Osteonectin regulates the extracellular matrix mineralization of osteoblasts through P38 signaling pathway

Abstract

Osteonectin binds strongly to type I collagen and hydroxyapatite and plays a crucial role in extracellular matrix mineralization. Previous studies have also shown that p38 signaling pathway is an important regulator for osteoblast mineralization. This study focused on the role of osteonectin in regulating extracellular matrix mineralization via the p38 signaling pathway. Osteoblasts were isolated and cultured from parietal bones of neonatal Sprague-Dawley rats. The gene and protein expressions of noncollagen proteins (BSP, bone sialoprotein; OCN, osteocalcin; OPN, osteopontin), p38 mitogen-activated protein kinase, and SIBLINGs (Small Integrin-Binding LIgand N-linked Glycoproteins) members (DMP1, dentine matrix protein 1, DSPP, dentin sialophosphoprotein, and MEPE, matrix extracellular phosphoglycoprotein) were detected by reverse-transcription quantitative polymerase chain reaction and western blot analysis. Alizarin red staining, intracellular calcium assay, and transmission electron microscopy were used to detect mineralization. Initially, by adding osteonectin at different concentrations in osteoblasts and detecting the above mineralization indexes, 1 µg/ml was determined to be the optima osteonectin concentration, which significantly increased gene expressions of BSP, OPN, OCN, DMP1, MEPE, DSPP, and p38 in osteoblasts, p38 and p-p38 protein expressions were also significantly increased, mineralized nodules were significantly enhanced; when added with SB203580 (a specific inhibitor for p38) these effects were inhibited. Furthermore, osteoblasts transfected with Ad-p38 also significantly upregulated the protein and gene expressions of noncollagens and SIBLINGs members, whereas transfection of p38-rhRNA showed the opposite effect. Our data suggest that osteonectin regulates the extracellular matrix mineralization of osteoblasts through the P38 signaling pathway.

Keywords: mineralization; osteoblasts (OB); osteonectin (SPARC); p38 MAPK signaling pathway.