Giardia Duodenalis-induced Intestinal Epithelial Cell Cycle Arrest And Apoptosis Involve Activation Of Endoplasmic Reticulum Stress

Giardia duodenalis,a zoonotic protozoan parasite that causes diarrhea and chronic gastroenteritis in the host,causes huge economic losses annually and poses a major threat to global public health security.Giardia infection can inhibit the proliferation of intestinal epithelial cells and induce cell death,resulting in intestinal epithelial cells damage.Giardia infection have clinical symptoms such as diarrhea,abdominal pain,vomiting and malnutrition among others.Metronidazole is the front-line treatment for Giardia infection,but drug resistance problems hinder its therapeutic efficacy.Therefore,it is very important to explore pathogenic mechanisms of Giardia infection in intestinal epithelial cells and find new potential targets for drug action.Recently,endoplasmic reticulum(ER)stress is considered to be an important mechanism involved in parasite infection and drug resistance.Pathogen infection can cause an imbalance of host cell homeostasis,accumulation of misfolded proteins in the ER causes ER stress.In order to restore ER homeostasis and prolong survival of cells,the unfolded protein response(UPR)is activated.When prolonged ER stress signals are too strong,ER homeostasis cannot be reestablished and cells promote cell death through UPR.Our previous study found that Giardia could induce ER stress in intestinal epithelial cells,but the molecular mechanisms remain unclear.In this study,a model of interaction between Giardia and intestinal epithelial cells in vitro was established to explore the mechanism of proliferation and death of intestinal epithelial cells regulated by ER stress induced by Giardia,providing a theoretical basis for further understanding of the pathogenesis of Giardia and seeking potential drug targets.In this study,an interaction model of Giardia with Caco-2 cells or HT-29 cells was established.CCK-8 was used to detect cell viability and determine the interaction ratio and duration of Giardia with cells.The results showed that Giardia could inhibit the activity of Caco-2 and HT-29 cells.The most significant inhibitory effect of cell activity was obtained when the ratio of Giardia to cells was 10:1.This ratio was applied in subsequent experiments.The cell cycle distribution was detected by flow cytometry after the treatment of Giardia.The results showed that the percentage of cells in the G0/G1 phase was increased(p < 0.01),and the percentage of cells in the S phase was decreased significantly(p < 0.05),suggesting that Giardia infection could arrest the cell cycle in G0/G1 phase.The m RNA and protein levels of cyclins and cyclin-dependent kinases(CDKs)were detected at different time points of Giardia stimulation.The results of q PCR and western blot showed a trend for reduction of m RNA and protein expression levels of cyclins(cyclin A,cyclin B,cyclin D,and cyclin E)and CDK proteins(CDK1,CDK2,CDK4 and CDK6)at 6 h with Giardia exposure in both Caco-2 and HT-29 cells(p < 0.05).To explore the mechanism of cell cycle arrest regulated by Giardia,the m RNA and protein levels of members of the transcription factors E2 F family and Cip/Kip family were detected by q PCR and western blot,respectively.The results showed that the m RNA and protein levels of E2F1,E2F2 and E2F3 were significantly down-regulated(p < 0.05),the m RNA and protein levels of E2F4 were up-regulated(p < 0.01),and the m RNA levels of E2F5,E2F6,E2F7 and E2F8 were up-regulated after stimulation of intestinal epithelial cells with Giardia exposure for 6 h(p < 0.05).The results of co-immunoprecipitation showed that the formation of E2F1-RB complex could be promoted by Giardia stimulating intestinal epithelial cells.During the interaction between Giardia and intestinal epithelial cells,m RNA and protein expression levels of p21 and p27,members of the Cip/Kip family that regulate cell cycle progression,were significantly increased(p < 0.05),while the expression level of p53 was significantly decreased(p < 0.05).These results suggest that Giardia inhibited cell cycle process by up-regulating the expression of p21 and p27,but it did not activate the p53/p21 pathway.Together these experiments demonstrated that Giardia induced intestinal epithelial cell cycle arrest by up-regulating expressions of p21 and p27 and regulating the activity of transcription factor E2 F,thereby regulating the expression of cyclins and CDKs.To explore the mechanism of cell cycle regulation that involves activation of ER stress,this study first detected the expression of ER stress marker Glucose-regulating protein 78(GRP78).The q PCR and western blot results showed that Giardia stimulation could significantly upregulate the m RNA and protein levels of GRP78(p < 0.05),which proved that Giardia could activate ER stress in intestinal epithelial cells.Western blot results showed that three UPR signaling pathways(IRE1,PERK and ATF6)were activated by Giardia exposure.The production of reactive oxygen species(ROS)was detected by ROS assay kit and showed that Giardia infection had increased levels of ROS.The results showed that ROS serve as an upstream regulator of UPR,and activated IRE1,PERK and ATF6 pathways.In order to further explore the mechanism of cell cycle regulation involving UPR,this study detected key molecules in the regulation of cell cycle after treatment with UPR pathway inhibitors.These results jointly demonstrated that Giardia up-regulated expressions of p21 and p27 by activating IRE1,PERK and ATF6 pathways,promoted the expression of E2 F transcriptional repressor factors,and inhibited the activity of E2 F transcriptional activators by forming E2F1-RB complex,thus down-regulating protein expressions of cyclins and CDKs and causing G0/G1 cell cycle arrest.UPR and ER-associated degradation(ERAD)are two key quality control mechanisms that maintain ER homeostasis.In this study,m RNA expression levels of ER chaperone proteins Pdia6,P58 ipk and ERO1 were up-regulated,and m RNA levels of EDEM,ERDj4 and