LeBlanc Tremaine, Nemere Lka
Department of Nutrition, Dietetic and Food Science, Utah State University, Logan, UT 84322-8700, USA.
Immunol Endocr Metab Agents Med Chem. 2014 Aug;14(2):55-66. doi: 10.2174/1871522214666140704171342.
We have shown that the 1,25D-MARRS receptor is necessary for the rapid, pre-genomic effects of 1,25(OH)D on phosphate and/or calcium absorption in chick intestines. However, a clear understanding of the proteins involved in the signaling mechanisms by which the 1,25D-MARRS receptor facilitates 1,25(OH)D-mediated phosphate or calcium uptake, as well as other cellular effects, is still under investigation. We used co-immunoprecipitation studies and mass spectroscopy to identify actin and keratin as proteins that interact with the 1,25D-MARRS receptor. Using confocal microscopy, we visualized 1,25(OH)D- MARRS receptor localizations relative to actin and/or keratin distribution in chick enterocytes. Cells cultured in media containing phenol red had the 1,25D-MARRS receptor and actin localized largely in the nucleus, which was dispersed upon addition of (OH) 1,25(OH)D. In the absence of phenol red, staining was cytoplasmic. Addition of steroid caused diminished staining at 10 s and 30 s, with a return of intensity between 1 and 5 min. Nuclear staining was observed after 1 min. We found that F-actin concentrations are maximal when 1,25D-MARRS receptor localizations within enterocytes are low suggesting that cyclical conversions of F-actin to G-actin are involved in the 1,25(OH)D-mediated redistribution of the 1,25D-MARRS receptor within the cell. We also found that keratin distribution remains constant with 1,25(OH)D exposure when Factin depolymerizes into G-actin, which suggests that actin and keratin work in concert to facilitate hormonemediated redistribution of the 1,25D-MARRS receptor. We subsequently investigated whether the cyclical redistribution was related to either 1,25(OH)D-stimulated phosphate or calcium uptake, but no congruent pattern was found.
我们已经证明，1,25D-MARRS受体对于1,25(OH)D对雏鸡肠道中磷酸盐和/或钙吸收的快速、基因组前效应是必需的。然而，对于1,25D-MARRS受体促进1,25(OH)D介导的磷酸盐或钙摄取以及其他细胞效应的信号传导机制中涉及的蛋白质，仍在进行研究。我们使用免疫共沉淀研究和质谱法鉴定出肌动蛋白和角蛋白是与1,25D-MARRS受体相互作用的蛋白质。利用共聚焦显微镜，我们观察了1,25(OH)D-MARRS受体相对于雏鸡肠细胞中肌动蛋白和/或角蛋白分布的定位。在含有酚红的培养基中培养的细胞，1,25D-MARRS受体和肌动蛋白主要定位于细胞核，添加1,25(OH)D后细胞核分散。在没有酚红的情况下，染色位于细胞质。添加类固醇后，在10秒和30秒时染色减弱，在1至5分钟之间强度恢复。1分钟后观察到细胞核染色。我们发现，当肠细胞内1,25D-MARRS受体定位较低时，F-肌动蛋白浓度最高，这表明F-肌动蛋白向G-肌动蛋白的周期性转化参与了1,25(OH)D介导的1,25D-MARRS受体在细胞内的重新分布。我们还发现，当F-肌动蛋白解聚为G-肌动蛋白时，角蛋白分布在1,25(OH)D暴露下保持不变，这表明肌动蛋白和角蛋白协同作用以促进激素介导的1,25D-MARRS受体的重新分布。我们随后研究了这种周期性重新分布是否与1,25(OH)D刺激的磷酸盐或钙摄取有关，但未发现一致的模式。
