角蛋白18及其磷酸化在HBV感染慢性肝病及肝细胞凋亡中的作用研究
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摘要
角蛋白18(keratin 18,K18)是构成成熟肝细胞骨架的主要中间纤维丝蛋白之一,其主要作用是保护肝细胞免受各种机械性和非机械性的损伤。K18的突变、结构的重组以及磷酸化的改变会影响肝细胞的完整性,引起肝细胞的损伤。此外,K18除了发挥抗机械性损伤作用之外还参与着重要的细胞信号传导作用,包括Fas/FasL、TRADD等相关的凋亡通路都与K18及其磷酸化有关。已有研究发现K18磷酸化水平在慢性丙型肝炎时出现增高,并且增高的程度与肝病进程呈正相关。在我国乙型肝炎病毒(HBV)感染是慢性肝病的最常见病因,关于HBV感染的肝病进展与K18及其磷酸化之间关系的研究在国内外未见报道。因此,研究K18及其磷酸化对揭示HBV感染慢性肝病肝损伤及肝细胞凋亡的机制具有重要意义,对于临床制定个体化的治疗方案、判定预后都具有重要的参考价值和临床应用前景。
p53是重要的促凋亡基因,p53及其介导的凋亡通路在HBV感染慢性肝病的肝细胞损伤中发挥着重要的作用。ASPP2(Apoptosis Stimulating Protein 2 of P53, ASPP2)是p53凋亡刺激蛋白家族的重要成员,能够与p53结合选择性地提高p53的促凋亡活性。我们在前期的工作中应用酵母双杂交技术发现,ASPP2能够与K18结合。这一发现对于揭示K18及其磷酸化与肝细胞凋亡的关系和探索其作用方式提供了重要线索。本研究围绕K18及其磷酸化在HBV感染慢性肝病和肝细胞凋亡中的作用及其作用机制进行了系统研究。
1、角蛋白18磷酸化在HBV感染慢性肝病中的作用及其意义
为了研究K18及其磷酸化在慢性乙型肝炎(chronic hepatitis B, CHB)病程进展中的变化及其意义,我们收集了40例慢性乙肝患者肝穿刺组织,21例肝硬化肝组织(其中静止性肝硬化11例,活动性肝硬化10例)和5例慢性重型肝炎肝组织,以12名健康肝移植供体肝组织作为对照。首先应用免疫荧光技术,分析了不同慢性肝病进程当中K18及其磷酸化在肝细胞内的表达和定位,结果发现,K18在正常肝组织中表达水平较高,呈网状分布;慢性肝炎中表达水平无显著变化;但在肝硬化肝组织和慢性重型肝炎肝组织中,其组织正常结构被破坏,许多新生胆管细胞中K18水平较高;K18 Ser33和Ser52在正常肝组织中磷酸化水平较低,但是随着肝损伤程度的增加,在新生胆管细胞中磷酸化水平明显增高。
应用Western Blotting的方法检测肝组织中K18磷酸化水平,分组分析K18磷酸化水平与慢乙肝病程进展的关系。
(1)将肝组织按照疾病进展分为正常组织,慢性肝炎和肝硬化组。结果发现,K18 Ser52磷酸化水平在正常肝组织中较低,而慢性肝炎组增高,至肝硬化组织达到最高水平;K18 Ser33磷酸化水平在慢性肝炎和肝硬化两组均显著高于正常组织,但两组之间无显著性差异。
(2)进一步将40例慢性乙型肝炎患者的肝组织按照Ishak组织学评分的高低分为3组,即轻度损伤(MiH)组、中度损伤(MeH)组、重度损伤(AdH)组,肝硬化组织按照炎症程度分为静止性肝硬化和活动性肝硬化,五组之间进行比较研究其K18磷酸化水平的差异。结果发现,K18 Ser52磷酸化水平随着肝病进程而逐渐增加,从正常肝组织到活动性肝硬化,相邻两组之间有显著性差异。K18 Ser52磷酸化水平变化趋势是:正常组织< MiH < MeH < AdH <静止性肝硬化<活动性肝硬化。K18 Ser33位磷酸化水平从正常组织到活动性肝硬化亦呈上升趋势,在慢性肝炎肝组织中,从轻度损伤至重度损伤,K18 Ser33磷酸化增加明显(p<0.005),但在静止性肝硬化组织中磷酸化水平较低,其水平与MiH组相当(p=0.750),而慢肝中重度损伤组磷酸化水平与活动性肝硬化相当(p=0.715),即K18 Ser33磷酸化水平变化趋势是:正常组织< MiH≈静止性肝硬化< MeH < AdH≈活动性肝硬化。
(3)为了进一步探究K18磷酸化与整体肝组织损伤的关系,我们分析了K18磷酸化与临床上最常用的反映肝功能损伤的生化指标谷丙转氨酶(ALT)之间的关系。将本研究中所有慢性乙肝患者的肝组织根据其ALT水平进行分组(1,ALT<40U/L;2,ALT:40~200 U/L;3,ALT>200 U/L),分别与正常肝组织进行比较。结果显示K18 Ser52磷酸化在正常肝组织组、ALT<40U/L组、40~200 U/L组和>200 U/L组中的均值分别为0.73,0.72,1.77和2.71。其中后两组与前两组比较均有显著性差异,而正常组织和ALT<40 U/L组比较无显著性差异(p=0.976),表明在HBV感染但肝功能未受损伤的肝组织中K18 Ser52磷酸化水平并不增加。提示K18 Ser52磷酸化水平反映了肝组织的损伤情况,但与HBV感染本身并无直接联系。而K18 Ser33位磷酸化水平随着肝功能水平变化增高明显,在四组中均值分别为1.02,1.96,2.53和3.00,各组之间存在显著性差异。值得注意的是,在ALT<40 U/L组,K18 Ser33位磷酸化水平也出现增高,与正常相比差异显著。这一结果提示我们,K18 Ser33位磷酸化可能与HBV感染有关系。
(4)为了进一步研究HBV感染对于K18磷酸化水平的影响,我们选取了HBV感染但是肝功能水平正常的慢性乙肝携带者的肝组织标本,冰冻切片后分别进行了HBsAg抗体和K18两种磷酸化抗体的双染免疫荧光检测,结果发现,HBsAg在HBV感染的肝细胞胞浆中表达,呈现大面积红染,而在正常对照肝组织中未见染色。K18 Ser33磷酸化恰恰在HBV感染的细胞中出现异常增高,而K18 Ser52磷酸化水平仍然较低,与周围细胞无差异。
本研究表明:K18磷酸化水平是慢性乙型肝炎肝损伤的重要指标;K18 Ser52位磷酸化是慢性乙型肝炎肝病进展的判定指标;K18 Ser33位磷酸化可能是HBV感染的早期现象,并且是肝组织炎症损伤程度的判定指标。
2、角蛋白18与p53凋亡刺激蛋白2(ASPP2)在肝细胞凋亡中的作用研究
我们前期的研究发现,K18能够和p53凋亡刺激蛋白(ASPP2)结合。为进一步证实这种结合关系并研究其对于肝细胞凋亡的意义,我们从三个方面进行了研究。
(1)K18及其磷酸化和ASPP2在肝细胞凋亡中的作用研究:应用免疫荧光双染发现,在正常HepG2细胞中,K18和ASPP2均表达在细胞浆中,给予药物处理的细胞发生凋亡时,ASPP2进入到细胞核中,发生核转位。应用不同浓度的甲基甲烷磺酸盐(MMS)作用于HepG2细胞,用Annexin V/PI染色流式细胞法检测细胞凋亡水平,研究K18及其磷酸化和ASPP2在肝细胞凋亡不同状态下的水平变化。结果发现,K18总体水平随着药物浓度增加而降低,ASPP2和K18 Ser52位磷酸化水平随着药物浓度增加而增加,K18 Ser33磷酸化在低浓度药物作用下水平增高,但在高浓度药物作用下反而降低。以上结果说明,ASPP2在肝细胞发生凋亡时出现核转位,从而进入到细胞核中参与p53介导的细胞凋亡通路,而K18的Ser33或Ser52磷酸化水平的变化与肝细胞凋亡有密切关系。
