Effect of siRNA PERK on fluoride-induced osteoblastic differentiation in OS732 cells

The purpose of this work is to study the action of fluoride on osteoblastic function through knocking down double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) mRNA in OS732 cells (human osteoblast-like cell line). The previous researches had demonstrated that fluoride induced endoplasmic reticulum (ER) stresses in other cells or tissues. PERK as one branch of UPR to combat ER stress played a role in mediating the proliferation and differentiation of osteoblast. The mechanism of skeletal fluorosis by which fluoride regulated osteoblast was not fully defined. We used the real-time PCR and small interfering RNA techniques to determine the expression PERK signaling and osteoblastic and osteoclastic differentiation-related factors and investigated the role of PERK signaling in fluoride-stimulated osteoblastic function. Cells transfected with 50 nM small interfering RNA (siRNA)-PERK showed effectively decreased protein and gene expression of PERK and reduced protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2). Meantime, cells transfected with siRNA significantly decreased the protein level of alkaline phosphatase (ALP) and nuclear factor kappa B ligand (RANKL) in cells under fluoride exposure. It suggested that knockdown of PERK expression hardly stimulated osteoblastic and osteoclastic early differentiation induced by fluoride. Conversely, there were littler effect of siRNA PERK on expression of Runt-related transcription factor 2 (RUNX2) and osteoprotegerin (OPG) in cells, but fluoride exposure markedly stimulated their expression. This study proved that the mechanism underlying fluoride induced osteoblastic and osteoclastic differentiation possible was due to activation of ALP and RANKL mediated by PERK in OS732 cells.