Inhibition of PCSK9 Transcription by Berberine Involves Down-regulation of Hepatic HNF1 α Protein Expression through the Ubiquitin-Proteasome Degradation Pathway
Inhibition of PCSK9 Transcription by Berberine Involves Down-regulation of Hepatic HNF1 α Protein Expression through the Ubiquitin-Proteasome Degradation Pathway
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DOI：
10.1074/jbc.m114.597229
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发表时间：
2015-02-13
影响因子：
4.8
通讯作者：
Liu, Jingwen
中科院分区：
文献类型：
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作者：
Dong, Bin;Li, Hai;Liu, Jingwen
我们先前的体外研究已经证实，肝细胞核因子1α(HNF1α)是PCSK9基因表达的专有反式激活因子，并证明其参与了天然降胆固醇化合物黄连素(BBR)抑制PCSK9基因表达的功能。在这项研究中，我们研究了BBR抑制HNF1α介导的PCSK9转录的机制。给高脂血症小鼠和仓鼠服用BBR可降低循环PCSK9浓度和肝脏PCSK9 mRNA水平，但不影响HNF1α的基因表达。然而，与对照组相比，BBR组大鼠肝脏HNF1α蛋白水平显著降低。以HepG2细胞为模型系统，我们得到了BBR促进HNF1α蛋白降解的证据。通过应用抑制剂选择性地阻断泛素蛋白酶体系统(UPS)和自噬-溶酶体途径，我们发现Bafilmycin A1处理不影响HepG2细胞中HNF1α蛋白的含量，但UPS抑制剂Bortezomib和MG132可剂量依赖性地增加HNF1α蛋白的含量。Bortezomib治疗提高了HNF1α和PCSK9细胞水平，同时降低了低密度脂蛋白受体蛋白。此外，在BBR或过表达泛素的细胞中，HNF1α蛋白显示出多泛素化梯形图案。通过在细胞中表达GFP-HNF1α融合蛋白，我们观察到阻断UPS导致GFP-HNF1α在细胞质中积聚。重要的是，我们发现BBR对HNF1α蛋白和PCSK9基因转录的抑制作用可以被蛋白酶体抑制剂消除。总之，我们以BBR为探针的研究揭示了泛素诱导HNF1α蛋白酶体降解对PCSK9调控的新方面。
Our previous in vitro studies have identified hepatocyte nuclear factor 1 alpha (HNF1 alpha) as an obligated trans-activator for PCSK9 gene expression and demonstrated its functional involvement in the suppression of PCSK9 expression by berberine (BBR), a natural cholesterol-lowering compound. In this study, we investigated the mechanism underlying the inhibitory effect of BBR on HNF1 alpha-mediated PCSK9 transcription. Administration of BBR to hyperlipidemic mice and hamsters lowered circulating PCSK9 concentrations and hepatic PCSK9 mRNA levels without affecting the gene expression of HNF1 alpha. However, hepatic HNF1 alpha protein levels were markedly reduced in BBR-treated animals as compared with the control. Using HepG2 cells as a model system, we obtained evidence that BBR treatment let to accelerated degradation of HNF1 alpha protein. By applying inhibitors to selectively block the ubiquitin proteasome system (UPS) and autophagy-lysosomal pathway, we show that HNF1 alpha protein content in HepG2 cells was not affected by bafilomycin A1 treatment, but it was dose-dependently increased by UPS inhibitors bortezomib and MG132. Bortezomib treatment elevated HNF1 alpha and PCSK9 cellular levels with concomitant reductions of LDL receptor protein. More-over, HNF1 alpha protein displayed a multiubiquitination ladder pattern in cells treated withBBRor overexpressing ubiquitin. By expressing GFP-HNF1 alpha fusion protein in cells, we observed that blocking UPS resulted in accumulation of GFP-HNF1 alpha in cytoplasm. Importantly, we show that the BBR reducing effects on HNF1 alpha protein and PCSK9 gene transcription can be eradicated by proteasome inhibitors. Altogether, our studies using BBR as a probe uncovered a new aspect of PCSK9 regulation by ubiquitin- induced proteasomal degradation of HNF1 alpha.