(2)ASPP2与K18结合功能区域的确定:采用重组的GST-mASPP2,ASPP2 N-末端和C-末端融合蛋白分别与K18融合蛋白进行免疫共沉淀。实验结果发现ASPP2的N-末端能够与K18结合。进一步将ASPP2的N-末端逐步截短,表达了ASPP2 N-末端的截短小片段融合蛋白,再次与K18进行体外免疫沉淀反应,结果发现,ASPP2的N-末端位于83-105个氨基酸残基之间正是与K18结合的关键部位,而其下游的氨基酸残基则与结合能力有关系。应用293T细胞分别高效转染K18-pcDNA4或K18 S33A、K18 S52A,提取细胞总蛋白应用GST-mASPP2融合蛋白进行免疫沉淀反应,结果发现,ASPP2能够与K18结合,但在转染突变K18的细胞中结合降低,在转染S33A突变K18的细胞中结合最少。提示这种结合可能与33位的丝氨酸磷酸化有关系。为了研究这种结合与肝病进展的关系,我们提取了活动性肝硬化和静止性肝硬化肝组织的总蛋白,利用免疫共沉淀技术分析了K18和ASPP2的结合水平与肝病进展的关系,结果发现,ASPP2在静止性肝硬化中与K18的结合更强,在活动性肝硬化结合显著减少。说明,在肝组织受到严重损伤时,ASPP2与K18的结合减少。
(3)K18与ASPP2结合作用方式的初步研究:将细胞共转染ASPP2和K18或S33A,S52A定点突变的K18,给予细胞药物刺激,转染S33A K18的细胞发生凋亡时,ASPP2全部进入到细胞核中,而转染K18和S52A K18的细胞发生凋亡时,ASPP2部分进入到核中,大部分仍然位于细胞浆中,说明K18 Ser33位磷酸化与ASPP2的核转位有关。应用报告基因分析法研究K18对ASPP2-p53及其调控的下游分子Bax转录活性的影响。结果发现:ASPP2的存在能够显著提高p53刺激的Bax-luc报告基因活性,当加入过量野生型K18后,Bax-luc报告基因活性大大减低,提示K18可能参与了抑制ASPP2的促p53转录激活作用;当用K18 S33A质粒替代K18后,Bax-luc报告基因活性水平与ASPP2单独存在时大致相同。这一结果再次提示K18 Ser33位磷酸化能够促进K18与ASPP2的结合,从而间接抑制ASPP2与p53的结合而发挥凋亡抑制作用。
上述实验研究表明:K18磷酸化在肝细胞凋亡中发挥着重要的作用;正常情况下K18在细胞浆中以Ser33位磷酸化依赖的方式结合ASPP2,在细胞遇到轻度损伤性刺激时,K18 Ser33和Ser52位磷酸化水平均出现增高,使得ASPP2与K18的结合水平增加;但当细胞受到严重刺激时,K18 Ser33位磷酸化水平反而降低,导致ASPP2与K18的结合变得松弛,ASPP2进入到细胞核与p53结合中发挥其促凋亡的作用。
Keratin 18 (K18) is one of the major intermediate filament proteins forming the cytoskeleton of human mature hepatocytes, and its main function is to protect hepatocytes from mechanical and non-mechanical injuries. Mutations, structural reorganization, and changes in phosphorylation in K18 may affect the integrity of hepatocytes and cause their injury. In addition to its role in resisting mechanical injury, K18 also participates in some key signal transduction activities, for example, apoptosis pathways such as Fas/FasL, TRADD have been found to be related to K18 phosphorylation. Studies have shown that K18 phosphorylation level increases in chronic liver diseases with hepatitis C virus (HCV) infection, and the degree of increase is positively associated with liver disease progression. In China , hepatitis B virus (HBV) infection is the most common cause of chronic liver diseases, but there has been no report on the relation of K18 and its phosphorylation to chronic liver diseases with HBV infection. Therefore the study of this relationship will help to reveal the mechanisms of liver injury and hepatocyte apoptosis in chronic liver injury with HBV infection, and it can also be valuable for clinicians in making individualized treatment plan and prognosis.
P53 is an important pro-apoptosis gene; p53-mediated apoptosis pathway plays an important role in hepatocytes injury in chronic liver diseases with HBV infection. Apoptosis Stimulating Protein 2 of P53 (ASPP2) is an important member of the p53 apoptosis stimulating proteins family and it can bind with p53 and selectively increase the pro-apoptotic activity of p53. In our previous work, we found by yeast two-hybrid technique that ASPP2 can bind with K18, which provides a key clue in revealing the relationship between K18 phosphorylation and hepatocyte apoptosis and explore the underlying mechanism. The current study systemically investigated the role of K18 and its phosphorylation in HBV infection related chronic liver diseases and hepatocyte apoptosis and its mechanism of action.
