Two step model for the fusion of macrophages induced by 1alpha, 25-dihydroxyvitamin D_3

我们报道了1α，25-二羟基维生素D_3[1α，25(OH)_2D_3]在3天内直接诱导小鼠肺泡巨噬细胞融合，融合率高达70-80%。从那时起，我们一直在研究细胞融合作为破骨细胞形成的模型系统的机制。在这项研究中，我们检测了可能参与融合的蛋白质。我们还研究了钙离子在1α，25(OH)_2D_3.1α，25(OH)_2D_3诱导的巨噬细胞融合过程中的作用。白介素4(IL-4)通过一种涉及亚精胺的机制类似地诱导巨噬细胞融合。IL-4.2对这3种蛋白的表达也有诱导作用。我们发现融合过程可以分为两个阶段：依赖1α，25(OH)_2D_3的启动阶段(0-18小时)和依赖钙的进程阶段(18-72小时)。巨噬细胞用1α，25(OH)2D3在低钙(0.13 mm)介质中处理48小时，未见融合。当换成正常钙(1.85摩尔)时，巨噬细胞在30分钟内开始融合。3.1α，25(OH)_2D_3通过依赖亚精胺的机制诱导钙依赖酶谷氨酰胺转氨酶的动态合成。用转谷氨酰胺酶抑制剂半胺处理巨噬细胞，在抑制转谷氨酰胺酶活性的同时，也抑制了融合。IL-4还通过依赖亚精胺的机制提高转谷氨酰胺酶的活性。综上所述，1α，25(OH)_22D3首先诱导亚精胺的合成，然后在启动阶段诱导转谷氨酰胺酶。这种依赖钙的酶似乎催化了负责细胞融合的生物反应。细胞外的钙离子而不是细胞内的钙离子在进展阶段起着重要的作用。

We reported that 1alpha,25-dihydroxyvitamin D_3[1alpha,25(OH)_2D_3] directly induces fusion of mouse alveolar macrophages at a high rate of 70-80% within 3 days. Since then, we have been examining the mechanism of cell fusion as a model system of osteoclast formation. In this study, we examined proteins possibly involved in the fusion. We also examined the role of calcium ions on the progression of macrophage fusion induced by 1alpha,25(OH)_2D_3.1.1alpha,25(OH)_2D_3 enhanced the synthesis of spermidine, which was followed by the synthesis of specific 3 proteins (98, 78 and 50 kD) responsible for the fusion. Interleukin 4 (IL-4) similarly induced macrophage fusion by a mechanism involving spermidine. These 3 proteins were also induced by IL-4.2. We found that the fusion process could be divided into two phases: the 1alpha,25(OH)_2D_3-dependent priming phase (0-18 hr) and the calcium-dependent progression phase (18-72 hr). When macrophages were treated with 1alpha,25(OH)_2D_3 in a low calcium (0.13 mill) medium for 48 hr, no fusion occurred. When the medium was changed to normal calcium (1.85 mill) medium, the macrophages began to fuse within 30 min. The extracellular, but not intracellular calcium ions appeared to be involved in this progression phase.3.1alpha,25(OH)_2D_3 induced the dynthesis of transglutaminase, a calcium-dependent enzyme, by a spermidine-dependent mechanism. The treatment of macrophages with cystamine, an inhibitor of transglutaminase, inhibited the fusion in parallel with the inhibition of transglutaminase activity. IL-4 also increased transglutaminase activity by a spermidine-dependent mechanism.In conclusion, 1alpha,25(OH)_22D_3 first induces spermidine synthesis, which in turn induces transglutaminase in the priming phase. This calcium-dependent enzyme appears to catalyze biological reactions responsible for cell fusion. The extracellular, but not intracellular calcium ions play an important role in the progression phase.