3'‑Phosphoadenosine 5'‑phosphosulfate synthetase 2 (PAPSS2) has been shown to be important in the development of normal skeletal structure. The aim of the present study was to evaluate the role of PAPSS2 in the differentiation of chondrocytes as well as their mechanisms. Using RNA interference‑mediated via a lentivirus and a retrovirus, PAPSS2 gene silence and overexpression in ATDC5 chondrogenic cells were performed. Chondrocyte differentiation and chondrogenic‑related gene markers associated with extracellular matrix formation were noted. The mRNA and protein expression for Wnt4, β‑catenin and SOX9 genes were observed. The PAPSS2 transcript expression levels progressively decline in ATDC5‑induced chondrocyte‑like cells during differentiation. Silencing of PAPSS2 expression had a significantly attenuating effect on cell differentiation and decreased expression of collagen II and X. In contrast, over‑expression of PAPSS2 promoted the differentiation of ATDC5 chondrogenic cells. The mRNA expression levels of Wnt4 and SOX9 decreased significantly in PAPSS2 knock down cells vs. control cells. However, this expression was increased in the cells over‑expressing PAPSS2. These data indicate that PAPSS2 regulates aggrecan activity as well as cell differentiation. The findings favor a mechanism by which PAPSS2 induces differentiation in ATDC5 cells via direct regulation of early signaling events that promote formation of collagenous matrix components. This control is probably mediated via extracellular matrix formation Wnt/β‑catenin signaling pathways.

Several studies have indicated that PAPSS2 (3′-phosphoadenosine-5′-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.