锌指蛋白Zbtb20肝细胞特异性基因敲除小鼠对CCL_4肝损伤的敏感性及其机理研究
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摘要
锌指蛋白Zbtb20是一种含有POZ/BTBC2H2锌指结构的转录因子,可能在多种器官和组织的发育和功能调节中具有非常广泛和重要的作用。Zbtb20在出生后的肝脏中开始上调表达,到出生后8周达到成年水平。为了深入研究Zbtb20的体内生物学作用,我们利用Cre/LoxP条件基因打靶技术,成功建立了Zbtb20的肝细胞特异性基因敲除小鼠模型。通过对该小鼠模型的体内以及体外实验研究表明,Zbtb20是调控肝脏甲胎蛋白基因转录的重要转录抑制因子。然而目前还不清楚Zbtb20对肝细胞的体内物质代谢和解毒功能有何影响。
为了揭示Zbt20基因缺失后的肝细胞的物质代谢和解毒能力,我们建立了四氯化碳(CCl4)所致的急性肝损伤和慢性肝纤维化模型,系统比较了该基因敲除小鼠与正常对照小鼠的急性肝损伤程度、损伤后组织修复能力和慢性肝纤维化程度,并在此基础上对肝脏参与CCl4代谢的关键酶一细胞色素P450 2E1 (Cyp2El)的蛋白、mRNA、hnRNA表达水平进行分析,最后对影响Cyp2E1基因表达的因素进行初步分析,取得以下结果：
一、Zbtb20肝细胞特异性基因敲除小鼠对CCl4急性肝损伤具有抵抗作用。
具体表现在以下三方面：(1)组织学分析表明：单次注射CCl4后,Zbtb2O基因敲除小鼠和正常对照小鼠肝损伤高峰期均出现在注射后24 h,然而无论是在损伤高峰期还是此后的48 h和72 h,Zbtb20基因敲除肝组织的坏死面积均明显低于正常对照组,而且能很快得到修复。(2)TUNEL分析表明CCl4注射后72 h,Zbtb20基因敲除肝组织中的凋亡细胞数显著低于正常对照组。(3)CCl4注射后24 h、48 h和72 h, Zbtb2O肝细胞特异性基因敲除小鼠的血清谷丙转氨酶(ALT)和谷草转氨酶(AST)水平均显著低于正常对照小鼠。而注射橄榄油(溶剂组)后,Zbtb20肝细胞特异性基因敲除小鼠的肝组织结构、凋亡细胞数及血清ALT和AST水平与正常对照小鼠相比均无显著差异。以上结果表明Zbtb20肝细胞特异性基因敲除小鼠对CCl4的急性肝毒作用具有低敏感性。
二、CCl4急性肝损伤后Zbtb20缺失肝细胞未出现增殖活性异常增高现象。
为了检测肝细胞的增殖能力,我们在给小鼠注射CCl4后不同时间点进行了BrdU掺入实验,结果显示：CCl4注射后24h, Zbtb20基因敲除和正常对照肝组织中出现少量散在分布的BrdU日性细胞,在数量上两者没有显著性差异,提示在损伤的同时修复机制已开始启动。CCl4注射后48 h,Zbtb20敲除和正常对照肝组织中肝细胞增殖出现高峰,并且前者的增殖细胞比后者少,可能与其损伤程度轻有关。为了进一步证实BrdU的分析结果,我们又对肝组织进行了细胞增殖标记蛋白Ki67和CNA的免疫组化标记,结果与BrdU标记结果一致。溶剂组的Zbtb20基因敲除肝组织仅有很少BrdU阳性细胞,与正常对照小鼠相比无显著性差异,表明大部分肝细胞处于静息期。以上检测结果排除了Zbtb20基因敲除肝细胞在注射CCl4后发生异常高增殖的可能性,提示该小鼠对CCl4的低敏感性可能是由于其他机制造成的。
三、Zbtb20肝细胞特异性基因敲除小鼠对CCl4慢性肝纤维化作用具有低敏感性。
小鼠连续6周注射CCl4(每周2次)后,取肝组织进行HE和胶原纤维染色分析。染色结果显示正常对照肝组织在肝中央静脉周围区可见到肝细胞坏死、大量炎性细胞浸润和结缔组织增生,部分肝小叶结构紊乱,Sirius Red染色显示广泛的桥接纤维间隔形成“蜂巢”样结构。与对照小鼠相比,Zbtb20譬因敲除肝组织炎症反应较轻,肝小叶结构基本完整,桥接纤维间隔较少,相对胶原纤维面积较小。说明Zbt20基因敲除肝脏对CCl4慢性肝纤维化作用同样具有抵抗性。
四、Zbtb20基因敲除肝组织中Cyp2E1 mRNA和蛋白表达水平下调,而Cyp2E1 hnRNA水平没有改变。
CCl4的急慢性损伤实验结果表明Zbtb20基因敲除肝组织对CCl4的敏感性远远低于正常对照组,为了进一步揭示其分子机理,我们对影响CC]4体内活化的关键酶Cyp2E1的蛋白表达水平进行检测。Western Blot结果显示,Zbtb20基因敲除肝组织中Cyp2E1蛋白的相对表达量与正常对照相比下调了60%。接着我们又对Cyp2E1蛋白的表达进行免疫组化分析。结果显示在Zbtb20基因敲除肝组织中该蛋白的表达仍遵循肝小叶中心区强而门管区弱的区域性特征,但阳性标记区域比对照组明显缩小。荧光定量PCR检测结果表明Zbtb20基因敲除肝组织中Cyp2E1 mRNA水平下调了50%,而其hnRNA的水平与对照组相比没有显著性差异。提示该基因的表达在转录后水平受抑。
五、Zbtb20基因敲除肝组织中HNF1αmRNA表达水平和β-catenin的活化水平没有改变。
为了进一步揭示Zbtb20基因敲除肝组织中Cyp2E1 mRNA水平下调的分子机制,我们检测了目前已知的在该基因转录激活中起主要作用的转录因子HNF1α的mRNA水平,结果显示,其mRNA表达水平与正常对照没有显著差异。β-catenin被报道可以影响Cyp2E1 mRNA的转录,因此我们又检测了该蛋白的活化形式(activeβ-catenin)在Zbtb20基因敲除肝组织中的表达水平,发现与正常对照也没有显著差异。鉴于Zbtb20基因敲除肝组织中Cyp2E1 hnRNA表达水平没有改变,影响该分子转录的HNF1αmRNA和activeβ-catenin水平均与对照小组没有显著差异,因此我们推测造成Cyp2E1 mRNA下调的机制可能发生在转录后水平。
六、Zbtb20肝细胞特异性基因敲除小鼠血清胰岛素水平没有改变,甲状腺素反应基因Spot 14 mRNA表达水平没有明显变化。
为了揭示Zbt20基因敲除肝组织中Cyp2E1 mRNA下调的转录后机制,我们检测了与其mRNA稳定性相关的血浆胰岛素和T3水平,并进一步检测了肝组织中的甲状腺素反应基因Spot14(S14)的表达水平。检测结果显示,Zbtb20肝细胞特异性基因敲除小鼠血浆胰岛素水平与正常对照小鼠没有统计学差异；血浆甲状腺素T3水平比对照组略高但肝组织中的甲状腺素反应基因Spot 14的表达水平与对照小鼠没有统计学差异。
总结以上实验结果,我们得出以下结论：Zbtb20肝细胞特异性基因敲除后引起Cyp2E1 mRNA和蛋白水平下调,从而导致该小鼠对CCl4诱导的急性肝毒作用和慢性肝纤维化作用产生抵抗性。导致该小鼠肝细胞中Cyp2El mRNA水平下调的机制可能发生在转录后水平。以上结果表明Zbtb20除了调控甲胎蛋白基因转录外,还可能参与调控肝细胞的物质代谢和解毒功能。
