Elsevier

Kidney International

Volume 58, Issue 2, August 2000, Pages 559-568
Kidney International

Hormones – Cytokines – Signaling
Identification of 25-hydroxyvitamin D3 1α-hydroxylase gene expression in macrophages

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

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 × 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.

Keywords

vitamin D3
interferon-γ
THP-1 cells
RNase protection assay

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