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)

Tissue macrophages from patients with granuloma-forming disease, most notably sarcoidosis, express a 25-hydroxyvitamin D-1-hydroxylase which can produce in vivo sufficient quantities of the active vitamin D metabolite 1,25-dihydroxyvitamin D to cause hypercalcemia. In contrast to the NADPH-dependent cytochrome P450-linked mixed function oxidase which is normally only expressed in significant quantity in proximal renal tubular cells and regulated in an endocrine fashion, the mitochondrial-based 1-hydroxylase in the macrophage [1] is stimulated in a paracrine mode by cytokines (i.e., IFN-gamma) and lipopolysaccharide (LPS) [2] requires an extracellular source of L-arginine for full basal expression and [3] can be regulated in an intracrine fashion by nitric oxide (NO). In these experiments we employed inducible nitric oxide synthase (iNOS)-free, intact mitochondria preparations from the avain macrophage-like cell line HD-11, which constitutively express the 1-hydroxylase, and nonenzymatically-generated NO to investigate NO-mediated autoregulation of the macrophage 1-hydroxylase. Sodium nitroprusside (SNP)- or S-nitroso-N-acetyl-penicillamine (SNAP)-induced up-regulation of the 1-hydroxylase required the presence of either NADPH or NADP in the reaction mixture, while NO-induced inhibition of mitochondrial 1,25-(OH)2D3 synthesis was NO-dependent and NADP/NADPH-independent. These data suggest NO has bifunctional effects on the macrophage 1-hydroxylase. At relatively high concentrations NO competes with O2 for enzyme binding, inhibiting hormone synthesis. At lower production levels, NO serves as a source of reducing equivalents for the enzyme by providing for the reduction of NADP to NADPH.

We have recently described the existence of a cytochrome P450-associated, mitochondrial-based 25-hydroxyvitamin D (25-OHD)-1-hydroxylation reaction in the chick macrophage-like cell line HD-11. Considering that this reaction is regulated by the same set of factors (ie. interferon-gamma, lipopolysaccharide, and glucocorticoids) that modulate expression of the macrophage nitric oxide synthase (mac NOS), we investigated the possibility that endogenous nitric oxide (NO) production may be linked to 1,25-dihydroxyvitamin D3 (1,25-(OH)2D) synthesis by HD-11 cells in vitro. To test this hypothesis we investigated the effects excluding from the extracellular medium the essential amino acid L-arginine, substrate for endogenous NO production, on the basal and stimulated expression of the HD-11 cell 25-OHD-1-hydroxylation reaction. Depletion of L-arginine from the extracellular medium for as little as 6 h resulted in a significant decrease (p < 0.02) in basal 1,25-(OH)2D synthesis; after 15 h in an L-arginine-free environment hormone production was reduced to < 10% of basal levels without any adverse affect on cell viability. Reintroduction of L-arginine, but not D-arginine, into the extracellular medium restored 1,25-(OH)2D3 synthetic capacity fully if done after < or = 6 h of incubation in the absence of L-arginine. Competitive inhibition of NOS with Nw-nitro-L-arginine methyl ester (p < 0.002) and Nw-nitro-L-arginine (p < 0.02) significantly inhibited 1,25-(OH)2D synthesis, indicating that macrophage NO generating capacity is functionally linked to endogenous synthesis of the active vitamin D metabolite.

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