REGULATED PRODUCTION AND INTRACRINE ACTION OF 1,25-DIHYDROXYVITAMIN D-3 IN THE CHICK MYELOMONOCYTIC CELL-LINE HD-11
REGULATED PRODUCTION AND INTRACRINE ACTION OF 1,25-DIHYDROXYVITAMIN D-3 IN THE CHICK MYELOMONOCYTIC CELL-LINE HD-11
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DOI：
10.1210/en.134.6.2567
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发表时间：
1994-06-01
期刊：
影响因子：
4.8
通讯作者：
SHANY, S
中科院分区：
文献类型：
--
作者：
ADAMS, JS;REN, SY;SHANY, S
为了更好地理解单核细胞/巨噬细胞谱系细胞肾外产生活性维生素D代谢物，我们研究了v-myc转化的鸡骨髓单核细胞系HD-11中的25-羟基维生素D（25 OHD）-1-羟基化反应;该细胞系中的1-羟基化反应对25-羟基化维生素D底物具有高亲和力，定位于线粒体，并与细胞色素P450活性相关。在这项研究中，我们证明了HD-11细胞1-羟化反应在体外不受大多数细胞外调节因子，调节肾25 OHD-1-羟化酶在体内的表达。细胞外钙和磷酸盐浓度增加50%，肾1，25-二羟基维生素D [1，25（OH）（2）D]合成的生理抑制事件，不降低HI）-11细胞1-羟基化反应的基础表达，细胞外钙和磷酸盐浓度减少50%，体内1-羟基化酶的刺激信号，增加1，25-（OH）（2）D-3合成体外PTH和PTH相关肽的受体饱和浓度同样没有影响。与此相反，巨噬细胞刺激剂脂多糖[P < 0.001，最大有效浓度（EC（100））为25 μ g/ml]和Interfelon-γ以剂量依赖性方式显著刺激HD-11 L-羟基化反应（P < 0.001，EC（100）为1000 IU/ml）和胰岛素样生长因子-I（P < 0.01，EC（100）为15 nM），刺激的等级顺序为干扰素-γ>脂多糖>胰岛素样生长因子-I。地塞米松（大于或等于10 nM）和细胞色素P450抑制剂（EC（100），20 μ M），酮康唑，克霉唑，甲萘醌，都显着抑制HD-11细胞的L-羟基化反应。阻断电子传递到P450相关酶的萘醌甲萘醌是两种完整细胞中反应的最有效抑制剂（基础表达的3 +/- 1%; P小于或等于0.002）和用铁氧还蛋白、还原酶、O-2和NADPH重建HD-11细胞线粒体提取物后（基础值的5 +/- 1%; P ≤ 0.02）。我们还表明，从底物250 HD（3）产生的1，25-（OH）（2）D-3似乎对内源性（内分泌）对HD-11细胞生长的抑制作用;将HD-11细胞与已知使1，25-（OH）（2）D-3产生减少约50%的浓度的酮康唑（10 μ M）一起孵育，25-（OH）（2）D-3（EC（100），100 nM）。这些结果表明，巨噬细胞25 OHD（3）-1-羟基化反应的表达1）依赖于细胞色素P450的功能完整性，2）最有效地由炎性细胞活性的细胞外介质调节，3）能够发挥内分泌生长抑制作用。
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)(2)D] synthesis, did not decrease basal expression of the HI)-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)(2)D-3 synthesis in. vitro. Receptor-saturating concentrations of PTH and PTH-related peptide were similarly without effect. In contrast, the HD-11 l-hydroxylation reaction was significantly stimulated in a dose-dependent fashion by the macrophage stimulatory agents lipopolysaccharide [P < 0.001 at a maximum effective concentration (EC(100)) of 25 mu g/ml] and interfelon-gamma (P < 0.001 at EC(100) of 1000 IU/ml) and by insulin-like growth factor-I (P < 0.01 at EC(100) of 15 nM) with the rank order of stimulation being interferon-gamma > lipopolysaccharide > insulin-like growth factor-I. Dexamethasone ( greater than or equal to 10 nM) and the cytochrome P450 inhibitors (EC(100), 20 mu M), ketoconazole, clotrimazole, and menadione, all significantly inhibited the HD-11 cell l-hydroxylation reaction. The napthoquinone 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 less than or equal to 0.002) and after reconstitution of HD-11 cell mitochondrial extracts with a ferredoxin, reductase, O-2, and NADPH (5 +/- 1% of basal; P less than or equal to 0.02). We have also shown that 1,25-(OH)(2)D-3 produced from substrate 250HD(3) appears to exert an endogenous (intracrine) inhibitory effect on HD-11 cell growth; incubation of HD-11 cells with a concentration of ketoconazole (10 mu M) known to reduce 1,25-(OH)(2)D-3 production by roughly 50% restored 50% of the growth deficit induced by 1,25-(OH)(2)D-3 (EC(100), 100 nM). These results suggest that expression of the macrophage 25OHD(3)-1-hydroxylation reaction is 1) dependent upon functional integrity of the cell's compli ment of cytochrome P450, 2) regulated most effectively by extracellular mediators of inflammatory cell activity, and 3) capable of exerting an intracrine growth inhibitory effect.