Bland R, Walker E A, Hughes S V, Stewart P M, Hewison M
Department of Medicine, Institute of Clinical Research, University of Birmingham, United Kingdom.
Endocrinology. 1999 May;140(5):2027-34. doi: 10.1210/endo.140.5.6683.
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.
维生素D的活性形式1,25 - 二羟基维生素D3（1,25-(OH)2D3）的循环水平取决于肾线粒体细胞色素P450酶25 - 羟基维生素D3 - 1α - 羟化酶（1α - 羟化酶）的活性。1,25-(OH)2D3主要在肾近端小管产生，在肾功能不全和肾脏疾病中1α - 羟化酶活性受损。1α - 羟化酶的表达和活性受到甲状旁腺激素、钙、磷和1,25-(OH)2D3自身血清水平的严格调控。因此，在人肾组织中对1α - 羟化酶进行表征已被证明很困难。在本研究中，我们对猿猴病毒40转化的人近端小管细胞系HKC - 8中组成型1α - 羟化酶的表达进行了表征。使用正相和反相高效液相色谱对这些细胞中[3H]25 - 羟基维生素D3（25OHD3）代谢的初步分析显示，产物峰与真实的1,25-(OH)2D3以及24,25 - 二羟基维生素D3（24,25-(OH)2D3）一致。酶动力学研究表明，HKC - 8细胞中合成1,25-(OH)2D3的米氏常数为120 nmol/升25OHD3，最大反应速度为21 pmol/h/mg蛋白。酮康唑处理可抑制该活性，但二苯基苯二胺处理则无此作用。对HKC - 8细胞RNA的逆转录 - 聚合酶链反应分析显示，转录本大小与角质形成细胞和人近端小管细胞原代培养物中观察到的相似，并且通过蛋白质印迹分析检测到了蛋白质。用福司可林（10 μmol/升，24小时）处理可上调1,25-(OH)2D3的合成，而用1,25-(OH)2D3（10 nmol/升，24小时）处理则下调其合成。HKC - 8细胞中的1α - 羟化酶活性也对钙浓度敏感。在低钙（0.5 mmol/升）条件下生长的细胞中1α - 羟化酶诱导了4.8倍，而用含有高钙（2 mmol/升）的培养基处理则显著抑制了1,25-(OH)2D3的产生。这些数据表明，钙对近端小管细胞的直接作用可能是肾1,25-(OH)2D3产生调节的一个重要特征。
