TIP30 Induces P53-depended Apoptosis By Sensing Cellular Oxidative Stress

Backgroud and ObjectiveLiver as the main detoxic organ,abundant of mitochondria and lysosome,is active in metabolism.As byproducts of normal aerobic metabolism,reactive oxygen species act as second messager in various signal transduction pathways maintaining the cell senescence and death.The hepatocellular carcinoma cells evade the cell senescence and death predominantly due to the deregulated expression of the tumor suppressor gene.TIP30,also called CC3 or HTIP2,is a putative tumor suppressor.Recently,the decreased expression of TIP30 was observed in hepatocellular carcinoma(HCC) surgical specimens and cell lines.Consistent with these observations,genetically engineered mice deficient in Tip30 gene had a high incidence of HCC and other tumors.These data suggest that TIP30 plays an important role in the suppression of hepatocarcinogenesis.The suppressive effects of TIP30 on tumor development could partially be ascribed to its proapoptotic property.A recent crystal structure analysis indicated the potential of TIP30 acting as a metabolic sensor linked to its proapoptotic property.The protein structures most closely related to TIP30 are members of the shortchain dehydrogenase reductase family.In addition,a TIP30 homodimer was present in the crystal across a 2-fold axis,which involved a disulfide bridge via Cys172 residues and a PEG 600 molecule shared between two TIP30 monomers.Because cysteine thiol-disulfide exchanges are crucial for sensing intracellular levels of ROS and signal transduction,TIP30 therefore might be oxidized with disulfide bridge formation under oxidative stress and executes its antitumor activities.Although gene expression is crucially modulated by transcription,the essential contributions of posttranscriptional events are becoming increasingly recognized.Unlike transcription modulation that relied upon new molecular synthesis,mRNA turnover is primarily controlled as an early immediate response to various cellular stimuli through the association of RNA-binding proteins with specific RNA sequences.The RNA-binding protein HuR prominently regulates gene expression through binding to mRNAs that contain AU-rich elements(ARE) located in the 3'-untranslated regions(3'-UTR).HuR has emerged as a key regulator of genes that are central to the stress response,cell growth,and proliferation,such as p21,c-fos,c-myc,p53,cyclin A,and cyclin B1.HuR is shown to promote the stability of target transcripts and to enhance target mRNA translation in response to agents causing DNA damage.HuR is predominantly nuclear in unstimulated cells and translocates to the cytoplasm in response to various stimuli,which are intimately linked to its effects on target mRNAs.Importinβ2,also known as transportin,was found to participate in the nuclear import of HuR.TIP30 was recently found to form complex with multiple karyopherins of the importinβfamily in a RanGTP insensitive manner,by which it inhibited nuclear import and induced apoptosis.Thus,TIP30 might regulate HuR distribution through interaction with importinβ.We have previously found that TIP30 might induce apoptosis through elevation of p53 protein expression,which was not due to stabilization of p53 protein.In this study,we show that TIP30 is oxidized with formation of intermolecular disulfide bridge under oxidative stress,resulting in cytoplasmic accumulation of HuR,stabilization of p53 mRNA, and induction of mitochondria mediated apoptosis.Methods(1) ROS level was analyzed by flow cytometry using an H₂O₂-sensitive fluorescent dye,dichlorodihy rofluorescein diacetate.(2) Apoptosis was examined by assessing nuclear changes indicative of apoptosis using the DNA-binding dye Hoechst 33342.Tetramethylrhodamine ethylester (TMRE) to assess mitochondrial membrane potential changes.(3) Total cell lysate was prepared in radioimmunoprecipitation assay buffer. Proteins at the same amount were separated by 10%to 15%SDS-PAGE and transferred onto polyvinylidene difluoride membranes.To monitor TIP30 redox state,protein extracts were separated by nonreducing SDS-PAGE withoutβ-mercaptoethanol.We also conducted FRET assay to investigate whether TIP30 forms homodimer under oxidative stress in living cells.(4) mRNA expression was determined by quantitative reverse transcription-PCR (RT-PCR) using the LightCycler system.(5) Nuclear and cytoplasmic extracts were made using NE-PER Nuclear and Cytoplasmic Extraction Reagent and HuR distribution was measured by Western blotting.(6) We then used a siRNA to decrease the mRNA expression.(7) Coimmunoprecipitation analysis to test the protein-protein or protein-mRNA interaction. Results(1) The intracellular ROS level was much higher in 5 HCC cells tested than that in normal liver cell HL7702.Infection of Ad-TIP30 caused significant increase of cell death in HepG2 cells which had high intracellular ROS level,compared to Ad-GFP infection.Whereas no obvious difference was found in HL7702 cells which had low intracellular ROS level.Treatment with antioxidant NAC greatly suppressed TIP30-induced cell death in HepG2 cells.In contrast, TIP30-induced cell death significantly increased in HL7702 cells with the addition of H₂O₂.