CKII is involved in the regulation of Ikaros and ALP activity induced by L‐ascorbic acid

Abstract

L-ascorbic acid (AsA) is an essential supplement for the differentiation of a wide variety of cells in culture and induces osteoblastic differentiation through the formation of collagen matrix and the induction of the alkaline phosphatase (ALP) activity in some osteogenic cells. Despite their widespread nutritional use, the precise mechanism of AsA in osteoblastogenesis remains unclear. In order to investigate the effect of AsA on the differentiation of osteoblast, this study examined the expression of RANKL, OPG (osteoprotegerin), collagen synthesis and ALP activity in osteoblast-like cells (MG63), assessed by RT-PCR and western blot analysis. The results showed that an AsA treatment slightly down-regulated OPG mRNA expression, whereas the mRNA expression of RANKL and collagen were unaffected. AsA significantly increased the ALP activity after 6 days, which was inhibited by DRB, a casein kinase II (CKII) inhibitor. In addition, Western blot analysis showed that AsA induced the activation of CKII. EMSA analysis also indicated that AsA dose-dependently decreased the DNA binding affinity of the Ikaros transcription factor, which is a bifunctional differentiation factor. Moreover, the cells treated with AsA and DRB showed increased Ikaros activity compared with the AsA treatment only. Overall, these results suggest that AsA stimulates osteoblast differentiation through the enhancement of ALP activity and the suppression of Ikaros activity. Moreover, this process might be related to CKII regulation.