Nuclear Factor-kappa B Regulates Adenine Nucleotide Translocator 1 Gene Transcription And Leads To Mitochondrial Dysfunctions

BackgroundMitochondria are responsible for supplying cytoplasmic adenosine triphosphate(ATP)to support cellular activities and are the main intracellular source of reactive oxygen species(ROS).Increasing evidence implicated the accompanied role of mitochondrial dysfunction in age-related neurodegenerative disorders,including Alzheimer's disease(AD),Parkinson's disease(PD)and Huntington's disease(HD).AD is the most common form of dementia.It is characterized by extracellular deposition of a specific protein,beta-amyloid peptide fibrils,and is accompanied by extensive loss of neurons.This inflammatory response in neurons(neuroinflammation)includes activation of microglia,astrocytes,macrophages and lymphocytes,resulting in the release of inflammatory mediators such as cytokines,chemokines.neurotransmitters and ROS.The presence of neuroinflammation and oxidative stress is also a common feature in neurodegenerative diseases.Thus,the mechanisms underlying mitochondrial dysfunctions and inflammation signaling require clarification.Adenine nucleotide translocator 1(ANTI),located in the inner mitochondrial membrane,accounts for up to 10 percent of total mitochondrial protein contents.ANT1 plays a key role in catalysing the exchange of cytosolic adenosine diphosphate(ADP)to intramitochondrial ATP for cellular energy supply,basal uncoupling or proton leak and the mitochondrial permeability transition pore(mPTP)complex.Thus,ANT1 is of great importance in mitochondrial functions.Mitochondria are important targets of pro-inflammatory cytokines.and interrelated factors may contribute to the mitochondrial dysfunction associated with inflammation.nuclear factor kappa B(NF-?B)is a critical regulator of genes involved in immuno-inflammatory responses,tumorigenesis and apoptosis.Inactivated NF-?B is sequestered in cytoplasm and bounded to the I?B family of inhibitor proteins,while the activated NF-?B translocate into the nucleus to bind with target genes when activated.Tumour necrosis factor ?(TNFa)and lipopolysaccharide(LPS),the common NF-?B activators,had been reported to cause mitochondrial dysfuctions.Previous study showed that TNFa could repress ANTI expression,but the mechanism remains unclear.In conclusion,we focused on the regulation of ANTI in NF-?B signaling pathway.This subject may provide new insights in the pathogenesis of neurodegenerative disorders and new therapy in these diseases.ObjectiveThe aim of' this study was to investigate the molecular regulatory mechanism of ANT1 gene by NF-?B.Materials and methods1.NF-?B regulates the ANTI mRNA expression.1.1 HEK293 cells were transfected by pIKK,pNF-KB and their controls pcDNA3.1mychis to elevate the expression of IKK and NF-?B or not.The whole RNA was extracted by TRIzol and RT-PCR was applied to examine the mRNA expression of ANTI,TNF?,I?B? by their specific primers.?-actin was used as a reference gene.1.2 NF-?B signaling inhibitor JSH-23 or activator H2O2 was added into HEK293 cells.The whole RNA was extracted by TRIzol and RT-PCR was applied to examine the mRNA expression of ANT1,I?B? by their specific primers.?-actin was used as a reference gene.2.NF-?B regulates the ANTI protein expression.2.1 HEK293 cells were transfected by pNF-KB and added by NF-cB signaling inhibitor JSH-23.The whole protein were extracted and WB was applied to examine the endogenous ANTI and p65 protein expression level.(3-actin was used as a reference gene.2.2 T98G cells were induced by NF-?B signaling activator TNFa(10ng/ml)for 0,90,180 and 360min.The nuclear protein were extracted and EMSA was applied to examine the NF-?B signaling activities.Meanwhile,the whole RNA was extracted and Real-time PCR was used to examine the ANTI mRNA expression level.2.3 T98G cells were induced by NF-?B signaling activator TNFa(10ng/ml)for 0,90,180 and 360min.The whole protein were extracted and WB was applied to examine the endogenous ANT1,I?B? and COX-IV protein expression level.?