RNA-binding Protein HuR Regulates XIAP MRNA Stability And Translation In IEC-6 After Polyamine Depletion And Glycyrrhizic Acid Treatment

IntroductionMaintenance of intestinal mucosal epithelial integrity is a balance between cell proliferation,growth arrest,and apoptosis.The inhibitor of apoptosis proteins(IAPs), including XIAP,c-IAP1 and c-IAP2,function as potent natural suppressor of apoptosis and play a critical role in the control of intestinal epithelial cell(IEC) apoptosis,but the exact mechanisms by which IAP expression is regulated at the molecular level remain elusive. The RNA-binding protein HuR binds to many labile mRNAs beating U-or AU-rich elements(AREs) and modulates their stability and translation.Given the computationally predicted HuR-hits in the 3'-untranslated region(3'UTR) and coding region(CR) of XIAP mRNA,this study determined if HuR interacts with the XIAP mRNA and regulates its expression posttranscriptionally in IECs.METHODS1.Plasmids Construction and transient transfectionsThe complete coding region of XIAP without star codon was amplified and the resulting PCR fragment was cloned downstream of the EGFP coding region in plasmid pEGFP-N1(BD Biosciences).The complete 3'UTR of XIAP was amplified by PCR and the resulting PCR fragment was cloned uptream of the EGFP coding region in plasmid pEGFP-C1(BD Biosciences).Transient transfection was performed with Lipofectamine reagent(Invitrogen).2.RNA interferenceFor HuR RNAi analysis,a siRNA pool directed to the coding region of HuR (HuR-siRNA) and the corresponding control siRNA(Ctrl-siRNA) were purchased from Dharmacon.For XIAP RNAi analysis,The XIAP-siRNA and the corresponding C-siRNA were synthesized and purchased from Invitrogen.For each 60-mm cell culture dish,10μl of the 20μM stock siRNA was mixed with 300μl of Opti-MEM medium(Invitrogen).This mixture was gently added to a solution containing 15μl of Lipofectamine 2000 in 300μl of Opti-MEM.The solution was incubated for 20 min at room temperature and gently overlaid onto monolayer of cells in 3 ml of medium.3.Preparation of Synthetic RNA Transcripts and biotin pulldown assaycDNA from IEC-6 cells was used as a template for PCR amplification of the coding region(CR) and 3'-UTR of XIAP.The 5'-primers contained the T7 RNA polymerase promoter sequence(T7):were used to amplify XIAP 3'-UTR and CR,PCR-amplified products were used as templates to transcribe biotinylated RNAs by using T7 RNA polymerase in the presence of biotin-cytidine.For pulldown assays,biotinylated transcripts (15pmol) were incubated with 120μg of cytoplasmic lysate for 30 min at room temperature. Complexes were isolated with paramagnetic streptavidin-conjugated Dynabeads(Dynal, Oslo,Norway) and analyzed by Western blot analysis.4.Immunoprecipitation(IP) of RNP complexesFor IP of ribonucleoprotein complexes(endogenous RNA and RBPs) to assess the association of endogenous HuR with endogenous XIAP mRNAs,whole-cell lysates from treated or untreated cells were divided into two equal parts,and incubated for 2h at room temperature with Protein-A Sepharose beads precoated with IgG1 or anti-HuR antibody. The beads were washed five times with NT2 buffer,incubated with RNase-free DNaseⅠ(15 min,30℃) to digest DNA,washed twice with NT2 buffer,further incubated with 100μl NT2 buffer containing 0.1%SDS and 0.5 mg/ml Proteinase K(30 min,55℃) to digest the proteins bound to the beads,and centrifuged at 4℃.RNA in the supernatant was precipitated in the presence of glycoblue and extracted by using phenol and chloroform. The resulting RNA was used in RT followed by PCR analysis to detect the presence of XIAP mRNA.5.RT-PCR and Real-Time PCR AnalysisTotal RNA was isolated by using RNeasy mini kit(Qiagen,Valencia,CA) and used in reverse transcription and PCR amplification reactions as described(Liu et al.,2005).The levels of glyceraldehyde-3-phosphate