Study Of The Functions And Mechanisms Of VPS33B In Hepatocyte Polarity

The hepatocyte is a kind of highly polarized epithelial cell,and defects in hepatocyte polarization result in pathophysiological consequences,such as hepatitis,liver cirrhosis and liver cancer,etc.Arthrogryposis,renal dysfunction and cholestasis(ARC)syndrome is an autosomal recessive disorder involving multiple organs dysfunction.In terms of the liver,the hepatocytes lack polarity,some patients have hepatitis,which may develop liver cancer.But the functions and mechanisms of VPS33B in hepatocyte polarity and related diseases are still unclear.We examined the expression and distribution of VPS33B in human HCC samples.Our results showed that expression of VPS33B exhibited various degrees of reduction in HCC samples,as the tumors malignancy degree increased.Reduction of VPS33B was further confirmed in 2 hepatocarcinoma mouse models,including anti-CD137 mAb-treated HBV-transgenic mice and diethylnitrosamine(DEN)-induced liver cancer model.These results suggest that VPS33B may be involved in the occurrence and development of hepatitis and liver cancer.To further investigate the physiological function and molecular mechanism of VPS33B in liver,we generated the liver-specific Vps33b knockout mice(Vps33b~f/f/f Alb-Cre~+)and performed experiments using mice at the age of 1,4,8 and 12 months.We found that the liver-specific Vps33b knockout mice at the age of 1 month showed no difference in appearance,weight and structure of liver.Some indices related to liver function and blood lipid were measured with biochemical methods,and we found that VPS33B deficiency could lead to liver damage and aberrant plasma cholesterol from the age of 1 month.For further study,we analyzed the expression and location of molecules involved in cholesterol transport in hepatocytes.We showed that polarity disorder happened in Vps33b deficient hepatocytes.APOE,responsible for the transfer of cholesterol to the cytoplasm tended to locate at the hepatocyte canalicular membrane,and the expression of its receptor LRP1 decreased slightly.ABCG8,involved in the secretion of cholesterol to bile duct,was abnormally distributed in the cytoplasm.the mice at the age of 12 months exhibited the more pathologic features,including increase of relative liver weight,hepatomegaly,inflammatory cells infiltration and multiple tumor nodules.The incidence of liver cancer is about 42.9.Immunohistochemistry and western blotting results demonstrated that,similarly to ARC syndrome patients,adhesion junction protein E-cadherin expression was decreased.VPS33B is widely expressed in many fetal and adult tissues,and it may function in different tissues and cells by binding to various proteins.Immunoprecipitation experiment and mass spectra were performed using HepG2 cells to assess the interacting proteins with VPS33B.We found that VPS33B associated with VPS16B,SEC22B and FLOT1,these proteins may play a role in the hepatocyte polarization in the form of complex.For the first time,our study analyzed the impact of the Vps33b knockout on the liver function,and revealed that VPS33B may play important roles in hepatocyte polarization by binding to some proteins,liver-specific deletion of Vps33b affected the cholesterol transport in hepatocytes and further cause hepatitis and liver cancer.Our study provides important clues of the functions of VPS33B and the molecular mechanisms of liver disease.