The Roles Of PKCδ Activation In Endoplasmic Reticulum Stress And Hepatocyte Steatosis

Objective：Nonalcoholic fatty liver disease (NAFLD) is currently a common public healthproblem, which includes simple steatosis, non-alcoholic steatohepatitis(NASH)， andterminal stages such as liver cirrhosis, hepatic failure and even hepatocellularcarcinoma."Two hit hypothesis" is the most widely accepted theory for the pathogenesis ofNAFLD. The "first hit" is insulin resistance, which leads to liver steatosis, while the"second hit" includes endoplasmic reticulum stress(ERS) and mitochondrial dysfunctionwhich underlies the progression from fatty liver to NASH. Therefore, the molecular basis ofERS is under intensive investigations at present.Binding immunoglobulin protein (Bip), namely GRP78or HspA5, an importantmolecular chaperone located in the endoplasmic reticulum, is considered as a molecularmarker for ERS. Previous studies has demonstrate that splicing XBP-1(X-box bindingprotein1) can interact with endoplasmic reticulum stress response element(ERSE) andinduce the transcriptions of not only Bip and C/EBP homologous protein (CHOP) genes,but also ER degradation-enhancing-mannosidase-like protein (EDEM) gene. Therefore,XBP-1is considered as another indicator for ERS.PKC (protein kinase C) is a lipid-dependent serine/threonine protein kinase located inthe endochylema of mammalian cells, including at least12subtypes. PKCδ is a member ofthe novel PKC (nPKC) family and is mainly expressed by hepatocyte.Unlike cPKC andaPKC, PKCδ can be activated by diacylglycerol (DAG), the metabolite of free fatty acids,to promote apoptosis of hepatocytes via activating the c-jun N-terminal kinase (JNK) andCHOP. It has been reported that PKCδ activation is tightly associated with the pathogenesisof diabetes and other related diseases. Insulin resistance is markedly improved in the liversof mice with systematic or liver specific knocked out of Prkcd gene, which encodes thePCKδ. Moreover, liver and plasma triacylglycerol levels and epididymal fat mass were reduced in Prkcd/mice. These findings indicate that PKCδ may be involved in hepatocytesteatosis, however it remains unknown whether PKCδ activation can contribute to ERS andNASH. Therefore, we examined the roles of PKCδ activation in hepatocyte steatosis andERS. Our findings may shed light on the identification of novel therapeutic target forpreventing the progression from fatty liver to NASH.Methods：1. Establishement of an in vitro hepatocyte steatosis model using L02cells.1.1.1. Human liver cell line L02was cultured in Dulbecco’s Modified Eagle’sMedium (DMEM), supplemented with10%fetal bovine serum (FBS). Cells were deprivedof FBS overnight and then treated with1%FAF-BSA,0.5mM long chain free fatty acids(LCFA), oleic acid/palmitate for2h,4h,8h, or16h to induce steatosis. Control cells weremaintained in culture medium with vehicle only.1.2Oil red O staining showed intracellular lipid droplets of each group would indicatethe degree of steatosis.1.3The intracellular triglycerides(TG) content of each group was tested by TGdetection kits.2Examination of mRNA levels of PKCδ, Bip and XBP-1s by Real-time PCR.3Detection of PKCδ, Bip and XBP-1s expression by Western blot.4The effect of PKCδ activation on ERS, as indicated by expressions of Bip andXBP-1s.4.1Knockdown of PKCδ by siRNA transfection.4.2Detection of ERS by expressions of Bip and XBP-1s mRNA.4.3Detection of ERS by expressions of Bip and XBP-1s protein.5Detection of hepatocyte steatosis in cells with PKCδ knockdown.6Using SPSS18.0software to the experimental data for statistical analysis.Results：1Successful establishement of hepatocyte steatosis model using L02cells by oleicacid/palmitic acid (2:1) treatment.Oil red O staining showed time-dependent increases in fatty acid orange lipid dropletsin the cells. ImageJ2X analysis demonstrated that the average LD area per cell, whichindicated the abundance of lipid droplets of fatty acid group, was significantly increased in L02cells treated with oleic acid/palmitic acid (2:1)(P <0.01). The increases of intracellularTG content increased with extended incubation with oleic acid and palmitic acid treatment.2Compared with the control group, the L02cells undergoing steatosis showedtime-dependent increases in the mRNA levels of PKCδ, Bip and XBP-1s(P <0.05).3The expression of PKCδ, Bip and XBP-1s proteins.Western blot analysis demonstrated that the levels of PKCδ, Bip, and XBP-1s proteinswere higher in fatty acid group than the control group. Moreover, mixed fatty acid inducedThr505phosphorylation of PKCδ, which correlated with its full activation. The levels ofphospho-PKCδ were increased at early stage of the hepatocytes steatosis, reached itshighest abundance at4h post treatment and decreased after that.4The effect of PKCδ knockdown on the expression of Bip and XBP-1s.Real-time PCR and Western blot showed that Bip and XBP-1s expression wassignificantly lower in the cells transfected with PKCδ-siRNA than control group (P <0.05).5The effect of PKCδ knockdown on the steatosis of L02cells.Oil red O staining revealed that the percentage of steatosis cells in PKCδ-siRNAtransfection group was lower than control treated cells (P <0.01). The intracellular TGcontent in PKCδ-siRNA transfection group was lower than control treated cells(P <0.05).Conclusion：1We observed concomitant increases in the levels of Bip and XBP-1s expression andPKCδ activation during the process of hepatocyte steatosis.2PKCδ knockdown attenuated ERS, as indicated by the levels of Bip and XBP-1s, aswell as the steatosis of L02cells.3PKCδ might be a key molecule regulating both hepatocellular lipid overload andERS in the pathogenesis of NASH. Aberrant expression of PKCδ may contribute to lipidaccumulation and thus result in ERS.