Pigment epithelium derived factor regulates human Sost/Sclerostin and other osteocyte gene expression via the receptor and induction of Erk/GSK-3beta/beta-catenin signaling

Mutations in Serpinf1 gene which encodes pigment epithelium derived factor (PEDF) lead to osteogenesis imperfecta type VI whose hallmark is defective mineralization. We reported that PEDF suppressed expression of Sost/Sclerostin and other osteocyte related genes in mineralizing osteoblast cultures and suggested that this could be part of the mechanisms by which PEDF regulates matrix mineralization (Li et al. J Cellular Phys. 2014). We have used a long-term differentiated mineralizing osteoblast culture (LTD) to define mechanisms by which PEDF regulates osteocyte gene expression. LTD cultures were established by culturing human osteoblasts in an osteogenic medium for 4 months followed by analysis of osteocytes related genes and encoded proteins. LTD cells synthesized Sclerostin, matrix extracellular pbosphoglycoprotein (MEPE) and dentin matrix protein (DMP-1) and their synthesis was reduced by treatment with PEDF. Treatment of the cultures with PEDF induced phosphorylation of Erk and glycogen synthase kinase 3-beta (GSK-3 beta), and accumulation of nonphosphorylated beta-catenin. Inhibition of Erk activation and neutralizing antibodies to the pigment epithelium derived receptor (PEDF-R) suppressed GSK-3 beta phosphorylation and accumulation of nonphosphorylated beta-catenin in presence of PEDF. Topflash assays demonstrated that PEDF activated luciferase reporter activity and this activity was inhibited by treatment with Erk inhibitor or neutralizing antibodies to PEDF-R. Dickkopf-related protein 1 treatment of the cells in presence of PEDF had minimal effect suggesting that GSK-3 beta phosphorylation and accumulation of nonphosphorylayted beta-catenin may not involve LRP5/6 in osteocytes. Taken together, the data demonstrate that PEDF regulates osteocyte gene expression through its receptor and possible involvement of Erk/GSK-3 beta/beta-catenin signaling pathway.