CRIF1 Modulates Oxidative Stress In Bone Marrow Mesenchymal Stem Cells After Irradiation Through PKC-δ/NRF2 Signaling Pathway

Background:With the wide application of nuclear technology,the risk of acute radiation injury is increasing.There is an urgent need to solve problems such as radiation protection,treatment for radiation sickness and side effects prevention of tumor radiotherapy.The body’s exposure to a large dose irradiation in a short time period may cause acute radiation sickness(ARS).According to the exposure dose,pathological changes and clinical characteristics,ARS can be divided into the hematopoietic,gastrointestinal and cerebrovascular syndromes.After exposure to doses of 1-9Gy,hematopoietic-ARS(h-ARS)may be obtained,while after exposure to higher radiation levels(6-9Gy),a hematopoietic stem cells(HSCs)transplant may be required because HSCs are extremely sensitive to radiation.However,previous studies indicated that this treatment does not significantly improve survival.Transplanted HSCs might effectively replenish HSC quantity,but these cells could not effectively exert the hematopoietic function in a radiation-destroyed hematopoietic microenvironment.Therefore,the destruction of the hematopoietic microenvironment in the bone marrow upon irradiation might be the main cause for HSC transplantation failure.Bone marrow mesenchymal stem cells(BMMSCs)are a major component of the hematopoietic microenvironment,providing physical support and secreting a variety of cytokines for regulating HSC self-renewal,differentiation,and retention.BMMSCs are more resistant to radiation than HSCs,and this relative radiation resistance of BMMSCs is generally thought to be an important contributing factor in their ability to rescue hematopoiesis after radiation damage.The mechanisms of BMMSCs radio-resistance may involve efficient DNA damage recognition,double strand break repair,and apoptosis evasion.However,the molecular mechanisms underlying BMMSCs radiation tolerance have not been fully elucidated.Upon exposure to ionizing radiation,intracellular reactive oxygen species(ROS)attack cellular macromolecules,and DNA double strand break especially represents a critical cellular injury with many pathogenetic consequences.Nuclear factor E2 p45-related factor 2(NRF2)is a major stress-response transcription factor that activates a variety of cytoprotective and antioxidant genes,playing a crucial role in defense against ROS-mediated cellular injury.Under normal conditions,NRF2 is constantly ubiquitinated by the KEAP1-Cul3 ubiquitin ligase E3 complex,resulting in proteasomal degradation in the cytoplasm.External stresses induce the dissociation of NRF2 from KEAP1,resulting in NRF2 nuclear translocation and the activation of its downstream target genes through binding to antioxidant-responsive element(ARE)motifs.Previous studies found that oxidative stress-induced covalent modification of KEAP1 and PKC-mediated phosphorylation of serine 40 in NRF2 both contribute to the dissociation of NRF2 from KEAP1.Therefore,NRF2 may be important for BMMSCs radiation resistance due to its potent regulatory effect during oxidative stress.However,few studies to date have clarified the relationship between NRF2 and BMMSCs radiation resistance.CR6-interacting factor 1(CRIF1)is a multifunctional protein that was first identified as a regulator of cell cycle and growth.This protein is also known to interact with some transcription factors,and is a constitutive protein of the large mitoribosomal subunit.CRIF1 deficiency leads to various defects including the deterioration of mitochondrial function,which results in high levels of mitochondrial ROS.Here,we show that CRIF1 plays an important role in modulating BMMSCs oxidative stress after radiation,and explore the regulating molecular mechanisms of NRF2 signaling pathway by CRIF1.Materials and Methods:1.Primary human BMMSCs were derived from bone marrow samples obtained from healthy donors informed consent by density-gradient centrifugation method;the cultured cells were characterized;2.Since h-ARS was usually induced by up to 9 Gy dose irradiation,in this research cultured BMMSCs were irradiated with 9 Gy by Co-60 at a dose rate of 700 c Gy/min.While the radiation tolerance of mice was less than human beings,according to reported literatures,Nrf2-/-mice were exposed to total body irradiation(TBI)at 5 Gy by