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


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 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.


RESULTS
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%).


CONCLUSIONS
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.
Monkawa
T
T
Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
Yoshida
T
T
Hayashi
M
M
Saruta
T
T
D000906:Antibodies; D000970:Antineoplastic Agents / Q000494:pharmacology; D015152:Blotting, Northern; D015153:Blotting, Western; D002116:Calcitonin / Q000494:pharmacology; D002117:Calcitriol / Q000494:pharmacology; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D053493:Cholestanetriol 26-Monooxygenase; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000494:pharmacology; D018076:DNA, Complementary; D003609:Dactinomycin / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000201:enzymology*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010281:Parathyroid Hormone / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D012260:Ribonucleases; D013250:Steroid Hydroxylases / Q000032:analysis / Q000235:genetics* / Q000276:immunology
D000906:Antibodies; D000970:Antineoplastic Agents; D002120:Calcium Channel Agonists; D018076:DNA, Complementary; D019384:Nucleic Acid Synthesis Inhibitors; D010281:Parathyroid Hormone; D012333:RNA, Messenger; D003609:Dactinomycin; D007371:Interferon-gamma; D002116:Calcitonin; D000242:Cyclic AMP; D013250:Steroid Hydroxylases; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase; D012260:Ribonucleases; D002117:Calcitriol