Scharla S H, Strong D D, Mohan S, Baylink D J, Linkhart T A
Mineral Metabolism Laboratory, J. L. Pettis Memorial Veterans' Hospital, Loma Linda, California 92373.
Endocrinology. 1991 Dec;129(6):3139-46. doi: 10.1210/endo-129-6-3139.
1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] induces differentiation and inhibits proliferation in many cell types including bone cells. These effects may be mediated by the modulation of the insulin-like growth factor (IGF) regulatory system. Therefore we investigated the effects of 1,25-(OH)2D3 on transcript and protein levels of both IGF-I and IGF binding proteins (IGFBPs) in clonal mouse osteoblasts. Subconfluent cultures were treated in serum-free medium with 1,25-(OH)2D3. Secreted IGF-I was measured using a RIA under conditions eliminating the interference of IGFBPs. 1,25-(OH)2D3 (10(-11)-10(-8) M) inhibited IGF-I release in a dose dependent manner at 24 h (maximally to 30 +/- 5% of control, mean +/- SEM of seven independent experiments). In a time course study IGF-I increased in the media of control cultures over a 48-h period, while IGF-I secretion was completely prevented from 6 h onward in 1,25-(OH)2D3 treated cultures. Northern blot analysis revealed four IGF-I transcripts of 0.9, 1.8, 4.4, and 7.5 kilobases (kb). 1,25-(OH)2D3 decreased levels of the 7.5 kb IGF-I transcript from 4-48 h, with maximal inhibition occurring at 24 h (25% of control). Western ligand blots of the culture medium demonstrated secretion of a 25-kilodalton IGFBP, which comprised greater than or equal to 90% of the secreted IGFBPs. The 25-kilodalton IGFBP had previously been shown to have sequence similarity with IGFBP-4, a binding protein which inhibits the action of IGFs on bone cells. 1,25-(OH)2D3 treatment increased secretion of IGFBP-4 up to 14-fold over 24 h. 1,25-(OH)2D3 also increased IGFBP-4 (2.2 kb) transcript levels within 30 min, with the maximal stimulation of 8-fold occurring after 8 h. [3H]Thymidine incorporation into cells was inhibited by 1,25-(OH)2D3 both under basal and serum-stimulated conditions. Our results are consistent with the hypothesis that the effects of 1,25-(OH)2D3 on osteoblast proliferation may be mediated in part by decreased levels of IGF-I and increased concentrations of inhibitory IGFBP-4. It is proposed that this alteration in the IGF system may be an important functional autocrine or paracrine switch in the transition of osteoblasts from states of proliferation to differentiation.
1,25 - 二羟基维生素D3 [1,25 - (OH)2D3]可诱导多种细胞类型（包括骨细胞）分化并抑制其增殖。这些作用可能是通过调节胰岛素样生长因子（IGF）调节系统介导的。因此，我们研究了1,25 - (OH)2D3对克隆小鼠成骨细胞中IGF - I和IGF结合蛋白（IGFBPs）转录水平和蛋白水平的影响。亚汇合培养物在无血清培养基中用1,25 - (OH)2D3处理。在消除IGFBPs干扰的条件下，使用放射免疫分析法测量分泌的IGF - I。1,25 - (OH)2D3（10(-11)-10(-8) M）在24小时时以剂量依赖性方式抑制IGF - I释放（最大抑制至对照的30±5%，七个独立实验的平均值±标准误）。在一项时间进程研究中，对照培养物培养基中的IGF - I在48小时内增加，而在1,25 - (OH)2D3处理的培养物中，从6小时起IGF - I分泌完全被抑制。Northern印迹分析显示有0.9、1.8、4.4和7.5千碱基（kb）的四种IGF - I转录本。1,25 - (OH)2D3在4 - 48小时降低7.5 kb IGF - I转录本水平，在24小时时出现最大抑制（对照的25%）。培养基的Western配体印迹显示分泌一种25千道尔顿的IGFBP，其占分泌的IGFBPs的90%或更多。先前已表明25千道尔顿的IGFBP与IGFBP - 4具有序列相似性，IGFBP - 4是一种抑制IGFs对骨细胞作用的结合蛋白。1,25 - (OH)2D3处理在24小时内使IGFBP - 4分泌增加高达14倍。1,25 - (OH)2D3在30分钟内也增加IGFBP - 4（2.2 kb）转录水平，在8小时后出现最大8倍的刺激。在基础和血清刺激条件下，1,25 - (OH)2D3均抑制[3H]胸腺嘧啶掺入细胞。我们的结果与以下假设一致，即1,25 - (OH)2D3对成骨细胞增殖的影响可能部分是由IGF - I水平降低和抑制性IGFBP - 4浓度增加介导的。有人提出，IGF系统的这种改变可能是成骨细胞从增殖状态向分化状态转变过程中的一个重要功能性自分泌或旁分泌开关。
