The Role Of Keratin 18 And Its Phosphorylation In HBV Infection Induced Chronic Liver Diseases And Hepatocyte Apoptosis

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.