Gamblin G T, Liberman U A, Eil C, Downs R W, DeGrange D A, Marx S J
J Clin Invest. 1985 Mar;75(3):954-60. doi: 10.1172/JCI111796.
1,25(OH)2D3 induces 25(OH)D3-24-hydroxylase (24-OHase) in cultured skin fibroblasts from normal subjects. We evaluated 24-OHase induction by 1,25(OH)2D3 in skin fibroblasts from 10 normal subjects and from four unrelated patients with hereditary resistance to 1,25(OH)2D or vitamin D-dependent rickets type II (DD II). Fibroblasts were preincubated with varying concentrations of 1,25(OH)2D3 for 15 h and were then incubated with 0.5 microM [3H]25(OH)D3 at 37 degrees C for 30 min; lipid extracts of the cells were analyzed for [3H]24,25(OH)2D3 by high performance liquid chromatography and periodate oxidation. Apparent maximal [3H]24,25(OH)2D3 production in normal cell lines was 9 pmol/10(6) cells per 30 min and occurred after induction with 10(-8) M 1,25(OH)2D3. 24-OHase induction was detectable in normal fibroblasts at approximately 3 X 10(-10) M 1,25(OH)2D3. [3H]24,25(OH)2D3 formation after exposure to 1,25(OH)2D3 was abnormal in fibroblasts from all four patients with DD II. In fibroblasts from two patients with DD II, [3H]24,25(OH)2D3 formation was unmeasurable (below 0.2 pmol/10(6) cells per 30 min) at 1,25(OH)2D3 concentrations up to 10(-6) M. Fibroblasts from the other two patients with DD II required far higher than normal concentrations of 1,25(OH)2D3 for detectable [3H]24,25(OH)2D3 induction. In one, [3H]24,25(OH)2D3 production reached 2.9 pmol/10(6) cells per 30 min at 10(-6) M 1,25(OH)2D3 (30% normal maximum at 10(-6) M 1,25(OH)2D3). In the other, [3H]24,25(OH)2D3 production achieved normal levels, 7.3 pmol/10(6) cells per 30 min after 10(-6) M 1,25(OH)2D3. The two patients whose cells had a detectable 24-OHase induction by 1,25(OH)2D3 showed a calcemic response to high doses of calciferols in vivo. Our current observations correlate with these two patients' responsiveness to calciferols in vivo and suggest that their target organ defects can be partially or completely overcome with extremely high concentrations of 1,25(OH)2D3. The two patients whose cells showed no detectable 24-OHase induction in vitro failed to show a calcemic response to high doses of calciferols in vivo.
(a) the measurement of 24-OHase induction by 1,25(OH)2D3 in cultured skin fibroblasts is a sensitive in vitro test for defective genes in the 1,25(OH)2D effector pathway. (b) This assay provides a useful tool for characterizing the target tissue defects in DD II and predicting response to calciferol therapy.
1,25(OH)₂D₃可诱导正常受试者培养的皮肤成纤维细胞中的25(OH)D₃-24-羟化酶（24-OHase）。我们评估了1,25(OH)₂D₃对10名正常受试者以及4名患有遗传性1,25(OH)₂D抵抗或II型维生素D依赖性佝偻病（DD II）的无关患者的皮肤成纤维细胞中24-OHase的诱导作用。将成纤维细胞与不同浓度的1,25(OH)₂D₃预孵育15小时，然后在37℃下与0.5微摩尔[³H]25(OH)D₃孵育30分钟；通过高效液相色谱和高碘酸盐氧化分析细胞的脂质提取物中的[³H]24,25(OH)₂D₃。正常细胞系中[³H]24,25(OH)₂D₃的表观最大产量为每30分钟9皮摩尔/10⁶个细胞，在10⁻⁸M 1,25(OH)₂D₃诱导后出现。在正常成纤维细胞中，约3×10⁻¹⁰M 1,25(OH)₂D₃时可检测到24-OHase的诱导。所有4名DD II患者的成纤维细胞在暴露于1,25(OH)₂D₃后[³H]24,25(OH)₂D₃的形成均异常。在两名DD II患者的成纤维细胞中，在高达10⁻⁶M的1,25(OH)₂D₃浓度下，[³H]24,25(OH)₂D₃的形成无法测量（低于每30分钟0.2皮摩尔/10⁶个细胞）。另外两名DD II患者的成纤维细胞需要远高于正常浓度的1,25(OH)₂D₃才能检测到[³H]24,25(OH)₂D₃的诱导。其中一名患者在10⁻⁶M 1,25(OH)₂D₃时，[³H]24,25(OH)₂D₃产量达到每30分钟2.9皮摩尔/10⁶个细胞（在10⁻⁶M 1,25(OH)₂D₃时为正常最大值的30%）。另一名患者在10⁻⁶M 1,25(OH)₂D₃后，[³H]24,25(OH)₂D₃产量达到正常水平，即每30分钟7.3皮摩尔/10⁶个细胞。其细胞可被1,25(OH)₂D₃检测到24-OHase诱导的两名患者在体内对高剂量的维生素D显示出血钙反应。我们目前的观察结果与这两名患者在体内对维生素D的反应性相关，并表明他们的靶器官缺陷可以用极高浓度的1,25(OH)₂D₃部分或完全克服。在体外未检测到24-OHase诱导的两名患者在体内对高剂量的维生素D未显示出血钙反应。
（a）在培养的皮肤成纤维细胞中测量1,25(OH)₂D₃对24-OHase的诱导是检测1,25(OH)₂D效应途径中缺陷基因的敏感体外试验。（b）该检测为表征DD II中的靶组织缺陷和预测对维生素D治疗的反应提供了有用的工具。
