Regulation of 24,25-dihydroxyvitamin D-3 production by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1-34 in a cloned monkey kidney cell line (JTC-12).
Regulation of 25-hydroxyvitamin D-3 24-hydroxylase by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1-34 (PTH1-34) was investigated using a cloned monkey kidney cell line, JTC-12. Treatment of the cells with 1,25-dihydroxyvitamin D-3 markedly enhanced the conversion of [3H]-25-hydroxyvitamin D-3 into a more polar metabolite. The metabolite was identified as 24,25-dihydroxyvitamin D-3 by normal phase and reverse phase high-performance liquid chromatography and periodate oxidation. The 24-hydroxylase activity appeared to follow Michaelis-Menten kinetics, and 1,25-dihydroxyvitamin D-3 treatment increased the Vmax of 24-hydroxylase from 33 to 95 pmol/h per 10(6) cells without affecting the apparent Km value of the enzyme (220 nM in control vs. 205 nM in 1,25-dihydroxyvitamin D-3 treated cells). The enzyme activity reached a maximum between 4 and 8 h of treatment with 1,25-dihydroxyvitamin D-3. The dose of 1,25-dihydroxyvitamin D-3 required to cause a half-maximal stimulation was about 3 X 10(-10) M. The 1,25-dihydroxyvitamin D-3-induced increase in 24-hydroxylase was almost completely inhibited by the presence of 1 microM cycloheximide. Treatment of the cells with PTH1-34 caused a dose-dependent increase in cyclic AMP production. Half-maximal stimulation of cyclic AMP production was obtained at about 5 X 10(-9) M PTH1-34. When 2.4 X 10(-9) M PTH1-34 was added after 1,25-dihydroxyvitamin D-3 treatment, the 1,25-dihydroxyvitamin D-3-stimulated 24-hydroxylase was inhibited to 70.7 +/- 2.9% of control. Higher concentrations of PTH1-34 caused less inhibition of the enzyme activity. When cyclic AMP was added instead of PTH1-34, the enzyme activity was also suppressed significantly. These results indicate that, in JTC-12 cells, 1,25-dihydroxyvitamin D-3 stimulates 24-hydroxylase in a dose- and time-dependent manner by increasing the Vmax of the enzyme through a mechanism dependent upon new protein synthesis, and suggest that PTH1-34 inhibits the 1,25-dihydroxyvitamin D-3-induced stimulation of 24-hydroxylase through its effect on cyclic AMP production.
D015650:24,25-Dihydroxyvitamin D 3; D000818:Animals; D002117:Calcitriol / Q000494:pharmacology*; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System; D004100:Dihydroxycholecalciferols / Q000096:biosynthesis*; D004789:Enzyme Activation / Q000187:drug effects; D000882:Haplorhini; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology*; D013250:Steroid Hydroxylases / Q000378:metabolism; D019379:Teriparatide; D065668:Vitamin D3 24-Hydroxylase
D004100:Dihydroxycholecalciferols; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D019379:Teriparatide; D015650:24,25-Dihydroxyvitamin D 3; D003577:Cytochrome P-450 Enzyme System; D003513:Cycloheximide; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D002117:Calcitriol