of RNAi and performed real-time RT-PCR (Fig. 2E), Western blotting (Figs. 2F, G), and immunofluorescent staining (Fig. 2H) to assess overexpression. Unlike wild-type cells, we found that the expression of PAPSS2 protein increased in cells infected with virus. As shown in Figs. 2E–G, the overexpression of PAPSS2 both on the mRNA and protein levels was detectable in cell lines stably transfected with PBMN-I-GFP-PAPSS2 but not in cells transfected with empty vector. As shown in Fig/3A, mineralization signals were detected in transfected MC3T3-E1 cells. Different signals were observed in cells transfected with empty vector. ALP activity and cell mineralization were both much higher in cells over- expressing PAPSS2 than in cells transfected with empty vector at both 7 and 14 days after induction by OS media. Quantization of mineralization with Alizarin red staining indicated that the mineralization of the overexpression group and silencing group were significantly different from those of the control (P,0.05). As demonstrated in Fig. 3C, ALP activity of PAPSS2 overexpression and silence was significantly different (P,0.05) from the value of control group after incubation for 7 d. PAPSS2 overexpression- transfected cells had much higher ALP activity and mineralization than cells transfected with empty vector. These data indicate that PAPSS2 promotes ALP activity and mineralization of osteoblastic cells. Effects of PAPSS2 gene on the expression of osteoblast- specific marker The osteoblast-associated molecules Col I and OPN are considered osteoblast differentiation markers because their ex- pression levels increase during osteoblast differentiation [1,20]. To determine the effects of PAPSS2 overexpression and silencing on osteoblast differentiation in MC3T3-E1 cells, mRNA levels of Col1 and OPN were examined using quantitative real-time PCR. The mRNA levels of Col1 and OPN were changed after treatment, which lasted 7 days after infection. The signals of Col1 and OPN were stronger than control values in cells overexpressing PAPSS2 and weaker than control values in PAPSS2-silenced cells (Fig. 4A). The protein expression of Col1 and OPN (Fig. 4C, D) was confirmed by immunofluorescence of MC3T3 cells. Results showed that the expression of type 1 collagen and OPN was attenuated in PAPSS2-silenced cells and increased in PAPSS2-overexpressing cells (Fig. 4B). These results PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e43475