1. The function and significance of K18 phosphorylation in HBV infection related chronic liver diseases:
To investigate changes of K18 phosphorylation in the progression of chronic B type hepatitis (CHB) and its significance, liver tissue specimens were collected from 12 healthy donors (used as control), 40 chronic hepatitis B patients, 21 cirrhosis patients (11 inactive, 10 active) and 5 patients of severe chronic hepatitis. The cellular location of K18 expression and its phosphorylation level in different stages of CHB was first analyzed with immnofluorescence staining. In normal liver tissues, K18 expression level was high and distribute in a net form. Its expression level did not change much in chronic hepatitis. However in cirrhosis and chronic severe hepatitis, normal structure of liver tissue was damaged and K18 was expressed at high levels in proliferative bile duct epithelial cells. The phosphorylation of K18 Ser-33 and Ser-52 was low in normal liver tissues but as liver injury intensified the level increased markedly in proliferative bile duct epithelial cells.
Western Blotting was used to analyze K18 phosphorylation in liver tissues.
(1) Liver tissues were divided into normal, chronic hepatitis and cirrhosis. K18 Ser52 phosphorylation showd low level in normal liver, increased in chronic hepatitis and reached high level in cirrhosis. K18 Ser-33 phosphorylation in CHB and cirrhosis were both higher than normal tissue, but showed no evident difference between the two groups.
(2) Chronic hepatitis was further divided into three groups (minimal histological lesions, MiH; medium lesions, MeH; advanced lesions, AdH) and cirrhosis were divided into inactive cirrhosis (inactive-LC) and active cirrhosis (active-LC). K18 phosphorylations were compared among the five groups. K18 Ser-52 phosphorylation was increased along with the progression of liver disease. From normal liver to active cirrhosis, there are distinctive differences between consecutive groups. The relative level of Ser52 phosphorylation follows the trend of Ser52 normal < MiH < MeH < AdH < inactive LC < active LC. K18 Ser33 phosphorylation also displayed increasing trend from normal tissue to active cirrhosis. From MiH to AdH in chronic hepatitis, K18 Ser33 phosphorylation increased evidently (p<0.005). But K18 Ser33 phosphorylation showed low level in inactive-LC and was almost equivalent to MiH(P = 0.750). K18 Ser33 phosphorylation level in AdH was also equivalent to active-LC (P = 0.715). The relative level of Ser33 phosphorylation follows the trend of normal < MiH≈inactive LC < MeH < AdH≈active LC.