Co-60 at the same dose rate as before;3.BMMSCs were transfected with Crif1 sh RNA lentiviral vector or empty vectors as control,knocking down protein expression levels of CRIF1;4.The expression levels of target proteins in the cells were detected by western blotting analysis;5.The relative m RNA expression levels of target gens of BMMSCs were detected using the RNA isolation,c DNA synthesis and quantitative-PCR;6.Immunocytochemistry analysis was used for observing the sub-cellular localization of CRIF1 and NRF2 in BMMSCs by laser confocal microscope;7.Immunoprecipitation and co-immunoprecipitation were used for detecting the interaction of CRIF1 and PKC-δ in BMMSCs;8.Annexin-V/PI staining for cell detecting apoptosis was analysised by flow cytometry;9.SA-β-gal staining was used for detecting cell senescence;10.The levels of intracellular ROS were quantified using the DCFH-DA or DHE probes;11.The GSH content was determined in BMMSCs by the GSH assay kit;12.The counts and the ROS levels of quadri-color labeled BMMSCs in Nrf2-/-mice wereanalysised by flow cytometry;the expression of CRIF1 in these cells were measured by indirect FACS.Results:1.Radiation increased oxidative stress in BMMSCs,as well as induced cell senescence1.1 Intracellular ROS levels of BMMSCs increased immediately after 9 Gy irradiation.ROS levels appeared to peak within 4 h after irradiation,and 24 h later levels had recovered to a degree,but remained elevated compared to the normal state.Meanwhile,9 Gy of irradiation remarkably elevated the glutathione(GSH)content in BMMSCs.These results suggested that oxidative stress in BMMSCs significantly increased at the early phase after high-dose irradiation.1.2 The apoptotic cell rate increased only from 8.29% to 9.45% after radiation as revealed by Annexin V and PI staining,suggesting that BMMSCs were relatively radioresistant.1.3 The cell senescence indicated by SA-β-gal staining showed that 9 Gy of irradiation at 72 h significantly increased BMMSCs senescence.Several proteins related to cell senescence were detected by WB after radiation at 48 h and 72 h.The results showed that protein levels of RB,p53 and p21 significantly increased(but no remarkable change of p16 was observed),which confirmed that cell senescence was induced by irradiation.2.Knockdown of CRIF1 exacerbated BMMSCs oxidative stress after irradiation2.1 CRIF1-knockdown BMMSCs presented higher intracellular ROS levels after irradiation than the control cells at all time points,especially at 4 h,and even 24 h after irradiation the ROS levels were still notably higher than resting state levels.Additionally,GSH content rose higher after irradiation in CRIF1-deficient BMMSCs than in the control cells.2.2 The cell apoptosis rate significantly increased in CRIF1-deficient BMMSCs after irradiation at 48 h compared to control cells.2.3 Furthermore,the proportion of senescent cells significantly increased in CRIF1-deficient BMMSCs after irradiation compared to the proportions in control cells.The proteins(RB,p53,and p21)related to cell senescence significantly increased in CRIF1-deficient BMMSCs compared to levels in control cells after irradiation,with the exception of p16.3.Radiation not only elevated protein levels of NRF2 and CRIF1,but also induced their nuclear accumulation.3.1 NRF2 and CRIF1 protein levels increased significantly at early phases following irradiation,especially from 4-8 h after radiation.NRF2 began to return to baseline levels at 12 h,however,there was no obvious decrease of CRIF1 up to 24 h after radiation.3.2 We also observed that NRF2 downstream target proteins and m RNAs such as Gclc and Ggt1 showed similar expression patterns as NRF2 in BMMSCs after irradiation.3.3 To assess the effect of irradiation on nuclear translocation of NRF2 and CRIF1,the cytoplasm and nuclear fraction proteins were extracted and detected by western blotting and subcellular localization was observed using immunocytochemistry.The western blotting results and fluorescence images showed that the nuclear translocation of NRF2 and CRIF1 markedly increased upon radiation,and these two proteins were co-localized in BMMSCs nuclei after radiation.These data indicated that CRIF1 is likely to be involved in NRF2 regulation due to its protein level and nuclear translocation in BMMSCs after irradiation.3.4 We then used a Nrf2-/-mice model to evaluate the relationship