Role Of ER Stress In The Mechanism Of Skeletal Fluorosis

Skeletal fluorosis is complex and diverse pathological changes, and itspathogenesis has plagued the medical fiels for many years, a lot of academicdisagreement appears. Recently, it was demonstrtated that high bone turnoverand active osteoblast function play a key role in the occurance and developmentof skeletal fluorosis through in vivo and in vitro experiments. The boneturnover and ostelblast function had been recognized as the target for probinginto the skeletal fluorosis. The endoplasmic reticulum (ER) stress is the keyto study the relationship between the active osteoblast function and oxidativestress. The ER is a central organelle of each eukaryotic cell as the place oflipid synthesis, protein folding and protein maturation. Proteins of the plasmamembrane, secreted proteins as well as proteins of the Golgi apparatus andlysosomes fold into their tertiary and quaternary structure in the ER. ER stressis induced by accumulation of unfolded protein aggregates (unfolded proteinresponse, UPR) or by excessive protein traffic. We copied the skeletal fluorosismodel via rats drinking tap water containing100mg F-/L for12weeks. To observeendoplasmic reticulum stress in bone tissue of fluorosis rats and furtherexplores the pathogenesis of skeletal fluorosis. The immunohistochemicalstaining showed that the anti-BiP antibody positive osteoblasts was much morein the low calcium diet and coexposure to fluoride groups than that in thecontrol, and coexposure to fluoride elevated the positive cells compared tothat of only low calcium diet group. RT-PCR analysis indicated mRNA expressionof Bip/GAPDH in bone tissue is markedly higher in bone of low-calcium groupand100mgF-/L plus low-calcium group compared with the control. The expressionof Xbp1/GAPDH in bone tissue significantly increased in100mgF-/L pluslow-calcium groups compared with the control and the low-calcium group; andCHOP/GAPDH in bone tissue significantly increased in100mgF-/L plus low-calcium groups compared with the control. It showed the accelerated osteogenetic actionin fluorosis rats, accompanied by oxidative stress and bone endoplasmicreticulum stress, and both were likely involved in the pathogenesis of skeletalfluorosis.We utilized the OS732cell line (human osteoblast-like cell line) as thein vitro model of osteoblast exposed to fluoride. Osteoblasts and osteoclastsare responsible for the turnover between bone formation and bone resorption,respectively. In this study, fluoride largely stimulated the expression ofcbfa1, a critical transcription factor for osteoblast differentiation andbone formation.Meantime, we examined the key signaling molecules that regulatedosteoclastogenesis, RANKL and OPG. The bone resorption rate is affected bythebalance of RANKL and OPG. The OPG and RANKL expressions were various in OS732cells exposed to different dose and period of fluoride, which may indirectlyreflected the diversity of fluoride action on bone tissue. In other words, thealtered expression of cbfa1, OPG and RANKL induced by the different doses andtime of fluoride exposure may preliminarily interpret the complex picture ofskeletal responses to fluoride exposure.The PRK-like ER kinase (PERK) signaling, one branch of unfolded proteinresponse (UPR) to combat ER stress, is a transcription factor needed forosteoblast proliferation and differentiation. Here we studied the effect offluoride on PERK signaling genes and x-box binding protein1(xbp-1) in OS732cells. We found that early and continuous fluoride exposure increased thebinding immunoglobulin protein (BiP) expression and activated the PERKsignaling pathway, resulting in activation of transcription factor4(ATF4)and nuclear factor erythroid2-related factor2(Nrf2). These results showedfluoride impelled a distinctive ER stress response in OS732cells, primarilyby activating PERK and PERK-dependent signaling. Little effects were viewedfor activating xbp-1, a common target of the other two canonical sensors ofER stress, ATF6and IRE1. In this study the altered expression of bone turnover genes were consistent with activation of ER stress and PERK signaling. Thisstudy proved that PERK signaling play major roles in action of fluoride onosteoblast, and suggested that bone response in skeletal fluorosis might bedue in part to PERK signaling pathway.