Zbtb20 is a member of zinc finger protein family containing BTB/POZ and C2H2 domains. It has been suggested that Zbtb20 plays a variety of important roles in multiple systems. Zbtb20 mRNA was developmentally activated in postpartum liver reaching a plateau at the age of 8 weeks. To determine its physiological functions in vivo, we have previously generated hepatocyte-specific Zbtb20 knockout mice (LZB20KO) by the Cre/loxP approach and demonstrated that Zbtb20 functions as a transcriptional repressor regulating the developmental expression of the alpha-fetoprotein (AFP) gene in the liver. However, little is known about whether Zbtb20 plays any roles in additional aspects of liver functions such as drug metabolism and detoxification.
To define whether Zbtb20 plays a role in liver-specific metabolic functions, we examined the impact of both acute and chronic exposure to carbon tetrachloride (CCl4) in LZB20KO mice. We then examined the hnRNA, mRNA and protein expression level of cytochrome P4502E1 (Cyp2El), the major enzyme whose products underlie CCl4-induced injury. We also analyzed the mRNA expression level of hepatocyte nuclear factor la (Hnfl a) and the activation ofβ-catenin, which has been previously reported to regulate Cyp2e1 transcription. At last, we detected the serum insulin and L-3,3,5-triiodothyronine (T3) level, which has been shown to affect cyp2el mRNA stability. Our main findings are described as follows:
1. LZB20KO mice were resistant to CCl4-induced acute hepatic damage.
(1) Histological analysis data showed that the peak of injury was reached 24 h after a single dose of CCl4 injection in both LZB20KO and control mice. However, the LZB20KO mice exhibited attenuated liver injury by showing a significant decrease in liver necrosis area and accelerated hepatolobular repair. (2) LZB20KO livers showed a significant decrease in TUNEL+apoptotic cells 72h after CCl4 administration when compared with control mice. (3) Serum ALT and AST levels in LZB20KO mice were also significant lower than that in control mice 24,48 and 72h after CCI4 challenge, which paralleled the histopathological findings. There were no significant differences in histology, apoptotic cells and serum ALT and AST levels between LZB20KO and control mice after olive oil (serve as vehicle) injection. These data indicate that LZB20KO mice were less sensitive to CCl4-induced acute hepatic damage.
2. Accelerated hepatolobular repair after CC14 administration in LZB20KO mice was not due to an increased proliferation.
Hepatocyte proliferation was determined by BrdU incorporation assay. A relatively small number of BrdU+cells were scattered in the liver in both LZB20KO and control mice 24h after CCl4 challenge, with no significant differences between the two groups. The peak hepatocyte proliferation occurred at 48 h in both LZB20KO and control mice, but the number of BrdU+cells in LZB20KO mice was great lower than that in control mice, probably due to alleviated hepatic damage. This result was confirmed by immunohistochemical staining of Ki67 and PCNA, two proliferation markers. There were only a few BrdU+cells in both LZB20KO and control liver after olive oil injection, with no significant differences between the two, suggesting most cells are normally in the resting phase. Thus, the accelerated tissue repair after CCl4 exposure was not likely due to an increased proliferation in LZB20KO mice.