dehydrogenase(GAPDH) PCR product were assessed to monitor the even RNA input in RT-PCR samples.Real-time quantitative PCR (Q-PCR) was performed using 7500-Fast Real-Time PCR Systems(Applied Biosystems, Foster City,CA) with specific primers,probes,and software(Applied Biosystems).The levels of XIAP mRNA were quantified by Q-PCR analysis and normalized by GAPDH levels.6.Immunofluorescence StainingImmunofluorescence was performed as described(Li et al.,2001a b) with minor changes(Vielkind and Swierenga,1989).Cells were fixed using 3.7%formaldehyde,and the rehydrated samples were incubated overnight at 4℃with primary antibody anti-XIAP diluted 1:300 in blocking buffer and then incubated with secondary antibody conjugated with Alexa Fluor-594(Molecular Probes,Eugene,OR) for 2 h at room temperature.After rinsing,slides were incubated with 1μM TO-PRO3(Molecular Probes) for 10 min to stain nuclei,rinsed again,mounted,and viewed through a Zeiss confocal microscope(model LSM510,Thornwood,NY).Images were processed using PhotoShop software(Adobe,San Jose,CA). Results:1.RNA-binding protein HuR binds to endogenous and recombinant XIAP mRNAFirst,we assayed the existence of such RNPs by employing biotin pulldown analysis. Biotinylated transcripts spanning either the coding region(CR) or the 3'UTR of the XIAP mRNA were prepared and incubated with using whole lysates from IEC-6 cells or CDX-2 cells.After pulldown using streptavidin-coated beads,Western blot analysis revealed thin HuR associated specifically with XIAP CR and 3'UTR transcripts.Second,we examined whether HuR binding to the XIAP CR and 3'UTR is mediated through the specific sites containing predicted hits of the HuR motif.Partial biotinylated transcripts spanning various fragments were prepared and their binding with HuR was tested in pulldown assays.HuR was found to bind F-2 of CR and F-4 of 3'UTR,two transcripts that contained HuR motif hits.Third,RNP immunoprecipitation(IP) analysis was performed to study the association of endogenous HuR and endogenous XIAP mRNA using cytoplasmic fractions of IEC-6 and CDX-2 cells.The RNP complexes immunoprecipitated using anti-HuR antibody did contain endogenous XIAP mRNA,as measured by RT-PCR analysis. Importantly,the XIAP mRNA was undetectable in nonspecific IgG1 IPs Together,these findings support the notion that cytoplasmic HuR specifically binds to the XIAP mRNA.2.HuR overexpression increases XIAP mRNA stability and translationTo further define the role of HuR in regulating XIAP posttranscription,we examined the effect of overexpressing HuR on the XIAP mRNA stability in control IEC-6 cells.The adenoviral vector containing the corresponding human HuR cDNA was used to overexpress HuR.HuR protein level increased with Ad-HuR infection in IEC-6 cells.The control adenovirus without exogenous HuR cDNA(Ad-null) failed to induce HuR.To determine if the induction of HuR has effect on XIAP mRNA stability,we measured the XIAP mRNA half-life after overexpression of HuR induced byinfection of Ad-HuR for 48h.As the results shown,overexpression of HuR significantly increased the stability of XIAP mRNA in IEC-6 cells.In control cells infected with Ad-null,the mRNA levels declined rapidly after inhibition of gene transcription by addition of actinomycin D,While,the stability of XIAP mRNA was dramatically increased by overexpreeion of HuR.HuR has also been implicated in controlling the translation of target mRNAs.To test this possibility,we constructed the plasmid pEGFP C1-XIAP 3'UTR by inserting XIAP 3'UTR into the plasmid pEGFP C1 after its reporter gene EGFP coding region,IEC-6 cells were infected with Ad-HuR or Ad-null for 24h,then transfected with pEGFP C1 XIAP 3'UTR or control plasmid pC1 vector,whole cell lysates were collected after tranfection for 48h,and reporter EGFP protein levels were detected by western blot.Comparing to control pasmid,the reporter