Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.
Drug Metab Dispos. 2012 Oct;40(10):2031-40. doi: 10.1124/dmd.112.046748. Epub 2012 Jul 24.
Human UDP-glucuronosyltransferase (UGT) 1A1 is a critical enzyme responsible for detoxification and metabolism of endogenous and exogenous lipophilic compounds such as bilirubin. The present study shows how cyclin-dependent kinase (CDK) inhibitor roscovitine stimulated the expression of UGT1A1 in HepG2 cells. Pregnane X receptor (PXR)-mediated transactivation of UGT1A1 reporter gene was more prominently enhanced by roscovitine, compared with the basal-, constitutive androstane receptor (CAR)-, and aryl hydrocarbon receptor-mediated activities. We determined the regulatory mechanism of UGT1A1 expression through PXR's stimulation by roscovitine. Although phosphomimetic mutations at Thr290 and Thr408 retained the PXR protein in cytoplasm and attenuated the induction of UGT1A1 expression by both roscovitine and rifampicin, a mutation at Ser350 specifically reduced the activity of PXR induced by roscovitine. Immunoprecipitation analysis revealed that the T290D but not T408D mutant protein remained in cytoplasm by forming a complex with heat shock protein 90 and cytoplasmic CAR retention protein, whereas treatment with proteasome inhibitor MG-132 accumulated the T408D mutant protein in cytoplasm. Transfection with anti-CDK2 small interfering RNA (siRNA) but not anti-CDK1 or CDK5 siRNA led to enhanced expression of UGT1A1. S350D yellow fluorescent protein-PXR fusion protein could translocate from cytoplasm to nucleus similar to the wild-type protein but was detected as an acetylated protein, whose binding with retinoid X receptor (RXR) and histone deacetylase was impaired. Cotransfection with coactivator steroid receptor coactivator (SRC) 2 but not SRC-1 partly recovered its PXR activity. These results indicate that roscovitine stimulated the expression of UGT1A1 by inhibiting CDK2, which phosphorylated PXR at Ser350 to suppress binding with RXR and coactivator and maintain the acetylation of PXR protein.
人 UDP-葡糖醛酸基转移酶（UGT）1A1 是一种关键酶，负责解毒和代谢内源性和外源性亲脂化合物，如胆红素。本研究表明，细胞周期蛋白依赖性激酶（CDK）抑制剂罗沙替丁如何刺激 HepG2 细胞中 UGT1A1 的表达。与基础、组成型激活受体（CAR）和芳烃受体介导的活性相比，罗沙替丁更显著增强了 pregnane X 受体（PXR）介导的 UGT1A1 报告基因的反式激活。我们通过罗沙替丁刺激 PXR 确定了 UGT1A1 表达的调节机制。尽管 Thr290 和 Thr408 的磷酸模拟突变将 PXR 蛋白保留在细胞质中，并减弱了罗沙替丁和利福平诱导的 UGT1A1 表达，但 Ser350 的突变特异性降低了罗沙替丁诱导的 PXR 活性。免疫沉淀分析表明，T290D 但不是 T408D 突变蛋白通过与热休克蛋白 90 和细胞质 CAR 保留蛋白形成复合物而留在细胞质中，而用蛋白酶体抑制剂 MG-132 处理则使 T408D 突变蛋白在细胞质中积累。转染抗 CDK2 小干扰 RNA（siRNA）而不是抗 CDK1 或 CDK5 siRNA 导致 UGT1A1 的表达增强。S350D 黄色荧光蛋白-PXR 融合蛋白可以像野生型蛋白一样从细胞质转位到细胞核，但被检测为乙酰化蛋白，其与视黄酸 X 受体（RXR）和组蛋白去乙酰化酶的结合受到损害。与共激活子甾体受体共激活子（SRC）2 共转染而不是 SRC-1 部分恢复了其 PXR 活性。这些结果表明，罗沙替丁通过抑制 CDK2 刺激 UGT1A1 的表达，CDK2 使 PXR 在 Ser350 处磷酸化，从而抑制与 RXR 和共激活子的结合，并维持 PXR 蛋白的乙酰化。