(3) To further investigate the relationship of K18 phosphorylation and the total injury in liver tissue, K18 phosphorylation was analyzed to compare with the most commonly used clinical marker ALT. 40 CHB livers were divided into 3 subgroups (1, ALT < 40 U/L; 2, 40≤ALT <200 U/L; 3, ALT≥200 U/L). The mean relative levels of Ser52 phosphorylation in normal control liver and the three groups were: 0.73, 0.72, 1.77 and 2.71. Ser52 phosphorylation was equivalent in the control and subgroup 1 (P = 0.976), while significantly increased subgroups 1 and 2 (P < 0.001), and subgroups 2 and 3 (P < 0.001) . The positive correlation between levels of phosphorylation and ALT activities suggested that the phosphorylation of Ser52 in K18 was progressively related to liver injuries but had no direct relation with HBV infection. K18 Ser33 phosphorylation increased along with ALT, the mean relative level of the four subgroups were: 1.02, 1.96, 2.53 and 3.00. It was notable that K18 Ser33 phosphorylation increased in subgroup 2 and had significant difference to normal liver. This results indicated that K18 Ser33 phosphorylation might be related to HBV infection, therefore, K18 Ser-33 still increased in non-injured liver.
(4) To further investigate the effect of HBV infection on K18 phosphorylation, a liver specimen respectively from a randomly selected inactive HBV carrier and a healthy donor was sectioned, and probed for HBsAg and phosphorylated Ser33 or Ser52. Strong signals of phosphorylated Ser33 were restricted to hepatocytes with strong positive detection of HBsAg. However, only basal levels of Ser33 phosphorylation were detected in the control. Basal levels of Ser52 phosphorylation were observed in both HBV positive and healthy samples. These results reiterated the conclusion that phosphorylation at Ser33 in K18 was an indicator of HBV infection.
This study indicated: K18 phosphorylations were important injure markers of CHB. K18 Ser52 phosphorylation was a marker of progression of CHB; K18 Ser33 phosphorylation may be an early indicator of HBV infection and marker of liver inflammation.
2. Study of the function of Keratin 18 and p53 apoptosis stimulate protein ASPP2 in hepatocyte apoptosis
K18 was found in our previous work to bind to ASPP2 by Yeast two-hybrid. To further confirm the interaction of the two proteins and investigate the significance, we studied from three aspects.
(1) Study of the function of K18 phosphorylation and ASPP2 in apoptosis of hepatocytes: It was found by immnofluorescence double-staining that ASPP2 and K18 both located in cytoplasm in normal cells. But when cells were treated with drugs and induced into apoptosis, ASPP2 entered into nucleus. HepG2 cells were then treated with MMS with different concentration, apoptosis were stainedby Annexin V/PI and analyzed by Flow cytometry. It was found that total K18 decreased but ASPP2 and K18 Ser52 phosphorylation increased along with the increase of cell apoptosis level. Ser33 phosphorylatoin increased under low level MMS, but decreased under high level MMS. The results above indicated: when hepatocytes were apoptosis, ASPP2 entered into nucleus to participate in p53-mediated apoptosis pathway. K18 Ser33 or Ser52 phosphorylation had close relationship with hepatocytes apoptosis.
(2) Determination of binding domain of ASPP2 and K18: Recombination GST-mASPP2, ASPP2 N-terminal and C-terminal fusion proteins were prepared to Co-IP with K18 fusion protein in vitro. It was found that ASPP2 N-terminal can bind with K18. Then ASPP2 N-terminal was further truncated. The small fragment fusion proteins were expressed to Co-IP with K18. 83-105aa of ASPP2 N-terminal was found to be the key domain of binding. The followed amino acid residues of N-terminal were correlated with binding ability. 293T cells were transfected with K18 ,S33A K18 or S52A K18, then total protein were extracted to Co-IP with ASPP2 in semi-in vivo. It was found that ASPP2 could combine with K18, but the hybrid signal was much lower in cells transfected with mutated K18. The binding signal in cells transfected with S33A K18 was the lowest, which suggested that Ser33 phosphorylation was related with binding of ASPP2 and K18. To investigate the relationship of the combination with the progression of liver disease, total protein of active cirrhosis and inactive cirrhosis tissues were extracted. Then the relationship between the combination of ASPP2 and K18 and the progression of liver disease were analyzed by Co-IP. It was found that ASPP2 binded with K18 much more in inactive cirrhosis, but the hybrid signal was barely detected in active cirrhosis. It indicated that binding of ASPP2 and K18 decreased in serious injury in liver.
(3) Investigation of relationship between combination of K18 and ASPP2 and p53 apoptosis pathway: HepG2 cells were co-transfected with ASPP2 and K18 or S33A K18 or S52A K18. When cells were treated with drugs, ASPP2 entered into nucleus totally in apoptosis cells co-transfected with K18 S33A and ASPP2, but entered into nucleus much less in apoptosis cells co-transfected with K18 S52A and ASPP2 or K18 and ASPP2. This result indicated that K18 Ser33 phosphorylation had close relationship with ASPP2 nucleus translocation. Effect of K18 on ASPP2-p53 and its regulated Bax transcription activity were studied by report gene analysis. It was found that ASPP2 increased the activity of Bax-luc report gene but the activity decreased when excessive wild K18 was added, which indicated that K18 may inhibit the pro-apoptotic effect of ASPP2. When K18 was replaced with S33A K18, Bax report gene activity was almost the same with transfected with ASPP2 alone. This result again confirmed that K18 Ser33 phosphorylation could significantly enhance the combination of K18 and ASPP2, therefore indirectly inhibit ASPP2 binding with p53 and exert its apoptosis inhibition function.