3. LZB20KO mice exhibited an alleviated CCl4-induced chronic hepatic fibrosis.
Fibrosis was induced by injection of CCl4 twice each week for 6 weeks. Livers were then subjected to picro-sirius staining for showing collagen I deposition. The staining showed that collagen accumulation was greatly subdued in LZB20KO mice as compared with that in control mice. Thus, LZB20KO mice were also resistant to CCU-induced chronic hepatic fibrogenesis.
4. The expression of Cyp2El mRNA and protein was down-regulated in LZB20KO liver.
In order to reveal the mechanism underlying LZB20KO mice'resistance to CCl4 hepatotoxicity, we analyzed the expression of Cyp2E1, a key enzyme responsible for the activation of CCl4 in livers. The relative expression level of Cyp2El protein in LZB20KO liver was decreased by 60%determined by Western Blot, as compared to those in controls. Immunohistochemistry showed that CYP2E1 were still expressed in centrilobular pattern, but the positive area was signifivantly decreased in LZB20KO liver compared to control livers. The relative expression level of Cyp2El mRNA in LZB20KO liver was also decreased by 50%, while the expression of Cyp2E1 hnRNA was not changed, as determined by Real-time quantitative PCR. This data indicate that the down-regulation of Cyp2E1 mRNA in LZB20KO livers occurs most likely at post transcriptional levels.
5. Normal expression of Hnfla and activation ofβ-catenin in LZB20KO liver.
To reveal the mechanism underlying the down-regulation of Cyp2El mRNA expression in LZB20KO liver, we examined the mRNA expression of Hnfla.which is a key activator for Cyp2El transcription. There was no significant difference in Hnfla mRNA levels between LZB20KO and control livers. We next analyzed the activation ofβ-catenin, which has been previously reported to regulate Cyp2el transcription. No obvious difference was observed between LZB20KO and control livers. These data suggest that the down-regulation of Cyp2E1 mRNA expression in LZB20KO liver occurs most likely at post transcriptional levels.
6. Normal serum insulin level and expression of spot14 mRNA in LZB20KO liver.
To investigate possible post-transcriptional mechanism underlying the down-regulation of Cyp2E1 mRNA expression in LZB20KO liver, we detected the serum insulin and T3 level, which has been shown to affect cyp2el mRNA stability. There was no significant difference in serum insulin level between LZB20KO and control livers. The serum T3 level was slightly higher but the expression of spot 14 mRNA, a T3 responsive gene, was not changed in LZB20KO when compared to control mice.
In conclusion, we showed that the liver-specific ablation of Zbtb20 resulted in the down-regulation of Cyp2El mRNA and protein expression. Consequently, these mice were resistant to CCl4-induced acute hepatic damage and chronic fibrosis. The down-regulation of Cyp2E1 mRNA in LZB20KO livers occurs most likely at post transcriptional levels.These findings strongly suggest that in addition to regulating AFP transcription, Zbtb20 may also control drug metabolism and detoxification in the liver.
