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Constitutive Expression of 25-Hydroxyvitamin D₃-1-Hydroxylase in a Transformed Human Proximal Tubule Cell Line: Evidence for Direct Regulation of Vitamin D Metabolism by Calcium¹

Department of Medicine, Institute of Clinical Research, University of Birmingham, Birmingham, United Kingdom B15 2TT

Address all correspondence and requests for reprints to: Dr. M. Hewison, Department of Medicine, Queen Elizabeth Hospital, Edgbaston, Birmingham, United Kingdom B15 2TH. E-mail: m.hewison{at}bham.ac.uk

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 D₃-1-hydroxylase (1-hydroxylase). Production of 1,25-(OH)₂D₃ occurs predominantly in the renal proximal tubule, with 1-hydroxylase activity being impaired in renal insufficiency and renal disease. The expression and activity of 1-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 1-hydroxylase in human renal tissue has proved difficult. In this study we have characterized constitutive 1-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 K_m 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 µmol/liter, 24 h) and was down-regulated by 1,25-(OH)₂D₃ (10 nmol/liter, 24 h). 1-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 1-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.

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