The studies above indicated: K18 and its phosphorylation played important role in hepatocytes apoptosis; under normal circumstances, K18 in cytoplasm combined with ASPP2 in a Ser33 phosphorylation-dependent manner. When cells were suffered with mild injury, K18 Ser33 and Ser52 phosphorylation both increased to enhance the binding of ASPP2 and K18, But when cells suffered from serious injury, K18 Ser33 phosphorylation decreased to make ASPP2 depart from K18 and enter into nucleus to exert its pro-apoptosis function.
p53是重要的促凋亡基因,p53及其介导的凋亡通路在HBV感染慢性肝病的肝细胞损伤中发挥着重要的作用。ASPP2(Apoptosis Stimulating Protein 2 of P53, ASPP2)是p53凋亡刺激蛋白家族的重要成员,能够与p53结合选择性地提高p53的促凋亡活性。我们在前期的工作中应用酵母双杂交技术发现,ASPP2能够与K18结合。这一发现对于揭示K18及其磷酸化与肝细胞凋亡的关系和探索其作用方式提供了重要线索。本研究围绕K18及其磷酸化在HBV感染慢性肝病和肝细胞凋亡中的作用及其作用机制进行了系统研究。
1、角蛋白18磷酸化在HBV感染慢性肝病中的作用及其意义
为了研究K18及其磷酸化在慢性乙型肝炎(chronic hepatitis B, CHB)病程进展中的变化及其意义,我们收集了40例慢性乙肝患者肝穿刺组织,21例肝硬化肝组织(其中静止性肝硬化11例,活动性肝硬化10例)和5例慢性重型肝炎肝组织,以12名健康肝移植供体肝组织作为对照。首先应用免疫荧光技术,分析了不同慢性肝病进程当中K18及其磷酸化在肝细胞内的表达和定位,结果发现,K18在正常肝组织中表达水平较高,呈网状分布;慢性肝炎中表达水平无显著变化;但在肝硬化肝组织和慢性重型肝炎肝组织中,其组织正常结构被破坏,许多新生胆管细胞中K18水平较高;K18 Ser33和Ser52在正常肝组织中磷酸化水平较低,但是随着肝损伤程度的增加,在新生胆管细胞中磷酸化水平明显增高。
应用Western Blotting的方法检测肝组织中K18磷酸化水平,分组分析K18磷酸化水平与慢乙肝病程进展的关系。
(1)将肝组织按照疾病进展分为正常组织,慢性肝炎和肝硬化组。结果发现,K18 Ser52磷酸化水平在正常肝组织中较低,而慢性肝炎组增高,至肝硬化组织达到最高水平;K18 Ser33磷酸化水平在慢性肝炎和肝硬化两组均显著高于正常组织,但两组之间无显著性差异。
(2)进一步将40例慢性乙型肝炎患者的肝组织按照Ishak组织学评分的高低分为3组,即轻度损伤(MiH)组、中度损伤(MeH)组、重度损伤(AdH)组,肝硬化组织按照炎症程度分为静止性肝硬化和活动性肝硬化,五组之间进行比较研究其K18磷酸化水平的差异。结果发现,K18 Ser52磷酸化水平随着肝病进程而逐渐增加,从正常肝组织到活动性肝硬化,相邻两组之间有显著性差异。K18 Ser52磷酸化水平变化趋势是:正常组织< MiH < MeH < AdH <静止性肝硬化<活动性肝硬化。K18 Ser33位磷酸化水平从正常组织到活动性肝硬化亦呈上升趋势,在慢性肝炎肝组织中,从轻度损伤至重度损伤,K18 Ser33磷酸化增加明显(p<0.005),但在静止性肝硬化组织中磷酸化水平较低,其水平与MiH组相当(p=0.750),而慢肝中重度损伤组磷酸化水平与活动性肝硬化相当(p=0.715),即K18 Ser33磷酸化水平变化趋势是:正常组织< MiH≈静止性肝硬化< MeH < AdH≈活动性肝硬化。
(3)为了进一步探究K18磷酸化与整体肝组织损伤的关系,我们分析了K18磷酸化与临床上最常用的反映肝功能损伤的生化指标谷丙转氨酶(ALT)之间的关系。将本研究中所有慢性乙肝患者的肝组织根据其ALT水平进行分组(1,ALT<40U/L;2,ALT:40~200 U/L;3,ALT>200 U/L),分别与正常肝组织进行比较。结果显示K18 Ser52磷酸化在正常肝组织组、ALT<40U/L组、40~200 U/L组和>200 U/L组中的均值分别为0.73,0.72,1.77和2.71。其中后两组与前两组比较均有显著性差异,而正常组织和ALT<40 U/L组比较无显著性差异(p=0.976),表明在HBV感染但肝功能未受损伤的肝组织中K18 Ser52磷酸化水平并不增加。提示K18 Ser52磷酸化水平反映了肝组织的损伤情况,但与HBV感染本身并无直接联系。而K18 Ser33位磷酸化水平随着肝功能水平变化增高明显,在四组中均值分别为1.02,1.96,2.53和3.00,各组之间存在显著性差异。值得注意的是,在ALT<40 U/L组,K18 Ser33位磷酸化水平也出现增高,与正常相比差异显著。这一结果提示我们,K18 Ser33位磷酸化可能与HBV感染有关系。
(4)为了进一步研究HBV感染对于K18磷酸化水平的影响,我们选取了HBV感染但是肝功能水平正常的慢性乙肝携带者的肝组织标本,冰冻切片后分别进行了HBsAg抗体和K18两种磷酸化抗体的双染免疫荧光检测,结果发现,HBsAg在HBV感染的肝细胞胞浆中表达,呈现大面积红染,而在正常对照肝组织中未见染色。K18 Ser33磷酸化恰恰在HBV感染的细胞中出现异常增高,而K18 Ser52磷酸化水平仍然较低,与周围细胞无差异。
本研究表明:K18磷酸化水平是慢性乙型肝炎肝损伤的重要指标;K18 Ser52位磷酸化是慢性乙型肝炎肝病进展的判定指标;K18 Ser33位磷酸化可能是HBV感染的早期现象,并且是肝组织炎症损伤程度的判定指标。
2、角蛋白18与p53凋亡刺激蛋白2(ASPP2)在肝细胞凋亡中的作用研究
我们前期的研究发现,K18能够和p53凋亡刺激蛋白(ASPP2)结合。为进一步证实这种结合关系并研究其对于肝细胞凋亡的意义,我们从三个方面进行了研究。
