Identification of 25-hydroxyvitamin D3 1α-hydroxylase gene expression in macrophages

Toshiaki Monkawa, Tadashi Yoshida, M. Hayashi, T. Saruta

Research output: Contribution to journalArticle

90 Citations (Scopus)

Abstract

Background. The 25-hydroxyvitamin D3 1α-hydroxylase (1α-hydroxylase) is almost exclusively expressed in the kidney. However, 1α-hydroxylase activities have been observed in some extrarenal tissues, including inflammatory cells of the monocyte/macrophage lineage. In sarcoidosis, macrophage 1α-hydroxylase causes overproduction of 1,25-(OH)2D3, resulting in hypercalcemia. In this study, we investigated the regulation of macrophage 1α-hydroxylase at a molecular level. Methods. 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 1α-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 1α-hydroxylase. We investigated the regulation of 1α-hydroxylase mRNA expression by RNase protection assay. Results. Northern blot and immunoblot analyses confirmed the expression of 1α-hydroxylase in THP-1 cells at the mRNA and protein levels. Although parathyroid hormone and calcitonin, known stimulators of renal 1α-hydroxylase, did not affect the expression of 1α-hydroxylase mRNA, 8-Br-cAMP (5 x 10-4 mol/L) increased the expression of 1α-hydroxylase mRNA in THP-1 cells (198 ± 9%). 1,25-(OH)2D3, known as a suppressor of renal 1α-hydroxylase, did not affect the expression of 1α-hydroxylase mRNA. By contrast, 1,25-(OH)2D3 markedly increased the expression of 25-hydroxyvitamin D3 24-hydroxylase mRNA. Interferon-γ (2000 IU/mL) increased the expression of 1α-hydroxylase mRNA in differentiated THP-1 cells (922 ± 25 %). Conclusions. The present results suggest that 1α-hydroxylase activity in macrophages is mediated by the same enzyme as in kidney). Interferon-γ treatment increases macrophage 1α-hydroxylase levels via directly increasing gene expression of this enzyme.

Original languageEnglish
Pages (from-to)559-568
Number of pages10
JournalKidney International
Volume58
Issue number2
DOIs
Publication statusPublished - 2000

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Calcifediol
Mixed Function Oxygenases
Macrophages
Gene Expression
Messenger RNA
Kidney
Northern Blotting
Interferons

Keywords

  • Interferon-γ THP-1 cells
  • RNase protection assay
  • Vitamin D

ASJC Scopus subject areas

  • Nephrology

Cite this

Identification of 25-hydroxyvitamin D3 1α-hydroxylase gene expression in macrophages. / Monkawa, Toshiaki; Yoshida, Tadashi; Hayashi, M.; Saruta, T.

In: Kidney International, Vol. 58, No. 2, 2000, p. 559-568.

