Department of Nutrition and Food Sciences and the Center for Integrated BioSystems, Utah State University, Logan, UT, United States.
Steroids. 2010 Apr;75(4-5):307-13. doi: 10.1016/j.steroids.2010.01.004. Epub 2010 Jan 14.
We undertook studies to determine which isotype(s) of protein kinase C (PKC) is/are activated by ligand binding to the 1,25D(3)-MARRS receptor (ERp57/PDIA3) and subsequent stimulation of phosphate uptake. Isolated intestinal epithelial cells from vitamin D-replete chicks were exposed to 1,25(OH)(2)D(3) for 1, 3, or 5min, thoroughly chilled, homogenized, and P(2) fractions (20,000xg post-nuclear pellet) prepared. Western analyses with anti-pan PKC revealed steroid-stimulated redistribution to P(2) membranes 1min after hormone. Using this time point, cells were treated with vehicle, 130-, 300- or 650-pM hormone. Western blots with anti-PKCalpha exhibited redistribution to membranes in a biphasic dose-response curve: slightly stimulated at the lowest dose, maximal at 300pM 1,25(OH)(2)D(3), and equivalent to control levels at the highest dose, paralleling hormone-mediated phosphate uptake. Westerns with anti PKCbeta also revealed hormone-mediated differences, while those with anti PKCgamma did not. RNAi studies were then performed with siRNA against PKCalpha or PKCbeta. Untransfected cells treated with hormone for 7min exhibited enhanced (32)P uptake relative to vehicle controls. Cells transfected with either active siRNA revealed decreased (32)P uptake in both controls (relative to untransfected controls), and hormone treated cells. However, control and transfected cells treated with hormone had equivalent levels of uptake. Western blot analyses confirmed decreased immunoreactivity in transfected cells. Chemical PKCalpha (safingol) and PKCbeta ([3-(1-(3-Imidazol-1-ylpropyl)-1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione] blockers also confirmed the results from siRNA and demonstrated decreased (32)P uptake in cells treated with 1,25(OH)(2)D(3) plus blockers in comparison with cells treated with 1,25(OH)(2)D(3) alone. Thus, PKCalpha and PKCbeta are both involved in steroid-stimulated phosphate uptake.
我们进行了研究，以确定配体与 1,25D(3)-MARRS 受体（ERp57/PDIA3）结合并随后刺激磷酸盐摄取时，哪种（哪些）蛋白激酶 C（PKC）同工型被激活。用 1,25(OH)(2)D(3) 处理维生素 D 充足的雏鸡的分离肠上皮细胞 1、3 或 5min，彻底冷却，匀浆，并制备 P(2)级分（核后 20,000xg 沉淀）。用抗泛 PKC 的 Western 分析显示激素后 1min 类固醇刺激重新分布到 P(2)膜。使用这个时间点，用载体、130-、300-或 650-pM 激素处理细胞。用抗 PKCalpha 的 Western blot 显示在双相剂量反应曲线上向膜的重新分布：在最低剂量下略有刺激，在 300pM 1,25(OH)(2)D(3) 时最大，与最高剂量时的对照水平相当，与激素介导的磷酸盐摄取平行。用抗 PKCbeta 的 Western blot 也显示了激素介导的差异，而用抗 PKCgamma 的 Western blot 则没有。然后用 siRNA 对 PKCalpha 或 PKCbeta 进行 RNAi 研究。用激素处理 7min 的未转染细胞显示出相对于载体对照的增强的 (32)P 摄取。用活性 siRNA 转染的细胞显示在对照（相对于未转染对照）和激素处理的细胞中，(32)P 摄取减少。然而，用激素处理的对照和转染细胞具有等效的摄取水平。Western blot 分析证实转染细胞中的免疫反应性降低。化学 PKCalpha（鲨肝醇）和 PKCbeta ([3-(1-(3-咪唑基-1-丙基)-1H-吲哚-3-基)-4-苯胺基-1H-吡咯并[2,5-d] 酮] 阻滞剂也证实了 siRNA 的结果，并表明用 1,25(OH)(2)D(3) 加阻滞剂处理的细胞中的 (32)P 摄取减少与单独用 1,25(OH)(2)D(3) 处理的细胞相比。因此，PKCalpha 和 PKCbeta 都参与了类固醇刺激的磷酸盐摄取。
