1,25-Dihydroxyvitamin D 3 stimulates both alkaline phosphatase gene transcription and mRNA stability in human bone cells

Abstract

We have previously reported that 1,25(OH) 2D 3 stimulated the cellular alkaline phosphatase (ALP) activity and increased the steady-state level of ALP mRNA in a human osteosarcoma cell line (TE-85), under serum-free conditions. To define the molecular mechanism by which 1,25(OH) 2D 3 acts to stimulate ALP activity, the time courses of the increases in ALP activity and in the steady-state ALP mRNA level in response to 1,25(OH) 2D 3 were evaluated. 1,25(OH) 2D 3 progressively increased the steady-state level of ALP mRNA from 5 to 24 h of treatment, at which time a plateau was observed. In contrast, no significant increase in ALP-specific activity was detected until after 10 h of treatment, at which time the activity increased linearly with time up to 72 h. These time courses are consistent with the premise that the increased ALP activity was the result of increased gene expression. Nuclear runoff analysis indicated that the transcription rate of the ALP gene was more than five-fold higher in the 1,25(OH) 2D 3-treated cells than in the control cells. In addition, it was found that 1,25(OH) 2D 3 treatment increased ALP mRNA stability. The 1,25(OH) 2D 3-induced increase in ALP mRNA stability was not due to an interaction of the 1,25(OH) 2D 3—receptor complex with the ALP mRNA, since the removal of 1,25(OH) 2D 3 did not abolish its stabilizing effect. In the presence of cycloheximide, the stabilizing effect of 1,25(OH) 2D 3 was abolished, suggesting that a 1,25(OH) 2D 3-inducible protein factor was involved. Based on these findings, we have proposed a model in which 1,25(OH) 2D 3 stimulated ALP activity in human bone cells through mechanisms involving both (1) increased transcription of the ALP gene and (2) increased stability of ALP mRNA, an effect which requires the de novo synthesis of a protein, a putative ALP mRNA “stabilizing factor.”