(2) We then investigated the redox forms of the TIP30 in normal liver cell HL7702 using the cysteine-trapping method.Inspection of the Western blotting under non-reducing conditions revealed a faint anti-TIP30 stained higher molecular weight(HMW) band at various low doses of H₂O₂,whose presence correlated with TIP30 oxidation.The absence of this band under reducing conditions indicated a probable intermolecular disulfide linkage of TIP30 and HMW proteins.We further analyzed the redox forms of His-tagged TIP30 in HepG2 cells,in which TIP30 was almost undetectable,by immuno-blotting with an anti-His antibody.Again,a distinct slower mobility band was found after exposure to various concentrations of H₂O₂.The mutant containing Cys172 substituted into alanine lost its ability to form oxidized TIP30 band.The data indicates the formation of a TIP30 mixed-disulfide,involving Cys172,under oxidative stress.Upon H₂O₂ exposure,the enhanced fluorescence from cytoplasm was clearly detected in the FRET channel after correction for bleed-through of CFP and YFP.The interaction between TIP30-CFP and YFP-TIP30 was disrupted upon antioxidant NAC treatment.(3) Introduction of TIP30 resulted in increases of p53 mRNA compared to mock treatment in HepG2 cells harboring wild type p53 gene,which was augmented by the addition of H₂O₂ and attenuated by the addition of NAC.Western blotting confirmed the regulatory effects of TIP30 on p53.Consistent with the expression of p53,the transactivation activity of p53,as detected by p21,puma, mdm2 and bax mRNA expression,was enhanced upon ectopic expression of TIP30 in HepG2 cells,which was significantly augmented by oxidant H₂O₂ and blocked by antioxidant NAC.(4) To investigate whether TIP30-induced cell death was p53-dependent in HepG2 cells,we generated a HepG2 cell line stably expressing p53 shRNA.Depletion of p53 greatly attenuated TIP30-induced apoptosis.Moreover,introduction of TIP30 resulted in a significant loss of mitochondrial membrane potential in HepG2 cells,but not in Hep3B cells harboring p53 gene null.Depletion of p53 blocked TIP30-induced mitochondrial membrane potential decrease in HepG2 cells.(5) Assays were carried out to measure the stability of p53 mRNA by monitoring the rate of p53 mRNA clearance in HepG2 cells where transcription was halted by actinomycin D.Introduction of TIP30 significantly enhanced the stability of p53 mRNA.Addition of H₂O₂ significantly enhanced TIP30-induced stabilization of p53 mRNA,whereas NAC treatment reduced the stabilization. Use of biotinylated p53 transcripts in RNA pull-down assays revealed and association of the p53 mRNA 3'-UTR with HuR in lysates prepared from Ad-GFP-infected cells.The association significantly increased when using lysates prepared from Ad-TIP30-infected cells.In contrast,no such complexes were found when using biotinylated p53 coding region transcript(devoid of AU-rich regions).The in vivo association of endogenous p53 mRNA and HuR in HepG2 cells was analyzed through immunoprecipitation of HuR under the conditions that preserved its association with target mRNAs.RT-PCR analysis revealed the presence of endogenous p53 mRNA in the material immunoprecipitated with anti-HuR antibodies,but not with nonspecific antibodies(IgG1).The p53 mRNA was more abundant in Ad-TIP30-infected cell lysates.The association of HuR with p21 mRNA,which also contains ARE in its 3'-UTR,was also enhanced upon introduction of TIP30.We then used small interfering RNA(siRNA) targeting the HuR mRNA,which caused decreased expression of HuR mRNA,to examine the role of HuR in regulation of p53 mRNA expression following TIP30 transduction.TIP30-induced p53-upregulation and cell death was greatly attenuated by HuR depletion in HepG2 cells.(6) To further investigate the mechanism by which TIP30 promotes the association of HuR with p53 mRNA 3'-UTR,the expression of HuR was examined. Whole-cell HuR levels did not change with ectopic TIP30 expression in HepG2 cells.In contrast,its cytoplasmic presence significantly increased upon TIP30 introduction.This cytoplasmic accumulation of HuR was enhanced by addition of H₂O₂ and blocked by NAC treatment.We constructed a TIP30 mutant(TIP30F187A,F188A,G189A),named TIP30M6,in which the amino acids(residues 95-98,141-142 and 187-189 of TIP30) predicted to interact with Importinβ2 were substituted into alanine.TIP30M6 lost the capability to induce cytoplasmic HuR accumulation.Co-immunoprecipitation analysis showed that HuR interacted with His-Importinβ2 by immunoprecipitation with anti-His antibody in the cytoplasmic extracts of HepG2 cells.When TIP30 was introduced,accompanying with the interaction of TIP30 with Importinβ2,the interaction of Importinβ2 with HuR was decreased.Whereas TIP30M6, which was unable to form complex with Importinβ2,did not block the interaction of Importinβ2 with HuR.Moreover,treatment with NAC blocked the interaction of TIP30 with Importinβ2.ConclusionsReactive oxygen species(ROS) and cellular oxidant stress have long been associated with cancer.Here we demonstrate that TIP30,also called CC3, regulates p53 mRNA stability and induces apoptosis by sensing of intracellular oxidative stress in human hepatocellular carcinoma(HCC) cells.Our results suggest that TIP30 is involved in cellular oxidative stress surveillance and induces apoptosis through stabilization of p53 mRNA in HCC cells.