1 alpha,25-dihydroxyvitamin D3 stimulation of osteopontin expression in rat clonal dental pulp cells

Osteopontin (OPN) is a major phosphorylated non-collagenous protein isolated from bone. Rat clonal dental-pulp cell lines RPC-C2A and RDP4-1 produce and secrete OPN as a principal phosphoprotein. 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] is a potent calcitropic hormone which regulates calcified tissue metabolism including the synthesis of extracellular matrix proteins. The effects of 1,25(OH)2D3 on the expression of OPN mRNA and the synthesis of OPN protein by pulp cells in vitro were investigated. In RPC-C2A cells, 1,25(OH)2D3 markedly stimulated synthesis of both [32PO4]- and [35S]-methionine-labelled OPN. Phosphorylated OPN synthesis increased dose-dependently and showed a maximum level at 48 h after addition of 10(-11)-10(-7) M 1,25(OH)2D3. Similar stimulation was also observed in RDP4-1 cells. Northern hybridization analysis revealed that 1,25(OH)2D3 greatly increased the level of OPN mRNA in both pulp cell lines. Examination of the time course of the effects of 1,25(OH)2D3 on the level of OPN mRNA in RPC-C2A cells by dot-blot analysis showed that stimulation was detectable at 24 h and reached a maximum at 48 h after exposure to 10(-7)M 1,25(OH)2D3. These findings indicate that 1,25(OH)2D3 stimulates the production of dental-pulp OPN by a mechanism that involves de novo synthesis and transcriptional control.