Osteoadherin is upregulated by mature osteoblasts and enhances their in vitro differentiation and mineralization.

During the process of differentiation, osteoblasts commit through strictly controlled checkpoints under the influence of several growth factors, cytokines, and extracellular matrix (ECM) proteins. The mineralized tissue-specific ECM component osteoadherin (OSAD) belongs to the small leucine-rich repeat protein family of proteoglycans. Proteoglycans modulate cellular behavior either through the attached glycosaminoglycan chains or by direct protein-protein interactions via the core protein sequences. Leucine-rich repeats have been shown to directly interact with cell-surface receptors such as epidermal growth factor receptor, blocking its ability to bind its ligand. In the present study, we investigated the influence of OSAD on the behavior and maturation of MC3T3E1 osteoblasts. OSAD overexpression and repression clones were created by stably transfecting with plasmids coding for either mouse OSAD cDNA or small-hairpin RNA, targeted against mouse OSAD. Overexpression of OSAD resulted in an increase of osteoblast differentiation features, such as increased alkaline phosphatase (ALP) activity and increased in vitro mineralization, as well as reduced proliferation and migration. Bone sialoprotein (BSP) levels were unchanged, while upregulation of osteocalcin (OC) and osteoglycin (OGN) was observed. Conversely, repression of OSAD expression resulted in increased cell proliferation and migration. BSP and OC were unaffected, while OGN was downregulated. ALP activity was reduced, though no change in in vitro mineralization was observed. We conclude that OSAD overexpression enhanced the differentiation and maturation of osteoblasts in vitro.