Extracellular signal-regulated kinase 1/2 is involved in ascorbic acid-induced osteoblastic differentiation in periodontal ligament cells

Background: Periodontal ligament (PDL) cells possess osteoblast-like properties and play key roles in periodontal regeneration. Previously, we have reported that ascorbic acid promotes the osteoblastic differentiation of PDL cells by modulating the type I collagen-integrin interaction. However, the signaling pathway activated following collagen-integrin interaction is still unclear. In this study, we examined the involvement of extracellular signal-regulated kinase (ERK)1/2 in the expression of osteoblastic marker genes such as the osteoblast-specific transcriptional factor runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) in PDL cells. Methods: PDL cells were cultured on a conventional or type I collagen-coated dish in the presence or absence of ascorbic acid and examined for ALP activity and osteoblastic marker genes. For detection of ERK1/2, cells were plated on a Petri (non-adhesive) dish or type I collagen-coated dish, and Western blot analysis was performed. The effect of the ERK1/2 inhibitor on osteoblastic marker gene expression was examined. Results: Ascorbic acid increased gene expression of Runx2, ALP, and OCN. A combination of ascorbic acid and type I collagen remarkably upregulated Runx2, ALP, and OCN gene expression and ALP activity. Western blot analysis revealed an increased level of ERK1/2 phosphorylation in cells plated on type I Collagen. An ERK1/2 inhibitor suppressed ascorbic acid-induced ALP and OCN gene expression, whereas Runx2 was not affected in PDL cells. Conclusion: These results indicate that ERK1/2 is involved in ascorbic acid-induced osteoblastic differentiation during PDL cell attachment to type I Collagen.