Parathyroid hormone regulates 25-hydroxyvitamin D3-24-hydroxylase mRNA by altering its stability

The up-regulation of the 25-hydroxyvitamin D-3-24-hydroxylase by 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3] is well established and occurs at the transcriptional level through two vitamin D response elements in the promoter of the gene. However, the mechanism of down-regulation of the 24-hydroxylase by parathyroid hormone (PTH) has not yet been elucidated. To study the mechanism of PTH action, we used AOK-B50 cells, a porcine kidney-cell line with stably transfected opossum PTH receptor in which both the 24-hydroxylase mRNA and activity are down-regulated by PTH. Cells dosed with 1,25(OH)(2)D-3 at 0 h, and subsequently at 0, 1, 2, or 4 h with 100 nM of PTH, showed levels of 24-hydroxylase mRNA equivalent to 72.6, 65.3, 57.2, and 37.1%, respectively, of the levels found in cells dosed with 1,25(OH)2D3 only. All cells were collected 7 h after the initial 1,25(OH)(2)D-3 dose. This pattern of expression indicated that PTH does not act by repressing transcription but rather by making the mRNA for 24-hydroxylase susceptible to degradation. At least 1 h is required for PTH to act. Further RNA and protein syntheses are required for PTH to act. However, the sites and mechanism whereby PTH causes 24-hydroxylase mRNA degradation are unknown. Because the untranslated regions of genes can determine the stability of its transcripts, we studied the 5' untranslated region and the 3' untranslated region of the rat 24-hydroxylase gene by using reporter-gene strategy to identify possible PTH sites of action. None was found, suggesting that the destabilization site is elsewhere in the coding region.