Koyama H, Inaba M, Nishizawa Y, Ohno S, Morii H
Second Department of Internal Medicine, Osaka City University Medical School, Japan.
J Cell Biochem. 1994 Jun;55(2):230-40. doi: 10.1002/jcb.240550210.
Effects of protein kinase C (PKC) inhibitor and activator on 1,25(OH)2D3-induced gene expression were examined in rat intestinal epithelial cells, IEC-6 cells. A potent PKC inhibitor, H-7 (20 microM), completely abated 1,25(OH)2D3-induced 24-hydroxylase gene expression at 3 and 6 h. The effect of H-7 was dose dependent with IC50 around 5 microM. Other protein kinase inhibitors, HA-1004 and H-89 (20 microM), had no effects. Furthermore, the activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) potentiated the effect of 1,25(OH)2D3 by 1 h. TPA appeared to exert its effect at a transcriptional step, since mRNA stability was not affected by TPA treatment. At 3 h after the treatment of the cells with H-7 and TPA, vitamin D receptor (VDR) contents estimated by 3H-1,25(OH)2D3 binding capacity were 72.4 and 63.2% of vehicle-treated cells without significant changes of binding affinities, suggesting that the effect of H-7 and TPA was not the result of changes in VDR content or its binding affinity. In conclusion, PKC is involved in 1,25(OH)2D3-induced 24-hydroxylase gene expression in IEC-6 cells between 1,25(OH)2D3-VDR binding and VDR-induced gene transactivation.
在大鼠肠上皮细胞IEC-6细胞中检测了蛋白激酶C（PKC）抑制剂和激活剂对1,25(OH)₂D₃诱导的基因表达的影响。一种有效的PKC抑制剂H-7（20微摩尔）在3小时和6小时时完全消除了1,25(OH)₂D₃诱导的24-羟化酶基因表达。H-7的作用呈剂量依赖性，IC50约为5微摩尔。其他蛋白激酶抑制剂HA-1004和H-89（20微摩尔）则没有作用。此外，12-O-十四烷酰佛波醇-13-乙酸酯（TPA）激活PKC在1小时时增强了1,25(OH)₂D₃的作用。TPA似乎在转录步骤发挥作用，因为mRNA稳定性不受TPA处理的影响。在用H-7和TPA处理细胞3小时后，通过³H-1,25(OH)₂D₃结合能力估计的维生素D受体（VDR）含量分别为未处理细胞的72.4%和63.2%，结合亲和力没有显著变化，这表明H-7和TPA的作用不是VDR含量或其结合亲和力变化的结果。总之，PKC在IEC-6细胞中1,25(OH)₂D₃诱导的24-羟化酶基因表达中，在1,25(OH)₂D₃-VDR结合和VDR诱导的基因反式激活之间发挥作用。
