Role Of ER Stress And PERK Signaling Pathway In The Mechanism Of Skeletal Fluorosis

Background:The mechanism of how osteoblasts were activated and how bone turnover wasaccelerated in chronic fluorine poisoning is a key issue to be solved in thepathogenesis of endemic fluorosis. In previous research, we found that the proteinrelated to endoplasmic reticulum stress (ERS) including binding immunoglobulinprotein (BiP), protein disulfide isomerase (PDI), proteasome26S ATP enzyme andthioredoxin increased in osteoblasts exposure to fluorosis. Through culturingosteoblasts in the presence of fluoride in vitro, combined with fluorosis animal, theaim of our current study was to observe the expression of double-strandedRNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK),ATF4and Nrf2, and to detect the changes of regulatory factors related to osteoblastproliferation and differentiation. We analyzed the effect of PERK on osteoblastproliferation, differentiation and bone formation. The role of endoplasmic reticulumstress and PERK signaling pathway in osteoblast activation and bone turnoveracceleration induced by fluoride was discussed. This project was helpful to find newfactors and signaling pathway in skeletal fluorosis never studied before, and wouldprovide new insights into the prevention strategies of skeletal fluorosis.Methods:Animal experiments in vivo:100Wistar rats were divided evenly into controlgroup, low-fluoride group and high-fluoride group, according to the weight. Fluoridewas given by gavage to rats, with NaF (2.21kg/L) dissolved in distilled water, equal to100mg/mL fluoride. Rats in low-fluoride group were given fluoride by gavage, with10mg F-/kg per day, while high-fluoride group20mg F-/kg per day, and control group gavaged with distilled water. After given fluoride1,2and3month later, rats weretreated with ether to collect blood and separate serum for biochemical testing,separately. One tibia was taken to test bone mineral density, using Hologic DiscoveryWA absorptiometry. One femur was treated with10%EDTA, conventionaldehydration, embedded in paraffin, and made into pathological section at last. Theother femur was used to conduct Real time-PCR to test the expression of relatedgenes.MC3T3-E1experiments in vitro: Cells were implanted into96-well plates for1,3,7and14day, and each period was divided into control group and0.1,1.0,2.0,4.0,8.0,16.20,32and64mg F-/L group, using CCK-8for cell proliferation assay.According to the trend of cell activity, we selected2,8and20mgF-/L asrepresentative concentration. Cells were implanted into24-well plates and dividedinto control group,2,8and20mgF-/L group for staining of ALP and mineralizednodules. We used immunofluorescence assay to detect BiP expression in differentfluorine conditions. For knocking down of BiP and PERK, we appliedON-TARGETplus siRNA BiP and PERK to transfected MC3T3-E1and analyzedgene expression related to osteogenesis and endoplasmic reticulum stress byRealtime-PCR and Western blot.Results:Animal experiments results:1. Using gavage to copy fluorosis animal models showed low mortality rate andhigh modulus, and presented the complete process from skeletal fluorosis to dentalfluorosis.2. Results of bone pathology, bone density and osteoblasts related genesexpression confirmed pathological changes in skeletal fluorosis that osteoclastdominated early, osteoblast/osteoclast actived metaphase and osteoblast dominatedfinally.3. Fluoride caused unfolded protein response, and up-regulated PERK, ATF6andXbp1. Changes of PERK signal and bone resorption enhancement was consistent, and changes of Nrf2was consistent with osteogenic factor.MC3T3-E1experiments results:1. Fluoride had dual effect on osteoblast that low-dose fluoride stimulated cellactivity, while high-dose fluoride suppressed cell activity.2. Low-dose fluoride stimulated osteogenic factors expression in osteoblast whilehigh-dose fluoride suppressed them, which changes were bilateral.3. Fluoride significantly stimulated BiP and UPR signaling pathway expression.Down-regulation of BiP and PERK suppressed UPR pathway, and osteogenictranscription factors expression were decreased.Conclusions：1. This experiment copied skeletal fluorosis by intragastric administration, whichcharacterized the acceleration of bone turnover in animal model. Pathological changeswere bone resorption in early and bone formation in laterly.2. Fluoride caused unfolded protein response, including PERK, ATF6and Xbp1to participate in the process of bone metastasis. PERK was consistent withthe osteoclastic transcription factor, and Nrf2was consistent with theosteogenic transcription factors, which demonstrated that PERK and Nrf2had somekind of correlation with bone turnover and acceleration in fluorosis.3. Experiment using osteoblast exposured to fluoride in vitro further clarifiedPERK and its downstream factor Nrf2played an important role in bone formation andresorption enduced by fluoride, and proved endoplasmic reticulum stress and PERKsignal pathway played an important role in the pathogenesis of skeletal fluorosis.