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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. 1999

R Bland, and E A Walker, and S V Hughes, and P M Stewart, and M Hewison
Department of Medicine, Institute of Clinical Research, University of Birmingham, United Kingdom.

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.

UI MeSH Term Description Entries
D007654 Ketoconazole Broad spectrum antifungal agent used for long periods at high doses, especially in immunosuppressed patients. Nizoral,R-41400,R41,400,R41400,R 41400
D007687 Kidney Tubules, Proximal The renal tubule portion that extends from the BOWMAN CAPSULE in the KIDNEY CORTEX into the KIDNEY MEDULLA. The proximal tubule consists of a convoluted proximal segment in the cortex, and a distal straight segment descending into the medulla where it forms the U-shaped LOOP OF HENLE. Proximal Kidney Tubule,Proximal Renal Tubule,Kidney Tubule, Proximal,Proximal Kidney Tubules,Proximal Renal Tubules,Renal Tubule, Proximal,Renal Tubules, Proximal,Tubule, Proximal Kidney,Tubule, Proximal Renal,Tubules, Proximal Kidney,Tubules, Proximal Renal
D007700 Kinetics The rate dynamics in chemical or physical systems.
D002112 Calcifediol The major circulating metabolite of VITAMIN D3. It is produced in the LIVER and is the best indicator of the body's vitamin D stores. It is effective in the treatment of RICKETS and OSTEOMALACIA, both in azotemic and non-azotemic patients. Calcifediol also has mineralizing properties. 25-Hydroxycholecalciferol,25-Hydroxyvitamin D 3,25-Hydroxycholecalciferol Monohydrate,25-Hydroxyvitamin D3,Calcidiol,Calcifediol Anhydrous,Calcifediol, (3 alpha,5Z,7E)-Isomer,Calcifediol, (3 beta,5E,7E)-Isomer,Calderol,Dedrogyl,Hidroferol,25 Hydroxycholecalciferol,25 Hydroxycholecalciferol Monohydrate,25 Hydroxyvitamin D 3,25 Hydroxyvitamin D3,Anhydrous, Calcifediol,Monohydrate, 25-Hydroxycholecalciferol
D002117 Calcitriol The physiologically active form of vitamin D. It is formed primarily in the kidney by enzymatic hydroxylation of 25-hydroxycholecalciferol (CALCIFEDIOL). Its production is stimulated by low blood calcium levels and parathyroid hormone. Calcitriol increases intestinal absorption of calcium and phosphorus, and in concert with parathyroid hormone increases bone resorption. 1 alpha,25-Dihydroxycholecalciferol,1 alpha,25-Dihydroxyvitamin D3,1, 25-(OH)2D3,1,25(OH)2D3,1,25-Dihydroxycholecalciferol,1,25-Dihydroxyvitamin D3,1 alpha, 25-dihydroxy-20-epi-Vitamin D3,1,25(OH)2-20epi-D3,1,25-dihydroxy-20-epi-Vitamin D3,20-epi-1alpha,25-dihydroxycholecaliferol,Bocatriol,Calcijex,Calcitriol KyraMed,Calcitriol-Nefro,Decostriol,MC-1288,MC1288,Osteotriol,Renatriol,Rocaltrol,Silkis,Sitriol,Soltriol,Tirocal,1 alpha,25 Dihydroxyvitamin D3,1,25 Dihydroxycholecalciferol,1,25 Dihydroxyvitamin D3,1,25 dihydroxy 20 epi Vitamin D3,Calcitriol Nefro,D3, 1 alpha,25-Dihydroxyvitamin,D3, 1,25-Dihydroxyvitamin,D3, 1,25-dihydroxy-20-epi-Vitamin,KyraMed, Calcitriol,MC 1288
D002118 Calcium A basic element found in nearly all tissues. It is a member of the alkaline earth family of metals with the atomic symbol Ca, atomic number 20, and atomic weight 40. Calcium is the most abundant mineral in the body and combines with phosphorus to form calcium phosphate in the bones and teeth. It is essential for the normal functioning of nerves and muscles and plays a role in blood coagulation (as factor IV) and in many enzymatic processes. Coagulation Factor IV,Factor IV,Blood Coagulation Factor IV,Calcium-40,Calcium 40,Factor IV, Coagulation
D002460 Cell Line Established cell cultures that have the potential to propagate indefinitely. Cell Lines,Line, Cell,Lines, Cell
D002461 Cell Line, Transformed Eukaryotic cell line obtained in a quiescent or stationary phase which undergoes conversion to a state of unregulated growth in culture, resembling an in vitro tumor. It occurs spontaneously or through interaction with viruses, oncogenes, radiation, or drugs/chemicals. Transformed Cell Line,Cell Lines, Transformed,Transformed Cell Lines
D002851 Chromatography, High Pressure Liquid Liquid chromatographic techniques which feature high inlet pressures, high sensitivity, and high speed. Chromatography, High Performance Liquid,Chromatography, High Speed Liquid,Chromatography, Liquid, High Pressure,HPLC,High Performance Liquid Chromatography,High-Performance Liquid Chromatography,UPLC,Ultra Performance Liquid Chromatography,Chromatography, High-Performance Liquid,High-Performance Liquid Chromatographies,Liquid Chromatography, High-Performance
D004791 Enzyme Inhibitors Compounds or agents that combine with an enzyme in such a manner as to prevent the normal substrate-enzyme combination and the catalytic reaction. Enzyme Inhibitor,Inhibitor, Enzyme,Inhibitors, Enzyme

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