Regulation Of Nrf1 And Nrf2 In Osteoclast Differentiation And The Role Of Nrf2 In Arsenic-induced Dysfunction Of Bone Metabolism

Objective:Environmental inorganic arsenic?iAs?pollution is a significant global problem for human populations.Natural sources of iAs in the soil and sedimentary rock can be released to drinking water and food.Exposure to iAs is associated with various diseases including cancer,hypertension,diabetes,cardio-vascular disorders,skin lesions,and metabolic disorders among others.The biokinetics of iAs accumulation are complex,and levels will decline in the liver,spleen,gastrointestinal tract approximately one month after an initial iAs exposure,but then begin to accumulate in the bone.This metalloid is known to replace phosphorus and can localize in bone matrix,where it may remain for years.Some epidemiological studies report that iAs exposure increases the risk of various bone disorders.Exposure to iAs?0.05 and 0.5 ppm,12 weeks?in the drinking water,at levels relevant to human drinking water exposure,can alter bone microstructure and bone mineral density?BMD?in rats.In vitro low concentrations of iAs?0.5 or 1?M?reduce osteoblast differentiation in bone marrow cells through an extracellular signal-regulated kinase?ERK?dependent signaling pathway.Osteoclast precursor cells produce hydrogen peroxide?H₂O₂?after exposure to low level iAs?2.5and 5?M?and thereby undergo differentiation.It is suspected that iAs exposure can increase bone resorption/remodeling by promoting osteoclast differentiation.Bone is a living and dynamic tissue that is constantly being remodeled.The remodeling process is a delicate balance between the activities of osteoblasts and osteoclasts.Interference with this balance results in a variety of diseases that affect bone integrity.Tipping the balance in favor of osteoclasts leads to pathological bone resorption,which is observed in osteoporosis,autoimmune arthritis and periodontitis.Osteoclasts are giant multinucleated bone-resorbing cells formed by the fusion of their mononuclear precursors as monocytes and macrophages.Osteoclast differentiation of precursors can be stimulated by the receptor activator of nuclear factor-?B ligand?RANKL?and macrophage colony-stimulating factor?M-CSF?produced by various types of cells,in particular osteoblasts.RANKL binding to RANK triggers the terminal differentiation into osteoclasts that takes place through intracellular kinase cascades and nuclear genetic programs coordinated by essential transcription factors such as c-Fos,NF-?B and nuclear factor of activated T cells,cytoplasmic 1?NFATc1?.NFATc1expression and activity in response to RANKL and TNF require intact NF-?B activation at the early stage of osteoclastogenesis.Osteoclasts are well known to contain abundant mitochondria but they are also able to rely on glycolytic?anaerobic?metabolism to generate the ATP needed to power their activity.Osteoclasts show impressive metabolic flexibility during the course of differentiation to meet the increased demand for adenosine triphosphate?ATP?.Osteoclast differentiation is associated with a metabolic reprogramming that is characterized by an increased expression of glucose and glutamine transporters,followed by glycolysis and glutaminolysis.Up-regulation of LDH activity during osteoclastogenesis promotes both glycolysis and mitochondrial respiration,consequently potentiating mature osteoclast formation via nuclear factor of activated T cell?NFAT?c1 signaling.Nuclear factor erythroid 2-related factor 1?NRF1,also known as NRF1?,a ubiquitously expressed CNC-bZIP protein,is an important regulator of the antioxidant response,proteasome homeostasis,genetic stability,mitochondrial respiration,inflammation,lipid metabolism and cell differentiation.NRF1 plays critical roles in regulating glucose metabolism,mitochondrial function,and insulin secretion.Silencing of Nrf1 in MIN6 cells altered expression of glucose metabolic enzymes,with induction of high-affinity hexokinase 1 and suppression of low-affinity glucokinase,which is associated with the elevated basal insulin release and reduced glucose responsiveness.Nrf1-deficient