The Functions Of SUMO Protease SENP3 On Activation Of Macrophage,Gene Expression Profile And Phenotype Of Mice

SUMO(small ubiquitin-related modifier)is an important ubiquitin like protein.SUMOylation affects stability,sub-cellular localization and transcription of proteins,thus regulating diverse biological events.SUMO is involved in many physiological activities and diseases such as tumor,diabetes,neurodegenerative diseases and brain injury.SUMOylation is a dynamic and reversible process.SUMO can be removed from its substrates by sentrin-specific proteases(SENPs)family.Our previous work has showed that SENP3,the only redox-sensitive SUMO2/3-specific protease in SENP family,accumulates under cellular redox stress.It can re-program the gene expression and promote the malignant behavior of cancer cells by de-SUMOylation of many molecules,especially transcription factors,under oxidative stress.Here,we focus the role of SENP3 in inflammation both in vitro and in vivo by using Helicobacter pylori(H.pylori)to induce macrophage activation at cellular level and gastrititis in mouse as model system,as well as combining related patient samples.We found SENP3 regulates the inflammatory signaling pathway in macrophage cytoplasm by de SUMOylating an important substrate protein.We also observed the significant changes in the progression of H.pylori-induced gastritis in SENP3 knocked down cells and mice.At the same time,we carried out the analysis of the digital gene expression profile after SENP3 knocked down in gastric cancer cell line SGC7901,trying to understand the complete effects of SENP3 on gene expression.We also made SENP3 macrophage-specific knockout mice to elucidate the function of SENP3 on macrophages and gastritis.SENP3 heterozygous knockout mice were applied to understand the role of SENP3 on the general growth,metabolism and disease development.Gastric carcinoma is caused by multiple factors and undergoes stepwise progression,which are closely associated with H.pylori-induced chronic inflammation.However,there is rarely report of(De)-SUMOylation process in gastritis and gastric carcinoma.Therefore,finding SUMO substrates in gastritis and gastric carcinoma and elucidate the mechanisms will greatly advance the treatment.Chronic inflammation causes cellular oxidative stress and adaptation.Increased Reactive Oxygen Species(ROS)and monocytes/macrophage activation-induced inflammation are observed in the inflammatory microenvironment.However,it is not clear whether ROS is involved in regulating protein post-translational modifications.Our previous studies revealed a positive correlation between number of macrophage and expression level of SENP3 in mesenchyme of human gastritis tissue infectd with H.pylori.This suggests that SENP3 in macrophages may be involved in the development of H.pylori-induced gastritis.Therefore,we explored whether SENP3-mediated oxidative stress response in macrophage contributes to macrophage activation and development of gastritis.In the first part of this study,we investigated the molecular mechanism of macrophage activation by SENP3,and revealed the role of SENP3 in gastritis.Using 1)H.pylori and macrophages co-culture system and 2)H.pylori-induced chronic gastritis modelwith C57 mice,we observed an H.pylori-induced SENP3 accumulation through ROS stimulation.We further proved that macrophages are activated by SENP3 elevation by knocking down SENP3.We then found that SENP3 promotes phosphorylation of JNK,which suggests that SENP3 may De-SUMO2/3 modification of MAP2 K in JNK signaling pathway.Moreover,we revelaed that JNK specific kinase MAP2 K of MKK7 can be modified by SUMO3,and K18 is the SUMO site confirmed by co-immunoprecipitation.To clarify how MKK7 SUMOylation regulates p-JNK,we applied the SUMO-MKK7 fusion protein immunoprecipitation experiments and proved that SUMO site of MKK7 was among the docking domain of MKK7 thus blocking the interaction of MKK7 and JNK.At last,we confirmed SENP3 promoting the development of gastritis in macrophage-specific knockout mice of SENP3.These findings demonstrate that SENP3 acts as a redox sensitive protein regulateing the post-translational modification of cytoplasmic molecules to promote macrophage activation and the development of gastritis.It provides us new evidence of the role of ROS in inflammation and lay the foundation for the follow-up studies of the relationship between ROS,inflammation and gastric carcinoma.Many substrates of SENP3 localized in nucleus,among which the majority are transcription factors.Our previous study found a series of substrates of SENP3 in gastric carcinoma and other tumors,including transcription factors FOXC2,SP1,STAT3 and p53.SENP3 De-SUMOylates these transcription factors thereby facilitating cancer development.Therefore,we started the second part of our study by knocking down SENP3 in gastric cancer cell SGC7901 and detect the expression profile of digital genes,so that we can reveal the complete inventory of genes that involed in development of SENP3-associated gastric cancer with additional emphasis on those which are transcription factors.1379 genes were found with altered expression level after SENP3 knocked down,including 318(23.1%)up-regulaed genes and 1052(76.9%)down-regulated ones.Most of them pertain to signaling pathways related to metabolism,ubiquitin-mediated proteolysis and protein processing in the endoplasmic reticulum.First,we investigated the signaling patway of SENP3-invloved/related ubiquitin-mediated proteolysis.Ubiquitin modification was decreased by knocking down SENP3 in SGC7901 and increased by SENP3 overexpression,which indicates that SENP3 affects protein ubiqutination through its regulatory role on ubiquitin enzymes.Mouse genome is the closest to that of human’s.Thus,gene KO mouse model has been a popular tool to study gene functions in human diseases.Since SENP3-/-mice died of early embryos,we introduced and bred SENP3+/-mice in our lab.We found SENP3+/-mice present developmental retardation with smaller body size compared to their wild type littermates.In the trird part,by recording and analizing the phenotypes of SENP3+/-mice,we characterize the biological function of SENP3 upon whole organism which shall lay the foundation for futher study the role of SENP3 in human diseases.