Hara Hirokazu, Yasunami Yoko, Adachi Tetsuo
Laboratory of Clinical Pharmaceutics, Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, 502-8585, Gifu, Japan.
Biochem Biophys Res Commun. 2002 Aug 9;296(1):182-8. doi: 10.1016/s0006-291x(02)00860-4.
Expression of cytochrome P450 3A4 (CYP3A4) is induced by 1,25-dihydroxyvitamin D(3)(1,25(OH)(2)D(3)) in Caco-2 cells. However, since a typical vitamin D responsive element has not been found in the 5(')-flanking region of the CYP3A4 gene, the mechanism of 1,25(OH)(2)D(3)-induced CYP3A4 mRNA expression is poorly understood. In the present study, we demonstrated that vitamin D receptor (VDR) is a critical factor for the induction using the antisense oligonucleotide technique. In addition, we found that treatment of Caco-2 cells with the protein kinase C (PKC) inhibitors, staurosporine and GF109203X, and the tyrosine kinase inhibitor, genistein, but not with the protein kinase A inhibitor, H-89, suppressed CYP3A4 mRNA induction by 1,25(OH)(2)D(3). The depletion of PKC by prolonged treatment with phorbol ester abolished the induction. On the other hand, protein kinase inhibitors used had no effects on the constitutive expression of VDR mRNA. Therefore, these observations suggest that 1,25(OH)(2)D(3)-induced CYP3A4 mRNA expression might be involved in phosphorylation events in addition to transcriptional regulation via VDR. However, 1,25(OH)(2)D(3) did not rapidly activate PKC in the Caco-2 cells used, while the treatment with staurosporine and GF109203X, but not genistein, decreased basal PKC activity by approximately 30% of the controls. Taken together, these findings suggest that the change in the phosphorylation state via PKC and tyrosine kinase might, at least in part, modulate 1,25(OH)(2)D(3)-induced CYP3A4 mRNA expression via VDR.
1,25 - 二羟基维生素D3（1,25(OH)2D3）可诱导Caco - 2细胞中细胞色素P450 3A4（CYP3A4）的表达。然而，由于在CYP3A4基因的5′侧翼区域未发现典型的维生素D反应元件，1,25(OH)2D3诱导CYP3A4 mRNA表达的机制尚不清楚。在本研究中，我们使用反义寡核苷酸技术证明维生素D受体（VDR）是诱导过程中的关键因素。此外，我们发现用蛋白激酶C（PKC）抑制剂星形孢菌素和GF109203X以及酪氨酸激酶抑制剂染料木黄酮处理Caco - 2细胞，而非用蛋白激酶A抑制剂H - 89处理，可抑制1,25(OH)2D3诱导的CYP3A4 mRNA表达。用佛波酯长时间处理使PKC耗竭可消除这种诱导作用。另一方面，所用的蛋白激酶抑制剂对VDR mRNA的组成性表达没有影响。因此，这些观察结果表明，1,25(OH)2D3诱导的CYP3A4 mRNA表达除了通过VDR进行转录调控外，可能还涉及磷酸化事件。然而，1,25(OH)2D3在所用的Caco - 2细胞中并未快速激活PKC，而用星形孢菌素和GF109203X处理（而非染料木黄酮）可使基础PKC活性降低至对照的约30%。综上所述，这些发现表明，通过PKC和酪氨酸激酶引起的磷酸化状态变化可能至少部分地通过VDR调节1,25(OH)2D3诱导的CYP3A4 mRNA表达。
