Reinhardt T A, Horst R L
U.S. Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Ames, IA 50010-0070, USA.
Mol Cell Endocrinol. 1994 May;101(1-2):159-65. doi: 10.1016/0303-7207(94)90230-5.
In this study, we examined the effect of activation of protein kinase C (PKC) pathways on the regulation of 1,25-dihydroxyvitamin D receptors (VDR) in rat osteosarcoma (ROS) 17/2.8 cells. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) resulted in a time- and dose-dependent increase in VDR expression in ROS cells. Treatment of ROS cells with 4alpha-phorbol 12,13-dedeconate, a PKC-inactive phorbol ester, had no effect on VDR expression. Oleoyl acetyl glycerol (OAG), a synthetic diacylglycerol, stimulated VDR up-regulation in ROS cells. The PKC inhibitors (H-7, staurosporin, and sphingosine) all blocked PMA-mediated up-regulation of VDR in a dose-dependent manner. We next examined the interaction of 1,25(OH)2D3 and PKC activation by PMA on the regulation of VDR in ROS cells. We found that PMA or 1,25(OH)2D3 treatment alone resulted in a 50 and 200% increase in VDR, respectively. PMA treatment alone resulted in a 50% increase in VDR protein and a marginal 20% increase in VDR mRNA. 1,25(OH)2D3 up-regulation of VDR was associated with a 2-fold increase in VDR mRNA. In contrast, co-treatment of ROS cells with PMA and 1,25(OH)2D3 resulted in a synergistic 10-fold induction of VDR mRNA and the appearance of a 7.2 kb VDR transcript. VDR protein was also synergistically up-regulated by combined PMA and 1,25(OH)2D3 treatment of ROS cells. Scatchard analysis demonstrated that the synergistic effect of PMA and 1,25(OH)2D3 on VDR protein expression was not associated with any change in the affinity of VDR for 1,25(OH)2D3. The synergistic effect of 1,25(OH)2D3 and PMA on VDR expression supports a link between PKC signal pathways and the function of VDR.
在本研究中，我们检测了蛋白激酶C（PKC）信号通路的激活对大鼠骨肉瘤（ROS）17/2.8细胞中1,25 - 二羟基维生素D受体（VDR）调控的影响。用佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）激活PKC，导致ROS细胞中VDR表达呈时间和剂量依赖性增加。用PKC失活的佛波醇酯4α - 佛波醇12,13 - 二癸酸酯处理ROS细胞，对VDR表达无影响。合成二酰甘油油酰乙酰甘油（OAG）可刺激ROS细胞中VDR上调。PKC抑制剂（H - 7、星形孢菌素和鞘氨醇）均以剂量依赖性方式阻断PMA介导的VDR上调。接下来，我们检测了1,25(OH)₂D₃与PMA激活PKC对ROS细胞中VDR调控的相互作用。我们发现，单独用PMA或1,25(OH)₂D₃处理分别使VDR增加50%和200%。单独用PMA处理使VDR蛋白增加50%，VDR mRNA略有增加20%。1,25(OH)₂D₃上调VDR与VDR mRNA增加2倍相关。相反，用PMA和1,25(OH)₂D₃共同处理ROS细胞导致VDR mRNA协同诱导增加10倍，并出现7.2 kb的VDR转录本。VDR蛋白也通过PMA和1,25(OH)₂D₃联合处理ROS细胞而协同上调。Scatchard分析表明，PMA和1,25(OH)₂D₃对VDR蛋白表达的协同作用与VDR对1,25(OH)₂D₃的亲和力变化无关。1,25(OH)₂D₃和PMA对VDR表达的协同作用支持了PKC信号通路与VDR功能之间的联系。
