PAPSS2 Promotes Alkaline Phosphates Activity and Mineralization of Osteoblastic MC3T3-E1 Cells by Crosstalk and Smads Signal Pathways Weizhuo Wang1*, Fang Li2, Kunzheng Wang1, Bin Cheng1, Xiong Guo3 1 Department of Orthopedics Surgery, the Second Affiliated Hospital, College of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi, People’s Republic of China, 2 Department of Prosthodontics, School of Stomatology, The Fourth Military University, Xi’an, Shaanxi, People’s Republic of China, 3 Faculty of Public Health, College of Medicine, Key Laboratory of Environment and Gene Related Diseases of Ministry Education, Xi’an Jiaotong University, Xi’an, Shaanxi, People’s Republic of China Abstract Several studies have indicated that PAPSS2 (39-phosphoadenosine-59-phosphosulfate synthetase 2) activity is important to normal skeletal development. Mouse PAPSS2 is predominantly expressed during the formation of the skeleton and cartilaginous elements of the mouse embryo and in newborn mice. However, the role and mechanism of PAPSS2 in bone formation remains largely unidentified. By analyzing the expression pattern of the PAPSS2 gene, we have found that PAPSS2 is expressed in bone tissue and bone formation. PAPSS2 transcripts increase during osteoblast differentiation and are in less level in RANKL-induced osteoclast like cells. By using lentivirus-mediated RNA interference (RNAi) technology, we knocked down PAPSS2 expression in MC3T3-E1 osteoblast. Silencing of PAPSS2 expression significantly decreases ALP activity and cell mineralization, inhibits expression of osteoblast marker osteopontin (OPN) and collagen I. Conversely, overexpression of PAPSS2 promotes the MC3T3-E1 to differentiate into osteoblast and mineralization. Moreover, compared to that in the control cells, the mRNA level and protein expression of phosphorylated Smad 2/3, which is a key transcriptional factor in the Smad osteoblast differentiation pathway, showed significant decreases in PAPSS2-silenced cells and increases in PAPSS2- overexpression cells. These results suggest that PAPSS2 might regulate osteoblast ALP activity and cell mineralization, probably through Smads signal pathways. Citation: Wang W, Li F, Wang K, Cheng B, Guo X (2012) PAPSS2 Promotes Alkaline Phosphates Activity and Mineralization of Osteoblastic MC3T3-E1 Cells by Crosstalk and Smads Signal Pathways. PLoS ONE 7(8): e43475. doi:10.1371/journal.pone.0043475 Editor: Brenda Smith, Oklahoma State University, United States of America Received April 18, 2012; Accepted July 20, 2012; Published August 16, 2012 Copyright: 2012 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project was supported by the Natural Scientific Fund of China (81001225), the international Co-operative Fund in Xian Jiaotong University, China and US (08143004), and the Fundamental Research Funds for the Central Universities (08140003). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: wangweizhuo@mail.xjtu.edu.cn Introduction Bone is a mineralized tissue that underlies multiple mechanical and metabolic functions of the skeleton [1,2]. Formation and maintenance of bone tissue are regulated in a sophisticated fashion by bone-forming osteoblasts and bone-resorbing osteoclasts. Many skeletal diseases, such as osteoporosis, Kashin-Beck disease, Paget’s disease of the bone, rheumatoid arthritis, and bone metastases all arise from an imbalance in the relative activities of osteoblasts and osteoclasts [3]. The proliferation and differentiation of those two cell types are controlled by various local growth factors, cytokines produced, in the bone and by systemic enzymes. The presence of PAPS (39-phosphoadenosine-59-phosphosulfate) is a prerequisite for catalytic efficiency in all sulfation reactions. In humans, PAPS is synthesized from ATP and SO422 by two isoforms of PAPS synthetase (PAPSS): PAPSS1 and PAPSS2. The PAPSS2 is one of the principal enzymes required for the sulfation of extracellular matrix molecules in bone formation and other tissues [4–6]. A truncation mutation of the human PAPSS2 gene was reported in a Pakistani family suffering from a novel form of spondyloepime- taphyseal dysplasia (SEMD) [7,8]. A homozygous PAPSS2 mutation (S475X) was identified in another large Pakistani family affected by SEMD, Pakistani type [8,9]. This manifested as disproportionately short stature with short, bowed lower limbs, enlarged knee joints, kyphoscoliosis, and generalized brachydac- tyly [7]. Chondroitin 6-O-sulfotransferase requires PAPS for catalytic activity, and the abnormal PAPSS2 gene that encodes chondroitin 6-O-sulfotransferase can cause SEMD, Omani type [10]. Humans lacking normal PAPSS2 activity exhibit long bone shortening and bowing, and also show degenerative joint disease, including evidence of knee joint arthrosis. Given the premature development of degenerative knee joint disease in the mutant mice and other similarities between SEMD mice and human lacking normal PAPSS2 activity, it has been proposed that this mutant represents a model of human PAPSS2 deficiency-associated arthrosis [11]. One of our previous studies included a microarray analysis that showed some correlation between PAPSS2 genes in individuals afflicted with endemic knee osteoarthritis and those with Kashin- Beck disease exhibiting shortened long bones and enlarged knee and finger joints [12,13]. Some features of the bone phenotype in the endemic osteoarthritis patient resemble those observed in SEMD, excepting that the changes in the bone changes were milder for our patients and the changes in the epiphyseal plates of the long bones and metaphyseal changes were detectable in our patients [14,15]. However, the absence of the pubertal growth PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e43475