EWS Is Required For The Development Of Hepatic Steatosis

F-atty liver diseases is caused by a variety of etiological factors which leads to excessive or abnormal lipid deposition in liver.It has been reported that NAFLD(non-acoholic fatty liver disease),also named hepatic steatosis,is a rapidly spreading globle normal problem.In developed countries of Europe,North America and Australia,the incidence of NAFLD is up to 20～40%.In China,NAFLD has been listed as the second liver disease and it just less than the viral hepatitis.Severe NAFLD also promotes liver fibrosis,liver cancers and type 11 diabetes.These data indicate that the prevention and treatment of NAFLD is becoming a significant combate for health.EWSR1(or EWS).Ewing sarcoma breakpoint region 1,is a proto-oncogene in Ewing sarcoma.However,we found that hepatic EWS expressed significantly higher in the patient with NAFLD and in HFD-fed,ob/ob and db/db mice and also in the FA(fatty acid)stimulated primary hepatocyte,AML12 and HepG2 cells.These results proposed that EWS plays an essential role in the development of NAFLD.In order to investigate this relationship.we had constructed an adenovirus for knock-down hepatic EWS.We found that EWS knock-down can obviously reduce lipid droplets and expression of lipid synthesis and storage related gene,and allievate glucose tolerance and insulin resistance and decrease AST/ALT level in liver of HFD,ob/ob and db/db mice.Those beneficial changes are good for liver’function.In contrast,overexpression of EWS significantly promoted hepatic lipid accumulation in mice fed with normal food.Thus,EWS is able to promote NAFLD or hepatic steatosis.Then.we found that EWS entered the nucleus and interacted with MED1 involving in the transcription of adipogenic genes in HFD fed mice and FA treated hepatocytes.These data indicated that downregulation of hepatic EWS is helpful to reverse NAFLD.Next,we found that degradation of hepatic EWS protein depended on the ubiquitin proteasome system mediated by the E3 ligase.Among them.Nedd4L may be the main E3 ligase for degradation of EWS.By its WW domains.Nedd4L specifically interacted with EWS which contains typical PY motif resulting in degradation of EWS.PY motifs in the 187th and 191st amino acid of EWS protein were indispensable for recognition by WW domains of Nedd4L.In a word.Nedd4L is a negative regulator of NAFLD by triggering ubiquitin-mediated degradation of EWS.To confirm this role,we constructed an adenovirus for hepatic Nedd4L overexpression(Ad-Nedd4L)which was injected into HFD fed mice,ob/ob and db/db mice.These results showed that overexpression of Nedd4L inhibited hepatic lipid depositon in mice,reduced expression of genes related to lipid synthesis and storage,and ameliorated glucose tolerance and insulin resistance in mice with NAFLD.and decreased hepatic AST and ALT level.Eventually,overexpression of Nedd4L was able to alleviate NAFLD.On the contrary,knock-down of hepatic Nedd4L of NFD mice may significantly enhance lipid droplet accumulation,and expression of lipid synthesis and storage related genes.Taken together,the inhibition of hepatic steatosis by Nedd4L has been demostrated in mice liver and hepatocytesInterestingly,we detected that endogenous EWS protein was upregulated with decreased Nedd4L expression in the liver of HFD fed mice and in FA-stimulated primary hepatocyte.We also found both interaction of EWS with Nedd4L and ubiquitination of EWS protein was weaken coupled with the aggravation of NAFLD.In a word,the above results suggested that the overexpression of Nedd4L in hepatocytes can effectively reverse hepatic steatosis.Whether Nedd4L might be a target for drugs of hepatic steatosis?In our study,most of the anti-NAFLD drugs reported in clinic can stimulate expression of Nedd4L in hepatocytes with different mechanisms.Among them,upregulation of Nedd4L was induced by rapamycin,metformin,uosolic,acid,isoglycyrrhizinate,orlistato and gmfibrozil through stabilizing the Nedd4L 3’-UTR and also was caused by atorvastatin,UDCA,sibutramine,vitamin E and fasting through activation of Nedd4L promoter activity.Accordingly,Nedd4L is a common target of the anti-NAFLD drugs suggesting that Nedd4L may be considered to design drug screening systems related to anti-steatosis.Transcriptional activation is a key step in the regulation of Nedd4L expression.In our study,we found that transcription factor AP-2 alpha was a important factor in Nedd4L transcriptional activation.Interestingly,atorvastatin,UDCA,sibutramine.vitamin E,including fasting can enhance combination of AP-2 alpha with NEDD4L promoter resulting in transcription activation of Nedd4L.However,fatty acid could significantly reduce combination AP-2 alpha with NEDD4L promoter leading to inhibition of Nedd4L mRNA expression.In addition,expression of AP-2 alpha was lower in human NAFLD slices and in liver of high-fat fed,ob/ob and db/db mice Overexpression of AP-2 alpha significantly promoted Nedd4L expression in primary hepatocyte.These results suggested that AP-2 alpha might reverse hepatic steatosis through the Nedd4L/EWS/MEDI cascade.In conclusion,oncogene EWS has a novel biological function in promoting NAFLD.and EWS enters nuclei and interacts with MED1 to promote the expression of adipogenic synthesis and storage related genes.Nedd4L,as a E3 ligase,shows reversal effect of the steatosis by degradation of EWS.Next,Nedd4L is a common target of anti-NAFLD drugs.AP-2 alpha is a key transcription factor in activation of Nedd4L transcription.Consequently,we propse that the AP-2 alpha/Nedd4L/EWS/MEDI cascade is the novel biological mechanism in NAFLD.AP-2 alpha,Nedd4L,EWS and MED1 are expected to be new target protein for screening anti-NAFLD drugs.