EGFP protein level increased in presence of pEGFP N1-XIAP 3'UTR, furthermore,EGFP level increased after overexpression of HuR.XIAP full length CR without star cordon was cloned into the plasmid pEGFP N1 by being inserted in front of the reporter gene EGFP.Comparing to the control pasmid,EGFP protein level increased in pEGFP N1 XIAP CR,furthermore,EGFP level increased after overexpression of HuR. Consistently,XIAP protein with Ad-HuR load increased and reached～2,～4,～10,and～12fold higher levels than control cells as Ad-HuR was used at 20,50,100,and 200 pfu/cell respectively.3.HuR silence decreases XIAP mRNA stability and translationThe above results showed that overexpression of HuR increased XIAP mRNA stability and expression.On the other hand,we investigated XIAP mRNA stability and expression after silencing HuR.siRNA targeting HuR was used to reduce HuR levels in control IEC-6 cells,XIAP mRNA was detected by using real time quantity PCR,and the protein level of XIAP and EGFP was detected by using western blot.Comparing to the control,after silencing HuR,stability of XIAP mRNA decreased and XIAP protein also decreased.The level of EGFP increased in the presentation of pC1 XIAP 3'UTR or pN1 XIAP CR,and these increased EGFP were prevent by HuR silence.These results indicated that silence of HuR can decrease XIAP mRNA stability and drcrease XIAP translation through both XIAP CR and 3'UTR..4.Polyamine depletion enhanced HuR binding to endogenous and recombinant XIAP mRNAFirst,we assayed the existence of such RNPs by employing biotin pulldown analysis. Biotinylated transcripts spanning either the coding region(CR) or the 3'UTR of the XIAP mRNA were prepared and incubated with using cell lysates prepared from either untreated or polyamine-deficient cells.After pulldown using streptavidin-coated beads,Western blot analysis revealed that HuR associated specifically with XIAP CR and 3'UTR transcripts. And the binding intensity increased significantly using lysates prepared from cells that were treated with DFMO for 4 d,but was reduced using lysates from cells had been treated with DFMO plus putrescine.Second,RNP immunoprecipitation(IP) analysis was performed to study the association of endogenous HuR,to endogenous XIAP mRNA using cytoplasmic fractions of untreated,DFMO-treated,or DFMO plus putrescine treated cells.The RNP complexes immunoprecipitated using anti-HuR antibody did contain endogenous XIAP mRNA,as measured by RT-PCR analysis.The association of endogenous XIAP mRNA with endogenous HuR increased significantly in cells treated with DFMO for 4 d,but was absent when testing lysates from cells treated with DFMO plus putrescine.Importantly,the XIAP mRNA was undetectable in nonspecific IgG1 IPs.5.Polyamine depletion increases XIAP mRNA stability and translationTo determine if polyamine depletion has influence on XIAP mRNA turnover,we measured the XIAP mRNA half-life under conditions of different polyamine levels. Depletion of cellular polyamines by DFMO significantly increased the stability of XIAP mRNA in IEC-6 cells.The reporter EGFP protein level,western blot was performed using total lysate from the control cells and polyamine deficient cells of transfection with pC1-XIAP 3'UTR,pN1XIAP CR,or control plasmid for 48 hour.Comparing to control pasmid,the reporter EGFP protein level increased in presence of pEGFP C1-XIAP 3'UTR or pN1XIAP CR.These results indicated that polyamines regulate the XIAP expression posttranscriptionally and that polyamine depletion induces mRNA stability and expression.6.HuR silencing abolishes increased XIAP mRNA stability and translation in polyamine deficient IEC-6 cellsSince there was interaction between HuR and XIAP mRNA and polyamine depletion enhanced cytoplasmic HuR,XIAP mRNA stability and translation,we hypothesize that increased stability of XIAP mRNA and XIAP protein were due to the increased