Sphingosine-1-phosphate/S1PR2-mediated signaling triggers Smad1/5/8 phosphorylation and thereby induces Runx2 expression in osteoblasts

Sphingosine-l-phosphate (SIP) is a signaling sphingolipid that also plays crucial roles in bone regeneration. Recently, we reported that the SIP receptors S1PR1 and S1PR2 were mainly expressed in osteoblast-like cells, and that the S1P/S1PR1 signaling pathway up-regulated osteoprotegerin and osteoblast differentiation. However, the involvement of S1P/S1PR2 signaling in osteoblast differentiation is not well understood. Here we investigate the role of S1P/S1PR2-mediated signaling in osteoblast differentiation and clarify the underlying signaling mechanisms. We found that an S1P/S1PR2/Gi-independent signaling pathway activated RhoA activity, leading to phosphorylation of Smad1/5/8 in mouse osteoblast-like MOT3-E1 cells and primary osteoblasts. Furthermore, this signaling pathway promoted nuclear translocation of Smad4, and increased the amount of Smad6/7 protein in the nucleus. SIP also up-regulated runt-related transcription factor 2 (Runx2) expression through S1PR2/RhoA/ROCK/Smad1/5/8 signaling. Moreover, we found that SIP partially triggered S1PR2/RhoA/ROCK pathway leading to bone formation in vivo. These findings suggest that SIP induces RhoA activity, leading to the phosphorylation of Smad1/5/8, thereby promoting Runx2 expression and differentiation in osteoblasts. Our findings describe novel molecular mechanisms in S1P/S1PR2-mediated osteoblast differentiation that could aid future studies of bone regeneration. (C) 2016 Elsevier Inc. All rights reserved.