为了揭示Zbt20基因缺失后的肝细胞的物质代谢和解毒能力,我们建立了四氯化碳(CCl4)所致的急性肝损伤和慢性肝纤维化模型,系统比较了该基因敲除小鼠与正常对照小鼠的急性肝损伤程度、损伤后组织修复能力和慢性肝纤维化程度,并在此基础上对肝脏参与CCl4代谢的关键酶一细胞色素P450 2E1 (Cyp2El)的蛋白、mRNA、hnRNA表达水平进行分析,最后对影响Cyp2E1基因表达的因素进行初步分析,取得以下结果：
一、Zbtb20肝细胞特异性基因敲除小鼠对CCl4急性肝损伤具有抵抗作用。
具体表现在以下三方面：(1)组织学分析表明：单次注射CCl4后,Zbtb2O基因敲除小鼠和正常对照小鼠肝损伤高峰期均出现在注射后24 h,然而无论是在损伤高峰期还是此后的48 h和72 h,Zbtb20基因敲除肝组织的坏死面积均明显低于正常对照组,而且能很快得到修复。(2)TUNEL分析表明CCl4注射后72 h,Zbtb20基因敲除肝组织中的凋亡细胞数显著低于正常对照组。(3)CCl4注射后24 h、48 h和72 h, Zbtb2O肝细胞特异性基因敲除小鼠的血清谷丙转氨酶(ALT)和谷草转氨酶(AST)水平均显著低于正常对照小鼠。而注射橄榄油(溶剂组)后,Zbtb20肝细胞特异性基因敲除小鼠的肝组织结构、凋亡细胞数及血清ALT和AST水平与正常对照小鼠相比均无显著差异。以上结果表明Zbtb20肝细胞特异性基因敲除小鼠对CCl4的急性肝毒作用具有低敏感性。
二、CCl4急性肝损伤后Zbtb20缺失肝细胞未出现增殖活性异常增高现象。
为了检测肝细胞的增殖能力,我们在给小鼠注射CCl4后不同时间点进行了BrdU掺入实验,结果显示：CCl4注射后24h, Zbtb20基因敲除和正常对照肝组织中出现少量散在分布的BrdU日性细胞,在数量上两者没有显著性差异,提示在损伤的同时修复机制已开始启动。CCl4注射后48 h,Zbtb20敲除和正常对照肝组织中肝细胞增殖出现高峰,并且前者的增殖细胞比后者少,可能与其损伤程度轻有关。为了进一步证实BrdU的分析结果,我们又对肝组织进行了细胞增殖标记蛋白Ki67和CNA的免疫组化标记,结果与BrdU标记结果一致。溶剂组的Zbtb20基因敲除肝组织仅有很少BrdU阳性细胞,与正常对照小鼠相比无显著性差异,表明大部分肝细胞处于静息期。以上检测结果排除了Zbtb20基因敲除肝细胞在注射CCl4后发生异常高增殖的可能性,提示该小鼠对CCl4的低敏感性可能是由于其他机制造成的。
三、Zbtb20肝细胞特异性基因敲除小鼠对CCl4慢性肝纤维化作用具有低敏感性。
小鼠连续6周注射CCl4(每周2次)后,取肝组织进行HE和胶原纤维染色分析。染色结果显示正常对照肝组织在肝中央静脉周围区可见到肝细胞坏死、大量炎性细胞浸润和结缔组织增生,部分肝小叶结构紊乱,Sirius Red染色显示广泛的桥接纤维间隔形成“蜂巢”样结构。与对照小鼠相比,Zbtb20譬因敲除肝组织炎症反应较轻,肝小叶结构基本完整,桥接纤维间隔较少,相对胶原纤维面积较小。说明Zbt20基因敲除肝脏对CCl4慢性肝纤维化作用同样具有抵抗性。
四、Zbtb20基因敲除肝组织中Cyp2E1 mRNA和蛋白表达水平下调,而Cyp2E1 hnRNA水平没有改变。
CCl4的急慢性损伤实验结果表明Zbtb20基因敲除肝组织对CCl4的敏感性远远低于正常对照组,为了进一步揭示其分子机理,我们对影响CC]4体内活化的关键酶Cyp2E1的蛋白表达水平进行检测。Western Blot结果显示,Zbtb20基因敲除肝组织中Cyp2E1蛋白的相对表达量与正常对照相比下调了60%。接着我们又对Cyp2E1蛋白的表达进行免疫组化分析。结果显示在Zbtb20基因敲除肝组织中该蛋白的表达仍遵循肝小叶中心区强而门管区弱的区域性特征,但阳性标记区域比对照组明显缩小。荧光定量PCR检测结果表明Zbtb20基因敲除肝组织中Cyp2E1 mRNA水平下调了50%,而其hnRNA的水平与对照组相比没有显著性差异。提示该基因的表达在转录后水平受抑。
五、Zbtb20基因敲除肝组织中HNF1αmRNA表达水平和β-catenin的活化水平没有改变。
为了进一步揭示Zbtb20基因敲除肝组织中Cyp2E1 mRNA水平下调的分子机制,我们检测了目前已知的在该基因转录激活中起主要作用的转录因子HNF1α的mRNA水平,结果显示,其mRNA表达水平与正常对照没有显著差异。β-catenin被报道可以影响Cyp2E1 mRNA的转录,因此我们又检测了该蛋白的活化形式(activeβ-catenin)在Zbtb20基因敲除肝组织中的表达水平,发现与正常对照也没有显著差异。鉴于Zbtb20基因敲除肝组织中Cyp2E1 hnRNA表达水平没有改变,影响该分子转录的HNF1αmRNA和activeβ-catenin水平均与对照小组没有显著差异,因此我们推测造成Cyp2E1 mRNA下调的机制可能发生在转录后水平。
六、Zbtb20肝细胞特异性基因敲除小鼠血清胰岛素水平没有改变,甲状腺素反应基因Spot 14 mRNA表达水平没有明显变化。
为了揭示Zbt20基因敲除肝组织中Cyp2E1 mRNA下调的转录后机制,我们检测了与其mRNA稳定性相关的血浆胰岛素和T3水平,并进一步检测了肝组织中的甲状腺素反应基因Spot14(S14)的表达水平。检测结果显示,Zbtb20肝细胞特异性基因敲除小鼠血浆胰岛素水平与正常对照小鼠没有统计学差异；血浆甲状腺素T3水平比对照组略高但肝组织中的甲状腺素反应基因Spot 14的表达水平与对照小鼠没有统计学差异。
总结以上实验结果,我们得出以下结论：Zbtb20肝细胞特异性基因敲除后引起Cyp2E1 mRNA和蛋白水平下调,从而导致该小鼠对CCl4诱导的急性肝毒作用和慢性肝纤维化作用产生抵抗性。导致该小鼠肝细胞中Cyp2El mRNA水平下调的机制可能发生在转录后水平。以上结果表明Zbtb20除了调控甲胎蛋白基因转录外,还可能参与调控肝细胞的物质代谢和解毒功能。
Zbtb20 is a member of zinc finger protein family containing BTB/POZ and C2H2 domains. It has been suggested that Zbtb20 plays a variety of important roles in multiple systems. Zbtb20 mRNA was developmentally activated in postpartum liver reaching a plateau at the age of 8 weeks. To determine its physiological functions in vivo, we have previously generated hepatocyte-specific Zbtb20 knockout mice (LZB20KO) by the Cre/loxP approach and demonstrated that Zbtb20 functions as a transcriptional repressor regulating the developmental expression of the alpha-fetoprotein (AFP) gene in the liver. However, little is known about whether Zbtb20 plays any roles in additional aspects of liver functions such as drug metabolism and detoxification.
