title: Constitutive expression of 25-hydroxyvitamin D3-1alpha-hydroxylase in a transformed human proximal tubule cell line: evidence for direct regulation of vitamin D metabolism by calcium.
abstract: Circulating levels of the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) are dependent on activity of the renal mitochondrial cytochrome P450 enzyme, 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-hydroxylase). Production of 1,25-(OH)2D3 occurs predominantly in the renal proximal tubule, with 1alpha-hydroxylase activity being impaired in renal insufficiency and renal disease. The expression and activity of 1alpha-hydroxylase are tightly regulated in response to serum levels of PTH, calcium, phosphate, and 1,25-(OH)2D3 itself. As a consequence of this, the characterization of 1alpha-hydroxylase in human renal tissue has proved difficult. In this study we have characterized constitutive 1alpha-hydroxylase expression in a simian virus 40-transformed human proximal tubule cell line, HKC-8. Initial analyses of [3H]25-hydroxyvitamin D3 (25OHD3) metabolism in these cells using straight and reverse phase HPLC revealed product peaks that coincided with authentic 1,25-(OH)2D3 as well as 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3). Enzyme kinetic studies indicated that the Km for synthesis of 1,25-(OH)2D3 in HKC-8 cells was 120 nmol/liter 25OHD3, with a maximum velocity of 21 pmol/h/mg protein. This activity was inhibited by treatment with ketoconazole, but not diphenyl phenylenediamine. RT-PCR analysis of RNA from HKC-8 cells revealed a transcript similar in size to that observed in keratinocytes and primary cultures of human proximal tubule cells, and protein was detected by Western blot analysis. Synthesis of 1,25-(OH)2D3 was up regulated by treatment with forskolin (10 micromol/liter, 24 h) and was down-regulated by 1,25-(OH)2D3 (10 nmol/liter, 24 h). 1Alpha-hydroxylase activity in HKC-8 cells was also sensitive to the concentration of calcium. Cells grown in low calcium (0.5 mmol/liter) showed a 4.8-fold induction of 1alpha-hydroxylase, whereas treatment with medium containing high levels of calcium (2 mmol/liter) significantly inhibited 1,25-(OH)2D3 production. These data suggest that direct effects of calcium on proximal tubule cells may be an important feature of the regulation of renal 1,25-(OH)2D3 production.
authors.affiliation: Department of Medicine, Institute of Clinical Research, University of Birmingham, United Kingdom.
authors.forename: R
authors.initials: R
authors.lastname: Bland
authors.forename: E A
authors.initials: EA
authors.lastname: Walker
authors.forename: S V
authors.initials: SV
authors.lastname: Hughes
authors.forename: P M
authors.initials: PM
authors.lastname: Stewart
authors.forename: M
authors.initials: M
authors.lastname: Hewison
mesh_terms: D015650:24,25-Dihydroxyvitamin D 3 / Q000378:metabolism; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D002112:Calcifediol / Q000378:metabolism; D002117:Calcitriol / Q000378:metabolism / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002461:Cell Line, Transformed; D002851:Chromatography, High Pressure Liquid; D005576:Colforsin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D007654:Ketoconazole / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000201:enzymology*; D007700:Kinetics; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014807:Vitamin D / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D014807:Vitamin D; D005576:Colforsin; D015650:24,25-Dihydroxyvitamin D 3; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D002117:Calcitriol; D002112:Calcifediol; D007654:Ketoconazole; D002118:Calcium