cytoplasmic HuR after polyamine depletion.To test the possibility,siRNA targeting HuR was used to reduce HuR levels in polyamine deficient cells,XIAP mRNA was detected by using real time quantity PCR,and the XIAP level was detected by using western blot,the EGFP protein was also detected after HuR silencing in presence of pC1 XIAP 3'UTR or pN1 XIAP CR in polyamine deficient cells.Silencing HuR completely prevented the increased stability of XIAP mRNA in polyamine-deficient cells,as after HuR-SiRNA transfetion,the stability of XIAP mRNA in polyamine deficient cells decreased with its half-life similar to that measured in control cellsFurthermore,in HuR-silenced cells,the increase in XIAP protein levels after polyamine depletion was also prevented and was reduced to the levels observed in control populations.These results strongly suggest that the increased XIAP mRNA stability and XIAP protein after polyamine depletion results from the enhanced interaction of HuR with XIAP mRNA.Comparing to control pasmid,the reporter EGFP C1 protein level increased～1.5 fold in presence of pEGFP C1-XIAP 3'UTR,EGFP N1 level increased～2 fold in presence of pN1XIAP CR,and HuR silencing prevent this increase. 7.XIAP and HuR are Crucial for Induced Resistance of Polyamine-deficient Cells to ApoptosisHere,we first examined the spontaneous apoptotic cell death without any challenge of apoptotic stimulators after inhibition of HuR expression by using siHuR in the absence of cellular polyamines.Transfection with siHuR for 48 h prevented the increased expression of HuR and XIAP in polyaminedeficient cells,failed to directly induce apoptosis.There were no apparent differences in cell viability in DFMO-treated cells transfected with C-siRNA compared with DFMO-treated cells transfected with siHuR.No morphological features of apoptosis or detectable levels of active caspase-3 protein were obtained in polyamine-deficient cells after HuR silencing.Second,we determined whether HuR silencing altered the polyamine depletion-mediated resistance to apoptosis after exposure to tumor necrosis factor-α(TNF-α) plus cycloheximide(CHX).When control cells were exposed to TNF-α/CHX for 4 h,morphological features characteristic of programmed cell death were observed.The assessments of apoptosis was confirmed by an increase in levels of active caspase-3 protein and its enzyme activity(Figure 9B,left) after treatment with TNF-α/CHX.Consistent with our previous studies,exposure of polyamine-deficient cells to the same doses of TNF-α/CHX caused no apoptosis.There were no differences in the morphological features and percentages of apoptotic cells when comparing cells treated with DFMO alone and DFMO treated cells exposed to TNF-α/CHX for 4 h.This increased resistance to TNF-α/CHX-induced apoptosis was not altered when polyamine-deficient cells were transfected with C-siRNA,but it was lost when HuR expression was silenced by siHuR. The percentages of apoptotic cells and levels of active caspase-3 protein and its enzyme activity in DFMO-treated cells transfected with siHuR were significantly increased compared with those observed in DFMO-treated cells transfected with C-siRNA after exposure to TNF-α/CHX.8.Glycyrrhizic acid regulate ATF-2 expression posttrancriptionly through induced HuRCytoplsmic HuR increased after glycyrrhizic acid treated for 48h.We detected the binding between HuR and ATF-2 mRNA by pull down and RNP IP,the results showed that the associaction between HuR and ATF-2 mRNA and ATF-2 expression increased after treatment.Conclusions:These results indicate that 1) HuR regulates the stability and translation of XIAP mRNA through interaction with both XIAP 3'UTR and CR and 2) HuR-mediated XIAP expression plays an important role in the regulation of IEC apoptosis.3) glycyrrhizic acid regulates the ATF-2 expression through increased HuR and binding between HuR and ATF-2.