(1)K18及其磷酸化和ASPP2在肝细胞凋亡中的作用研究:应用免疫荧光双染发现,在正常HepG2细胞中,K18和ASPP2均表达在细胞浆中,给予药物处理的细胞发生凋亡时,ASPP2进入到细胞核中,发生核转位。应用不同浓度的甲基甲烷磺酸盐(MMS)作用于HepG2细胞,用Annexin V/PI染色流式细胞法检测细胞凋亡水平,研究K18及其磷酸化和ASPP2在肝细胞凋亡不同状态下的水平变化。结果发现,K18总体水平随着药物浓度增加而降低,ASPP2和K18 Ser52位磷酸化水平随着药物浓度增加而增加,K18 Ser33磷酸化在低浓度药物作用下水平增高,但在高浓度药物作用下反而降低。以上结果说明,ASPP2在肝细胞发生凋亡时出现核转位,从而进入到细胞核中参与p53介导的细胞凋亡通路,而K18的Ser33或Ser52磷酸化水平的变化与肝细胞凋亡有密切关系。
(2)ASPP2与K18结合功能区域的确定:采用重组的GST-mASPP2,ASPP2 N-末端和C-末端融合蛋白分别与K18融合蛋白进行免疫共沉淀。实验结果发现ASPP2的N-末端能够与K18结合。进一步将ASPP2的N-末端逐步截短,表达了ASPP2 N-末端的截短小片段融合蛋白,再次与K18进行体外免疫沉淀反应,结果发现,ASPP2的N-末端位于83-105个氨基酸残基之间正是与K18结合的关键部位,而其下游的氨基酸残基则与结合能力有关系。应用293T细胞分别高效转染K18-pcDNA4或K18 S33A、K18 S52A,提取细胞总蛋白应用GST-mASPP2融合蛋白进行免疫沉淀反应,结果发现,ASPP2能够与K18结合,但在转染突变K18的细胞中结合降低,在转染S33A突变K18的细胞中结合最少。提示这种结合可能与33位的丝氨酸磷酸化有关系。为了研究这种结合与肝病进展的关系,我们提取了活动性肝硬化和静止性肝硬化肝组织的总蛋白,利用免疫共沉淀技术分析了K18和ASPP2的结合水平与肝病进展的关系,结果发现,ASPP2在静止性肝硬化中与K18的结合更强,在活动性肝硬化结合显著减少。说明,在肝组织受到严重损伤时,ASPP2与K18的结合减少。
(3)K18与ASPP2结合作用方式的初步研究:将细胞共转染ASPP2和K18或S33A,S52A定点突变的K18,给予细胞药物刺激,转染S33A K18的细胞发生凋亡时,ASPP2全部进入到细胞核中,而转染K18和S52A K18的细胞发生凋亡时,ASPP2部分进入到核中,大部分仍然位于细胞浆中,说明K18 Ser33位磷酸化与ASPP2的核转位有关。应用报告基因分析法研究K18对ASPP2-p53及其调控的下游分子Bax转录活性的影响。结果发现:ASPP2的存在能够显著提高p53刺激的Bax-luc报告基因活性,当加入过量野生型K18后,Bax-luc报告基因活性大大减低,提示K18可能参与了抑制ASPP2的促p53转录激活作用;当用K18 S33A质粒替代K18后,Bax-luc报告基因活性水平与ASPP2单独存在时大致相同。这一结果再次提示K18 Ser33位磷酸化能够促进K18与ASPP2的结合,从而间接抑制ASPP2与p53的结合而发挥凋亡抑制作用。
上述实验研究表明:K18磷酸化在肝细胞凋亡中发挥着重要的作用;正常情况下K18在细胞浆中以Ser33位磷酸化依赖的方式结合ASPP2,在细胞遇到轻度损伤性刺激时,K18 Ser33和Ser52位磷酸化水平均出现增高,使得ASPP2与K18的结合水平增加;但当细胞受到严重刺激时,K18 Ser33位磷酸化水平反而降低,导致ASPP2与K18的结合变得松弛,ASPP2进入到细胞核与p53结合中发挥其促凋亡的作用。
Keratin 18 (K18) is one of the major intermediate filament proteins forming the cytoskeleton of human mature hepatocytes, and its main function is to protect hepatocytes from mechanical and non-mechanical injuries. Mutations, structural reorganization, and changes in phosphorylation in K18 may affect the integrity of hepatocytes and cause their injury. In addition to its role in resisting mechanical injury, K18 also participates in some key signal transduction activities, for example, apoptosis pathways such as Fas/FasL, TRADD have been found to be related to K18 phosphorylation. Studies have shown that K18 phosphorylation level increases in chronic liver diseases with hepatitis C virus (HCV) infection, and the degree of increase is positively associated with liver disease progression. In China , hepatitis B virus (HBV) infection is the most common cause of chronic liver diseases, but there has been no report on the relation of K18 and its phosphorylation to chronic liver diseases with HBV infection. Therefore the study of this relationship will help to reveal the mechanisms of liver injury and hepatocyte apoptosis in chronic liver injury with HBV infection, and it can also be valuable for clinicians in making individualized treatment plan and prognosis.
