Cyclophilin A Regulates The Apoptosis Of A549 Cells By Stabilizing Twist1 Protein

Apoptosis is a programmed death in animal cells that maintains a balance between cell survival and death.Cell apoptosis has been considered an important mechanism to prevent the emergence of tumors for a long period of time,thus inducing apoptosis could contribute to cancer treatment.However,apoptosis signals are complex and are regulated at multiple levels.Among them,Twistl protein plays a crucial role in the signaling pathway of cell apoptosis.Twistl is a highly conserved member of the basic helical-loop-helix transcription factor subfamily of Twist.Increasing studies have shown that Twistl also serves as a key regulator of NF-κB-mediated cellular immunity and inflammatory responses.NF-κB pathway has been recognized as a canonical pro-inflammatory signaling pathway,while NF-κB inhibitors have been shown to induce apoptosis.However,CypA enhances NF-κB nuclear translocation and transcriptional activity,thereby increasing the production of proinflammatory cytokines.In addition,CypA facilitates to maintain the stability of the NF-κB subunit p65.Therefore,we speculate that CypA may be involved in the regulation of NF-κB-mediated Twist1.Therefore,this study mainly explores the stability of CypA on Twistl and its regulation of its intracellular function,further expanding the biological functions of Twistl post-translational modification and downstream target gene transcription,laying the foundation for the development of clinical methods for treating cancer with CypA and Twist1.The main findings are as follows:1.CypA promotes Twistl-p65 interaction and nuclear accumulationOur previous studies have demonstrated that CypA is critical for SeV-induced activation of the NF-κB signaling pathway.In addition,studies indicated that CypA interacts with p65,a subunit of NF-κB.However,p65 has been found to increase the nuclear expression of Twistl and interact with Twistl after TNF-α stimulation.In this study,we further explored whether CypA regulates Twistl expression via NF-κB signaling pathway.We first experimentally verified that CypA promotes TNF-α and IL-1β activating NF-κB signaling.Further,co-immunoprecipitation assay was used to showed that overexpression of CypA promoted Twistl-p65 interaction and complex formation.Wild-type A549 cells(A549/CypA+)and A549 cells with shRNA knockdown of CypA(A549/CypA-)cells were transfected with siRNA of p65(sip65),and the nuclear accumulation of Twistl-p65 complex was observed under a laser confocal microscope.The results suggest that CypA regulates the subcellular localization of Twistl via p65.These results collectively suggest that CypA increases Twist1-p65 complex formation and nuclear accumulation.2.CypA interacts with Twist1 to promote the transcriptional activity of Twist1It was found that overexpression of CypA significantly increased the transcriptional activity of Twist1 through the luciferase reporter system assay.However,Twist1 reporter luciferase activity was significantly inhibited in A549/CypA-or A549/CypA+cells after CsA treatment.At the protein expression level,Co-immunoprecipitation results showed that Twist1 interacts with endogenous CypA,and this interaction depends on the PPIase activity of CypA.At the level of post translational modification,nuclear and cytoplasmic translocation of Twist1 is an important manifestation.In this experiment,A549 cells were subjected to immunofluorescence analysis,and it was found that CypA can regulate the subcellular localization of Twist1.In general,the results indicate that CypA is a key regulator of Twist1 transcription activity.3.The regulation of CDH1 and CDH2 by CypA is dependent on Twist1qPCR assay using siRNA of Twist1(siTwist1)or negative control siRNA(siNeg)transfected A549/CypA+and A549/CypA-cells showed that CDH1 expression was significantly increased in A549/CypA-cells compared with A549/CypA+cells,in contrast,the expression of CDH2 was significantly decreased.This difference in CDH1 and CDH2 gene expression,however,it was not significant in siTwist1-transfected cells or in Twist1-expressing loss-of-function cells(Twist1-ΔDBD).Interestingly,it was significant in wild-type cells.These results indicate that Twist1 transcriptional activity is required in part for CypA-mediated regulation of CDH1 and CDH2 transcription.4.CypA mediates A549 cell apoptosis by regulating Twist1 proteinThe ability of the CypA-Twist1 signaling pathway to regulate A549 cell death was assessed by examining the viability of A549 cells in the presence of the protein synthesis inhibitor cycloheximide(CHX).CHX treatment significantly reduced the viability of A549/CypA-cells compared with A549/CypA+cells.However,the effect of CypA on cell viability was increased in Twist1-overexpressed A549/CypA-cells.Fluorescence microscopy was used to analyze the nucleus to determine whether the death of A549 cells was caused by apoptosis.In the absence of CHX,no significant apoptosis induction was detected,while CHX treatment significantly induced apoptosis of A549/CypA-cells,which showed brighter nuclear contraction,which is related to abnormal DNA chromatin condensation and nuclear fragmentation.Furthermore,overexpression of Twist1 reduced aberrant condensation of DNA in A549/CypA+cells,suggesting that the CypA-Twist1 signaling pathway regulates viability versus apoptosis in A549 cells.Furthermore,Caspase-3 levels were found to be significantly elevated in A549/CypA-cells compared with A549/CypA+cells experimentally,suggesting that the CypA-Twistl signaling pathway regulates viability and apoptosis of A549 cells.5.CypA regulates the phosphorylation and ubiquitination of TwistlBy examining Twistl phosphorylation in A549/CypA-cells and A549/CypA+cells,we found that knockdown of the CypA gene significantly reduced the phosphorylation at the Ser68 site of Twistl in a dose-dependent manner.However,overexpression of the CypA recombinant vector significantly increased the phosphorylation of the Ser68 site of Twistl in a dose-dependent manner,suggesting that CypA promotes the phosphorylation of the Twistl Ser68 site in A549 cells.Using co-immunoprecipitation assay,we found that overexpression of CypA significantly reduced the ubiquitination of Twist1.Furthermore,in co-immunoprecipitation experiments,CypA was found to attenuate K48-linked but not K63-linked ubiquitination of exogenous and endogenous Twist1.In summary,CypA stabilizes Twistl protein by inhibiting K48-linked ubiquitination of Twist1.