Monkawa T, Yoshida T, Hayashi M, Saruta T
Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
Kidney Int. 2000 Aug;58(2):559-68. doi: 10.1046/j.1523-1755.2000.00202.x.
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.
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.
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%).
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.
25 - 羟维生素D3 1α - 羟化酶（1α - 羟化酶）几乎仅在肾脏中表达。然而，在一些肾外组织中也观察到了1α - 羟化酶活性，包括单核细胞/巨噬细胞系的炎症细胞。在结节病中，巨噬细胞1α - 羟化酶导致1,25 - (OH)2D3过度产生，从而引起高钙血症。在本研究中，我们在分子水平上研究了巨噬细胞1α - 羟化酶的调控机制。
我们使用人单核细胞系THP - 1，该细胞系可通过佛波酯处理分化为巨噬细胞样细胞。通过Northern印迹法和免疫印迹法，使用针对与1α - 羟化酶14个C末端氨基酸对应的合成肽产生的抗体，检测THP - 1细胞中1α - 羟化酶的表达。我们通过核糖核酸酶保护试验研究1α - 羟化酶mRNA表达的调控。
Northern印迹和免疫印迹分析证实了THP - 1细胞中1α - 羟化酶在mRNA和蛋白质水平的表达。尽管已知的肾脏1α - 羟化酶刺激剂甲状旁腺激素和降钙素不影响1α - 羟化酶mRNA的表达，但8 - Br - cAMP（5×10 - 4 mol/L）增加了THP - 1细胞中1α - 羟化酶mRNA的表达（198±9%）。已知为肾脏1α - 羟化酶抑制剂的1,25 - (OH)2D3不影响1α - 羟化酶mRNA的表达。相比之下，1,25 - (OH)2D3显著增加了25 - 羟维生素D3 24 - 羟化酶mRNA的表达。干扰素 - γ（2000 IU/mL）增加了分化的THP - 1细胞中1α - 羟化酶mRNA的表达（922±25%）。
目前的结果表明，巨噬细胞中的1α - 羟化酶活性由与肾脏中相同的酶介导。干扰素 - γ处理通过直接增加该酶的基因表达来提高巨噬细胞1α - 羟化酶水平。