P53 is an important pro-apoptosis gene; p53-mediated apoptosis pathway plays an important role in hepatocytes injury in chronic liver diseases with HBV infection. Apoptosis Stimulating Protein 2 of P53 (ASPP2) is an important member of the p53 apoptosis stimulating proteins family and it can bind with p53 and selectively increase the pro-apoptotic activity of p53. In our previous work, we found by yeast two-hybrid technique that ASPP2 can bind with K18, which provides a key clue in revealing the relationship between K18 phosphorylation and hepatocyte apoptosis and explore the underlying mechanism. The current study systemically investigated the role of K18 and its phosphorylation in HBV infection related chronic liver diseases and hepatocyte apoptosis and its mechanism of action.
1. The function and significance of K18 phosphorylation in HBV infection related chronic liver diseases:
To investigate changes of K18 phosphorylation in the progression of chronic B type hepatitis (CHB) and its significance, liver tissue specimens were collected from 12 healthy donors (used as control), 40 chronic hepatitis B patients, 21 cirrhosis patients (11 inactive, 10 active) and 5 patients of severe chronic hepatitis. The cellular location of K18 expression and its phosphorylation level in different stages of CHB was first analyzed with immnofluorescence staining. In normal liver tissues, K18 expression level was high and distribute in a net form. Its expression level did not change much in chronic hepatitis. However in cirrhosis and chronic severe hepatitis, normal structure of liver tissue was damaged and K18 was expressed at high levels in proliferative bile duct epithelial cells. The phosphorylation of K18 Ser-33 and Ser-52 was low in normal liver tissues but as liver injury intensified the level increased markedly in proliferative bile duct epithelial cells.
Western Blotting was used to analyze K18 phosphorylation in liver tissues.
(1) Liver tissues were divided into normal, chronic hepatitis and cirrhosis. K18 Ser52 phosphorylation showd low level in normal liver, increased in chronic hepatitis and reached high level in cirrhosis. K18 Ser-33 phosphorylation in CHB and cirrhosis were both higher than normal tissue, but showed no evident difference between the two groups.
(2) Chronic hepatitis was further divided into three groups (minimal histological lesions, MiH; medium lesions, MeH; advanced lesions, AdH) and cirrhosis were divided into inactive cirrhosis (inactive-LC) and active cirrhosis (active-LC). K18 phosphorylations were compared among the five groups. K18 Ser-52 phosphorylation was increased along with the progression of liver disease. From normal liver to active cirrhosis, there are distinctive differences between consecutive groups. The relative level of Ser52 phosphorylation follows the trend of Ser52 normal < MiH < MeH < AdH < inactive LC < active LC. K18 Ser33 phosphorylation also displayed increasing trend from normal tissue to active cirrhosis. From MiH to AdH in chronic hepatitis, K18 Ser33 phosphorylation increased evidently (p<0.005). But K18 Ser33 phosphorylation showed low level in inactive-LC and was almost equivalent to MiH(P = 0.750). K18 Ser33 phosphorylation level in AdH was also equivalent to active-LC (P = 0.715). The relative level of Ser33 phosphorylation follows the trend of normal < MiH≈inactive LC < MeH < AdH≈active LC.
(3) To further investigate the relationship of K18 phosphorylation and the total injury in liver tissue, K18 phosphorylation was analyzed to compare with the most commonly used clinical marker ALT. 40 CHB livers were divided into 3 subgroups (1, ALT < 40 U/L; 2, 40≤ALT <200 U/L; 3, ALT≥200 U/L). The mean relative levels of Ser52 phosphorylation in normal control liver and the three groups were: 0.73, 0.72, 1.77 and 2.71. Ser52 phosphorylation was equivalent in the control and subgroup 1 (P = 0.976), while significantly increased subgroups 1 and 2 (P < 0.001), and subgroups 2 and 3 (P < 0.001) . The positive correlation between levels of phosphorylation and ALT activities suggested that the phosphorylation of Ser52 in K18 was progressively related to liver injuries but had no direct relation with HBV infection. K18 Ser33 phosphorylation increased along with ALT, the mean relative level of the four subgroups were: 1.02, 1.96, 2.53 and 3.00. It was notable that K18 Ser33 phosphorylation increased in subgroup 2 and had significant difference to normal liver. This results indicated that K18 Ser33 phosphorylation might be related to HBV infection, therefore, K18 Ser-33 still increased in non-injured liver.