-actin was used as a reference gene.2.4 Over-expressed ANTI plasmid p3xflag-cmv-ANT1 was transfected into T98G cell.And then TNF? was added for 0.90.180 and 360min.The whole protein were extracted and WB was applied to examine the exogenous ANTI protein expression level.?-actin was used as a reference gene.3.The transcriptional regulation of ANT1 gene by NF-?B.3.1 Jaspar software(Jaspar 2016)was used to predicte the potential NF-?B responsive elements in ANTI gene promoter.3.2 Deletions of plasmids encoding ANTI promoters(pANT1-lucs)were constructed.The 5' upstream region of human ANTI gene was obtained by PCR of genomic DNA isolated from BAC-human-rp4 and subcloned into promoterless pGL3-Basic vector.HEK293 cells were co-transfected by pNF-?B and pANT1-lucs and the luciferases were examined by Dual-luciferase assay.3.3 ChIP assay were used to identify the NREs in ANTI promoter.3.4 EMSA assay were used to identify the NREs in ANTI promoter.4.Statistical AnalysisSPSS was used to analysis statistics.Student's t tests and one-way ANOVA were applied to compare different groups.Differences were classified as significant at P<0.05.Results1.NF-?B decreases ANTI mRNA expression level.1.1 Over-expressed NF-?2B and IKK could decrease the endogenous ANT1 mRNA expression.Meanwhile,the mRNA expression of TNF? and I?B? the canonical target genes of NF-?B signaling pathway,were decreased.It is indicated that the activation of NF-?B signaling pathway could repress the transcription of ANTI gene.1.2 Activation or inhibition of NF-?B signaling pathway could decrease or increase the mRNA expression level of ANT1,indicating that ANT1 gene is one of the downstream gene of NF-?B signaling pathway.2.NF-?B decreases ANTI protein expression level.2.1 Consist with the mRNA expression,over-expressed NF-?B could decrease the endogenous protein expression of ANTI,while the NF-?B signaling inhibitor could increase the endogenous protein expression of ANT1.2.2 TNFa could activate the endogenous NF-?B signaling pathway,and the activity of nuclear translocation was increased at 90 min,consisting with the mRNA and protein expression level.2.3 TNFo? has no impact on exogenous ANT1 protein expression,indicating that NF-?B decrease the mRNA and protein expression of ANT1 by repressing the transcription of ANTI.3.The NREs locate at +1?+20bp and +41?+61 bp of ANTI promoter.3.1 Dual-luciferase assay showed that NF-?B could decrease the deletions of ANT1-lucs,and the potential NREs were narrowed to +1?+61 bp.ChIP assay also confirmed the combination of NREs and ANTI promoter.3.2 Jaspar software predicted the potential NREs were +2?+14bp.+20?+32bp and+49?+61 bp of ANTI promoter.3.3 EMSA assay identified the NREs were +1?+20bp and +41-+61bp of ANTI promoter.Conclusions1.NF-?B could decrease the mRNA and protein expression of ANT1.2.The transcription of ANTI was regulated by NF-?B,and ANTI is one of'downstream genes of NF-?B signaling pathway.3.NF-?B bound to ANTI prompter at +1?+20bp and +41?+61 bp.ObjectiveThe aim of this research was to identify the effects of NF-?B activation on mitochondrial functions.Materials and methods1.Cell transfections1.1 Transfections of T98G.T98G cells were transfected by ANTI knockdown plasmid(pANT1)and its control(pSiCON)using Lipofectamine 2000.Cells were collected after 72h and WB were applied to examine the knockdown level of endogenous ANTI expression,?-actin was used as a reference gene.1.2 Extraction of fetal rat neurons.Primary neurons were isolated from 17-18 days embryos of rat.Rat forebrains were minced with scalpels and digested in StemPro Accutase cell dissociation reagent.Cells were cultured for 5-7 days before use.1.3 Transfections of fetal rat neurons.ANTI knockdown lentivirus(LV-SiANT1)and its control(LV-SiCON)were transfected in fetal rat neurons for 72h.WB was applied to examine the knockdown level of endogenous ANTI protein expression.?-actin was used as a reference gene.2.(Cell apoptosis-TNF?