Hrd1 involved in ERAD were up-regulated detected by q PCR,proving that Giardia can activate the ERAD pathway in intestinal epithelial cells.Ufd1 acts as a ubiquitin-recognizing protein that promotes the clearance of misfolded proteins via the ERAD pathway.Ufd1 maintains Skp2 stability by antagonizing Skp2 ubiquitination,which mediates p21 and p27 degradation.This study investigated whether Giardia infection regulates the expression of p21 and p27 through Ufd1-Skp2.The results showed that the m RNA levels of Ufd1 and Skp2 could be down-regulated after Giardia stimulated intestinal epithelial cells.Skp2 knockdown in intestinal epithelial cells was performed by si RNA interference,and the results showed that Skp2 knockdown promoted the accumulation of p21 and p27,suggesting that Giardia can induce the up-regulation of p21 and p27 protein levels in intestinal epithelial cells through the Ufd1-Skp2 pathway.In order to explore the mechanism of ER stress involved in the regulation of apoptosis,this study first detected the levels of apoptosis marker proteins CASP3 and PARP cleavage.The results showed that the levels of CASP3 and PARP cleavage were increased by Giardia exposure,that is,the levels of cl-CASP3 and cl-PARP increased significantly(p < 0.05).At the same time,Acridine orange(AO)/Ethidium bromide(EB)results also showed that Giardia stimulation could significantly increase the number of apoptotic cells.The IRE1,PERK and ATF6 pathways were inhibited respectively,and the levels of key apoptosis proteins CASP3 and PARP cleavage and the number of apoptotic cells were detected.The results showed that inhibition of IRE1 pathway could increase the levels of CASP3 and PARP cleavage and the number of apoptotic cells,and inhibition of PERK or ATF6 pathway could decrease the levels of CASP3 and PARP cleavage and the number of apoptotic cells compared with Giardia stimulation alone.In order to further explore the mechanism of IRE1 regulating apoptosis,this study examined activation of AKT and JNK pathways.Western blot results showed that Giardia stimulation could up-regulate the phosphorylation levels of AKT and JNK.Inhibition of IRE1 pathway could reduce the phosphorylation of AKT and increase the phosphorylation of JNK,thus increasing the levels of CASP3 and PARP cleavage and the number of apoptotic cells.These results suggested that activation of PERK and ATF6 pathways promoted apoptosis during Giardia infection,and the activation of the IRE1 pathway partially inhibited apoptosis by increasing the phosphorylation level of AKT and decreasing the phosphorylation level of JNK.The secreted proteins of Giardia may play a key role in the pathogenesis of Giardia.During the interaction between Giardia and intestinal epithelial cells,the expressions of Peptidyl-prolyl cis-trans isomerase B(PPIB)and Pyridoxamine 5’phosphate oxidase(PNPO)were significantly increased,but the specific roles were not clear.In this study,recombinant PPIB and PNPO expressed in prokaryotes were used to interact with intestinal epithelial cells to explore mechanisms of secreted proteins PPIB and PNPO in regulating cell cycle and apoptosis.The results showed that the recombinant proteins PPIB and PNPO could induce ROS accumulation,up-regulate the expression of GRP78,activate IRE1,PERK and ATF6 pathways,and effectively reduce the expressions of key cell cycle proteins cyclin D,cyclin E,CDK2,CDK4 and CDK6.The levels of CASP3 and PARP cleavage were increased.In order to explore mechanisms of ER stress regulation of cell cycle induced by recombinant PPIB and recombinant PNPO,the expression levels of cell cycle key proteins were detected by western blot in intestinal epithelial cells treated by ROS inhibitor,IRE1 inhibitor,PERK inhibitor and ATF6 inhibitor.The results indicated that recombinant PPIB and PNPO could regulate expressions of p21 and p27 through ROS accumulation and activation of UPR response,thus regulating key molecules of cell cycle.In order to explore the mechanism of regulating apoptosis by recombinant PPIB and PNPO,the levels of apoptosis marker proteins CASP3 and PARP cleavage were detected in intestinal epithelial cells after treatment with IRE1 inhibitor,PERK inhibitor and ATF6 inhibitor.The results showed that both recombinant PPIB and PNPO proteins promoted apoptosis through PERK and ATF6 pathways,while inhibited apoptosis through IRE1 pathway.The mechanisms of apoptosis regulated by AKT and JNK pathways were detected after cells treated with IRE1 inhibitor,AKT inhibitor and JNK inhibitor.The results showed that recombinant PPIB and PNPO activated IRE1 pathway to up-regulate phosphorylation of AKT and reduce phosphorylation of JNK,thus inhibiting apoptosis.These results indicated that mechanisms of recombinant PPIB and PNPO activating ER stress and regulating cell cycle and apoptosis was consistent with that of Giardia,suggesting that the secreted proteins PPIB and PNPO may be potential key virulence proteins of Giardia.In conclusion,this study demonstrated that Giardia infection activated UPR response by could inducing ROS accumulation.The three UPR signaling pathways(IRE1,PERK and ATF6)all regulated the protein expressions of p21 and p27,and regulated the activity of E2 F transcription factors,thus regulating protein expressions of cyclins and CDKs,and inducing cell cycle arrest.ERAD activated by Giardia infection regulated the cell cycle through the Ufd1-Skp2 pathway,which regulated the protein expressions of p21 and p27.Activation of PERK and ATF6 could promote the occurrence of apoptosis,while activation of IRE1 pathway could up-regulate the phosphorylation of AKT and reduce the phosphorylation of JNK,thus inhibiting partial apoptosis.Giardia recombinant proteins PPIB and PNPO could activate ER stress and participate in the regulation of cell cycle and apoptosis,which may be potential virulence proteins of Giardia.This study provided a theoretical basis for the regulation of ER stress on the pathogenesis of Giardia,and provided a new direction for the study of potential virulence proteins of Giardia.