To define whether Zbtb20 plays a role in liver-specific metabolic functions, we examined the impact of both acute and chronic exposure to carbon tetrachloride (CCl4) in LZB20KO mice. We then examined the hnRNA, mRNA and protein expression level of cytochrome P4502E1 (Cyp2El), the major enzyme whose products underlie CCl4-induced injury. We also analyzed the mRNA expression level of hepatocyte nuclear factor la (Hnfl a) and the activation ofβ-catenin, which has been previously reported to regulate Cyp2e1 transcription. At last, we detected the serum insulin and L-3,3,5-triiodothyronine (T3) level, which has been shown to affect cyp2el mRNA stability. Our main findings are described as follows:
1. LZB20KO mice were resistant to CCl4-induced acute hepatic damage.
(1) Histological analysis data showed that the peak of injury was reached 24 h after a single dose of CCl4 injection in both LZB20KO and control mice. However, the LZB20KO mice exhibited attenuated liver injury by showing a significant decrease in liver necrosis area and accelerated hepatolobular repair. (2) LZB20KO livers showed a significant decrease in TUNEL+apoptotic cells 72h after CCl4 administration when compared with control mice. (3) Serum ALT and AST levels in LZB20KO mice were also significant lower than that in control mice 24,48 and 72h after CCI4 challenge, which paralleled the histopathological findings. There were no significant differences in histology, apoptotic cells and serum ALT and AST levels between LZB20KO and control mice after olive oil (serve as vehicle) injection. These data indicate that LZB20KO mice were less sensitive to CCl4-induced acute hepatic damage.
2. Accelerated hepatolobular repair after CC14 administration in LZB20KO mice was not due to an increased proliferation.
Hepatocyte proliferation was determined by BrdU incorporation assay. A relatively small number of BrdU+cells were scattered in the liver in both LZB20KO and control mice 24h after CCl4 challenge, with no significant differences between the two groups. The peak hepatocyte proliferation occurred at 48 h in both LZB20KO and control mice, but the number of BrdU+cells in LZB20KO mice was great lower than that in control mice, probably due to alleviated hepatic damage. This result was confirmed by immunohistochemical staining of Ki67 and PCNA, two proliferation markers. There were only a few BrdU+cells in both LZB20KO and control liver after olive oil injection, with no significant differences between the two, suggesting most cells are normally in the resting phase. Thus, the accelerated tissue repair after CCl4 exposure was not likely due to an increased proliferation in LZB20KO mice.
3. LZB20KO mice exhibited an alleviated CCl4-induced chronic hepatic fibrosis.
Fibrosis was induced by injection of CCl4 twice each week for 6 weeks. Livers were then subjected to picro-sirius staining for showing collagen I deposition. The staining showed that collagen accumulation was greatly subdued in LZB20KO mice as compared with that in control mice. Thus, LZB20KO mice were also resistant to CCU-induced chronic hepatic fibrogenesis.
4. The expression of Cyp2El mRNA and protein was down-regulated in LZB20KO liver.
In order to reveal the mechanism underlying LZB20KO mice'resistance to CCl4 hepatotoxicity, we analyzed the expression of Cyp2E1, a key enzyme responsible for the activation of CCl4 in livers. The relative expression level of Cyp2El protein in LZB20KO liver was decreased by 60%determined by Western Blot, as compared to those in controls. Immunohistochemistry showed that CYP2E1 were still expressed in centrilobular pattern, but the positive area was signifivantly decreased in LZB20KO liver compared to control livers. The relative expression level of Cyp2El mRNA in LZB20KO liver was also decreased by 50%, while the expression of Cyp2E1 hnRNA was not changed, as determined by Real-time quantitative PCR. This data indicate that the down-regulation of Cyp2E1 mRNA in LZB20KO livers occurs most likely at post transcriptional levels.
5. Normal expression of Hnfla and activation ofβ-catenin in LZB20KO liver.
To reveal the mechanism underlying the down-regulation of Cyp2El mRNA expression in LZB20KO liver, we examined the mRNA expression of Hnfla.which is a key activator for Cyp2El transcription. There was no significant difference in Hnfla mRNA levels between LZB20KO and control livers. We next analyzed the activation ofβ-catenin, which has been previously reported to regulate Cyp2el transcription. No obvious difference was observed between LZB20KO and control livers. These data suggest that the down-regulation of Cyp2E1 mRNA expression in LZB20KO liver occurs most likely at post transcriptional levels.
6. Normal serum insulin level and expression of spot14 mRNA in LZB20KO liver.
To investigate possible post-transcriptional mechanism underlying the down-regulation of Cyp2E1 mRNA expression in LZB20KO liver, we detected the serum insulin and T3 level, which has been shown to affect cyp2el mRNA stability. There was no significant difference in serum insulin level between LZB20KO and control livers. The serum T3 level was slightly higher but the expression of spot 14 mRNA, a T3 responsive gene, was not changed in LZB20KO when compared to control mice.
In conclusion, we showed that the liver-specific ablation of Zbtb20 resulted in the down-regulation of Cyp2El mRNA and protein expression. Consequently, these mice were resistant to CCl4-induced acute hepatic damage and chronic fibrosis. The down-regulation of Cyp2E1 mRNA in LZB20KO livers occurs most likely at post transcriptional levels.These findings strongly suggest that in addition to regulating AFP transcription, Zbtb20 may also control drug metabolism and detoxification in the liver.
引文
1. Collins, T., J.R. Stone, and A.J. Williams, All in the family:the BTB/POZ, KRAB, and SCAN domains. Molecular& Cellular Biology,2001.21(11):p.3609-15.
2. Kelly, K.F. and J.M. Daniel, POZ for effect-POZ-ZF transcription factors in cancer and development. Trends in Cell Biology,2006.16(11):p.578-87.
3. Mitchelmore, C, et al., Characterization of two novel nuclear BTB/POZ domain zinc finger isoforms. Association with differentiation of hippocampal neurons, cerebellar granule cells, and macroglia. J Biol Chem,2002.277(9):p.7598-609.
4. Zhang, W., et al., Identification and characterization of DPZF, a novel human BTB/POZ zinc finger protein sharing homology to BCL-6. Biochem Biophys Res Commun,2001.282(4):p. 1067-73.
