Teillaud Christophe, Nemere Ilka, Boukhobza Florine, Mathiot Claire, Conan Nicole, Oboeuf Martine, Hotton Dominique, Macdougall Mary, Berdal Ariane
Laboratoire de Biologie-Oro-Faciale et Pathologie, INSERM EMI-U 0110-IFR 58, Université Paris 7, Centre de Recherches Biomédicales des Cordeliers, Escalier E, Paris Cedex 06, France.
J Cell Biochem. 2005 Jan 1;94(1):139-52. doi: 10.1002/jcb.20275.
The rapid, nongenomic effects of 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3 have been related to a 1,25D3-membrane associated, rapid response steroid binding protein or 1,25D3-[MARRS]bp, with a molecular weight of 65 kDa, in several tissues and species. Currently, no information is available concerning the nongenomic responses to 1alpha,25-(OH)2D3 in dental tissues. In order to investigate the expression of 1,25D3-[MARRS]bp in dental cells, in the presence or absence of 1alpha,25-(OH)2D3, we have used rabbit polyclonal antibodies directed against the N-terminus of the 1,25D3-[MARRS]bp (Ab099) that recognizes the 1alpha,25-(OH)2D3 binding protein in chick intestinal basolateral membranes and a mouse odontoblast-like cell line (MO6-G3). Western blotting and flow cytometric analyses with Ab099 specifically detected 1,25D3-[MARRS]bp in MO6-G3 cells. Moreover, 1,25D3-[MARRS]bp was up-regulated, in vivo, in differentiated dental cells. Electron microscopic analysis confirmed the plasma membrane localization of this binding protein and also showed its intracellular presence. Incubation of MO6-G3 cells with different doses of 1alpha,25-(OH)2D3 for 36 h resulted in an inhibition of 1,25D3-[MARRS]bp expression with a maximal effect at 50 nM steroid. In addition, the culture media of MO6-G3 cells contains immunoreactive 1,25D3-[MARRS]bp. Immunogold positive membrane vesicle-like structures are present in the extracellular matrix of MO6-G3 cells. Altogether, these results indicate that the 1,25D3-[MARRS]bp expression in MO6-G3 cells is modulated by 1alpha,25-(OH)2D3. In conclusion, this 1alpha,25-(OH)2D3 binding protein could play an important role in the rapid, nongenomic responses to 1alpha,25-(OH)2D3 in dental cells.
1α,25 - 二羟基维生素D3（1α,25-(OH)2D3）的快速、非基因组效应与一种分子量为65 kDa的1,25D3膜相关快速反应类固醇结合蛋白或1,25D3 - [MARRS]bp有关，在多个组织和物种中均有发现。目前，关于牙组织对1α,25-(OH)2D3的非基因组反应尚无相关信息。为了研究在有或无1α,25-(OH)2D3存在的情况下，1,25D3 - [MARRS]bp在牙细胞中的表达情况，我们使用了针对1,25D3 - [MARRS]bp N端的兔多克隆抗体（Ab099），该抗体可识别鸡肠基底外侧膜和小鼠成牙本质细胞样细胞系（MO6 - G3）中的1α,25-(OH)2D3结合蛋白。用Ab099进行的蛋白质印迹和流式细胞术分析在MO6 - G3细胞中特异性检测到了1,25D3 - [MARRS]bp。此外，在体内分化的牙细胞中，1,25D3 - [MARRS]bp表达上调。电子显微镜分析证实了这种结合蛋白在质膜上的定位，同时也显示了其在细胞内的存在。用不同剂量的1α,25-(OH)2D3处理MO6 - G3细胞36小时，导致1,25D3 - [MARRS]bp表达受到抑制，在50 nM类固醇时抑制作用最大。此外，MO6 - G3细胞的培养基中含有免疫反应性1,25D3 - [MARRS]bp。免疫金阳性的膜泡样结构存在于MO6 - G3细胞的细胞外基质中。总之，这些结果表明MO6 - G3细胞中1,25D3 - [MARRS]bp的表达受1α,25-(OH)2D3调节。综上所述，这种1α,25-(OH)2D3结合蛋白可能在牙细胞对1α,25-(OH)2D3的快速非基因组反应中发挥重要作用。