mouse livers,the Nrf1 deficiency leads to the reduced expression of the transcriptional coactivator genes Lipin1 and PGC-1?.Cold adaptation induces Nrf1 in BAT to increase proteasomal activity and that this is crucial for maintaining ER homeostasis and cellular integrity,specifically when the cells are in a state of high thermogenic activity.Nrf1 emerges as a guardian of brown adipocyte function,providing increased proteometabolic quality control for adapting to cold or to obesity.NRF1 interacted with C/EBP?provide an important role in the transcriptional regulation of DSPP in odontoblast.In undifferentiated odontoblasts,NRF1 and C/EBP?repress DSPP promoter activity individually and synergistically by cooperatively interacting with each other.In fully differentiated odontoblasts,the loss of interaction between NRF1 and C/EBP?results in an increased DSPP transcription.Ascorbic Acid can induce embryonic and mesenchymal stem cells to differentiation into osteoblasts.AA modulated osterix expression via a novel mechanism involving NRF1 nuclear translocation and NRF1 binding to ARE to activate genes critical for cell differentiation.However,the expression and function of NRF1 in osteoclastogenesis have not yet been reported.The binding of RANKL to its receptor RANK leads to the recruitment of TRAF6to the cytoplasmic domain of RANK,and thus results in the activation of several distinct signaling cascades such as NF-?B and MAPKs?including JNK,ERK,and p38?which are known to play crucial roles in osteoclastogenesis.Activation of the p38cascade results in downstream stimulation of events which help control expression of genes encoding for TRAP and cathepsin K,indicating the importance of this signaling cascades.It is now generally accepted that intracellular ROS can be produced to serve as second messengers in response to a variety of stimuli.ROS such as H₂O₂ and superoxide,act as intracellular signaling molecules following RANKL signaling during osteoclastogenesis.Nrf2 plays a protective role against xenotoxic stress through controlling the expression of genes encoding for many cytoprotective antioxidant enzymes,such as HO-1,NQO1,and G6PD.Intensifying intracellular ROS signaling by perturbation of cytoprotective antioxidant mechanisms is linked to stimulation of osteoclastogenesis.Nrf2 can inhibit RANKL-induced osteoclast differentiation by regulating cellular redox status through control of the expression of oxidative response genes.Indeed,one previous study showed that H₂O₂ is involved in the differentiation of osteoclastic precursor cells exposed to iAs.This is consistent with the present study where iAs stimulated Nrf2-KD cells to produce more ROS than cells expressing Nrf2normally,and this overproduction of ROS increased osteoclast differentiation.Methods:1.5-month-old female C57BL/6 mice were randomly divided into sham groups and OVX groups,which were further randomly assigned into control group?distilled water?and iAs exposure groups?5 ppm and 20 ppm,inorganic arsenite?iAs^??:inorganic arsenate?iAs^??=1:1?.Following 3 months of exposure to iAs,BMD of the mice were determined by the dual energy X-ray detector.RAW 264.7 cell line and bone marrow hematopoietic stem cells?BMHSC?primarily isolated from C57BL/6mice were used to study the in vitro effects of iAs on osteoclast differentiation and underlying mechanisms.2.We treated 40 week-old Nrf2^+/+and Nrf2^-/-mice with 5 ppm arsenic in the drinking water,which produces a blood arsenic level similar to humans living in areas where arsenic exposure is endemic.After 4 months,Micro-CT and dual-energy x-ray analysis revealed a drastic overall decrease in the bone volume with arsenic treatment in mice lacking Nrf2.RAW 264.7 cell line and bone marrow hematopoietic stem cells?BMHSC?primarily isolated from C57BL/6 mice were used to study the in vitro effects of iAs on osteoclast differentiation and underlying mechanisms.3.We treated 12 week-old Nrf1M???-KO and Nrf1-KI mice with OVX,after 8 weeks,Micro-CT and dual-energy x-ray analysis revealed a drastic decrease in bone volume in Nrf1?M??