5. Nielsen, J.V., et al., Hippocampus-like corticoneurogenesis induced by two isoforms of the BTB-zinc finger gene Zbtb20 in mice. Development,2007.134(6):p.1133-40.
6. Xie, Z., et al., Zinc finger protein ZBTB20 is a key repressor of alpha-fetoprotein gene transcription in liver. Proc Natl Acad Sci USA,2008.105(31):p.10859-64.
7. Sutherland, A.P., et al., Zinc finger protein Zbtb20 is essential for postnatal survival and glucose homeostasis. Mol Cell Biol,2009.29(10):p.2804-15.
8. Nielsen, J.V., et al., Zbtb20-Induced CA1 Pyramidal Neuron Development and Area Enlargement in the Cerebral Midline Cortex of Mice. Cereb Cortex,2009.
9. Slater, T.F., K.H. Cheeseman, and K.U. Ingold, Carbon tetrachloride toxicity as a model for studying free-radical mediated liver injury. Philos Trans R Soc Lond B Biol Sci,1985. 311(1152):p.633-45.
10. Weber, L.W., M. Boll, and A. Stampfl, Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Crit Rev Toxicol,2003.33(2):p.105-36.
11. Rao, P.S., R.S. Mangipudy, and H.M. Mehendale, Tissue injury and repair as parallel and opposing responses to CC14 hepatotoxicity:a novel dose-response. Toxicology,1997.118(2-3): p.181-93.
12. Mehendale, H.M., Role of hepatocellular regeneration and hepatolobular healing in the final outcome of liver injury. A two-stage model of toxicity. Biochem Pharmacol,1991.42(6):p. 1155-62.
13. Wong, F.W., W.Y. Chan, and S.S. Lee, Resistance to carbon tetrachloride-induced hepatotoxicity in mice which lack CYP2E1 expression. Toxicol Appl Pharmacol,1998.153(1): p.109-18.
14. Koop, D.R. and J.P. Casazza, Identification of ethanol-inducible P-450 isozyme 3a as the acetone and acetol monooxygenase of rabbit microsomes. J Biol Chem,1985.260(25):p. 13607-12.
15. Buhler, R., et al., Zonation of cytochrome P450 isozyme expression and induction in rat liver. Eur J Biochem,1992.204(1):p.407-12.
16. Hart, S.N., et al., Three patterns of cytochrome P450 gene expression during liver maturation in mice. Drug Metab Dispos,2009.37(1):p.116-21.
17. Matsunaga, N., et al., The molecular mechanism regulating 24-hour rhythm of CYP2E1 expression in the mouse liver. Hepatology,2008.48(1):p.240-51.
18. Liu, S.Y. and F.J. Gonzalez, Role of the liver-enriched transcription factor HNF-1 alpha in expression of the CYP2E1 gene. DNA Cell Biol,1995.14(4):p.285-93.
19. Ueno, T. and F.J. Gonzalez, Transcriptional control of the rat hepatic CYP2E1 gene. Mol Cell Biol,1990.10(9):p.4495-505.
20. Cheung, C, et al., Hepatic expression of cytochrome P450s in hepatocyte nuclear factor 1-alpha (HNFlalpha)-deficientmice. Biochem Pharmacol,2003.66(10):p.2011-20.
21. Sekine, S., et al., Liver-specific loss of beta-catenin blocks glutamine synthesis pathway activity and cytochrome p450 expression in mice. Hepatology,2006.43(4):p.817-25.
22. Truong, N.T., et al., Regulatory sequence responsible for insulin destabilization of cytochrome P4502B1 (CYP2B1) mRNA. Biochem J,2005.388(Pt 1):p.227-35.
23. Peng, H.M. and M.J. Coon, Regulation of rabbit cytochrome P4502E1 expression in HepG2 cells by insulin and thyroid hormone. Mol Pharmacol,1998.54(4):p.740-7.
24. Rosenberg, D.W., G.S. Drummond, and T.J. Smith, Depletion of cytochrome P-450 by thyroid hormone and cobalt-protoporphyrin IX in rat liver:evidence that susceptibility varies among forms of the heme protein. Pharmacology,1995.51(4):p.254-62.
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30. Ma, X., et al., Loss of steroid receptor co-activator-3 attenuates carbon tetrachloride-induced murine hepatic injury and fibrosis. Lab Invest,2009.89(8):p.903-14.
31. Gavrieli, Y., Y. Sherman, and S.A. Ben-Sasson, Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol,1992.119(3):p.493-501.
32. von Wasielewski, R., et al., Tyramine amplification technique in routine immunohistochemistry. J Histochem Cytochem,1997.45(11):p.1455-9.
33. Puchtler, H., F.S. Waldrop, and L.S. Valentine, Polarization microscopic studies of connective tissue stained with picro-sirius red FBA. Beitr Pathol,1973.150(2):p.174-87.
34. Kohler, C.U. and P.H. Roos, Focus on the intermediate state:immature mRNA of cytochromes P450-methods and insights. Anal Bioanal Chem,2008.392(6):p.1109-22.
35. Sugimoto, K., et al., Quantitative analysis of thyroid-stimulating hormone messenger RNA and heterogeneous nuclear RNA in hypothyroid rats. Brain Res Bull,2007.74(1-3):p.142-6.
36. Sims, F.H. and P. Rautanen, Serum arginino-succinate lyase:observations on the sensitivity and specificity of this test in the detection of minimal hepatocellular damage. Clin Biochem, 1975.8(3):p.213-21.
37. Gratzner, H.G., Monoclonal antibody to 5-bromo-and 5-iododeoxyuridine:A new reagent for detection of DNA replication. Science,1982.218(4571):p.474-5.