(10ng/ml)was added inT98G cells for 0,90.180 and 360 min.WB was applied to examine the protein expression of cleaved-caspase3 and total-caspase3.P-actin was used as a reference gene.3.Determination of ATP-ADP exchange rate in T98G cells.-TNFa treated and pANT1 or pSiANT1 transfected T98G were permeated by digitonion.After the addition of ADP(2mM)and Magnesium Green K+ salt(1?M),Magnesium Green fluorescence was recorded in Varioskan flash instruments respectively using 505nm(Ex)and 535 nm(Em).4.Determination of ATP levels of T98G and neurons.T98G cells were transfected by pSiANT1 and pSiCON and fetal rat neurons were infected by lentivirus LV-SiANT1 and LV-SiCON.ATP determination kit and 20/20 Glomax luminometer were applied to examine the ATP levels.5.Determination of mPTP opening levels of T98G and neurons.Calcin-AM(2?M)was added in T98G and neurons for 30min at 37?.After washing cells by PBS,CoCl2(1mM)was added for another 30min.After washing cells for three times,half of the cells were left as is,and the other half were treated with 500 ?M CaCl2,ionomycin(5?M),CATR(1?M)or BKA(5?M)to trigger or inhibit mPTP opening for another 30 min.Fluorescence was recorded in Varioskan flash instruments respectively using 494nm(Ex)and 517nm(Em).which indicated the mPTP opening level.6.Ca2+-induced mitochondrial swelling in T98G cells TNFa(10ng/ml)was added inT98G cells for 3h and T98G cells were transfected by pSiANT1 and pSiCON for 48h.Mitochondria were isolated and resuspended in mitochondrial swelling buffer.After the addition of CaCl2(100?M).the absorbance at 540 nm was recorded for 600s indicating the swelling of mitochondria.7.Determination of mitochondrial membrane potential(??m)of T98G cells and neurons.TNF?(10ng/ml)was added for 3h,T98G cells were transfected by pSiANT1 and pSiCON and fetal rat neurons were infected by lentivirus LV-SIANT1 and LV?SiCON.After the addition of TMRM(50nM)for 90min at 37?,fluorescence microscope was applied to examine the TMRM.8.Determination of mitochondrial ROS of T98G cells and neurons.TNF?(10ng/ml)was added for 3h,T98G cells were transfected by pSiANT1 and pSiCON and fetal rat neurons were infected by lentivirus LV-SiANT1 and LV-SiCON.After the addition of DCFH-DA(5?M)or DHE(5?M)for 20 or 30 min at 37?,FACScan flow cytometer was applied to examine the ROS production.Results1.WB results showed that pSiANT1 and LV-SiANT1 could decrease the endogenous ANTI protein expression to 50%.2.WB results showed that the addition of TNFa in T98G for 0.90,180 and 360 min had no effect on cell apoptosis.3.The determination of ATP-ADP exchange rate showed that the rate in ANT1 over-expressed T98G was much higher than control,while the rates were decreased in ANTI knockdown and TNFa treated T98G cells.4.Compared to control groups.the ATP levels were decreased in ANT1 knockdown and TNF? treated T98G cells.Meanwhile,the ATP levels were decreased in ANTI knockdown and TNFa treated neuronal cells.5.Compared to control groups,the mPTP levels were decreased in ANTI knockdown and TNF? treated T98G cells.Meanwhile,the ATP levels were decreased in ANT1 knockdown and TNFa treated neuronal cells.6.Compared to control groups,the Ca2+-induced mitochondrial swelling levels were decreased in ANTI knockdown and TNFa treated T98G cells.It is indicated that the decrement of the Ca2+-induced mitochondrial swelling was caused by the decrease of TNFa-induced ANTI expression.7.Compared to control groups,the mitochondrial membrane potential(??m)were increased in ANT1 knockdown or TNFa treated T98G cells and ANTI knockdown or TNF? treated neuronal cells.8.Compared to control groups,the ROS production were increased in ANTI knockdown or TNFa treated T98G cells and ANTI knockdown or TNFa treated neuronal cells.Conclusions1.Activation of NF-?B could decrease the mitochondrial ATP-ADP exchange rate.2.Activation of NF-?B could decrease the cellular ATP level.3.Activation of NF-?B could decrease the mitochondrial mPTP opening.4.Activation of NF-?B could decrease the Ca2+-induced mitochondrial swelling.5.Activation of NF-?B could increase the mitochondrial membrane potential.6.Activation of NF-?B could increase the cellular ROS production.