Research output: Contribution to journalArticle

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abstract = "Background. The 25-hydroxyvitamin D3 1α-hydroxylase (1α-hydroxylase) is almost exclusively expressed in the kidney. However, 1α-hydroxylase activities have been observed in some extrarenal tissues, including inflammatory cells of the monocyte/macrophage lineage. In sarcoidosis, macrophage 1α-hydroxylase causes overproduction of 1,25-(OH)2D3, resulting in hypercalcemia. In this study, we investigated the regulation of macrophage 1α-hydroxylase at a molecular level. Methods. 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 1α-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 1α-hydroxylase. We investigated the regulation of 1α-hydroxylase mRNA expression by RNase protection assay. Results. Northern blot and immunoblot analyses confirmed the expression of 1α-hydroxylase in THP-1 cells at the mRNA and protein levels. Although parathyroid hormone and calcitonin, known stimulators of renal 1α-hydroxylase, did not affect the expression of 1α-hydroxylase mRNA, 8-Br-cAMP (5 x 10-4 mol/L) increased the expression of 1α-hydroxylase mRNA in THP-1 cells (198 ± 9{\%}). 1,25-(OH)2D3, known as a suppressor of renal 1α-hydroxylase, did not affect the expression of 1α-hydroxylase mRNA. By contrast, 1,25-(OH)2D3 markedly increased the expression of 25-hydroxyvitamin D3 24-hydroxylase mRNA. Interferon-γ (2000 IU/mL) increased the expression of 1α-hydroxylase mRNA in differentiated THP-1 cells (922 ± 25 {\%}). Conclusions. The present results suggest that 1α-hydroxylase activity in macrophages is mediated by the same enzyme as in kidney). Interferon-γ treatment increases macrophage 1α-hydroxylase levels via directly increasing gene expression of this enzyme.",
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N2 - Background. The 25-hydroxyvitamin D3 1α-hydroxylase (1α-hydroxylase) is almost exclusively expressed in the kidney. However, 1α-hydroxylase activities have been observed in some extrarenal tissues, including inflammatory cells of the monocyte/macrophage lineage. In sarcoidosis, macrophage 1α-hydroxylase causes overproduction of 1,25-(OH)2D3, resulting in hypercalcemia. In this study, we investigated the regulation of macrophage 1α-hydroxylase at a molecular level. Methods. 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 1α-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 1α-hydroxylase. We investigated the regulation of 1α-hydroxylase mRNA expression by RNase protection assay. Results. Northern blot and immunoblot analyses confirmed the expression of 1α-hydroxylase in THP-1 cells at the mRNA and protein levels. Although parathyroid hormone and calcitonin, known stimulators of renal 1α-hydroxylase, did not affect the expression of 1α-hydroxylase mRNA, 8-Br-cAMP (5 x 10-4 mol/L) increased the expression of 1α-hydroxylase mRNA in THP-1 cells (198 ± 9%). 1,25-(OH)2D3, known as a suppressor of renal 1α-hydroxylase, did not affect the expression of 1α-hydroxylase mRNA. By contrast, 1,25-(OH)2D3 markedly increased the expression of 25-hydroxyvitamin D3 24-hydroxylase mRNA. Interferon-γ (2000 IU/mL) increased the expression of 1α-hydroxylase mRNA in differentiated THP-1 cells (922 ± 25 %). Conclusions. The present results suggest that 1α-hydroxylase activity in macrophages is mediated by the same enzyme as in kidney). Interferon-γ treatment increases macrophage 1α-hydroxylase levels via directly increasing gene expression of this enzyme.

AB - Background. The 25-hydroxyvitamin D3 1α-hydroxylase (1α-hydroxylase) is almost exclusively expressed in the kidney. However, 1α-hydroxylase activities have been observed in some extrarenal tissues, including inflammatory cells of the monocyte/macrophage lineage. In sarcoidosis, macrophage 1α-hydroxylase causes overproduction of 1,25-(OH)2D3, resulting in hypercalcemia. In this study, we investigated the regulation of macrophage 1α-hydroxylase at a molecular level. Methods. 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 1α-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 1α-hydroxylase. We investigated the regulation of 1α-hydroxylase mRNA expression by RNase protection assay. Results. Northern blot and immunoblot analyses confirmed the expression of 1α-hydroxylase in THP-1 cells at the mRNA and protein levels. Although parathyroid hormone and calcitonin, known stimulators of renal 1α-hydroxylase, did not affect the expression of 1α-hydroxylase mRNA, 8-Br-cAMP (5 x 10-4 mol/L) increased the expression of 1α-hydroxylase mRNA in THP-1 cells (198 ± 9%). 1,25-(OH)2D3, known as a suppressor of renal 1α-hydroxylase, did not affect the expression of 1α-hydroxylase mRNA. By contrast, 1,25-(OH)2D3 markedly increased the expression of 25-hydroxyvitamin D3 24-hydroxylase mRNA. Interferon-γ (2000 IU/mL) increased the expression of 1α-hydroxylase mRNA in differentiated THP-1 cells (922 ± 25 %). Conclusions. The present results suggest that 1α-hydroxylase activity in macrophages is mediated by the same enzyme as in kidney). Interferon-γ treatment increases macrophage 1α-hydroxylase levels via directly increasing gene expression of this enzyme.

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