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- Title
Identification of 25-hydroxyvitamin D3 1alpha-hydroxylase gene expression in macrophages.
- Authors
Monkawa, Toshiaki; Yoshida, Tadashi; Hayashi, Matsuhiko; Saruta, Takao; Monkawa, T; Yoshida, T; Hayashi, M; Saruta, T
- Abstract
<bold>Background: </bold>The 25-hydroxyvitamin D3 1alpha-hydroxylase (1alpha-hydroxylase) is almost exclusively expressed in the kidney. However, 1alpha-hydroxylase activities have been observed in some extrarenal tissues, including inflammatory cells of the monocyte/macrophage lineage. In sarcoidosis, macrophage 1alpha-hydroxylase causes overproduction of 1,25-(OH)2D3, resulting in hypercalcemia. In this study, we investigated the regulation of macrophage 1alpha-hydroxylase at a molecular level.<bold>Methods: </bold>We used the human monocytic cell line THP-1, which can be differentiated into macrophage-like cells by treatment with phorbol ester. The expression of 1alpha-hydroxylase in THP-1 cells was examined by Northern blotting and immunoblotting using an antibody raised against a synthetic peptide corresponding to the 14 C-terminal amino acids of 1alpha-hydroxylase. We investigated the regulation of 1alpha-hydroxylase mRNA expression by RNase protection assay.<bold>Results: </bold>Northern blot and immunoblot analyses confirmed the expression of 1alpha-hydroxylase in THP-1 cells at the mRNA and protein levels. Although parathyroid hormone and calcitonin, known stimulators of renal 1alpha-hydroxylase, did not affect the expression of 1alpha-hydroxylase mRNA, 8-Br-cAMP (5 x 10-4 mol/L) increased the expression of 1alpha-hydroxylase mRNA in THP-1 cells (198 +/- 9%). 1,25-(OH)2D3, known as a suppressor of renal 1alpha-hydroxylase, did not affect the expression of 1alpha-hydroxylase mRNA. By contrast, 1,25-(OH)2D3 markedly increased the expression of 25-hydroxyvitamin D3 24-hydroxylase mRNA. Interferon-gamma (2000 IU/mL) increased the expression of 1alpha-hydroxylase mRNA in differentiated THP-1 cells (922 +/- 25%).<bold>Conclusions: </bold>The present results suggest that 1alpha-hydroxylase activity in macrophages is mediated by the same enzyme as in kidney. Interferon-gamma treatment increases macrophage 1alpha-hydroxylase levels via directly increasing gene expression of this enzyme.
- Subjects
GENE expression; MACROPHAGES; CELL differentiation; RNA analysis; ANTINEOPLASTIC agents; CALCITONIN; CELL lines; COMPARATIVE studies; CYCLIC adenylic acid; DNA; ENZYME inhibitors; ESTERASES; GENES; GENETIC techniques; IMMUNOBLOTTING; IMMUNOGLOBULINS; INTERFERONS; RESEARCH methodology; MEDICAL cooperation; NUCLEOTIDE separation; OXIDOREDUCTASES; PARATHYROID hormone; RESEARCH; VASOCONSTRICTORS; WESTERN immunoblotting; EVALUATION research; CHOLECALCIFEROL; CALCITRIOL; DACTINOMYCIN; PHARMACODYNAMICS; PHYSIOLOGY
- Publication
Kidney International, 2000, Vol 58, Issue 2, p559
- ISSN
0085-2538
- Publication type
journal article
- DOI
10.1046/j.1523-1755.2000.00202.x