Posttranscriptional regulation of osteocalcin mRNA in clonal osteoblast cells by 1,25-dihydroxyvitamin D3

Abstract

A well-established action of 1,25(OH)2D3 in bone is stimulation of osteocalcin synthesis. Here we show that osteocalcin mRNA regulation by 1,25(OH)2D3 in clonal osteoblast cells (ROS 17/2.8), besides occurring at a transcriptional level, is also regulated by a posttranscriptional mechanism. Transcriptional "run-on" assays performed after 1, 3, 4, and 24 h of treatment with 1,25(OH)2D3 (50 nM) indicate an approximate twofold increase in the rate of osteocalcin transcription at each time point examined. Actinomycin D (AD) was used as a transcriptional inhibitor to measure the osteocalcin message half-life in ROS 17/2.8 cells. A 24-h treatment of ROS 17/2.8 cells with 1,25(OH)2D3 resulted in prolongation of osteocalcin half-life from 7.0 +/- 1.0 h in untreated cells to 28.0 +/- 0.7 h in the treated cells. Inhibition of protein synthesis by cycloheximide resulted in moderate stabilization of osteocalcin mRNA (t1/2 = 7.7 h in the absence and 14.0 h in the presence of cycloheximide) in ROS 17/2.8 cells. However, osteocalcin mRNA half-life in cells that had been treated with 1,25(OH)2D3 was not further prolonged in the presence of cycloheximide (t1/2 = 22.1 h in the absence and 22.6 h in the presence of cycloheximide). The osteocalcin poly(A) tail length was not altered by 1,25(OH)2D3 treatment and, therefore, may not play a role in 1,25(OH)2D3-induced mRNA stabilization. Osteocalcin mRNA expressed by a cytomegalovirus (CMV) promoter in transfected U937 cells was shown to be stabilized by 1,25(OH)2D3 (control cells, t1/2 = 5.0 +/- 0.6 h; treated cells, t1/2 = 22.0 +/- 0.2 h). This suggests that cellular events resulting in mRNA stabilization can operate independently from events that mediate transcriptional regulation of osteocalcin by 1,25(OH)2D3.