Constitutive expression of 25-hydroxyvitamin D-3-1 alpha-hydroxylase in a transformed human proximal tubule cell line: Evidence for direct regulationof vitamin D metabolism by calcium

Circulating levels of the active form of vitamin D, 1,25-dihydroxyvitamin D -3 (1,25-(OH)(2)D-3) are dependent on activity of the renal mitochondrial c ytochrome P450 enzyme, 25-hydroxyvitamin D-3-1 alpha-hydroxylase (1 alpha-h ydroxylase). Production of 1,25-(OH)(2)D-3 occurs predominantly in the rena l proximal tubule, with 1 alpha-hydroxylase activity being impaired in rena l insufficiency and renal disease. The expression and activity of 1 alpha-h ydroxylase are tightly regulated in response to serum levels of PTH, calciu m, phosphate, and 1,25(OH)(2)D-3 itself. As a consequence of this, the char acterization of 1 alpha-hydroxylase in human renal tissue has proved diffic ult. In this study we have characterized constitutive 1 alpha-hydroxylase e xpression in a simian virus 40-transformed human proximal tubule cell line, HKC-8. Initial analyses of [H-3]25-hydroxyvitamin D-3 (250HD(3)) metabolis m in these cells using straight and reverse phase HPLC revealed product pea ks that coincided with authentic 1,25-(OH)(2)D-3 as well as 24,25-dihydroxy vitamin D-3 (24,25-(OH)(2)D-3). Enzyme kinetic studies indicated that the K -m for synthesis of 1,25-(OH)(2)D-3 in HKC-8 cells was 120 nmol/liter 25OHD (3), with a maximum velocity of 21 pmol/h/mg protein. This activity was inh ibited by treatment with ketoconazole, but not diphenyl phenylenediamine. R T-PCR analysis of RNA from HKC-8 cells revealed a transcript similar in siz e 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)(2)D-3 was up regulated by treatment with forskolin (10 mu mol/l iter, 24 h) and was down-regulated by 1,25-(OH)(2)D-3 (10 nmol/liter, 24 h) . 1 alpha-Hydroxylase activity in HKC-8 cells was also sensitive to the con centration of calcium. Cells grown in low calcium (0.5 mmol/liter) showed a 4.8-fold induction of 1 alpha-hydroxylase, whereas treatment with medium c ontaining high levels of calcium (2 mmol/liter) significantly inhibited 1,2 5-(OH)(2)D-3 production. These data suggest that direct effects of calcium on proximal tubule cells may be an important feature of the regulation of r enal 1,25-(OH)(2)D-3 production.