-KO mice.RAW 264.7 cell line and bone marrow hematopoietic stem cells?BMHSC?primarily isolated from C57BL/6 mice were used to study the in vitro effects of Nrf1 on osteoclast differentiation and underlying mechanisms.Results:1.In the sham groups,the femoral BMD of the mice that have been exposed to 20 ppm of iAs for 3 months was substantially decreased?P=0.0539?compared to control mice.As expected,the BMD of the OVX groups was significant decreased compared to the sham control group?p<0.0539?.However,the BMD among the OVX groups showed no significant difference.In vitro studies using RAW 264.7 cells and BMHSC showed that iAs^?evidently affected the osteoclast differentiation in a concentration-dependent fashion.Low concentrations of iAs^?exposure significantly augmented osteoclast differentiation in the two cell models while high concentrations showed inhibitory effect.N-acetyl-cysteine pretreatment significantly abolished low-level iAs^?-induced augmentation of osteoclast differentiation in a concentration-dependent fashion.2.Here,we show that female mice lacking Nrf1 specifically in myeloid lineage?Nrf1?M??-KO?developed a worsened ovariectomy-induced osteoporosis,as determined by a Dual-energy x-ray absorptiometry and Micro-computed tomography.Notably,Nrf1?M??-KO mice exhibited an elevated osteoclast activity in the metaphysis of femurs,showing increased number of osteoclasts and tartrate-resistant acid phosphatase?TRAP?activity.The bone marrow monocytes?BMMs?isolated from Nrf1?M??-KO mice and RAW 264.7 cells with stable silencing of Nrf1?Nrf1-KD?showed osteoclast lineage commitment,resulting in augmented RANKL-induced osteoclast differentiation.In response to RANKL and M-CSF treatment,BMMs from Nrf1?M??-KO mice and Nrf1-KD RAW 264.7 cells showed significant increases in TRAP activity,expression of osteoclast-specific genes and bone-absorbing activity.Mechanistic studies revealed that Nrf1 deficiency in mononuclear precursor cells disturbed the cell metabolism,leading to elevated glycolysis and increased ROS production followed by elevated expression of NF-?B and NFATc1.The RANKL-induced osteoclastogenesis and induction of NFATc1 in Nrf1-deficient cells were substantially mitigated by glycolytic inhibition and antioxidant.3.Deficiency of Nrf2 in RAW 264.7 cells or bone marrow-derived macrophages?BMMs?promoted arsenic-induced osteoclast differentiation.Lack of Nrf2 increases arsenic-induced ROS levels and phosphorylation of p38.N-Acetyl-cysteine and SB203580 pretreatment essentially abolished arsenic-induced phosphorylation of p38and reversed arsenic-induced increased osteoclast differentiation in Nrf2 deficiency.Conclusion:1.The inhibitory effect of iAs on bone metabolism is dependent on the availability of ovary function,suggesting that iAs may interfere with estrogen metabolism and/or function to disturb bone metabolism.Oxidative stress induced by iAs exposure stimulates osteoclast differentiation,and increased osteoclast differentiation may be involved in the reduction of BMD caused by chronic iAs exposure.These preliminary findings suggest that antioxidant intervention may be an effective approach to prevent osteoporosis induced by chronic iAs exposure.2.These findings identify NRF1 as a critical suppressor for the RANKL stimulation of osteoclastogenesis by targeting NFATc1 signaling.Nrf1 is a potential negative regulator of osteoclastogenesis.Nrf1 also acts as a negative regulator in RANKL-induced osteoclastogenesis by down-regulating NFATc1,in part,through accelerating glycolytic and oxidative metabolism.3.Loss of Nrf2 causes increased oxidative stress and enhanced susceptibility to arsenic-induced bone loss.Nrf2-KO mice are highly sensitive to bone loss due to drinking water iAs due to elevated osteoclastogenesis.Further study in vitro demonstrated that iAs increased osteoclastogenesis through RANKL-mediated ROS production and phosphorylation of p38,and this was inhibited by Nrf2-mediated up-regulation of cytoprotective enzymes.These findings strongly suggest that suppressed expression of functional Nrf2 in osteoclast progenitor cells may be a risk factor in iAs-induced bone loss and related bone diseases.