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8. Geerts, A., History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells. Semin Liver Dis,2001.21(3):p.311-35.
9. Gressner, O.A., et al., Changing the pathogenetic roadmap of liver fibrosis? Where did it start; where will it go? J Gastroenterol Hepatol,2008.23(7 Pt 1):p.1024-35.
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12. Roberts, B.J., et al., Ethanol induces CYP2E1 by protein stabilization. Role of ubiquitin conjugation in the rapid degradation ofCYP2El. J Biol Chem,1995.270(50):p.29632-5.
13. Takeda, K., et al., Rifampicin suppresses hepatic CYP2E1 expression and minimizes DNA injury caused by carbon tetrachloride in perivenular hepatocytes of mice. Alcohol Clin Exp Res,2000.24(4 Suppl):p.87S-92S.
14. Wong, F.W., W.Y. Chan, and S.S. Lee, Resistance to carbon tetrachloride-induced hepatotoxicity in mice which lack CYP2E1 expression. Toxicol Appl Pharmacol,1998.153(1): p.109-18.
15. Zangar, R.C., et al., Cytochrome P4502E1 is the primary enzyme responsible for low-dose carbon tetrachloride metabolism in human liver microsomes. Chem Biol Interact,2000.125(3): p.233-43.
16. Gonzalez, F.J., The 2006 Bernard B. Brodie Award Lecture. Cyp2el. Drug Metab Dispos,2007. 35(1):p.1-8.
17. Koop, D.R. and J.P. Casazza, Identification of ethanol-inducible P-450 isozyme 3a as the acetone and acetol monooxygenase of rabbit microsomes. J Biol Chem,1985.260(25):p. 13607-12.
18. Buhler, R., et al., Zonation of cytochrome P450 isozyme expression and induction in rat liver. Eur J Biochem,1992.204(1):p.407-12.
19. Hart, S.N., et al., Three patterns of cytochrome P450 gene expression during liver maturation in mice. Drug Metab Dispos,2009.37(1):p.116-21.
20. Matsunaga, N., et al., The molecular mechanism regulating 24-hour rhythm of CYP2E1 expression in the mouse liver. Hepatology,2008.48(1):p.240-51.
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23. Ueno, T. and F.J. Gonzalez, Transcriptional control of the rat hepatic CYP2E1 gene. Mol Cell Biol,1990.10(9):p.4495-505.
24. Cheung, C, et al., Hepatic expression of cytochrome P450s in hepatocyte nuclear factor 1-alpha (HNFlalpha)-deficient mice. Biochem Pharmacol,2003.66(10):p.2011-20.
25. Sekine, S., et al., Liver-specific loss of beta-catenin blocks glutamine synthesis pathway activity and cytochrome p450 expression in mice. Hepatology,2006.43(4):p.817-25.
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29. Woodcroft, K.J., M.S. Hafner, and R.F. Novak, Insulin signaling in the transcriptional and posttranscriptional regulation ofCYP2El expression. Hepatology,2002.35(2):p.263-73.
30. Moncion, A., et al., Identification of a 16-nucleotide sequence that mediates post-transcriptional regulation of rat CYP2E1 by insulin. J Biol Chem,2002.277(48):p. 45904-10.
31. Truong, N.T., et al., Regulatory sequence responsible for insulin destabilization of cytochrome P4502B1 (CYP2B1) mRNA. Biochem J,2005.388(Pt 1):p.227-35.
32. Tierney, D.J., A.L. Haas, and D.R. Koop, Degradation of cytochrome P4502E1:selective loss after labilization of the enzyme. Arch Biochem Biophys,1992.293(1):p.9-16.
33. Song, B.J., et al., Induction of rat hepatic N-nitrosodimethylamine demethylase by acetone is due to protein stabilization. J Biol Chem,1989.264(6):p.3568-72.
34. Eliasson, E.,I. Johansson, and M. Ingelman-Sundberg, Substrate-, hormone-, and cAMP-regulated cytochrome P450 degradation. Proc Natl Acad Sci U S A,1990.87(8):p. 3225-9.
2. Kelly, K.F. and J.M. Daniel, POZ for effect-POZ-ZF transcription factors in cancer and development. Trends in Cell Biology,2006.16(11):p.578-87.
3. Mitchelmore, C, et al., Characterization of two novel nuclear BTB/POZ domain zinc finger isoforms. Association with differentiation of hippocampal neurons, cerebellar granule cells, and macroglia. J Biol Chem,2002.277(9):p.7598-609.
4. Zhang, W., et al., Identification and characterization of DPZF, a novel human BTB/POZ zinc finger protein sharing homology to BCL-6. Biochem Biophys Res Commun,2001.282(4):p. 1067-73.
5. Nielsen, J.V., et al., Hippocampus-like corticoneurogenesis induced by two isoforms of the BTB-zinc finger gene Zbtb20 in mice. Development,2007.134(6):p.1133-40.
6. Xie, Z., et al., Zinc finger protein ZBTB20 is a key repressor of alpha-fetoprotein gene transcription in liver. Proc Natl Acad Sci USA,2008.105(31):p.10859-64.
7. Sutherland, A.P., et al., Zinc finger protein Zbtb20 is essential for postnatal survival and glucose homeostasis. Mol Cell Biol,2009.29(10):p.2804-15.
8. Nielsen, J.V., et al., Zbtb20-Induced CA1 Pyramidal Neuron Development and Area Enlargement in the Cerebral Midline Cortex of Mice. Cereb Cortex,2009.
