Effects of ascorbic acid, calcitriol, and retinoic acid on the differentiation of preosteoblasts

Abstract

The responses of the immortalized rat preosteoblast UMR‐201‐10B to ascorbic acid (AA), 1,25(OH)₂D₃ (calcitriol), and retinoic acid (RA) were examined. UMR‐201‐10B cells have an undetectable basal alkaline phosphatase (ALP) activity that is induced after 24 h of treatment with 10⁻⁶ M RA (4.64 ± 0.06 μmol/h/mg of protein). The addition of 10⁻⁸ M calcitriol resulted in a slight induction of ALP activity after 72 h (0.43 ± 0.07 μmol/h/mg of protein). When calcitriol was added to RA, however, over the same period ALP activity was enhanced significantly compared with treatment with RA alone (RA and calcitriol, 12.29 ± 0.86 μmol/h/mg of protein). Treatment with AA (50 μg/ml) alone had no effect on ALP activity but increased RA‐induced ALP activity to 6.78 ± 0.28 μmol/h/mg of protein at 24 h. In contrast, AA inhibited calcitriol‐induced ALP activity after 7 days of combined treatment with calcitriol (calcitriol, 7.73 ± 0.16 μmol/h/mg of protein; AA and calcitriol, 1.44 ± 0.06 μmol/h/mg of protein). Individually, RA and calcitriol induced mRNA expression for ALP, matrix‐gla protein (MGP), and osteopontin (OP). The steady state level of pro‐α1(I) collagen mRNA also was increased significantly by treatment with RA and AA individually. The combination of RA and calcitriol had a synergistic effect on ALP, OP, and especially MGP mRNA expression but significantly reduced the expression of pro‐α1(I) collagen mRNA. AA enhanced the effect of RA on the expression of pro‐α1(I) collagen, MGP, and ALP mRNAs as well as the effect of calcitriol on OP and MGP. The addition of AA to RA resulted in a decrease in the steady state level of OP, whereas its cotreatment with calcitriol caused a decrease in pro‐α1(I) collagen and ALP mRNA. In conclusion, these studies identify RA, calcitriol, and AA as regulators of differentiated osteoblast function.