between NRF2 and CRIF1 in BMMSCs after 5 Gy of TBI.The results agreed with our hypothesis that quadri-color labeled BMMSCs were decreased in Nrf2-/-mice compared to control mice,meanwhile the ROS levels in these cells were significantly elevated in Nrf2-knockout mice.However,the results of indirect fluorescent staining showed that CRIF1 expression increased in BMMSCs after 5 Gy TBI,whether mice were deficient in NRF2 or not.4.Knockdown of CRIF1 resulted in reduced NRF2 protein level and nuclear translocation in BMMSCs after radiative injury4.1 NRF2 protein levels were decreased in CRIF1-deficient BMMSCs irrespective of irradiation,however,q PCR results indicated that the relative m RNA expression of Nrf2 was not significantly changed in CRIF1-deficient BMMSCs,suggesting that CRIF1-knockdown affects NRF2 at the protein level.4.2 After irradiation,NRF2 protein levels in CRIF1-deficient BMMSCs were less than NRF2 levels in control cells at all time points.Moreover,the peak time point of NRF2 expression appeared earlier,at 4 h instead of at 8 h in the control.4.3 The cytoplasmic and nuclear protein levels determined by western blotting and subcellular localization observed using immunocytochemistry consistently indicated that NRF2 nuclear translocation was markedly reduced in CRIF1-deficient BMMSCs after irradiation compared to nuclear translocation in control cells.4.4 Furthermore,the increased degrees of NRF2-dependent proteins(HO1,GGT1 and GCLC)expression in CRIF1-deficient BMMSCs were also decreased after irradiation,and NRF2 target genes such as Ho1,Ggt1 and Gclc were also decreased.These results demonstrated that CRIF1-knockdown aggravated oxidative stress in BMMSCs after irradiation and influenced stem cell death,which might be concerned with NRF2 pathway.5.Interaction between CRIF1 and PKC-δ contributed to NRF2 Ser40 phosphorylation and NRF2 stability.5.1 The phosphorylation of NRF2 Ser40 by PKC-δ is a critical mechanism for releasing NRF2 from KEAP1 and translocating it into the nucleus.To determine the mechanism underlying CRIF1-mediated regulation of protein expression level and subcellular localization of NRF2,we first determined the level of NRF2 Ser40 phosphorylation after irradiation.As expected,NRF2 Ser40 phosphorylation was significantly reduced in CRIF1-deficient BMMSCs compared to control cells when without irradiation.Similarly,NRF2 Ser40 phosphorylation in CRIF1-deficient BMMSCs significantly decreased compared to phosphorylation in control cells at all time points after irradiation.5.2 Co-IP experiments demonstrated that PKC-δ and CRIF1 interacted with each other.5.3 To verify the role of CRIF1 in PKC-δ activity,three PKC agonists were utilized.PMA was used as a potent agonist,and Prostratin and Oleic acid were used as moderately efficient agonists.After agonist exposure,we found that NRF2 Ser40 phosphorylation significantly increased in control cells while phosphorylation was suppressed in CRIF1-deficient BMMSCs.Meanwhile,expression of the NRF2-induced protein HO1 followed the same pattern.Taken together,our results suggested that CRIF1 likely co-activates PKC-δ and can regulate oxidative stress of BMMSCs after irradiation,mainly through the PKC-δ/NRF2 pathway.Conclusions:1.At the early phase after 9 Gy of irradiation,the oxidative stress in BMMSCs was aggravated.Intracellular ROS levels and GSH contents were both significantly raised;the cell senescence significantly increased,but the cell apoptosis did not obviously change;The proteins(RB,p53,p21)related to cell senescence were significantly increased at 72 h after irradiation,with the exception of p16.2.CRIF1 knocking down aggravated oxidative stress in BMMSCs after 9 Gy irradiation than controls,and cell apoptosis and cell senescence than control cells;the proteins(RB,p53,p21)related to cell senescence significantly increased in CRIF1-deficient BMMSCs compared to levels in control cells at 72 h after irradiation,with the exception of p16.3.CRIF1 affected the NRF2 protein expression on the protein level,and acted as a factor facilitating the nuclear translocation of NRF2 after radiation;4.CRIF1 regulated the nuclear translocation of NRF2 by co-activating PKC-δ to phosphorylate NRF2 Ser40.In a word,co-activation of PKC-δ by CRIF1 modulates the oxidative stress in BMMSCs after irradiation by phosphorylating NRF2 Ser40,to alleviate radiative injury in BMMSCs.