9. Slater, T.F., K.H. Cheeseman, and K.U. Ingold, Carbon tetrachloride toxicity as a model for studying free-radical mediated liver injury. Philos Trans R Soc Lond B Biol Sci,1985. 311(1152):p.633-45.
10. Weber, L.W., M. Boll, and A. Stampfl, Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Crit Rev Toxicol,2003.33(2):p.105-36.
11. Rao, P.S., R.S. Mangipudy, and H.M. Mehendale, Tissue injury and repair as parallel and opposing responses to CC14 hepatotoxicity:a novel dose-response. Toxicology,1997.118(2-3): p.181-93.
12. Mehendale, H.M., Role of hepatocellular regeneration and hepatolobular healing in the final outcome of liver injury. A two-stage model of toxicity. Biochem Pharmacol,1991.42(6):p. 1155-62.
13. Wong, F.W., W.Y. Chan, and S.S. Lee, Resistance to carbon tetrachloride-induced hepatotoxicity in mice which lack CYP2E1 expression. Toxicol Appl Pharmacol,1998.153(1): p.109-18.
14. Koop, D.R. and J.P. Casazza, Identification of ethanol-inducible P-450 isozyme 3a as the acetone and acetol monooxygenase of rabbit microsomes. J Biol Chem,1985.260(25):p. 13607-12.
15. Buhler, R., et al., Zonation of cytochrome P450 isozyme expression and induction in rat liver. Eur J Biochem,1992.204(1):p.407-12.
16. Hart, S.N., et al., Three patterns of cytochrome P450 gene expression during liver maturation in mice. Drug Metab Dispos,2009.37(1):p.116-21.
17. Matsunaga, N., et al., The molecular mechanism regulating 24-hour rhythm of CYP2E1 expression in the mouse liver. Hepatology,2008.48(1):p.240-51.
18. Liu, S.Y. and F.J. Gonzalez, Role of the liver-enriched transcription factor HNF-1 alpha in expression of the CYP2E1 gene. DNA Cell Biol,1995.14(4):p.285-93.
19. Ueno, T. and F.J. Gonzalez, Transcriptional control of the rat hepatic CYP2E1 gene. Mol Cell Biol,1990.10(9):p.4495-505.
20. Cheung, C, et al., Hepatic expression of cytochrome P450s in hepatocyte nuclear factor 1-alpha (HNFlalpha)-deficientmice. Biochem Pharmacol,2003.66(10):p.2011-20.
21. Sekine, S., et al., Liver-specific loss of beta-catenin blocks glutamine synthesis pathway activity and cytochrome p450 expression in mice. Hepatology,2006.43(4):p.817-25.
22. Truong, N.T., et al., Regulatory sequence responsible for insulin destabilization of cytochrome P4502B1 (CYP2B1) mRNA. Biochem J,2005.388(Pt 1):p.227-35.
23. Peng, H.M. and M.J. Coon, Regulation of rabbit cytochrome P4502E1 expression in HepG2 cells by insulin and thyroid hormone. Mol Pharmacol,1998.54(4):p.740-7.
24. Rosenberg, D.W., G.S. Drummond, and T.J. Smith, Depletion of cytochrome P-450 by thyroid hormone and cobalt-protoporphyrin IX in rat liver:evidence that susceptibility varies among forms of the heme protein. Pharmacology,1995.51(4):p.254-62.
25. Tierney, D.J., A.L. Haas, and D.R. Koop, Degradation of cytochrome P4502E1:selective loss after labilization of the enzyme. Arch Biochem Biophys,1992.293(1):p.9-16.
26. Roberts, B.J., et al., Ethanol induces CYP2E1 by protein stabilization. Role of ubiquitin conjugation in the rapid degradation of CYP2E1. J Biol Chem,1995.270(50):p.29632-5.
27. Song, B.J., et al., Induction of rat hepatic N-nitrosodimethylamine demethylase by acetone is due to protein stabilization. J Biol Chem,1989.264(6):p.3568-72.
28. Eliasson, E., I. Johansson, and M. Ingelman-Sundberg, Substrate-, hormone-, and cAMP-regulated cytochrome P450 degradation. Proc Natl Acad Sci U S A,1990.87(8):p. 3225-9.
29. Hopman, A.H., F.C. Ramaekers, and E.J. Speel, Rapid synthesis of biotin-, digoxigenin-, trinitrophenyl-, and fluorochrome-labeled tyramides and their application for In situ hybridization using CARD amplification. J Histochem Cytochem,1998.46(6):p.771-7.
30. Ma, X., et al., Loss of steroid receptor co-activator-3 attenuates carbon tetrachloride-induced murine hepatic injury and fibrosis. Lab Invest,2009.89(8):p.903-14.
31. Gavrieli, Y., Y. Sherman, and S.A. Ben-Sasson, Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol,1992.119(3):p.493-501.
32. von Wasielewski, R., et al., Tyramine amplification technique in routine immunohistochemistry. J Histochem Cytochem,1997.45(11):p.1455-9.
33. Puchtler, H., F.S. Waldrop, and L.S. Valentine, Polarization microscopic studies of connective tissue stained with picro-sirius red FBA. Beitr Pathol,1973.150(2):p.174-87.
34. Kohler, C.U. and P.H. Roos, Focus on the intermediate state:immature mRNA of cytochromes P450-methods and insights. Anal Bioanal Chem,2008.392(6):p.1109-22.
35. Sugimoto, K., et al., Quantitative analysis of thyroid-stimulating hormone messenger RNA and heterogeneous nuclear RNA in hypothyroid rats. Brain Res Bull,2007.74(1-3):p.142-6.
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