Document Detail


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.
MedLine Citation:
PMID:  10218951     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
R Bland; E A Walker; S V Hughes; P M Stewart; M Hewison
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Endocrinology     Volume:  140     ISSN:  0013-7227     ISO Abbreviation:  Endocrinology     Publication Date:  1999 May 
Date Detail:
Created Date:  1999-05-06     Completed Date:  1999-05-06     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0375040     Medline TA:  Endocrinology     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  2027-34     Citation Subset:  AIM; IM    
Affiliation:
Department of Medicine, Institute of Clinical Research, University of Birmingham, United Kingdom.
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MeSH Terms
Descriptor/Qualifier:
24,25-Dihydroxyvitamin D 3 / metabolism
25-Hydroxyvitamin D3 1-alpha-Hydroxylase / antagonists & inhibitors,  genetics*,  metabolism
Blotting, Western
Calcifediol / metabolism
Calcitriol / metabolism,  pharmacology
Calcium / metabolism*
Cell Line
Cell Line, Transformed
Chromatography, High Pressure Liquid
Enzyme Inhibitors / pharmacology
Forskolin / pharmacology
Gene Expression*
Humans
Ketoconazole / pharmacology
Kidney Tubules, Proximal / enzymology*
Kinetics
RNA, Messenger / analysis
Reverse Transcriptase Polymerase Chain Reaction
Vitamin D / metabolism*
Chemical
Reg. No./Substance:
0/Enzyme Inhibitors; 0/RNA, Messenger; 1406-16-2/Vitamin D; 19356-17-3/Calcifediol; 32222-06-3/Calcitriol; 40013-87-4/24,25-Dihydroxyvitamin D 3; 65277-42-1/Ketoconazole; 66428-89-5/Forskolin; 7440-70-2/Calcium; EC 1.14.-/25-Hydroxyvitamin D3 1-alpha-Hydroxylase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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