(4) To further investigate the effect of HBV infection on K18 phosphorylation, a liver specimen respectively from a randomly selected inactive HBV carrier and a healthy donor was sectioned, and probed for HBsAg and phosphorylated Ser33 or Ser52. Strong signals of phosphorylated Ser33 were restricted to hepatocytes with strong positive detection of HBsAg. However, only basal levels of Ser33 phosphorylation were detected in the control. Basal levels of Ser52 phosphorylation were observed in both HBV positive and healthy samples. These results reiterated the conclusion that phosphorylation at Ser33 in K18 was an indicator of HBV infection.
This study indicated: K18 phosphorylations were important injure markers of CHB. K18 Ser52 phosphorylation was a marker of progression of CHB; K18 Ser33 phosphorylation may be an early indicator of HBV infection and marker of liver inflammation.
2. Study of the function of Keratin 18 and p53 apoptosis stimulate protein ASPP2 in hepatocyte apoptosis
K18 was found in our previous work to bind to ASPP2 by Yeast two-hybrid. To further confirm the interaction of the two proteins and investigate the significance, we studied from three aspects.
(1) Study of the function of K18 phosphorylation and ASPP2 in apoptosis of hepatocytes: It was found by immnofluorescence double-staining that ASPP2 and K18 both located in cytoplasm in normal cells. But when cells were treated with drugs and induced into apoptosis, ASPP2 entered into nucleus. HepG2 cells were then treated with MMS with different concentration, apoptosis were stainedby Annexin V/PI and analyzed by Flow cytometry. It was found that total K18 decreased but ASPP2 and K18 Ser52 phosphorylation increased along with the increase of cell apoptosis level. Ser33 phosphorylatoin increased under low level MMS, but decreased under high level MMS. The results above indicated: when hepatocytes were apoptosis, ASPP2 entered into nucleus to participate in p53-mediated apoptosis pathway. K18 Ser33 or Ser52 phosphorylation had close relationship with hepatocytes apoptosis.
(2) Determination of binding domain of ASPP2 and K18: Recombination GST-mASPP2, ASPP2 N-terminal and C-terminal fusion proteins were prepared to Co-IP with K18 fusion protein in vitro. It was found that ASPP2 N-terminal can bind with K18. Then ASPP2 N-terminal was further truncated. The small fragment fusion proteins were expressed to Co-IP with K18. 83-105aa of ASPP2 N-terminal was found to be the key domain of binding. The followed amino acid residues of N-terminal were correlated with binding ability. 293T cells were transfected with K18 ,S33A K18 or S52A K18, then total protein were extracted to Co-IP with ASPP2 in semi-in vivo. It was found that ASPP2 could combine with K18, but the hybrid signal was much lower in cells transfected with mutated K18. The binding signal in cells transfected with S33A K18 was the lowest, which suggested that Ser33 phosphorylation was related with binding of ASPP2 and K18. To investigate the relationship of the combination with the progression of liver disease, total protein of active cirrhosis and inactive cirrhosis tissues were extracted. Then the relationship between the combination of ASPP2 and K18 and the progression of liver disease were analyzed by Co-IP. It was found that ASPP2 binded with K18 much more in inactive cirrhosis, but the hybrid signal was barely detected in active cirrhosis. It indicated that binding of ASPP2 and K18 decreased in serious injury in liver.
(3) Investigation of relationship between combination of K18 and ASPP2 and p53 apoptosis pathway: HepG2 cells were co-transfected with ASPP2 and K18 or S33A K18 or S52A K18. When cells were treated with drugs, ASPP2 entered into nucleus totally in apoptosis cells co-transfected with K18 S33A and ASPP2, but entered into nucleus much less in apoptosis cells co-transfected with K18 S52A and ASPP2 or K18 and ASPP2. This result indicated that K18 Ser33 phosphorylation had close relationship with ASPP2 nucleus translocation. Effect of K18 on ASPP2-p53 and its regulated Bax transcription activity were studied by report gene analysis. It was found that ASPP2 increased the activity of Bax-luc report gene but the activity decreased when excessive wild K18 was added, which indicated that K18 may inhibit the pro-apoptotic effect of ASPP2. When K18 was replaced with S33A K18, Bax report gene activity was almost the same with transfected with ASPP2 alone. This result again confirmed that K18 Ser33 phosphorylation could significantly enhance the combination of K18 and ASPP2, therefore indirectly inhibit ASPP2 binding with p53 and exert its apoptosis inhibition function.
The studies above indicated: K18 and its phosphorylation played important role in hepatocytes apoptosis; under normal circumstances, K18 in cytoplasm combined with ASPP2 in a Ser33 phosphorylation-dependent manner. When cells were suffered with mild injury, K18 Ser33 and Ser52 phosphorylation both increased to enhance the binding of ASPP2 and K18, But when cells suffered from serious injury, K18 Ser33 phosphorylation decreased to make ASPP2 depart from K18 and enter into nucleus to exert its pro-apoptosis function.
引文
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