Regulated production and intracrine action of 1,25-dihydroxyvitamin D3 in the chick myelomonocytic cell line HD-11.
To better understand the extrarenal production of active vitamin D metabolites by cells of the monocyte/macrophage lineage, we investigated the 25-hydroxyvitamin D (25OHD)-1-hydroxylation reaction in the v-myc-transformed chick myelomonocytic cell line HD-11; the 1-hydroxylation reaction in this cell line has a high affinity for 25-hydroxylated vitamin D substrates, is localized to mitochondria, and is associated with cytochrome P450 activity. In this study we demonstrated that the HD-11 cell 1-hydroxylation reaction in vitro is not affected by the majority of extracellular regulatory factors that modulate expression of the renal 25OHD-1-hydroxylase in vivo. A 50% increase in extracellular calcium and phosphate concentrations, physiological inhibitory events for renal 1,25-dihydroxyvitamin D [1,25-(OH)2D] synthesis, did not decrease basal expression of the HD-11 cell 1-hydroxylation reaction, nor did a 50% decrease in extracellular calcium and phosphate concentrations, stimulatory signals for the 1-hydroxylase in vivo, increase 1,25-(OH)2D3 synthesis in vitro. Receptor-saturating concentrations of PTH and PTH-related peptide were similarly without effect. In contrast, the HD-11 1-hydroxylation reaction was significantly stimulated in a dose-dependent fashion by the macrophage stimulatory agents lipopolysaccharide [P < 0.001 at a maximum effective concentration (EC100) of 25 micrograms/ml] and interferon-gamma (P < 0.001 at EC100 of 1000 IU/ml) and by insulin-like growth factor-I (P < 0.01 at EC100 of 15 nM) with the rank order of stimulation being interferon-gamma > lipopolysaccharide > insulin-like growth factor-I. Dexamethasone (> or = 10 nM) and the cytochrome P450 inhibitors (EC100, 20 microM), ketoconazole, clotrimazole, and menadione, all significantly inhibited the HD-11 cell 1-hydroxylation reaction. The naphthoquinone menadione, which blocks electron transfer to the P450-associated enzyme, was the most effective inhibitor of the reaction in both intact cells (3 +/- 1% of basal expression; P < or = 0.002) and after reconstitution of HD-11 cell mitochondrial extracts with a ferredoxin, reductase, O2, and NADPH (5 +/- 1% of basal; P < or = 0.02). We have also shown that 1,25-(OH)2D3 produced from substrate 25OHD3 appears to exert an endogenous (intracrine) inhibitory effect on HD-11 cell growth; incubation of HD-11 cells with a concentration of ketoconazole (10 microM) known to reduce 1,25-(OH)2D3 production by roughly 50% restored 50% of the growth deficit induced by 1,25-(OH)2D3 (EC100, 100 nM).(ABSTRACT TRUNCATED AT 400 WORDS)
Adams
J S
JS
Division of Endocrinology and Metabolism, Cedars-Sinai Medical Center, University of California School of Medicine, Los Angeles 90048.
Ren
S Y
SY
Arbelle
J E
JE
Horiuchi
N
N
Gray
R W
RW
Clemens
T L
TL
Shany
S
S
D000818:Animals; D002112:Calcifediol / Q000378:metabolism; D002117:Calcitriol / Q000096:biosynthesis* / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002461:Cell Line, Transformed; D002645:Chickens; D003022:Clotrimazole / Q000494:pharmacology; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D016259:Genes, myc; D006098:Granulocytes / Q000378:metabolism*; D006900:Hydroxylation; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007371:Interferon-gamma / Q000494:pharmacology; D007654:Ketoconazole / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D009000:Monocytes / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D014812:Vitamin K / Q000494:pharmacology
D065607:Cytochrome P-450 Enzyme Inhibitors; D008070:Lipopolysaccharides; D010710:Phosphates; D014812:Vitamin K; D007334:Insulin-Like Growth Factor I; D007371:Interferon-gamma; D003577:Cytochrome P-450 Enzyme System; D002117:Calcitriol; D003022:Clotrimazole; D002112:Calcifediol; D007654:Ketoconazole; D002118:Calcium